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AbstractCaralluma europaea Guss. (C. europaea) is a medicinal plant used for cancer treatment. However, these treatments may be associated with complications that need to be investigated. This work aims to evaluate not only the chemical composition but also the hepatoprotective and anticancer properties of C. europaea extracts. The chemical constitution of the hydroethanolic extract was explored using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The hydroethanolic extract, flavonoids, and polyphenols-rich extract at 100, 15, and 50 mg/kg, respectively, were administered to acetaminophen-treated rats for seven days. We used Western blotting and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) to determine the protein and the mRNA levels of cancer stemness markers in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 exposed to increasing doses of C. europaea extracts. In silico analysis was used to evaluate the effects of phenolic compounds revealed in C. europaea on caspase-3 and HSP90, and on liver damage on CYP2E1. The primary phenolics detected by GC-MS and HPLC were ferulic acid and benzofurazan. The positive control group showed an increase in AST, ALT, ALP, triglycerides, and VLDL levels. C. europaea extracts demonstrated hepatoprotective effects by ameliorating acetaminophen-induced alterations of biochemical and hispathological parameters. Immunoblotting and RT-qPCR profiling of cancer stemness markers indicated a reduction in the expression levels of Oct-4 and Nanog proteins, as well as a reduction in the mRNA levels of CD133 by 50–60% and Sox2 by 80–90% in pancreatic cancer cells. Molecular docking showed that naringenin presented the highest docking Gscore on CYP2E1 (−8.199) and HSP90 (−7.742). In conclusion, C. europaea extracts could be considered as a safe and promising therapeutic strategy to sensitize pancreatic cancer cells to chemotherapy. [ABSTRACT FROM AUTHOR]

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25–50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY: Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility. [ABSTRACT FROM AUTHOR]

Objectives To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. Methods We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electronic medical records. Results PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. Conclusion Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement. [ABSTRACT FROM AUTHOR]

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n = 8; 5 ± 2 years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24 h after their first/naïve RE bout. Participants also engaged in 10 weeks of resistance training and donated VL biopsies before and 24 h after their last RE bout. VL biopsies were also examined in an acute cycling study (n = 7) and a study involving 2 weeks of leg immobilization (n = 20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3 h post‐RE (∼200%, P = 0.018) and returned to pre‐exercise levels by 6 h post‐RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3 h post‐RE (8.6 ± 6.3‐fold vs. 2.1 ± 0.7‐fold, P = 0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24 h post‐RE (+65% and +36%, P < 0.001); however, the last RE bout response was attenuated compared to the first bout (P = 0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2 weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P < 0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease‐associated muscle atrophy affect these outcomes is needed. Highlights: What is the central question of this study?How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation.What is the main finding and its importance?This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases. [ABSTRACT FROM AUTHOR]

Simple Summary: Non-small cell lung cancer (NSCLC) accounts for 80–85% of primary lung cancers. Radiation therapy is widely used to treat both primary NSCLC and lung cancer that has spread to the brain. However, radiotherapy responses are not durable and toxicity limits therapy. Therefore, there is an urgent need for new agents that can enhance the effectiveness of radiation therapy in lung cancer and reduce its toxicity. We find that AM-101, a benzodiazepine analog, enhances the effects of radiation and significantly improves the survival of mice with lung cancer brain-metastatic tumors. Additionally, AM-101 makes radiation treatment work better and slows down the growth of NSCLC subcutaneous xenograft tumors in mice. AM-101 activates the GABA(A) receptor in lung cancer cells, triggering selective autophagy by causing GABARAP to form multimers and stabilizing the mitochondrial receptor Nix. GABA(A) receptor activation may improve tumor control and allow for lower radiation doses, reducing toxicity. In non-small cell lung cancer (NSCLC) treatment, radiotherapy responses are not durable and toxicity limits therapy. We find that AM-101, a synthetic benzodiazepine activator of GABA(A) receptor, impairs the viability and clonogenicity of both primary and brain-metastatic NSCLC cells. Employing a human-relevant ex vivo 'chip', AM-101 is as efficacious as docetaxel, a chemotherapeutic used with radiotherapy for advanced-stage NSCLC. In vivo, AM-101 potentiates radiation, including conferring a significant survival benefit to mice bearing NSCLC intracranial tumors generated using a patient-derived metastatic line. GABA(A) receptor activation stimulates a selective-autophagic response via the multimerization of GABA(A) receptor-associated protein, GABARAP, the stabilization of mitochondrial receptor Nix, and the utilization of ubiquitin-binding protein p62. A high-affinity peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP–Nix multimerization axis that triggers autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. [ABSTRACT FROM AUTHOR]

BACKGROUND: A broad suite of bisphenol S (BPS) derivatives as alternatives for BPS have been identified in various human biological samples, including 4-hydroxyphenyl 4-isopropoxyphenylsulfone (BPSIP) detected in human umbilical cord plasma and breast milk. However, very little is known about the health outcomes of prenatal BPS derivative exposure to offspring. OBJECTIVES: Our study aimed to investigate the response of hepatic cholesterol metabolism by sex in offspring of dams exposed to BPSIP. METHODS: Pregnant ICR mice were exposed to 5μg/kg body weight (BW)/day of BPSIP, BPS, or E2 through drinking water from gestational day one until the pups were weaned. The concentration of BPSIP, BPS, or E2 in the plasma and liver of pups was determined by liquid chromatography-tandem mass spectrometry. Metabolic phenotypes were recorded, and histopathology was examined for liver impairment. Transcriptome analysis was employed to characterize the distribution and expression patterns of differentially expressed genes across sexes. The metabolic regulation was validated by quantitative real-time PCR, immunohistochemistry, and immunoblotting. The role of estrogen receptors (ERs) in mediating sex-dependent effects was investigated using animal models and liver organoids. RESULTS: Pups of dams exposed to BPSIP showed a higher serum cholesterol level, and liver cholesterol levels were higher in females and lower in males than in the controls. BPSIP concentration in the male liver was 1.22±0.25 ng/g and 0.69±0.27 ng/g in the female liver. Histopathology analysis showed steatosis and lipid deposition in both male and female offspring. Transcriptome and gene expression analyses identified sex-specific differences in cholesterol biosynthesis, absorption, disposal, and efflux between pups of dams exposed to BPSIP and those in controls. In vivo, chromatin immunoprecipitation analysis revealed that the binding of ERα protein to key genes such as Hmgcr, Pcsk9, and Abcg5 was attenuated in BPSIP-exposed females compared to controls, while it was enhanced in males. In vitro, the liver organoid experiments demonstrated that restoration of differential expression induced by BPSIP in key genes, such as Hmgcr, Ldlr, and Cyp7a1, to levels comparable to the controls was only achieved when treated with a combination of ERα agonist and ERβ; agonist. DISCUSSION: Findings from this study suggest that perinatal exposure to BPSIP disrupted cholesterol metabolism in a sex-specific manner in a mouse model, in which ERa played a crucial role both in vivo and in vitro. Therefore, it is crucial to systematically evaluate BPS derivatives to protect maternal health during pregnancy and prevent the transmission of metabolic disorders across generations [ABSTRACT FROM AUTHOR]

