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IntroductionVariants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization.MethodsWe described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling.ResultsANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways.DiscussionThis study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity.HighlightsANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

Purpose: In our previous study, we found that the Chk1 inhibitor prexasertib enhances the antitumour effect of the oral anticancer drug S-1 against pancreatic cancer cells. In this study, we investigated the effect of combining S-1 and ceralasertib, an oral inhibitor of ATR, which is located upstream of Chk1. Ceralasertib is currently being investigated in multiple clinical trials for various cancers. Methods: The cell-proliferation inhibitory effect was measured by MTT assay, using the pancreatic cancer cell lines BxPC-3, SUIT-2, PANC-1, and MIA PaCa-2, while apoptosis was measured by flow cytometry using PI/Annexin staining. The mechanism underlying the combined effect was analysed using western blotting, and the antitumor effect was analysed using a mouse xenograft model. Results: MTT assay revealed that the combination of S-1 and ceralasertib had a synergistic effect, leading to the suppression of cell proliferation. Measurement with PI/Annexin staining revealed that the combination of S-1 and ceralasertib induced apoptosis more efficiently than either drug alone. Western blotting results showed that ceralasertib inhibited S-1-induced activation of ATR and Chk1. The average estimated tumour volume after 3 weeks of administration was 601 mm3 in the S-1 group, 580 mm3 in the ceralasertib group, and 298 mm3 in the combination group. Conclusion: The combination of S-1 and ceralasertib demonstrated a high antiproliferative effect in inhibiting tumour growth in vitro. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is a heterogeneous ailment in both biological and clinical concepts. Numerous efforts have been devoted to discover natural compounds for combating cancer, which showed great potential in cancer management. Methylsulfonylmethane (MSM), an organosulfur dietary supplement, is utilized for improving various clinical conditions, particularly osteoarthritis. MSM can exert antitumor activity in a wide range of cancers. Objectives: The molecular mechanisms of action underlying antileukemic activity of MSM remain unclear. In this regard, we aimed to investigate the anticancer properties of MSM on human AML cell lines (U937 and HL60) with focus on underlying cell death mechanism. Methods: Anticancer activity of the MSM was examined employing MTT assay, Annexin V‐PE/7AAD staining, caspase3/7 activity test, and real‐time qPCR. Both cell lines were treated with different concentrations (50–400 mM) of MSM for 24 h. Pretreatment of the cells with a caspase inhibitor (i.e., Z‐VAD‐fmk) was performed for the assessment of apoptosis induction. Results: The results of MTT assay revealed that in both cell lines, the MSM markedly reduced cell viability in comparison to the control cells. Additionally, findings of Annexin V‐7AAD staining revealed that MSM induced apoptosis and activated caspase 3/7 in both cell lines markedly. Real‐time quantitative PCR results also supported the induction of apoptosis in AML cells. MSM altered the expression levels of various apoptotic genes (BAX, BAD, and BIM). Conclusion: Overall, our results indicated that MSM could induce apoptosis in AML cell lines in a dose‐dependent manner, which therefore could be utilized as an antileukemic agent. [ABSTRACT FROM AUTHOR]

Colorectal cancer is the second deadliest cancer worldwide. The side effects and resistance of existing chemotherapy drugs are the background for various studies looking for potential plant extracts as new anticancer agents. This study aims to determine the cytotoxic activity of simpor leaf extract from Belitung and its effect on apoptosis of WiDr colorectal cancer cells. The compound content in simpor leaf extract was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The cytotoxic activity of simpor leaf extract at different concentrations (25, 50 and 100 ppm) was determined by the MTT test and apoptosis induction by Annexin V/PI flow cytometry analysis. Based on the GC-MS chromatogram results, 13 different compounds were detected. Analysis of the relationship between extract concentration and the percentage of WiDr cell inhibition showed that an extract concentration of 100 ppm was the concentration with the highest percentage of inhibition (20.12 % ± 4.99) compared to concentrations of 50 ppm (13.95 % ± 2.32) and 25 ppm (15.90 % ± 1.38) with an inhibitory concentration value (IC50) of simpor leaf extract of 10.45 ppm. Induction of WiDr cell apoptosis by the extract was influenced by the concentration of simpor leaf extract, which was increase in the percentage of apoptotic cells from 16 % at a concentration of 25 ppm to 70.5 % at a concentration of 100 ppm. The results of this study show that simpor leaf extract from Belitung is able to induce apoptosis in WiDr colorectal cancer cells. [ABSTRACT FROM AUTHOR]

Rabies virus causes an estimated 59,000 annual fatalities worldwide and promising therapeutic treatments are necessary to develop. In this study, affinity tag-purification mass spectrometry was employed to delineate RABV glycoprotein and host protein interactions, and PDIA3/ERP57 was identified as a potential inhibitor of RABV infection. PDIA3 restricted RABV infection with follow mechanisms: PDIA3 mediated the degradation of RABV G protein by targeting lysine 332 via the selective macroautophagy/autophagy pathway; The PDIA3 interactor, AP3B1 (adaptor related protein complex 3 subunit beta 1) was indispensable in PDIA3-triggered selective degradation of the G protein; Furthermore, PDIA3 competitively bound with NCAM1/NCAM (neural cell adhesion molecule 1) to block RABV G, hindering viral entry into host cells. PDIA3 190-199 aa residues bound to the RABV G protein were necessary and sufficient to defend against RABV. These results demonstrated the therapeutic potential of biologics that target PDIA3 or utilize PDIA3 190-199 aa peptide to treat clinical rabies. Abbreviation: aa: amino acids; ANXA2: annexin A2; AP-MS: affinity tag purification-mass spectrometry; AP3B1: adaptor related protein complex 3 subunit beta 1; ATP6V1A: ATPase H+ transporting V1 subunit A; ATP6V1H: ATPase H+ transporting V1 subunit H; BafA1: bafilomycin A1; CHX: cycloheximide; co-IP: co-immunoprecipitation; DDX17: DEAD-box helicase 17; DmERp60: drosophila melanogaster endoplasmic reticulum p60; EBOV: Zaire ebolavirus virus; EV: empty vector; GANAB: glucosidase II alpha subunit; G protein: glycoprotein; GRM2/mGluR2: glutamate metabotropic receptor 2; HsPDIA3: homo sapiens protein disulfide isomerase family A member 3; IAV: influenza virus; ILF2: interleukin enhancer binding factor 2; KO: knockout; MAGT1: magnesium transporter 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MmPDIA3: mus musculus protein disulfide isomerase associated 3; NCAM1/NCAM: neural cell adhesion molecule 1; NGFR/p75NTR: nerve growth factor receptor; NGLY1: N-glycanase 1; OTUD4: OTU deubiquitinase 4; PDI: protein disulfide isomerase; PPIs: protein-protein interactions; RABV: rabies virus; RUVBL2: RuvB like AAA ATPase 2; SCAMP3: secretory carrier membrane protein 3; ScPdi1: Saccharomyces cerevisiae s288c protein disulfide isomerase 1; SLC25A6: solute carrier family 25 member 6; SQSTM1/p62: sequestosome 1; VSV: vesicular stomatitis virus. [ABSTRACT FROM AUTHOR]