The youcai mosaic virus (YoMV) can infect a diverse array of crop species, such as Raphanus sativus, Brassica napus, Solanum nigrum, and Rehmannia glutinosa, causing substantial economic damage. This study aimed to develop a rapid, sensitive, and economical diagnostic method for YoMV. We successfully expressed and purified the recombinant His-CPYoMV-YZ protein in E. coli BL21, which was used to immunize New Zealand White rabbits, generating high-titer polyclonal antibodies (PAb-CPYoMV-YZ). Additionally, a serological-based reverse transcription loop-mediated isothermal amplification (S-RT-LAMP) assay was refined, combining serological and molecular detection techniques to enhance practicality. Utilizing PAb-CPYoMV-YZ, we developed four techniques for detecting YoMV: Western blot, dot immunoblotting assay, enzyme-linked immunosorbent assay (ELISA), and S-RT-LAMP. YoMV isolates from various regions and hosts were analyzed. The results indicated that PAb-CPYoMV-YZ was highly effective in detecting YoMV across a range of hosts and isolates from diverse regions. This study fills an important gap in the serological detection of YoMV and provides a practical tool for on-site diagnosis and control of YoMV infection. [ABSTRACT FROM AUTHOR]

Cervical malignancy (CC) is the 2nd most prevalent malignancy among females, leading to cancer mortality. Primary detection of CC tumors results in an improved prognosis. CC is a malignant gynecological tumor, with few treatment options. New diagnostic and therapeutic agents are required to expand patient survival and quality of life. If CC tumors can be found at an early stage, the prognosis is much brighter. New diagnostic and therapeutic agents are needed to increase patient survival and quality of life. In this work, we performed whole-exome sequencing utilizing V5 (Illumina platform) 10 samples, 5 control and 5 CC tumour tissue, and we compared the results with transcriptome studies. KMT2C variations were shown to be among the most vicious in this analysis. From an Indian viewpoint, we found a plethora of SNVs and mutations, including those with known, unknown, and possible effects on health. Based on our findings, we know that the KMT2C gene is on chr. Seven and in exon 8, all three recognized variants are missense, synonymous, coding synonymous, non-coding variants, and GnomAD MAF (− 0.05). The variation at position (7:152265091, T > A, SNV 62478356) in KMT2C is unique, potent, and pathogenic. The missense coding transcript CIQTNF maps to chromosome 7 and displays T > C SNV. In addition, we performed single strand conformational polymorphism analysis on 64 samples and further confirmed them using Sanger sequencing to understand and verify the mutations. KMT2C shows a log FC value of − 1.16. Understanding emerging harmful mutations from an Indian viewpoint is facilitated by our bioinformatics-based, extensive correlation studies of WES analysis. Potentially harmful and new mutations were found in our preliminary analysis; among these ten top mutated genes, KMT2C and CIQTNF were altered in ten cases of CC with an Indian phenotype. [ABSTRACT FROM AUTHOR]

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/. NEW & NOTEWORTHY: GelBox is open-source software that was developed to enhance rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the effect of plasma‐activated Ringer's lactate solution (PAL) on oral squamous cell carcinoma (OSCC) cells and carcinogenic processes with a particular focus on iron and collagenous matrix formation. Materials and Methods: We used three OSCC cell lines, one keratinocyte cell line, and two fibroblast lines, and cell viability assays, immunoblotting, flow cytometry, and transmission electron microscopy were performed to evaluate the effect and type of cell death. The effect of PAL treatment on lysyl oxidase (LOX) expression was investigated in vitro and in vivo. Tamoxifen‐inducible Mob1a/b double‐knockout mice were used for the in vivo experiment. Results: PAL killed OSCC cells more effectively than the control nontumorous cells and suppressed cell migration and invasion. Ferroptosis occurred and the protein level of LOX was downregulated in cancer cells in vitro and in vivo. Additionally, PAL improved the survival rate of mice and suppressed collagenous matrix formation. Conclusions: We demonstrated that PAL specifically kills OSCC cells and that ferroptosis occurs in vitro and in vivo. Furthermore, PAL can prevent carcinogenesis and improve the survival rate of oral cancer, especially tongue cancer, by changing collagenous matrix formation via LOX suppression. [ABSTRACT FROM AUTHOR]

Background: Here, we evaluated whether the histone lysine demethylase 5B (JARID1B), is involved in osteogenic phenotype commitment of periodontal ligament cells (PDLCs), by considering their heterogeneity for osteoblast differentiation. Materials and Methods: Epigenetic, transcriptional, and protein levels of a gene set, involved in the osteogenesis, were investigated by performing genome‐wide DNA (hydroxy)methylation, mRNA expression, and western blotting analysis at basal (without osteogenic induction), and at the 3rd and 10th days of osteogenic stimulus, in vitro, using PDLCs with low (l) and high (h) osteogenic potential as biological models. Results: h‐PDLCs showed reduced levels of JARID1B, compared to l‐PDLCs, with significant inversely proportional correlations between RUNX2 and RUNX2/p57. Epigenetically, a significant reduction in the global H3K4me3 content was observed only in h‐PDLCs. Immunoblotting data reveal a significant reduction in the global H3K4me3 content, at 3 days of induction only in h‐PDLCs, while an increase in the global H3K4me3 content was observed at 10 days for both PDLCs. Additionally, positive correlations were found between global H3K4me3 levels and JARID1B gene expression. Conclusions: Altogether, our results show the crucial role of JARID1B in repressing PDLCs osteogenic phenotype and this claims to pre‐clinical protocols proposing JARID1B as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Background Objectives Methods Results Conclusions Anti‐p200 pemphigoid is a rare autoimmune subepidermal blistering disease. Although the phenomenon of epitope spreading has been reported to be common in anti‐p200 pemphigoid, the association between its clinical and immunoserological features has yet to be elucidated.Our aim was to compare the clinical and immunoserological characteristics of anti‐p200 pemphigoid patients with and without epitope spreading.We performed a retrospective cohort study encompassing 30 patients with anti‐p200 pemphigoid between January 2015 and December 2022. The clinical and immunoserological characteristics of anti‐p200 pemphigoid were analyzed using combined immunoserological assays.Epitope spreading was observed in 11 of 30 patients (36.7%) with anti‐p200 pemphigoid. Compared with patients in the non‐epitope spreading group, patients in the epitope spreading group showed more heterogeneous clinical presentations (P = 0.018), a higher proportion of mucosal involvement (P = 0.003), higher Bullous Pemphigoid Disease Area Index (BPDAI) scores for skin erosions/blisters (P = 0.018), mucosal erosions/blisters (P = 0.001), activity (P = 0.017) and total scores (P = 0.022), and required a higher initial dose of prednisone for disease control (P = 0.040).This study supported the idea that anti‐p200 pemphigoid was prone to epitope spreading. Anti‐p200 pemphigoid patients with epitope spreading are more likely to present heterogeneous clinical phenotypes, frequent mucosal involvement, and a more severe and recalcitrant disease course. [ABSTRACT FROM AUTHOR]

To investigate the effects of Cirsilineol on cell viability, pyroptosis, and inflammation in hypoxia/reoxygenation-induced renal tubular cells, we utilized the renal tubular epithelial cell line HK2. The in vitro renal injury model was induced by hypoxia/reoxygenation. Cell viability in hypoxia/reoxygenation-induced HK2 cells was determined using a methylthiazolyldiphenyltetrazolium bromide assay and lactate dehydrogenase releasing kit. The effects of Cirsilineol on inflammation and pyroptosis in HK2 cells were evaluated using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot assays. Our results demonstrated that Cirsilineol promoted survival in hypoxia/reoxygenation-induced tubular cells, inhibited inflammation, and reduced pyroptosis in these cells. Further investigation revealed that the protective effects of Cirsilineol are mediated by the suppression of the Toll-like receptor 4/nuclear factor kappa B pathway, which mitigates the cellular response to hypoxia/ reoxygenation injury. Our study suggests that Cirsilineol protects renal tubular epithelial cells from hypoxia/reoxygenation injury by inhibiting inflammation and pyroptosis, highlighting its potential therapeutic value in renal conditions characterized by ischemic injury. [ABSTRACT FROM AUTHOR]

Cervical cancer (CC) is a common gynecological malignancy, and improving cisplatin sensitivity has become a hot topic in CC chemotherapy research. Polyphyllin I (PPI), a potent bioactive compound found in Rhizoma Paridis, known for its anticancer properties, remains underexplored in CC resistance. In this study, we evaluated PPI's impact on cisplatin-resistant CC cells and elucidated its underlying mechanism. Our findings reveal that PPI enhances the sensitivity of cisplatin-resistant CC cells to the drug, promotes apoptosis, and inhibits cell migration. Mechanistically, PPI was found to regulate p53 expression and its target genes, and suppressing p53 expression reverses PPI's sensitizing effect in drug-resistant CC cells. In conclusion, PPI showed promise in sensitizing cisplatin-resistant human CC cells to cisplatin treatment, suggesting that it could serve as a potent adjunct therapy for cervical cancer, particularly for cases that have developed resistance to cisplatin, thereby providing a promising basis for further clinical investigation into PPI for enhancing the efficacy of existing chemotherapy regimens in resistant cervical cancer. [ABSTRACT FROM AUTHOR]

Highlights: SPI1 and SMAD5 are upregulated during MC3T3-E1 cell osteogenic differentiation. SPI1 enhances SMAD5 transcription. Icariin increases SMAD5 expression by upregulating SPI1. The SPI1/SMAD5 axis underlies the promoting effect of icariin on MC3T3-E1 cell osteogenic differentiation. Background: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation. Methods: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays. Results: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells. Conclusion: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells. In dexamethasone-stimulated murine pre-osteoblast MC3T3-E1 cells, icariin upregulates SPI1 and thus enhances SMAD5 transcription, leading to enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Objective: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. Results: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies. [ABSTRACT FROM AUTHOR]

Background: Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC). Methods: We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis. Results: Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%). Conclusions: The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing. [ABSTRACT FROM AUTHOR]

The adaption of plants to stressful environments depends on long-distance responses in plant organs, which themselves are remote from sites of perception of external stimuli. Jasmonic acid (JA) and its derivatives are known to be involved in plants' adaptation to salinity. However, to our knowledge, the transport of JAs from roots to shoots has not been studied in relation to the responses of shoots to root salt treatment. We detected a salt-induced increase in the content of JAs in the roots, xylem sap, and leaves of pea plants related to changes in transpiration. Similarities between the localization of JA and lipid transfer proteins (LTPs) around vascular tissues were detected with immunohistochemistry, while immunoblotting revealed the presence of LTPs in the xylem sap of pea plants and its increase with salinity. Furthermore, we compared the effects of exogenous MeJA and salt treatment on the accumulation of JAs in leaves and their impact on transpiration. Our results indicate that salt-induced changes in JA concentrations in roots and xylem sap are the source of accumulation of these hormones in leaves leading to associated changes in transpiration. Furthermore, they suggest the possible involvement of LTPs in the loading/unloading of JAs into/from the xylem and its xylem transport. [ABSTRACT FROM AUTHOR]

Objective To screen and identify new human sperm specific antigen epitopes from candidate sperm-specific antigen epitope peptides using phage display random peptide library technolog and bioinformatics methods. Methods Candidate sperm-specific epitope peptide monomers and trimers were synthesized by solid phase polypeptide synthesis. Dot immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used to detect the immune reaction of the peptides with anti-sperm antibody (ASA) positive serum. ELISA was used to detect antibody titers against candidate sperm-specific epitope peptides in serum of immunized mice. The effects of the sera of the mice immunized with the candidate sperm-specific epitope peptide on human spermatozoa were examined by computer aided sperm analysis. Results High performance liquid chromatography and mass spectrometry showed that the purity and structure of the synthesized candidate sperm-specific epitope peptide were in accordance with the design. Blot and ELISA results indicated that the sperm-specific epitope peptide with the amino acid sequence n-TRRIHRHMTIRR-c and its trimers displayed strong immune response to 17 of 35 ASA positive sera. The serum from mice immunized with the peptide and its trimers significantly attenuated the sperm motility parameters compared with serum from the control group (P < 0. 05), with trimeric-induced decrease in motility parameters being particularly significant. (P<0.01). Conclusion The dodecapeptide with amino acid sequence n-TRRIHRHMTIRR-c is a novel human sperm-specific epitope peptide. [ABSTRACT FROM AUTHOR]

Objective: Myositis‐associated autoantibodies (MAAs) have been associated with overlap myositis, certain disease manifestations such as interstitial lung disease (ILD), and worse prognosis in the idiopathic inflammatory myopathies. MAAs overall remain largely uncharacterized in patients with juvenile‐onset myositis. Moreover, it is unknown whether the number of MAAs is associated with disease severity. Methods: Patients with juvenile myositis in cross‐sectional natural history studies who underwent testing for myositis autoantibodies were included. Demographics, myositis autoantibodies, clinical characteristics, medications received, and outcomes of those with and without MAAs were compared. Multivariable logistic regression was performed to determine whether the number of MAAs detected was associated with severe disease features. Results: Among 551 patients, 36% had an MAA and 13% had more than one MAA. Among those who were MAA positive, there was a higher frequency of overlap myositis (18% vs 5.9%, P < 0.001). MAA positivity was associated with certain clinical features, including Raynaud phenomenon (odds ratio [OR] 2.44, 95% confidence interval [CI] 1.41–4.28) and ILD (OR 3.43, 95% CI 1.75–6.96), as well as a chronic disease course (OR 1.72, 95% CI 1.10–2.72) and mortality (OR 3.76, 95% CI 1.72–8.43). The number of MAAs was also associated with mortality (OR 1.83, 95% CI 1.16–2.86). Conclusion: MAAs were prevalent in a large cohort of patients with juvenile myositis. ILD, refractory disease, and mortality were associated with MAA positivity. Prospective studies are needed to determine whether early detection of MAAs may lead to improved outcomes for patients with juvenile myositis. [ABSTRACT FROM AUTHOR]