Simple Summary: Annexin A2 is a protein that plays a role in many important cell functions, but when its levels are not properly regulated, it can contribute to the development of cancer, particularly in the digestive system. This review looks at the current understanding of how Annexin A2 is involved in cancers of the digestive system, including its potential to help diagnose cancer, predict how a patient will respond to treatment, and even serve as a target for new therapies. Some early studies suggest that blocking this protein or stopping it from interacting with other molecules could slow down cancer growth, prevent the spread of cancer, and make cancer cells more responsive to treatment. By exploring this protein further, scientists hope to develop better tools to diagnose and fight cancer, ultimately benefiting society by improving survival rates and quality of life for patients. Annexin A2 (ANXA2) is a protein that is involved in many physiological and pathological cellular processes. There is compelling evidence that its dysregulated expression, be it up- or downregulation, contributes to the oncogenesis of various neoplasms, including those of the digestive system. The present review summarizes the current knowledge on the role of ANXA2 in the main tumors of the digestive system. The clinical significance of ANXA2 is primordial, due to its potential use as a diagnostic and prognostic biomarker, and as a part of therapeutic protocols. Certain preclinical studies have shown that inhibiting ANXA2 or disrupting its interactions with key molecules can suppress tumor growth, invasion, and metastasis, as well as increase the cancer cells' sensitivity to treatment in various cancers. Further research is needed to fully elucidate the complex role of ANXA2 in the carcinogenesis of tumors of the digestive system, and to translate these findings into clinical applications for improved diagnosis, prognosis, and treatment. [ABSTRACT FROM AUTHOR]

Morphogen gradients instruct cells to pattern tissues. Although the mechanisms by which morphogens transduce chemical signals have been extensively studied, the roles and regulation of the physical communication between morphogen-receiver cells remain unclear. Here, we show that the Wnt/ß-catenin-morphogen gradient, which patterns the embryonic anterior-posterior (AP) axis, generates intercellular tension gradients along the AP axis by controlling membrane cadherin levels in zebrafish embryos. This "mechano-gradient" is used for the cell competition-driven correction of noisy morphogen gradients. Naturally and artificially generated unfit cells, producing noisy Wnt/ß-catenin gradients, induce local deformation of the mechano-gradients that activate mechanosensitive calcium channels in the neighboring fit cells, which then secrete annexin A1a to kill unfit cells. Thus, chemo-mechanical interconversion-mediated competitive communication between the morphogen-receiver cells ensures precise tissue patterning. [ABSTRACT FROM AUTHOR]

This study investigates the role of Annexin A1 (ANXA1) in regulating T cell function and its implications in bone marrow adiposity in aplastic anaemia (AA). Utilizing single‐cell sequencing analysis, we compared bone marrow tissues from AA patients and healthy individuals, focusing on T cell subgroups and their impact on bone marrow pathology. Our findings reveal a significant activation of CD8+ T cells in AA, driven by reduced ANXA1 expression. This heightened T cell activity promotes adipogenesis in bone marrow‐derived mesenchymal stem cells via IFN‐γ secretion. Overexpression of ANXA1 was found to suppress this process, suggesting its therapeutic potential in AA treatment. The study highlights ANXA1 as a crucial regulator in the AA‐associated immune microenvironment and bone marrow adiposity. Key points: This study found that ANXA1 is significantly downregulated in AA and provides detailed insights into its critical role in the disease.The study demonstrates the excessive activation of CD8+ T cells in the progression of AA.The research shows that the overexpression of ANXA1 can effectively inhibit the activation of CD8+ T cells.The study confirms that overexpression of ANXA1 reduces the secretion of the cytokine IFN‐γ, decreases adipogenesis in bone marrow‐derived mesenchymal stem cells and may improve AA symptoms.This research provides new molecular targets for the treatment of AA. [ABSTRACT FROM AUTHOR]

Cervical cancer is a common malignant tumor in women with high morbidity and mortality. Chemotherapy drugs such as cisplatin (DDP) are easy to cause chemotherapy resistance and affect the therapeutic effect. Hence, it is critical to design new therapies that can reverse chemotherapy resistance and increase sensitivity to chemotherapy drugs. This study investigated the function of RecQ protein‐like 4 (RECQL4) in DDP‐resistant cervical cancer cells and its regulatory mechanism. By constructing DDP‐resistant Hela and CaSki cell lines, it was found that RECQL4 expression was elevated. RECQL4 knockdown is able to promote apoptosis, DNA damage, and increase the DDP sensitivity in cervical cancer cells. In vivo experiments have demonstrated that knockdown of RECQL4 suppresses tumor growth and promotes tumor apoptosis. Next, we investigated the potential regulatory relationship of RECQL4 to Annexin A2 (ANXA2). The results demonstrated that RECQL4 binds to ANXA2. Knockdown of RECQL4 downregulates the ANXA2 expression via promoting ubiquitination. Furthermore, we also found that ANXA2 overexpression partially abolished the role of RECQL4 knockdown in promoting apoptosis and DNA damage of cervical cancer cells, suggesting that RECQL4 plays a role in DDP sensitivity of cervical cancer cells by mediating ANXA2. In conclusion, the present study suggests that knocking down RECQL4 reduces DDP sensitivity in cervical cancer cells by modulating ANXA2. Targeting RECQL4 therapy may be a new strategy to improve chemosensitivity of cervical cancer cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the effectiveness of the Gaussian and Flat‐Top profiles in proliferation and differentiation of mesenchymal stem cell of buccal fat pad. Based on the timing schedule and type of laser handpieces, the cells were assigned to a control group with no radiation, and two irradiation test groups (980 nm) with Flat‐Top (F) (power of 1.1 W, beam area of 1 cm2) and standard Gaussian (G) (power of 0.7 W, beam area of 0.5 cm2) handpieces. Each test group was divided into three subgroups, receiving one time (60 J/cm2), two times (120 J/cm2), and three times (180 J/cm2) irradiation. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) and Annexin V tests were performed. The Alizarin Red staining and polymerase chain reaction tests were done both at the beginning and the end of the first and second weeks. The degree of mineralization and expression of osteogenic markers, RUNX2, OCN, and OPN were evaluated. Based on the MTT and Annexin V test results, both test groups outperformed the control group in degrees of cell proliferation during the first day of laser irradiation (p < 0.05). After one and two times irradiation, the expression of osteogenic markers in the test groups was significantly higher than the control group. PBM with Flat‐Top and Gaussian handpieces can enhance ossification and cell differentiation regardless of the type of handpieces. [ABSTRACT FROM AUTHOR]