Mitochondrial genes can be artificially relocalized in the nuclear genome in a process known as allotopic expression, such is the case of the mitochondrial cox2 gene, encoding subunit II of cytochrome c oxidase (C c O). In yeast, cox2 can be allotopically expressed and is able to restore respiratory growth of a cox2-null mutant if the Cox2 subunit carries the W56R substitution within the first transmembrane stretch. However, the COX2W56R strain exhibits reduced growth rates and lower steady-state C c O levels when compared to wild-type yeast. Here, we investigated the impact of overexpressing selected candidate genes predicted to enhance internalization of the allotopic Cox2W56R precursor into mitochondria. The overproduction of Cox20, Oxa1, and Pse1 facilitated Cox2W56R precursor internalization, improving the respiratory growth of the COX2W56R strain. Overproducing TIM22 components had a limited effect on Cox2W56R import, while overproducing TIM23-related components showed a negative effect. We further explored the role of the Mgr2 subunit within the TIM23 translocator in the import process by deleting and overexpressing the MGR2 gene. Our findings indicate that Mgr2 is instrumental in modulating the TIM23 translocon to correctly sort Cox2W56R. We propose a biogenesis pathway followed by the allotopically produced Cox2 subunit based on the participation of the 2 different structural/functional forms of the TIM23 translocon, TIM23MOTOR and TIM23SORT, that must follow a concerted and sequential mode of action to insert Cox2W56R into the inner mitochondrial membrane in the correct Nout–Cout topology. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs), which function as chaperones, are activated in response to various environmental stressors. In addition to their role in diverse aspects of protein production, HSPs protect against harmful protein-related stressors. Calycosin exhibits numerous beneficial properties. This study aims to explore the protective effects of calycosin in the heart under heat shock and determine its underlying mechanism. H9c2 cells, western blot, TUNEL staining, flow cytometry, and immunofluorescence staining were used. The time-dependent effects of heat shock analyzed using western blot revealed increased HSP expression for up to 2 h, followed by protein degradation after 4 h. Hence, a heat shock damage duration of 4 h was chosen for subsequent investigations. Calycosin administered post-heat shock demonstrated dose-dependent recovery of cell viability. Under heat shock conditions, calycosin prevented the apoptosis of H9c2 cells by upregulating HSPs, suppressing p-JNK, enhancing Bcl-2 activation, and inhibiting cleaved caspase 3. Calycosin also inhibited Fas/FasL expression and activated cell survival markers (p-PI3K, p-ERK, p-Akt), indicating their cytoprotective properties through PI3K/Akt activation and JNK inhibition. TUNEL staining and flow cytometry confirmed that calycosin reduced apoptosis. Moreover, calycosin reversed the inhibitory effects of quercetin on HSF1 and Hsp70 expression, illustrating its role in enhancing Hsp70 expression through HSF1 activation during heat shock. Immunofluorescence staining demonstrated HSF1 translocation to the nucleus following calycosin treatment, emphasizing its cytoprotective effects. In conclusion, calycosin exhibits pronounced protective effects against heat shock-induced damages by modulating HSP expression and regulating key signaling pathways to promote cell survival in H9c2 cells. [ABSTRACT FROM AUTHOR]

Hyperglycemia induces chronic stresses, such as oxidative stress and endoplasmic reticulum (ER) stress, which can result in β -cell dysfunction and development of Type 2 Diabetes Mellitus (T2DM). Ginsenoside Rk1 is a minor ginsenoside isolated from Ginseng. It has been shown to exert anti-cancer, anti-inflammatory, anti-oxidant, and neuroprotective effects; however, its effects on pancreatic cells in T2DM have never been studied. This study aims to examine the novel effects of Ginsenoside Rk1 on ER stress-induced apoptosis in a pancreatic β -cell line MIN6 and HFD-induced diabetic pancreas, and their underlying mechanisms. We demonstrated that Ginsenoside Rk1 alleviated ER stress-induced apoptosis in MIN6 cells, which was accomplished by directly targeting and activating insulin-like growth factor 1 receptor (IGF-1R), thus activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Bcl-2-associated agonist of cell death (Bad)-B-cell lymphoma-2 (Bcl-2) pathway. This pathway was also confirmed in an HFD-induced diabetic pancreas. Meanwhile, the use of the IGF-1R inhibitor PQ401 abolished this anti-apoptotic effect, confirming the role of IGF-1R in mediating anti-apoptosis effects exerted by Ginsenoside Rk1. Besides, Ginsenoside Rk1 reduced pancreas weights and increased pancreatic insulin contents, suggesting that it could protect the pancreas from HFD-induced diabetes. Taken together, our study provided novel protective effects of Ginsenoside Rk1 on ER stress-induced β -cell apoptosis and HFD-induced diabetic pancreases, as well as its direct target with IGF-1R, indicating that Ginsenoside Rk1 could be a potential drug for the treatment of T2DM. [ABSTRACT FROM AUTHOR]

BACKGROUND: Although sodium-glucose cotransporter-2 inhibitors have been shown to improve cardiovascular outcomes in general, little is presently known about any sex-specific changes that may result from this therapy. We sought to investigate and quantify potential sex-specific changes seen with the sodium-glucose cotransporter-2 inhibitor canagliflozin (CAN) in a swine model of chronic myocardial ischemia. STUDY DESIGN: Eighteen Yorkshire swine underwent left thoracotomy with placement of an ameroid constrictor. Two weeks postop, swine were assigned to receive either control (F = 5 and M = 5) or CAN 300 mg daily (F = 4 and M = 4). After 5 weeks of therapy, swine underwent myocardial functional measurements, and myocardial tissue was sent for proteomic analysis. RESULTS: Functional measurements showed increased cardiac output, stroke volume, ejection fraction, and ischemic myocardial flow at rest in male swine treated with CAN compared with control male swine (all p < 0.05). The female swine treated with CAN had no change in cardiac function as compared with control female swine. Proteomic analysis demonstrated 6 upregulated and 97 downregulated proteins in the CAN female group compared with the control female group. Pathway analysis showed decreases in proteins in the tricarboxylic acidic cycle. The CAN male group had 639 upregulated and 172 downregulated proteins compared with control male group. Pathway analysis showed increases in pathways related to cellular metabolism and decreases in pathways relevant to the development of cardiomyopathy and to oxidative phosphorylation. CONCLUSIONS: Male swine treated with CAN had significant improvements in cardiac function that were not observed in female swine treated with CAN. Moreover, CAN treatment in male swine was associated with significantly more changes in protein expression than in female swine treated with CAN. The increased proteomic changes seen in the CAN male group likely contributed to the more robust changes in cardiac function seen in male swine treated with CAN. [ABSTRACT FROM AUTHOR]

The article titled "Correction to 'integrin‐linked kinase regulates endothelial cell nitric‐oxide synthase expression in hepatic sinusoidal endothelial cells'" by Shafiei et al. published in Liver International in April 2015 contains a correction regarding errors in the figures. The authors identified mistakes in the beta‐actin lanes of Figure 2A,C, and in the total AKT lane in Figure 6B. The revised figures are provided in the article. The authors apologize for the errors. [Extracted from the article]

Background: Intervertebral disc degeneration (IDD) is an increasingly important cause of low back pain (LBP) that results in substantial health and economic burdens. Inflammatory pathway activation and the production of reactive oxygen species (ROS) play vital roles in the progression of IDD. Several studies have suggested that phillyrin has a protective role and inhibits inflammation and the production of ROS. However, the role of phillyrin in IDD has not been confirmed. Purpose: The purpose of this study was to investigate the role of phillyrin in IDD and its mechanisms. Study design: To establish IDD models in vivo, ex-vivo, and in vitro to verify the function of phillyrin in IDD. Method: The effects of phillyrin on extracellular matrix (ECM) degeneration, inflammation, and oxidation in nucleus pulposus (NP) cells were assessed using immunoblotting and immunofluorescence analysis. Additionally, the impact of phillyrin administration on acupuncture-mediated intervertebral disc degeneration (IDD) in rats was evaluated using various techniques such as MRI, HE staining, S-O staining, and immunohistochemistry (IHC). Result: Pretreatment with phillyrin significantly inhibited the IL-1β-mediated reduction in the degeneration of ECM and apoptosis by alleviating activation of the NF-κB inflammatory pathway and the generation of ROS. In addition, in vivo and ex-vivo experiments verified the protective effect of phillyrin against IDD. Conclusion: Phillyrin can attenuate the progression of IDD by reducing ROS production and activating inflammatory pathways. [ABSTRACT FROM AUTHOR]