The present article demonstrates the regioselective synthesis, characterization, and biological evaluation of eighteen novel pyrazolo‐benzothiazole hybrid molecules 5a–5r. We have utilized β,β‐ditosyloxy ketone protocol to synthesize these hybrid molecules. The synthesized compounds were tested for their in‐vitro antiproliferative activities using MTT assay against breast cancer (MCF‐7), cervical cancer (HeLa), and Lung cancer (A549) cell lines. Hybrid molecules 5a, 5m, 5n, and 5o with IC50 values of 0.359 mM, 0.051 mM, 0.079 mM, and 0.259 mM respectively exhibited admirable growth inhibitory properties against MCF‐7 cancer cells which are even better than reference carboplatin drug having IC50 (0.439 mM). Compound 5k with IC50 value of 0.765 mM was found to be the most potent antiproliferative agent for the HeLa cancer cells. Moreover, hybrid molecule 5f with IC50 value of 0.706 mM exhibited better inhibitory activity against A549 cancer cells in comparison to the reference carboplatin drug having IC50 (0.805 mM). The mechanism of cellular toxicity was studied using the Annexin V‐FITC/PI double staining method and cell cycle assay. Molecular docking studies for all the synthesized compounds have also been performed in the binding pocket of VEGFR2 sites (PDB code: 4ASD). Finally, the ADMET profile of the potent molecules was investigated to predict their drug‐likeness behaviour. [ABSTRACT FROM AUTHOR]

Background: Malignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells. Results: We compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions. Conclusion: This study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis. [ABSTRACT FROM AUTHOR]

ABSTRACT The antimicrobial, antitumor and antioxidant potential of Co(II), Cu(II), Mn(II), Ni(II), Pd(II), and Pt(II) complexes with 2‐(((2‐chloro‐5‐(trifluoromethyl)phenyl)imino)methyl)phenol HL ligand was investigated. A molecular docking study was carried out to estimate the predicted binding affinity of the compounds to protein targets involved in proliferative and bacterial activities. Fluorescence quenching studies and synchronous spectra were used to examine the metal complexes interactions with CT‐DNA and BSA protein. The DNA binding study have revealed that the complexes are capable of binding with DNA via intercalative mode. The antimicrobial activity of the ligand and metal complexes was studied against three Gram‐positive bacteria, two Gram‐negative bacteria, and three strains of fungi. The best antibacterial effect was demonstrated by the Pt(II) complex on the Staphylococcus aureus strain, and as for the inhibitory effect on fungi, it was stronger on the Candida albicans strain after treatment with the Co(II) complex. The in vitro antiproliferative activity of ligand and complexes was analyzed using MTT, Annexin V/PI, and cell cycle assays. The in vitro results showed that, except for Pd(II) complex, where slight effects were observed, the MCF‐7 line was resistant to the activity of the complexes. In the case of A549 cells, Cu(II) and Pd(II) complexes showed a dose‐dependent antitumor activity, confirmed by both the Alamar blue and the Annexin V/PI analysis. Finally, the antioxidant activity of the compounds was examined by ABTS and DPPH methods. Antioxidant investigation showed that the Ni(II) complex possesses a remarkable ability to trap the cation of the ABTS+ radical (IC50 value 9.35 μM). [ABSTRACT FROM AUTHOR]

To improve the potential of lupeol against cancer cells, a privileged structure, thiazolidinedione, was introduced into its C-3 hydroxy group with ester, piperazine-carbamate, or ethylenediamine as a linker, and three series of thiazolidinedione-conjugated compounds (6a–i, 9a–i, and 12a–i) were prepared. The target compounds were evaluated for their cytotoxic activities against human lung cancer A549, human breast cancer MCF-7, human hepatocarcinoma HepG2, and human hepatic LO2 cell lines, and the results revealed that most of the compounds displayed improved potency over lupeol. Compound 12i exhibited significant activity against the HepG2 cell line, with an IC50 value of 4.40 μM, which is 9.9-fold more potent than lupeol (IC50 = 43.62 μM). Mechanistic studies suggested that 12i could induce HepG2 cell apoptosis, as evidenced by AO/EB staining and annexin V-FITC/propidium iodide dual staining assays. Western blot analysis suggested that compound 12i can upregulate Bax expression, downregulate Bcl-2 expression, and activate the mitochondria-mediated apoptotic pathway. Collectively, compound 12i is worthy of further investigation to support the discovery of effective agents against cancer. [ABSTRACT FROM AUTHOR]

Treatment options for advanced head and neck squamous cell carcinoma (HNSCC) are limited and often cause severe toxicity and debilitating long-term impacts. Developing effective and safer treatments is warranted. Several plant extracts have shown their effectiveness, but a comprehensive comparison between plant extracts in HNSCC has not been reported. Our aim was to investigate the effect of different plant extracts on the proliferation and viability of HNSCC cell lines. In addition, we investigated the efficacy of combining cytotoxic plant extracts with radiation. Since RT is a cornerstone in the treatment and management of HNSCC, it is desirable to enhance its efficacy through combination with cytotoxic agents that have minimal side effects. HNSCC cell lines were treated with various plant extracts at different concentrations. MTT assays were performed to identify the most potent anti-tumor plant extract. Colony-formation assays were performed to determine the radiosensitization effect. To investigate the effect on migration, transwell migration assays were performed. Annexin V staining was performed to analyze cell apoptosis. 6-gingerol resulted in the most significant dose-dependent inhibition in all cell lines compared to other plant extracts. Colony-formation assays showed a significant radiosensitizing effect when 6-gingerol was combined with radiation. In addition, the combination of 6-gingerol with radiation resulted in a significant decrease in HNSCC cell migration. Mechanistically, Annexin V staining showed that the combination of 6-gingerol and RT induces a synergistic apoptotic effect in MOC1, MOC2 and SCC9 cells compared to RT alone. In conclusion, 6-gingerol enhances the effect of radiation in HNSCC cell lines and could be a suitable candidate for combination therapy in HNSCC. [ABSTRACT FROM AUTHOR]

Cilia defects lead to scoliosis in zebrafish, but the underlying pathogenic mechanisms are poorly understood and may diverge depending on the mutated gene. Here, we dissected the mechanisms of scoliosis onset in a zebrafish mutant for the rpgrip1l gene encoding a ciliary transition zone protein. rpgrip1l mutant fish developed scoliosis with near- total penetrance but asynchronous onset in juveniles. Taking advantage of this asynchrony, we found that curvature onset was preceded by ventricle dilations and was concomitant to the perturbation of Reissner fiber polymerization and to the loss of multiciliated tufts around the subcommissural organ. Rescue experiments showed that Rpgrip1l was exclusively required in foxj1a- expressing cells to prevent axis curvature. Genetic interactions investigations ruled out Urp1/2 levels as a main driver of scoliosis in rpgrip1 mutants. Transcriptomic and proteomic studies identified neuroinflammation associated with increased Annexin levels as a potential mechanism of scoliosis development in rpgrip1l juveniles. Investigating the cell types associated with annexin2 over- expression, we uncovered astrogliosis, arising in glial cells surrounding the diencephalic and rhombencephalic ventricles just before scoliosis onset and increasing with time in severity. Anti- inflammatory drug treatment reduced scoliosis penetrance and severity and this correlated with reduced astrogliosis and macrophage/microglia enrichment around the diencephalic ventricle. Mutation of the cep290 gene encoding another transition zone protein also associated astrogliosis with scoliosis. Thus, we propose astrogliosis induced by perturbed ventricular homeostasis and associated with immune cell activation as a novel pathogenic mechanism of zebrafish scoliosis caused by cilia dysfunction. [ABSTRACT FROM AUTHOR]