Background. Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and potentially life-threatening mucocutaneous blistering diseases that clinically can resemble autoimmune bullous diseases. Moreover, it has been shown that autoantibodies against epidermal proteins are present in SJS/TEN. Objectives. To establish the presence of antibodies against desmosomal and hemidesmosomal proteins in confirmed SJS/TEN patients. Methods. Serum of SJS/TEN patients diagnosed based on clinical criteria, e.g., epidermal detachment with erosions and severe mucosal lesions, (suspicion of) a culprit drug, and matching histologic results was evaluated by various techniques, e.g., indirect immunofluorescence on monkey esophagus, salt split skin and rat bladder, immunoblotting (IB) and immunoprecipitation (IP), ELISAs against desmogleins and BP180, keratinocyte footprint assay, and keratinocyte binding assay. Results. A total of 28 patients were included in this study, 15 men and 13 women with a mean age of 56 years. In most patients, none of the serological tests were positive. In two patients, an elevated DSG3 titer was found suspicious for pemphigus vulgaris. Three patients had elevated NC16a titers, suggesting bullous pemphigoid. However, in all these patients, no other tests were positive and in these patients, the biopsy for direct immunofluorescence showed no evidence for an autoimmune bullous disease. Three patients showed reactivity against rat bladder rat bladder; these were, however, completely negative for A2ML1, envoplakin, and periplakin in the IB as well as the IP. Conclusions. Serological analysis for desmosomal and hemidesmosomal antibodies is reliable to rule an autoimmune bullous disease in patients with suspected SJS/TEN. However, one should not rely on one single test method since false positive results can occur. Moreover, this study also makes it less plausible that antibodies against desmosomal and/or hemidesmosomal components are involved in the pathogenesis of SJS/TEN. [ABSTRACT FROM AUTHOR]

We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160 kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40–50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males. [ABSTRACT FROM AUTHOR]

Objective Autoantibodies against MDA5 (melanoma differentiation-associated protein 5) serve as a biomarker for DM (dermatomyositis) and indicate a risk factor for interstitial lung disease (ILD). MDA5 is a protein responsible for sensing RNA virus infection and activating signalling pathways against it. However, little is known about the antigen epitopes on MDA5 autoantibodies. We aimed to determine the interaction of the MDA5 autoantibody–antigen epitope. Methods Cell-based assays (CBAs), immunoprecipitation-immunoblot assays, and various immunoblotting techniques were used in the study. Results We demonstrated that DM patient autoantibodies recognize MDA5 epitopes in a native conformation–dependent manner. Furthermore, we identified the central helicase domain （3Hel） formed by Hel1, Hel2i, Hel2, and pincer as the major epitopes. As proof of principle, the purified 3Hel efficiently absorbed MDA5 autoantibodies from patient sera through immunoprecipitation-immunoblot assay. Conclusion Our study uncovered the nature of the antigen epitopes on MDA5 and can provide guidance for diagnosis and a targeted therapeutic approach development. [ABSTRACT FROM AUTHOR]

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1–2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism. [ABSTRACT FROM AUTHOR]

Herbal supplements containing several types of plant sterols, vitamins, and minerals, are marketed for prostate health. In the majority of these supplements, the most abundant plant sterol is saw palmetto extract or its' principal component, beta-sitosterol. In terms of prostate health, previous work almost exclusively focused on the effects of beta-sitosterol on prostatic epithelium, with little attention paid to the effects on prostatic stroma. This omission is a concern, as the abnormal accumulation of collagen, or fibrosis, of the prostatic stroma has been identified as a factor contributing to lower urinary tract symptoms and dysfunction in aging men. To address whether beta-sitosterol may be promoting prostatic fibrosis, immortalized and primary prostate stromal fibroblasts were subjected to immunoblotting, immunofluorescence, qRT-PCR, ELISA, and image quantitation and analysis techniques to elucidate the effects of beta-sitosterol on cell viability and collagen expression and cellular localization. The results of these studies show that beta-sitosterol is nontoxic to prostatic fibroblasts and does not stimulate collagen production by these cells. However, beta-sitosterol alters collagen distribution and sequesters collagen within prostatic fibroblasts, likely in an age-dependent manner. This is a significant finding as prostate health supplements are used predominantly by middle aged and older men who may, then, be affected disproportionately by these effects. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin–proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin–proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae , although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1 , which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48 /p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired. [ABSTRACT FROM AUTHOR]

Background: Atypical hemolytic uremic syndrome (aHUS) can be associated with mutations, deletions, or hybrid genes in factor H-related (FHR) proteins. Methods: A child with aHUS was investigated. Genetics was assessed by Sanger and next generation sequencing. Serum FHR5 was evaluated by immunoblotting, ELISA, and by induction of rabbit red blood cell hemolysis in the presence/absence of recombinant human rFHR5. Mutagenesis was performed in HEK cells. Results: A heterozygous genetic variant in factor H-related protein 5 (CFHR5), M514R, was found in the child, who also had a homozygous deletion of CFHR3/CFHR1, and antibodies to factor H, as well as low levels of C3. Patient serum exhibited low levels of FHR5. In the presence of rabbit red blood cells, patient serum induced hemolysis which decreased when rFHR5 was added at physiological concentrations. Similar results were obtained using serum from the father, bearing the CFHR5 variant without factor H antibodies. Patient FHR5 formed normal dimers. The CFHR5 M514R variant was expressed in HEK cells and minimal secretion was detected whereas the protein level was elevated in cell lysates. Conclusions: Decreased secretion of the product of the mutant allele could explain the low FHR5 levels in patient serum. Reduced hemolysis when rFHR5 was added to serum suggests a regulatory role regarding complement activation on red blood cells. As such, low levels of FHR5, as demonstrated in the patient, may contribute to complement activation. [ABSTRACT FROM AUTHOR]

Background and Objectives: Data about myositis‑specific autoantibodies (MSAs) and myositis‑associated autoantibodies (MAAs) in Saudi patients are limited, and most studies have focused on anti-Jo1. This study aimed at reporting the MSAs and MAAs in Saudi population and their significance. Methods: This was a retrospective analysis of 190 Saudi patients investigated for idiopathic inflammatory myopathies(IIMs) between January 2019 and January 2023. Data for MSAs and MAAs were collected from medical records of patients. MSAs and MAAs were detected by line immunoblot. Results: Among the 190 sera tested, 47 yielded positive results for MSAs. There were 19 (40.4%) patients with IIMs, 20 (42.6%) with interstitial lung disease (ILD), and 8 (17%) with connective tissue diseases. Anti-signal recognition particle (SRP) was the most common MSA and was positive among 16 (34%) patients. Anti-PL-12 was the most frequent anti-synthetase antibody (21.3%) followed by anti‑PL‑7 (19.1%). Anti‑Jo1 was associated with Raynaud’s phenomenon (odds ratio [OR] = 9, 95% confidence interval [CI] = 1.3–60, P = 0.037) and with ILD (OR = 29, 95% CI = 2.4–351, P = 0.008) in patients with IIMs whereas anti-PL-7 was associated with ILD in the rest of the patients (OR = 6, 95% CI = 1.1–33, P = 0.021). MAAs were positive in 24 (51.1%) patients with anti-Ro52 as the most frequently detected antibody (29.8%). Conclusion: We confirm the association of MSAs with IIMs and ILD in the Saudi population. Anti‑SRP and anti-PL-12 were the most common MSAs. These observations should be validated by large-scale studies. [ABSTRACT FROM AUTHOR]