Background Objective Materials and methods Results Conclusions Infertility affects a significant percentage of couples worldwide, with male infertility contributing substantially in a considerable number of cases. Research indicates that oxidative stress is a critical factor impacting male fertility.To explore the relationship between semen static oxidation–reduction potential (sORP), sperm parameters, and validated biomarkers of oxidative stress in infertile men.This cross‐sectional study involved 202 men diagnosed with idiopathic male factor infertility and male partners from couples with unexplained infertility. Multivariable linear regression to query the associations between sORP, sperm parameters, and oxidative aggression biomarkers (lipid peroxidation, mitochondrial membrane potential, annexin V, and sperm DNA fragmentation).SORP has no linear association with any semen analysis parameter. Furthermore, its relationship with validated biomarkers of oxidative stress was inconsistent. sORP was inversely related to lipid peroxidation (multivariable linear regression coefficient: −0.64), positively associated with sperm DNA fragmentation (multivariable linear regression coefficient: 3.20), and unrelated to mitochondrial membrane potential or annexin V.There is no clear or consistent relationship between sORP and validated oxidative aggression biomarkers or sperm parameters. Our findings suggest that sORP is unlikely to be helpful in the evaluation of a male with idiopathic infertility. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between serum lymphocyte-activated gene-3 (LAG-3) combined with annexin A3 (ANXA3) and the efficacy of lenvatinib combined with transcatheter arterial chemoembolization (TACE) in patients with intermediate and advanced hepatocellular carcinoma (HCC). Methods: 147 patients with intermediate and advanced HCC were selected, and they were divided into remission group and non-remission group base on whether the combination of lenvatinib and TACE treatment whether to remission.The influencing factors of non-remission after treatment with lenvatinib combined with TACE in patients with inter-mediate and advanced HCC were analyzed by multifactorial logistic regression model. Methods: The objective remission rate of 147 patients with intermediate to advanced HCC treated with lenvatinib in combination with TACE was 36.05%, and the non-remission rate was 63.95%. The proportion of Child-Pugh grade B, the proportion of AFP≥400 μg/L, the proportion of poor differentiation, Barcelona liver cancer staging (BCLC) stage C, the proportion of vascular invasion and the levels of serum LAG-3 and ANXA3 in non-remission group were higher than those in remission group(P<0.05). Child-Pugh grade B, poor differentiation, BCLC stage C, vascular invasion and elevated levels of LAG-3 and ANXA3 were independent risk factors for non-remission after lenvatinib combined with TACE in patients with intermediate and advanced HCC (P<0.05). Conclusion: Elevated serum LAG-3 and ANXA3 levels are associated with non-remission after treatment in patients with intermediate- and advanced-stage HCC. [ABSTRACT FROM AUTHOR]

Aims: Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D‐electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high‐resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. Methods: We conducted a comprehensive approach to investigate the proteomic profile and disease‐associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age‐matched controls. Our methodology encompasses tandem mass tag (TMT)‐labeled liquid chromatography‐mass spectrometry (LC–MS/MS)‐based proteomics, protein–protein interaction (PPI) network analysis, weighted gene co‐expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. Results: A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy‐afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. Conclusions: Through the aggregation of expression data from dysferlinopathy‐impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors. [ABSTRACT FROM AUTHOR]

Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3‐oxygen–substituted isoflavone analogue (3‐benzyloxychromone, 3‐Boc) from the herb. Initially, we established cell line–derived xenograft mouse model using H1299 non–small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3‐Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3‐Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V‐FITC/PI apoptosis assays demonstrated that 3‐Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure‐based target prediction and MD simulation suggested that 3‐Boc potentially suppressed the activity of glycogen synthase kinase‐3β (GSK‐3β) by interacting with the ATP–binding site. Western blot analysis indicated that 3‐Boc triggered the phosphorylation of Serine 21 of GSK‐3α or Serine 9 of GSK‐3β in a time‐ and dose‐dependent manner. To investigate the dependency of GSK‐3β, we established GSK‐3β knockout in H1299 cells. Depletion of GSK‐3β enhanced 3‐Boc–induced cytotoxicity compared with wild‐type counterparts through activated c‐Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3‐oxygen–substituted chromone from the herb, which may have important implications for screening promising modulators of GSK‐3β and related signaling pathways in the treatment of cancer. Summary: A novel compound featured by 3‐Benzyloxychromone (3‐Boc), which was isolated from the stem of Celastrus orbiculatus Thunb, exhibited significant anticancer activity in NSCLC cells.3‐Boc was more likely to act as a potential ATP‐competitive inhibitor rather than an allosteric inhibitor of GSK‐3β.Depletion of GSK‐3β enhanced the sensitivity of NSCLC cells to 3‐Boc by activating the c‐Jun/ATF2 pathway. Significance Statement Natural products serve as good sources of lead molecules for anticancer drugs. We here characterized the anticancer effect of Celastrus orbiculatus Thunb. and reported a novel class of natural product featured by 3‐benzyloxychromone (3‐Boc). By targeting prediction and validation, we found that GSK‐3β was a potential target of 3‐Boc. Moreover, GSK‐3β ablation enhanced the sensitivity of NSCLC to 3‐Boc, which was dependent on the activation of the c‐Jun/ATF2 pathway. Our data further provided an experimental basis for the utilization and development of anticancer precursor from 3‐Boc found in the Celastrus species. [ABSTRACT FROM AUTHOR]

Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 μg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 μg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss. • Fluazuron induces genotoxicity in CHO-K1 and genotoxicity and apoptosis in cumulus cells. • Fluazuron reduces metaphase II and polar body (PB) formation, degenerates oocytes, and disorganizes PB chromatin. • Embryo development rate decreased significantly after cumulus-oocyte cells exposure to 50 μg fluazuron/mL. • Fluazuron does not alter the oxidative stress parameters assessed in bovine cumulus cells and oocytes. [ABSTRACT FROM AUTHOR]