Progressive nonfluent aphasia (PNFA) is a form of frontotemporal lobar degeneration (FTLD) caused by tau and transactive response DNA‐binding protein of 43 kDa (TDP‐43) accumulation. Here we report the autopsy findings of a 64‐year‐old right‐handed man with an atypical TDP‐43 proteinopathy who presented with difficulties with speech, verbal paraphasia, and dysphagia that progressed over the 36 months prior to his death. He did not show pyramidal tract signs until his death. At autopsy, macroscopic brain examination revealed atrophy of the left dominant precentral, superior, and middle frontal gyri and discoloration of the putamen. Spongiform change and neuronal loss were severe on the cortical surfaces of the precentral, superior frontal, and middle frontal gyri and the temporal tip. Immunostaining with anti‐phosphorylated TDP‐43 revealed neuronal cytoplasmic inclusions and long and short dystrophic neurites in the frontal cortex, predominantly in layers II, V, and VI of the temporal tip, amygdala, and transentorhinal cortex. Immunoblot analysis of the sarkosyl‐insoluble fractions showed hyperphosphorylated TDP‐43 bands at 45 kDa and phosphorylated C‐terminal fragments at approximately 25 kDa. The pathological distribution and immunoblot band pattern differ from the major TDP‐43 subtype and therefore may represent a new FTLD‐TDP phenotype. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic cancer (PC) is one of the most deadly human malignancies. Curcumin is a natural polyphenolic compound with wide-ranging pharmacological effects. Growing evidence suggests that curcumin has anticancer activity against PC, but the mechanism remains incompletely elucidated. This study aimed to investigate the effects and mechanisms of curcumin on the invasion and migration of PC cells. Methods: Effect of curcumin on tissue factor pathway inhibitor (TFPI)-2 mRNA expression in PC cells was initially identified using qRT-PCR. Cytotoxicity of curcumin was assessed with MTT assays and IC50 was calculated. Involvement of ERK and JNK pathways, as well as protein expression of TFPI-2 and epithelial–mesenchymal transition (EMT)-related markers, were detected using immunoblotting. Invasion and migration of PC cells were examined using Transwell assays. TFPI-2 expression was manipulated by transfection with siRNA and shRNA. Rescue assays were used to validate the effect of curcumin on cell invasion and migration via TFPI-2. Results: Curcumin increased the expression of TFPI-2 mRNA and protein in PC cells and attenuated cell invasion and migration. Curcumin also inhibited ERK and JNK pathways and EMT in PC cells. Knockdown of TFPI-2 partially reversed the inhibition of ERK and JNK pathways and EMT by curcumin. Mechanistically, curcumin upregulated TFPI-2, thereby inhibiting the ERK and JNK pathways, leading to the inhibition of EMT in PC cells. Conclusion: Collectively, curcumin inhibits ERK- and JNK-mediated EMT through upregulating TFPI-2, which in turn suppresses the migration and invasion of PC cells. These findings provide new insights into the antitumor mechanism of curcumin. [ABSTRACT FROM AUTHOR]

The microtubule motor dynein is critical for the assembly and positioning of mitotic spindles. In Caenorhabditis elegans , these dynein functions have been extensively studied in the early embryo but remain poorly explored in other developmental contexts. Here, we use a hypomorphic dynein mutant to investigate the motor's contribution to asymmetric stem cell–like divisions in the larval epidermis. Live imaging of seam cell divisions that precede formation of the seam syncytium shows that mutant cells properly assemble but frequently misorient their spindle. Misoriented divisions misplace daughter cells from the seam cell row, generate anucleate compartments due to aberrant cytokinesis, and disrupt asymmetric cell fate inheritance. Consequently, the seam becomes disorganized and populated with extra cells that have lost seam identity, leading to fatal epidermal rupture. We show that dynein orients the spindle through the cortical GOA-1Gα–LIN-5NuMA pathway by directing the migration of prophase centrosomes along the anterior–posterior axis. Spindle misorientation in the dynein mutant can be partially rescued by elongating cells, implying that dynein-dependent force generation and cell shape jointly promote correct asymmetric division of epithelial stem cells. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a common, highly lethal tumor. Herein, we reported that S-phase kinase-associated protein 2 (Skp2) is essential for the growth and aerobic glycolysis of ovarian cancer cells. Skp2 was upregulated in ovarian cancer tissues and associated with poor clinical outcomes. Using a customized natural product library screening, we found that xanthohumol inhibited aerobic glycolysis and cell viability of ovarian cancer cells. Xanthohumol facilitated the interaction between E3 ligase Cdh1 and Skp2 and promoted the Ub-K48-linked polyubiquitination of Skp2 and degradation. Cdh1 depletion reversed xanthohumol-induced Skp2 downregulation, enhancing HK2 expression and glycolysis in ovarian cancer cells. Finally, a xenograft tumor model was employed to examine the antitumor efficacy of xanthohumol in vivo. Collectively, we discovered that xanthohumol promotes the binding between Skp2 and Cdh1 to suppress the Skp2/AKT/HK2 signal pathway and exhibits potential antitumor activity for ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Dysregulation of estrogen receptor alpha (ER) activity has been implicated in the development of resistance to current treatment strategies. The purpose of this study was to profile novel coregulatory proteins to identify potential therapeutic targets. We identified TRIM33 as a novel interactor and regulator of ER activity and protein stability. These results provide rationale for further investigation of the role of TRIM33 in cancers exhibiting increased ER protein levels. Estrogen receptor alpha (ER)-positive breast cancer is responsible for over 60% of breast cancer cases in the U.S. Among patients diagnosed with early-stage ER+ disease, 1/3 will experience recurrence despite treatment with adjuvant endocrine therapy. ER is a nuclear hormone receptor responsible for estrogen-driven tumor growth. ER transcriptional activity is modulated by interactions with coregulators. Dysregulation of the levels of these coregulators is involved in the development of endocrine resistance. To identify ER interactors that modulate transcriptional activity in breast cancer, we utilized biotin ligase proximity profiling of ER interactomes. Mass spectrometry analysis revealed tripartite motif containing 33 (TRIM33) as an estrogen-dependent interactor of ER. shRNA knockdown showed that TRIM33 promoted ER transcriptional activity and estrogen-induced cell growth. Despite its known role as an E3 ubiquitin ligase, TRIM33 increased the stability of endogenous ER in breast cancer cells. TRIM33 offers a novel target for inhibiting estrogen-induced cancer cell growth, particularly in cases of endocrine resistance driven by ER (ESR1) gene amplification or overexpression. [ABSTRACT FROM AUTHOR]

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research. [ABSTRACT FROM AUTHOR]

Background: Condyloma acuminatum (CA), which is a highly contagious sexually transmitted illness generated by human papillomavirus (HPV) infection, is characterized by abnormal proliferation of keratinocytes resulting in verrucous lesions. Although solute carrier family 30 member 1 (ZNT1) is highly expressed in CA tissues, the function of ZNT1 in CA remains unclear. Methods: HPV transfection was performed in HaCaT to simulate the CA pathological environment. The mRNA and protein levels were monitored using RT-qPCR and immunoblotting. Cell viability was found using the MTT test. Cell invasion and migration were probed using the transwell and wound healing. Results: ZNT1 expression was up-regulated in CA tissues, and HPV transfection increased the expression of ZNT1. Overexpression of ZNT1 promoted the proliferation, migration and invasion of Human immortalized keratinocyte (HaCaT) transfected with HPV. Meanwhile, ZNT1 knockdown repressed the proliferation, migration and invasion of HaCaT that HPV transfected. Further research displayed that ZNT1 promoted the proliferation, migration and invasion of HaCaT transfected with HPV through the PI3K/Akt pathway. Conclusion: Our research confirmed that ZNT1 regulated the proliferation, migration and invasion of HaCaT transfected with HPV through the PI3K/Akt pathway, providing a new target for the effective remedy of CA. [ABSTRACT FROM AUTHOR]