Triptolide (TPL), a natural product extracted from Tripterygium wilfordii Hook F, exerts potential anti‐cancer activity. Studies have shown that TPL is involved in multiple cellular processes and signal pathways; however, its pharmaceutical activity in human colorectal cancer (CRC) as well as the underlying molecular mechanism remain elusive. In this study, the effects of TPL on HCT116 human colon cancer cells and CCD841 human colon epithelial cells are first evaluated. Next, the protein targets of TPL in HCT116 cells are identified through an activity‐based protein profiling approach. With subsequent in vitro experiments, the mode of action of TPL in HCT116 cells is elucidated. As a result, TPL is found to selectively inhibit HCT116 cell viability and migration. A total of 54 proteins are identified as the targets of TPL in HCT116 cells, among which, Annexin A1 (ANXA1) and Peroxiredoxin I/II (Prdx I/II) are picked out for further investigation due to their important role in CRC. The interaction between TPL and ANXA1 or Prdx I is confirmed, and it is discovered that TPL exerts inhibitory effect against HCT116 cells through binding to ANXA1 and Prdx I. The study reinforces the potential of TPL in the CRC therapy, and provides novel therapeutic targets for the treatment of CRC. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary arterial hypertension (PAH) is a serious complication of systemic lupus erythematosus (SLE) with increased mortality. A prothrombotic state may contribute to pathogenesis of SLE-PAH. Extracellular vesicles (EVs) are known to be associated with thrombosis. Here, we investigated circulating EVs and their associations with SLE-PAH. Methods: Eighteen SLE-PAH patients, 36 SLE-non-PAH patients, and 36 healthy controls (HCs) were enrolled. Flow cytometry was used to analyze circulating EVs from leukocytes (LEVs), red blood cells (REVs), platelets (PEVs), endothelial cells (EEVs), and Annexin V+ EVs with membrane phosphatidylserine (PS) exposure. Results: Plasma levels of all EV subgroups were elevated in SLE patients with or without PAH compared to HCs. Furthermore, plasma Annexin V+ EVs, LEVs, PEVs, REVs, EEVs, and Annexin V+ REVs were significantly elevated in SLE-PAH patients compared to SLE-non-PAH patients. Additionally, PAH patients with moderate/high SLE showed a significant increase in LEVs, PEVs, REVs, Annexin V+ EVs, and Annexin V+ REVs compared to SLE-non-PAH patients. However, PAH patients with inactive/mild SLE only exhibited elevations in Annexin V+ EVs, REVs, and Annexin V+ REVs. In the SLE-PAH patients, EEVs were positively correlated with pulmonary arterial systolic pressure, while PEVs and EEVs were positively correlated with right ventricular diameter. Moreover, the receiver operating characteristic curve indicated that Annexin V+ EVs, LEVs, PEVs, REVs, EEVs and Annexin V+ REVs could predict the presence of PAH in SLE patients. Importantly, multivariate logistic regression analysis showed that circulating levels of LEVs or REVs, anti-nRNP antibody, and serositis were independent risk factors for PAH in SLE patients. Discussion: Findings reveal that specific subgroups of circulating EVs contribute to the hypercoagulation state and the severity of SLE-PAH. Higher plasma levels of LEVs or REVs may serve as biomarkers for SLE-PAH. [ABSTRACT FROM AUTHOR]

Chronic wounds and burns are a worldwide healthcare problem that erodes patients’ well-being and healthcare systems. This silent and costly epidemic requires new, cost-efficient solutions to improve patients’ physical and economic welfare. Eschar-degrading vegetal and bacterial proteases have been utilized as a solution. However, these proteins are evolutionarily far from those present in human wound healing. Serine protease (SP) and annexin (ANX) proteins interact within the skin healing process. A homology-based identification pipeline can help in discovering selective human SP and ANX analogs in the epithelial tissue of the fast-healing species, Pangasius hypophthalmus. In the present work, we found 14 candidates for RT-PCR in P. hypophthalmus using homology inference. The genetically detected candidates were then structurally and sequentially analyzed to understand their possible relation to SPs and ANXs involved in human wound healing. A total of six TBLASTN/BLASTX candidates (four SPs and two ANXs) were detected in P. hypophthalmus skin. Structural analysis revealed that all SP candidates resembled human KLK4, KLK5, KLK6, and KLK8, whereas all ANX only resembled human ANXA4. Structure and sequence analysis revealed high conservation of ANX Ca2+ binding sites (GDXD) and SP catalytic triad (HDS) motifs. In addition, structural analysis revealed that SP substrate selectivity position 186 was the main difference between human KLK5 and P. hypophthalmus SPs. These findings may allow the proposal and testing of more selective formulations, broadening treatments beyond debridement.

AbstractBackgroundObjectiveMethodsResultsConclusionsTriptolide (TP) has been confirmed to possess many beneficial functions including anti-inflammation and immunosuppression.The present study aimed to explore the potential involvement of IL-2/IL-2R pathway in the immunosuppressive activities of TP.Cultured CTLL-2 cells were utilized to evaluate the potential benefits of TP. Then cell viability was determined by CCK-8 assay, IFN-γ level by ELISA assay, Annexin V-FITC/PI double-staining and CD25 expression by flow cytometry, and protein expression by western blotting. Additionally, rhIL-2–driven lymphocytes following ConA activation were investigated. The interactions of TP with IL-2 and IL-2Rα were investigated by binding assays and molecular dynamics simulations.TP treatment attenuated IFN-γ level and cell viability in both rhIL-2–induced CTLL-2 cells and rhIL-2–driven splenic lymphocytes. TP treatment increased cellular apoptosis/necrosis and cleaved PARP-1 level, while suppressed c-Myc level in rhIL-2–induced CTLL-2 cells. Additionally, TP treatment reduced CD25 expression on CTLL-2 cell surface. Notably, the phosphorylation protein levels in IL-2R signaling pathways were inhibited by TP exposure prior to rhIL-2 stimulation. SPR and BLI assays verified TP that directly bound to rhIL-2 and rmIL-2Rα, respectively. Molecular simulations suggested that TP bound at the interface of IL-2 and IL-2Rα near the hydrophobic patch composed of F62, L92 on IL-2 and L23, I46, V139 on IL-2Rα, resulting in decreased binding free energy between IL-2 and IL-2Rα.These findings collectively emphasized that TP interfered IL-2/IL-2Rα interactions, down-regulated IL-2Rα expression, and inhibited IL-2R signaling pathways activation, thereby leading to the immune cells being desensitized to rhIL-2 and exhibiting immunosuppressive properties. [ABSTRACT FROM AUTHOR]

Feline infectious peritonitis (FIP), a serious infectious disease in cats, has become a challenging problem for pet owners and the industry due to the lack of effective vaccinations and medications for prevention and treatment. Currently, most natural compounds have been proven to have good antiviral activity. Hence, it is essential to develop efficacious novel natural compounds that inhibit FIPV infection. Our study aimed to screen compounds with in vitro anti-FIPV effects from nine natural compounds that have been proven to have antiviral activity and preliminarily investigate their mechanisms of action. In this study, the CCK-8 method was used to determine the maximum noncytotoxic concentration (MNTC), 50% cytotoxic concentration (CC50), and 50% effective concentration (EC50) of natural compounds on CRFK cells and the maximum inhibition ratio (MIR) of the compounds inhibit FIPV. The effect of natural compounds on FIPV-induced apoptosis was detected via Annexin V-FITC/PI assay. Network pharmacology (NP), molecular docking (MD), and 4D label-free quantitative (4D-LFQ) proteomic techniques were used in the joint analysis the mechanism of action of the screened natural compounds against FIPV infection. Finally, Western blotting was used to validate the analysis results. Among the nine natural compounds, baicalin had good antiviral effects, with an MIR > 50% and an SI > 3. Baicalin inhibited FIPV-induced apoptosis. NP and MD analyses showed that AKT1 was the best target of baicalin for inhibiting FIPV infection. 4D-LFQ proteomics analysis showed that baicalin might inhibit FIPV infection by modulating the PI3K-AKT pathway and the apoptosis pathway. The WB results showed that baicalin promoted the expression of EGFR, PI3K, and Bcl-2 and inhibited the expression of cleaved caspase 9 and Bax. This study found that baicalin regulated the PI3K-AKT pathway and the apoptosis pathway in vitro and inhibited FIPV-induced apoptosis, thus exerting anti-FIPV effects. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IVDD) is a chronic degenerative disease involving the aging and loss of proliferative capacity of nucleus pulposus cells (NPCs), processes heavily dependent on mitochondrial dynamics and autophagic flux. This study finds that the absence of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) is associated with senescence‐related NPC degeneration, disrupting mitochondrial quality control. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation potential and produce extracellular vesicles containing cellular activators. Therefore, in this study, BMSCs are induced under hypoxic stimulation to deliver BNIP3‐rich extracellular vesicles to NPCs, thereby alleviating aging‐associated mitochondrial autophagic flux, promoting damaged mitochondrial clearance, and restoring mitochondrial quality control. Mechanistically, BNIP3 is shown to interact with the membrane‐bound protein annexin A2 (ANXA2), enabling the liberation of the transcription factor EB (TFEB) from the ANXA2‐TFEB complex, promoting TFEB nuclear translocation, and regulating autophagy and lysosomal gene activation. Furthermore, a rat model of IVDD is established and verified the in vivo efficacy of the exosomes in repairing disc injuries, delaying NPC aging, and promoting extracellular matrix (ECM) synthesis. In summary, hypoxia‐induced BMSC exosomes deliver BNIP3‐rich vesicles to alleviate disc degeneration by activating the mitochondrial BNIP3/ANXA2/TFEB axis, providing a new target for IVDD treatment. [ABSTRACT FROM AUTHOR]