Objective: The contribution of hypoxia to the pathophysiology of vascular smooth muscle cells (VSMCs) has not yet been fully elucidated. This study evaluated the effect of hypoxia on the phenotype and function of SMCs derived from the human normal great saphenous veins (NGSVs). Methods: Fifteen NGSV tissue samples were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, senescence, and the structure of cytoskeletal filaments in SMCs were observed. mRNA and protein expression of Bax, Bcl-2, caspase-3, matrix metalloproteinases (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was detected by fluorescent quantitative polymerase chain reaction and immunoblotting in the cobalt chloride (CoCl2) and the control groups. Results: A decrease in the number of cytoskeletal filaments was observed. mRNA and protein expression of Bas and caspase-3 was significantly decreased, while the quantity of proliferation, migration, adhesion, senescence, and mRNA and protein expression of Bcl-2, MMP-2, MMP-9, TIMP-1, and TIMP-2 in SMCs in the CoCl2 group were significantly increased compared with the control group. Conclusion: Under hypoxic conditions, the phenotype and function of SMCs derived from the human NGSVs were dysregulated, suggesting that VSMCs switch from the contractile phenotype to the secretory or synthetic phenotype, and more dedifferentiate, resulting in extracellular matrix deposition and apoptotic decrease through the intrinsic pathway. [ABSTRACT FROM AUTHOR]

• Swimming training and caffeine revert the increase of NF-kB phosphorylation in rat hippocampus induced by chronic fructose intake. • Swimming training decreases anxiety-like behavior in rats with metabolic syndrome. • Swimming and caffeine improve lipid profile and blood glucose clearance. Metabolic syndrome (MS) is a disorder that increasingly affects the world population, mainly because of changes in lifestyle and dietary habits. In this regard, both physical exercise and caffeine are low-cost and easily accessible therapies that separately have shown positive effects against metabolic disorders. Therefore, we hypothesized that physical exercise combined with caffeine could have a synergistic effect in the treatment of MS, risk factors, and cognitive deficits. Animals were divided into 8 groups and received fructose (15% w/v) or vehicle for 10 weeks. Swimming training and caffeine (6 mg/kg) started 4 weeks after fructose administration. Trained animals presented decreased body weight and visceral fat mass and increased soleus weight compared with untrained fructose-treated animals. Caffeine supplementation also prevented the gain of visceral fat mass induced by fructose. Furthermore, both treatments reversed fructose-induced decrease in glucose clearance over time and fructose-induced increase in 4-hydroxynonenal and nuclear factor-κB immunoreactivity. Physical training also improved the lipidic profile in fructose-treated animals (high-density lipoprotein, low-density lipoprotein, and triglycerides), improved short-term, long-term, and localization memory, and reversed the fructose-induced deficit in short-term memory. Physical training also increased nuclear factor erythroid 2-related factor 2 immunoreactivity per se. Considering that physical training and caffeine reversed some of the damages induced by fructose it is plausible to consider these treatments as alternative, nonpharmacological, and low-cost therapies to help reduce MS-associated risk factors; however, combined treatments did not show additive effects as hypothesized. Over 10 weeks, rats consumed water containing 15% fructose. During the last 6 weeks, animals underwent caffeine intake and swimming training. These treatments reduced fructose-induced anxiety-like behavior, hyperglycemia, body fat mass, 4-HNE and NF-κB immunoreactivity, and improved lipid profile impairments. Additionally, swimming training increased muscle mass and NRF2. Abbreviations: 4-HNE, 4-hydroxynonenal; NF-κB, nuclear factor κB); NRF2, nuclear factor erythroid 2-related factor 2). [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer‐associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation‐related molecules. Methods: Cluster analysis, immunoblotting, real‐time quantitative‐PCR, CRISPR‐Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA‐LPAR1 on B7‐H3 in tumor promotion of CAFs. Results: In this study, we found that LPAR1 and CD276 (B7‐H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7‐H3 up‐expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7‐H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non‐small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7‐H3. Conclusion: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7‐H3 targeting therapies. [ABSTRACT FROM AUTHOR]

Background: Usenamine A, a novel natural compound initially isolated from the lichen Usnea longissima, has exhibited promising efficacy against hepatoma in prior investigation. Nevertheless, the underlying mechanisms responsible for its antihepatoma effects remain unclear. Furthermore, the role of the AKT/mechanistic target of the rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3)/inhibitor of differentiation/DNA binding 1 (ID1) signaling axis in hepatocellular carcinoma (HCC), and the potential anti-HCC effects of drugs targeting this pathway are not well understood. Methods: CCK-8 assay was used to investigate the effects of usenamine A on the proliferation of human HCC cells. Moreover, the effects of usenamine A on the invasion ability of human HCC cells were evaluated by transwell assay. In addition, expression profiling analysis, quantitative real-time PCR, immunoblotting, immunohistochemistry (IHC) analysis, RNAi, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assay were used to explore the effects of usenamine A on the newly identified AKT/mTOR/STAT3/ID1 signaling axis in human HCC cells. Results: Usenamine A inhibited the proliferation and invasion of human HCC cell lines (HepG2 and SK-HEP-1). Through the analysis of gene expression profiling, we identified that usenamine A suppressed the expression of ID1 in human HCC cells. Furthermore, immunoprecipitation experiments revealed that usenamine A facilitated the degradation of the ID1 protein via the ubiquitin–proteasome pathway. Moreover, usenamine A inhibited the activity of STAT3 in human HCC cells. ChIP analysis demonstrated that STAT3 positively regulated ID1 expression at the transcriptional level in human HCC cells. The STAT3/ID1 axis played a role in mediating the anti-proliferative and anti-invasive impacts of usenamine A on human HCC cells. Additionally, usenamine A suppressed the STAT3/ID1 axis through AKT/mTOR signaling in human HCC cells. Conclusion: Usenamine A displayed robust anti-HCC potential, partly attributed to its capacity to downregulate the AKT/mTOR/STAT3/ID1 signaling pathway and promote ubiquitin–proteasome-mediated ID1 degradation. Usenamine A has the potential to be developed as a therapeutic agent for HCC cases characterized by abnormal AKT/mTOR/STAT3/ID1 signaling, and targeting the AKT/mTOR/STAT3 signaling pathway may be a viable option for treating patients with HCC exhibiting elevated ID1 expression. [ABSTRACT FROM AUTHOR]

Introduction: Cervical cancer (CC) is the fourth most common cancer type and a leading cause of cancer-related deaths in women worldwide. Its underlying molecular mechanisms are unclear. Cancer cell-derived extracellular vesicles (EVs) are involved in cancer development and progression by delivering regulatory factors, including microRNAs and long non-coding RNAs (lncRNAs). Methods: Here, we identified the EV lncRNA expression profiles associated with different developmental stages of CC using next-generation sequencing. EVs from the serum of patients with stages I–III CC and healthy donors were characterized using EV marker immunoblotting and transmission electron microscopy. Results: The EV concentration increases with progression of the disease. Most particles had a 100–250-nm diameter, and their sizes were similar in all groups. We identified many lncRNAs that were uniquely and differentially expressed (DE) in patients with different stages of CC. The pathway analysis results indicated that the upregulated DE EV lncRNAs abundant in stages I and II were associated with cell proliferation and inflammation and cancer progression pathways, respectively. LINC00941, LINC01910, LINC02454, and DSG2-AS1 were highly expressed, suggesting poor overall survival of CC patients. Interestingly, DSG2-AS1 was associated with the human papillomavirus infection pathway through AKT3, DLG1, and COL6A2 genes. Conclusion: This is the first study that reports the levels of EVs and their lncRNA contents change during cancer development, demonstrating the existence of a unique vesicle-mediated cell-to-cell communication network underlying cancer progression. [ABSTRACT FROM AUTHOR]