Choriocarcinoma is a malignant cancer that belongs to gestational trophoblastic neoplasia (GTN). Herein, serum metabolomic analysis was performed on 29 GTN patients and 30 healthy individuals to characterize the metabolic variations during GTN progression. Ultimately 24 differential metabolites (DMs) were identified, of which, Equol was down-regulated in GTN patients, whose VIP score is the 3rd highest among the 24 DMs. As an intestinal metabolite of daidzein, the anticancer potential of Equol has been demonstrated in multiple cancers, but not choriocarcinoma. Hence, human choriocarcinoma cell lines JEG-3 and Bewo were used and JEG-3-derived subcutaneous xenograft models were developed to assess the effect of Equol on choriocarcinoma. The results suggested that Equol treatment effectively suppressed choriocarcinoma cell proliferation, induced cell apoptosis, and reduced tumorigenesis. Label-free quantitative proteomics showed that 136 proteins were significantly affected by Equol and 20 proteins were enriched in Gene Ontology terms linked to protein degradation. Tripartite motif containing 21 (TRIM21), a E3 ubiquitin ligase, was up-regulated by Equol. Equol-induced effects on choriocarcinoma cells could be reversed by TRIM21 inhibition. Annexin A2 (ANXA2) interacted with TRIM21 and its ubiquitination was modulated by TRIM21. We found that TRIM21 was responsible for proteasome-mediated degradation of ANXA2 induced by Equol, and the inhibitory effects of Equol on the malignant behaviors of choriocarcinoma cells were realized by TRIM21-mediated down-regulation of ANXA2. Moreover, β-catenin activation was inhibited by Equol, which also depended on TRIM21-mediated down-regulation of ANXA2. Taken together, Equol may be a novel candidate for the treatment for choriocarcinoma. [ABSTRACT FROM AUTHOR]

Objective: To discuss the protective effect of carnosine (CAR) against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocyte (AS) injury, and to clarify its possible mechanism. Methods: The AS were divided into control group, model group (OGD/R group), OGD/R+CAR group (CAR group), and OGD/R+CAR+AMP-activated protein kinase (AMPK) activator AICAR group (CAR+AICAR group). MTT assay and green cyanine staining method were used to detect the survival rates and green cyanine staining positive rates of the AS in various groups; Annexin Ⅴ-FITC/PI method and flow cytometry were used to detect the apoptotic rates of the AS in various groups; Western blotting method was used to detect the expression levels of AMPK, phosphorylated AMPK (p-AMPK), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), microtubule-associated protein light chain 3B (LC3B), Beclin-1, and P62 proteins in the AS in various groups; immunofluorescence staining was used to observe the LC3B positive fluorescence intensities in the AS in various groups. Results : Compared with control group, the survival rate and green cyanine staining positive rate of the AS in OGD/R group were decreased (P<0. 01), the apoptotic rate of the AS was increased (P< 0. 01), the ratios of p-AMPK/AMPK and LC3BⅡ/LC3BⅠ and the expression level of Beclin-1 protein were increased (P<0. 01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased (P<0. 01). Compared with OGD/R group, the survival rate and green cyanine staining positive rate of the AS in CAR group were increased (P<0. 01), the apoptotic rate of the AS was decreased (P<0. 01), the ratios of p-AMPK/AMPK and LC3B Ⅱ/LC3B Ⅰ and the expression level of Beclin-1 protein were decreased (P<0. 01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were increased (P<0. 01). Compared with CAR group, the survival rate and green cyanine staining positive rate of the AS in CAR+AICAR group were decreased (P<0. 01), the apoptotic rate of the AS was increased (P<0. 01), the ratios of p-AMPK/AMPK and LC3B Ⅱ/ LC3BⅠ and the expression level of Beclin-1 protein were increased (P<0. 01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased (P<0. 01). The LC3B immunofluorescence staining results were consistent with the Western blotting results. Conclusion: CAR has the protective effect on injury of the AS induced by OGD/R, and its molecular mechanism may be related to the inhibition of the AMPK/mTOR signaling pathway, thereby inhibiting autophagy. [ABSTRACT FROM AUTHOR]