Objective: Autoantibodies are clinically useful in phenotyping patients with systemic sclerosis (SSc). Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS) and commonly impaired in SSc, suggesting that the SSc autoimmune response may target ENS antigens. We sought to identify novel anti‐ENS autoantibodies with an aim to clinically phenotype SSc GI dysfunction. Methods: Serum from a patient with SSc with GI dysfunction but without defined SSc‐associated autoantibodies was used for autoantibody discovery. Immunoprecipitations performed with murine myenteric plexus lysates were on‐bead digested, and autoantigens were identified by mass spectrometry. Prevalence was determined, and clinical features associated with novel autoantibodies were evaluated in a SSc cohort using regression analyses. The expression of gephyrin in human GI tract tissue was examined by immunohistochemistry. Results: We identified gephyrin as a novel SSc autoantigen. Anti‐gephyrin antibodies were present in 9% of patients with SSc (16/188) and absent in healthy controls (0/46). Anti‐gephyrin antibody–positive patients had higher constipation scores (1.00 vs 0.50, P = 0.02) and were more likely to have severe constipation and severe distention/bloating (46% vs 15%, P = 0.005; 54% vs 25%, P = 0.023, respectively). Anti‐gephyrin antibody levels were significantly higher among patients with severe constipation (0.04 vs 0.00; P = 0.001) and severe distention and bloating (0.03 vs 0.004; P = 0.010). Severe constipation was associated with anti‐gephyrin antibodies even in the adjusted model. Importantly, gephyrin was expressed in the ENS, which regulates gut motility. Conclusion: Gephyrin is a novel ENS autoantigen that is expressed in human myenteric ganglia. Anti‐gephyrin autoantibodies are associated with the presence and severity of constipation in patients with SSc. [ABSTRACT FROM AUTHOR]

Background: Commercially available D‐dimer assays use antibodies against human D‐dimer, with limited sensitivity and specificity data in companion animals. Objectives: To evaluate the immunoreactivity of D‐dimer in plasma of dogs, horses, and cats with commercially available antibodies to human D‐dimer. Animals: Plasma samples were collected from healthy dogs and horses, and from surplus feline plasma submitted for diagnostic purposes. Methods: Descriptive research study. A cross‐linked fibrin lysate was prepared from plasma samples, and SDS‐PAGE and immunoblotting were performed with a variety of commercially available antibodies to human D‐dimer. Results: The selected antibodies demonstrated variable reactivity with D‐dimer of each species. The monoclonal antibody DD44 bound canine D‐dimer with good specificity and sensitivity, but this antibody did not react with feline or equine D‐dimer. The polyclonal antibody D2D bound putative D‐dimer in dogs, cats, and horses with good specificity, and higher sensitivity compared to human D‐dimer. Conclusions and Clinical Importance: The variable performance of commercially available human D‐dimer assays between species is, in part, because of inter‐species variation in D‐dimer immunoreactivity. The use of these assays should follow validation studies. Monoclonal antibody DD44 could be a focus for the development of a canine‐specific assay. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focuses on finding improved treatment strategies for advanced or metastatic synovial sarcoma, which is a rare and aggressive type of soft tissue sarcoma. The researchers tested a combination of two drugs, peposertib and doxorubicin, to see if they could work together to kill synovial sarcoma cells more efficiently. The experiments conducted in cultured cancer cells and mouse models of synovial sarcoma demonstrated that when these drugs were used together, they had a significantly stronger effect against cancer cells compared to using either drug alone. It is noteworthy that the combination could successfully overcome resistance to doxorubicin monotherapy in a patient-derived tumor model. This study also shed light on the underlying molecular mechanisms of this combination effect. Overall, the findings suggest that combining peposertib with doxorubicin could be a promising treatment option for synovial sarcoma patients in the future. Synovial sarcoma is a rare and highly aggressive subtype of soft tissue sarcoma. The clinical challenge posed by advanced or metastatic synovial sarcoma, marked by limited treatment options and suboptimal outcomes, necessitates innovative approaches. The topoisomerase II (Topo II) inhibitor doxorubicin has remained the cornerstone systemic treatment for decades, and there is pressing need for improved therapeutic strategies for these patients. This study highlights the potential to enhance the cytotoxic effects of doxorubicin within well-characterized synovial sarcoma cell lines using the potent and selective DNA-PK inhibitor, peposertib. In vitro investigations unveil a p53-mediated synergistic anti-tumor effect when combining doxorubicin with peposertib. The in vitro findings were substantiated by pronounced anti-tumor effects in mice bearing subcutaneously implanted tumors. A well-tolerated regimen for the combined application was established using both pegylated liposomal doxorubicin (PLD) and unmodified doxorubicin. Notably, the combination of PLD and peposertib displayed enhanced anti-tumor efficacy compared to unmodified doxorubicin at equivalent doses, suggesting an improved therapeutic window—a critical consideration for clinical translation. Efficacy studies in two patient-derived xenograft models of synovial sarcoma, accurately reflecting human metastatic disease, further validate the potential of this combined therapy. These findings align with previous evidence showcasing the synergy between DNA-PK inhibition and Topo II inhibitors in diverse tumor models, including breast and ovarian cancers. Our study extends the potential utility of combined therapy to synovial sarcoma. [ABSTRACT FROM AUTHOR]

Simple Summary: By examining the detailed molecular oncogenic mechanisms of KLC1-ROS1 fusion, the function of a novel RTK fusion was investigated to discover novel therapeutic targets for glioma. When wild-type ROS1 and KLC1-ROS1 fusions were expressed in human glioma cell lines, immunoblotting revealed that KLC1-ROS1 fusion specifically activated the JAK2–STAT3 axis compared with wild-type ROS1. Immunoprecipitation revealed a more efficient association of KLC1-ROS1 fusion with JAK2 compared with wild-type ROS1. A mutagenesis study of KLC1-ROS1 fusion demonstrated the essential roles of both the KLC1 and ROS1 domains in the serum factor-independent constitutive activation of the fusion. In addition, KLC1-ROS1 fusion induced the upregulation of cell proliferation and invasion compared to wild-type ROS1 in a JAK-STAT-dependent manner under serum deprivation. Overall, our results suggested that molecules other than RTKs may serve as novel therapeutic targets for gliomas with RTK fusions. Here, we investigated the detailed molecular oncogenic mechanisms of a novel receptor tyrosine kinase (RTK) fusion, KLC1-ROS1, with an adapter molecule, KLC1, and an RTK, ROS1, discovered in pediatric glioma, and we explored a novel therapeutic target for glioma that possesses oncogenic RTK fusion. When wild-type ROS1 and KLC1-ROS1 fusions were stably expressed in the human glioma cell lines A172 and U343MG, immunoblotting revealed that KLC1-ROS1 fusion specifically activated the JAK2-STAT3 pathway, a major RTK downstream signaling pathway, when compared with wild-type ROS1. Immunoprecipitation of the fractionated cell lysates revealed a more abundant association of the KLC1-ROS1 fusion with JAK2 than that observed for wild-type ROS1 in the cytosolic fraction. A mutagenesis study of the KLC1-ROS1 fusion protein demonstrated the fundamental roles of both the KLC1 and ROS1 domains in the constitutive activation of KLC1-ROS1 fusion. Additionally, in vitro assays demonstrated that KLC1-ROS1 fusion upregulated cell proliferation, invasion, and chemoresistance when compared to wild-type ROS1. Combination treatment with the chemotherapeutic agent temozolomide and an inhibitor of ROS1, JAK2, or a downstream target of STAT3, demonstrated antitumor effects against KLC1-ROS1 fusion-expressing glioma cells. Our results demonstrate that KLC1-ROS1 fusion exerts oncogenic activity through serum-independent constitutive activation, resulting in specific activation of the JAK-STAT pathway. Our data suggested that molecules other than RTKs may serve as novel therapeutic targets for RTK fusion in gliomas. [ABSTRACT FROM AUTHOR]