The maternal -fetal interface is essential for successful embryo implantation and subsequent placental development. Many studies have showed that the maternal-fetal interface is rich in the annexin A (AnxA) family, and that AnxA family not only regulates the biological behavior of trophoblast cells at the maternal-fetal interface, but also participates in the metaphase reaction. Therefore, the aberrant expression of the AnxA family is associated with a range of adverse pregnancy outcomes, such as recurrent implantation failure and recurrent pregnancy loss. Clarifying the role of the AnxA family at the maternal-fetal interface will help to understand better the mechanism of action of AnxA leading to adverse pregnancy outcomes, and to provide new targets for the treatment of related diseases. The possible roles of the AnxA family at the maternal -fetal interface and in the related adverse pregnancy are summarized in this review, to show a potential direction of the treatment of adverse pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is a rising global burden, affecting one in four adults. Despite the increasing prevalence of NAFLD, the exact cellular and molecular mechanisms remain unclear, and effective therapeutic strategies are still limited. In vitro models of NAFLD are critical to understanding the pathogenesis and searching for effective therapies; thus, we evaluated the effects of free fatty acids (FFAs) on NAFLD hallmarks and their association with the modulation of Annexin A2 (ANXA2) and Keratin 17 (KRT17) in HepG2 cells. Our results show that oleic and palmitic acids can differentially induce intracellular lipid accumulation, cell death, and promote oxidative stress by increasing lipid peroxidation, protein carbonylation, and antioxidant defense depletion. Moreover, a markedly increased expression of inflammatory cytokines demonstrated the activation of inflammation pathways associated with lipotoxicity and oxidative stress. ANXA2 overexpression and KRT17 nuclear translocation were also observed, supporting the role of both molecules in the progression of liver disease. Taken together, these data provide insights into the interplay between ANXA2 and KRT17 in NAFLD, paving the way for understanding molecular mechanisms involved with the disease and developing new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Despite unquestionable advances in therapy, melanoma is still characterized by a high mortality rate. For years, high expectations have been raised by compounds of natural origin as a component of pharmacotherapy, particularly by triterpenes found in the bark of birch trees. In this study, 3,4-seco-dammara-4(29),20(21),24(25)-trien-3-oic acid (SDT) was isolated from buds of silver birch and its mechanisms of cell death induction, including apoptosis and autophagy, were determined. Cytotoxicity of SDT was evaluated by the cell viability test and clonogenic assay, whereas induction of apoptosis and autophagy was determined by annexin V staining and Western blot. The results revealed dose- and time-dependent reductions in viability of melanoma cells. Treatment of cells for 48 h led to an increase in the percentage of annexin V-positive cells, activation of caspase-8, caspase-9, and caspase-3, and cleavage of PARP, confirming apoptosis. Simultaneously, it was found that SDT increased the level of autophagy marker LC3-II and initiator of autophagy beclin-1. Pretreatment of cells with caspase-3 inhibitor or autophagy inhibitor significantly reduced the cytotoxicity of SDT and revealed that both apoptosis and autophagy contribute to a decrease in cell viability. These findings suggest that 3,4-seco-dammaranes may become a promising group of natural compounds for searching for anti-melanoma agents. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is a leading cause of blindness, yet its molecular mechanisms are unclear. Extracellular vesicles (EVs) contribute to dysfunction in DR, but the characteristics and functions of vitreous EVs are unclear. This study investigated the inflammatory properties of type 2 diabetic (db) vitreous EVs. EVs isolated from the vitreous of db and non-db donors were used for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold staining, Western blotting, and proteomic analysis by mass spectrometry. Intracellular uptake of vitreous EVs by differentiated macrophages was evaluated using ExoGlow membrane labeling, and the impact of EVs on macrophage (THP-1) activation was assessed by cytokine levels using RT-qPCR. NTA and TEM analysis of db and non-db vitreous EVs showed non-aggregated EVs with a heterogeneous size range below 200 nm. Western blot detected EV markers (Alix, Annexin V, HSP70, and Flotillin 1) and an upregulation of Cldn5 in db EVs. While the db EVs were incorporated into macrophages, treatment of THP-1 cells with db EVs significantly increased mRNA levels of TNFα and IL-1β compared to non-db EVs. Proteomic and gene enrichment analysis indicated pro-inflammatory characteristics of db EVs. Our results suggest a potential involvement of EC-derived Cldn5+ EVs in triggering inflammation, offering a novel mechanism involved and presenting a possible therapeutic avenue for DR. [ABSTRACT FROM AUTHOR]

Objective: To verify whether phosphoglycerate mutase 1 (PGAM1) allosteric inhibitor could enhance the sensitivity of colon cancer cells to oxaliplatin (OXA). Methods: OXA sensitivity and PGAM1 expression levels of four consensus molecular subgroups (CMS) of colon cancer cells were analyzed according to genomics of drug sensitivity in cancer (GDSC）and cancer cell line encyclopedia (CCLE) databases. CCK8 assay and Annexin V/PI staining were used to investigate the effect of PGAM1 allosteric inhibitor HKB99 on the proliferation and apoptosis of human colon cancer cell lines. CCK8 assay were performed to investigate the effect of HKB99 combined with OXA on the proliferation of colon cancer DLD-1 cells and cooperativity index was analyzed according to the result. Results: Firstly, GDSC and CCLE databases results showed that CMS1 and CMS4 colon cancer cells were less sensitive to OXA than CMS2 and CMS3 cells in terms of the sensitive cells proportion. Compared with, the expression of PGAM1 was higher in CMS1 and CMS4 cells than in CMS1 and CMS3 cells (P＜0. 05). HKB99 significantly inhibits the proliferation of SW480, SCUN2B and DLD-1 cells, with an IC50 of approximately 1μM. Annexin V/PI staining results showed that apoptosis of DLD-1 cells was significantly increased compared with Control group after HKB99 treatment (P＜0. 01). Results of CCK8 assay result showed that 1. 5μM HKB99 significantly enhanced the inhibitory effect of OXA on colon cancer DLD-1 cells compared with control group (IC50: 0. 37 vs 3. 26μM). The cooperativity index analysis result showed that the synergistic coefficient of OXA with HKB99 was less than 1 at the concentration between 1. 56μM and 25μM. Conclusions: In this study, we found that CMS1 and CMS4 colon cancer cells are less sensitive to OXA and higher level of PGAM1compared with CMS2 and CMS3 cells. An allosteric inhibitor of PGAM1, HKB99, enhances the sensitivity of colon cancer cells to OXA, which suggests that HKB99 may enhance the sensitivity of OXA chemotherapy and provide a potential treatment strategy for patients with individualized chemotherapy in colon cancer. [ABSTRACT FROM AUTHOR]

Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC. [ABSTRACT FROM AUTHOR]

Macrophages play pivotal roles in the regulation of inflammatory responses and tissue repair, making them a prime target for inflammation alleviation. However, the accurate and efficient macrophages targeting is still a challenging task. Motivated by the efficient and specific removal of apoptotic cells by macrophages efferocytosis, a novel biomimetic liposomal system called Effero‐RLP (Efferocytosis‐mediated Red blood cell hybrid Liposomes) is developed which incorporates the membrane of apoptotic red blood cells (RBCs) with liposomes for the purpose of highly efficient macrophages targeting. Rosiglitazone (ROSI), a PPARγ agonist known to attenuate macrophage inflammatory responses, is encapsulated into Effero‐RLP as model drug to regulate macrophage functions in DSS‐induced colitis mouse model. Intriguingly, the Effero‐RLP exhibits selective and efficient uptake by macrophages, which is significantly inhibited by the efferocytosis blocker Annexin V. In animal models, the Effero‐RLP demonstrates rapid recognition by macrophages, leading to enhanced accumulation at inflammatory sites. Furthermore, ROSI‐loaded Effero‐RLP effectively alleviates inflammation and protects colon tissue from injury in the colitis mouse model, which is abolished by deletion of macrophages from mice model. In conclusion, the study highlights the potential of macrophage targeting using efferocytosis biomimetic liposomes. The development of Effero‐RLP presents novel and promising strategies for alleviating inflammation. [ABSTRACT FROM AUTHOR]

AIM: To explore the influence of ethyl (2, 4, 6-trimethylbenzoyl) phenylphosphinate (TPOL) on cell apoptosis and its potential mechanism. METHODS: HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation, before treatment with various inhibitors, N-acetyl-L-cysteine (NAC), pifithrin-α and Z-DVED-FMK. Cell viability was measured by CCK-8 assay. Annexin V/propidium iodide staining was used to count the number of apoptotic cells. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels, and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry. The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot. RESULTS: TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner (P<0. 05), with a decrease in Bcl-2 and increases in Bax and cytochrome C (Cyto C), followed by up-regulation of activated caspase-9 and caspase-3, and the cleavage of PARP (P<0. 05). The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK (P<0. 01). TPOL also led to a rapid increase in ROS, a reduction in mitochondrial membrane potential, and the release of Cyto C (P< 0. 01), all of which could be reversed by the ROS scavenger NAC. Moreover, the TPOL-caused alterations in p21, p27, Rb, and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0. 05). The TPOL-induced changes in Bax, Bcl-2, cleaved caspase-9, activated caspase-3, and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α (P<0. 05). CONCLUSION: TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis. [ABSTRACT FROM AUTHOR]

Despite progress in the management of patients with retinal vascular and degenerative diseases, there is still an unmet clinical need for safe and effective therapeutic options with novel mechanisms of action. Recent mechanistic insights into the pathogenesis of retinal diseases with a prominent vascular component, such as retinal vein occlusion (RVO), diabetic retinopathy (DR) and wet age-related macular degeneration (AMD), may open up new treatment paradigms that reach beyond the inhibition of vascular endothelial growth factor (VEGF). Phosphatidylserine (PS) is a novel lipid target that is linked to the pathophysiology of several human diseases, including retinal diseases. PS acts upstream of VEGF and complement signaling pathways. Annexin A5 is a protein that targets PS and inhibits PS signaling. This review explores the current understanding of the potential roles of PS as a target and Annexin A5 as a therapeutic. The clinical development status of Annexin A5 as a therapeutic and the potential utility of PS-Annexin A5 as a theranostic pairing in retinal vascular conditions in particular is described. [ABSTRACT FROM AUTHOR]

Simple Summary: The greater wax moth, Galleria mellonella, is a significant pest in apiculture. This investigation explores the individual and combined effects of the ectoparasite Bracon hebetor (B.h.) and the entomopathogenic nematode Steinernema carpocapsae (S.c.) on G. mellonella larvae. We found that control larvae exhibited higher glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities compared to those treated with B.h., S.c., or the B.h. + S.c. combination. Conversely, malondialdehyde (MDA) levels displayed an opposite trend. Superoxide dismutase (SOD) activity was reduced in the B.h. and S.c. groups but significantly higher in the combined treatment. Cytochrome P450 activity increased in response to parasitism by B. hebetor. The Annexin V-FITC assay revealed decreased cell viability in parasitized groups (B.h. 79.4%, S.c. 77.3%, B.h. + S.c. 70.1%) compared to controls. DNA damage analysis demonstrated significant differences between groups, and SEM observations confirmed severe cuticle abnormalities or malformations in G. mellonella larvae. The greater wax moth, Galleria mellonella, is a significant pest in apiculture and a well-established model organism for immunological and ecotoxicological studies. This investigation explores the individual and combined effects of the ectoparasite Bracon hebetor (B.h.) and the entomopathogenic nematode Steinernema carpocapsae (S.c.) on G. mellonella larvae. We evaluated the activity of oxidative stress enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), malondialdehyde (MDA) levels, cytochrome P450 activity, cell viability using Annexin V-FITC, DNA damage via comet assay, and larval morphology through scanning electron microscopy (SEM). Control larvae exhibited higher GPx and GST activities compared to those treated with B.h., S.c., or the B.h. + S.c. combination. Conversely, MDA levels displayed the opposite trend. SOD activity was reduced in the B.h. and S.c. groups but significantly higher in the combined treatment. Cytochrome P450 activity increased in response to parasitism by B. hebetor. The Annexin V-FITC assay revealed decreased cell viability in parasitized groups (B.h. 79.4%, S.c. 77.3%, B.h. + S.c. 70.1%) compared to controls. DNA damage analysis demonstrated significant differences between groups, and SEM observations confirmed severe cuticle abnormalities or malformations in G. mellonella larvae. These findings highlight the complex interactions between B. hebetor, S. carpocapsae, and their host, G. mellonella. Additionally, they illuminate the intricate physiological responses triggered within the host larvae. [ABSTRACT FROM AUTHOR]

Background: GP‐2250, a novel analog of taurultam (TRLT), has emerged as a potent anti‐neoplastic drug; however, the mechanisms underlying its effects are not well understood. Here, we investigated the mechanism of action and the biological effects of GP‐2250 using in vitro and in vivo models. Methods: We carried out a series of in vitro (MTT assay, Annexin V/PI assay, colony formation assay, reverse‐phase protein array [RPPA], and HRLC/IC analysis) to determine the biological activity of GP‐2250 and investigate the mechanism of action. In vivo experiments were carried out to determine the therapeutic efficacy of GP‐2250 alone and in combination with standard‐of‐care drugs (e.g., paclitaxel, cisplatin, topotecan, and poly ADP‐ribose polymerase [PARP] inhibitors). Results: We investigated the cytotoxic effect of GP‐2250 in 10 ovarian cancer cell lines and found GP‐2250 combined with a PARP inhibitor had the greatest synergy. RPPA revealed that GP‐2250 inhibited hypoxia‐inducible factor‐1α, AKT, and mammalian target of rapamycin (mTOR) activation and expression. High‐resolution mass spectrometry revealed that hexokinase2 activity and protein expression were significantly reduced by GP‐2250 exposure. Furthermore, GP‐2250 reduced glycolysis and ATP synthesis in cancer cells. An in vivo pharmacodynamic experiment using the OVCAR8 mouse model demonstrated that 500 mg/kg GP‐2250 was effective in downregulating AKT and mTOR activation and expression. In the in vivo therapy experiment using an orthotopic mouse model, a combination of GP‐2250 with either PARP inhibitors or bevacizumab showed a significant reduction of tumor weights and nodules compared to those treated with a vehicle, control IgG groups, or monotherapy groups. Conclusions: Taken together, our data indicate that GP‐2250 exerts profound effects on tumor metabolism and, in combination with PARP inhibitors or bevacizumab, showed promising anti‐tumor efficacy. These findings could have implications for the clinical development of GP‐2250. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common cancer in the world. Despite considerable improvements in the treatment of this cancer, further research to discover novel and more effective agents is ongoing. In this study, possible cytotoxic and apoptotic properties of six benzothiazolopyrimidine derivatives were studied. To assess the IC50 values of these agents, MTT assay was performed on HCT 116, CT26, and NIH/3T3 cells. Moreover, cell death mechanism induced by studied compounds was evaluated by PI/annexin V staining. Then, based on molecular docking results and in vitro experiments, the compounds with the highest anticancer properties were further analyzed in vivo in a mouse model of CRC. MTT results indicated that BTP(1) and BTP(4) had the highest selective cytotoxicity on colorectal cancer cells. Furthermore, flow cytometry results demonstrated a considerable increase in the percentage of the early apoptotic cells in BTP(1)‐ and BTP(4)‐treated groups. In vivo studies confirmed the antitumor properties of the two compounds by a significant regression in tumor size of BTP(1)‐ and BTP(4)‐treated mice compared to control groups. Histopathological examination of tumor tissues showed an increased number of apoptotic cells in these two groups compared to the control animals. Additionally, hematoxylin and eosin staining of the spleen and liver of treated mice did not exhibit considerable tissue damage. Thus, BTP(1) and BTP(4) can be considered promising agents in the treatment of colorectal cancer, although further experiments are required to assess their mechanism of action before their application in clinical studies. [ABSTRACT FROM AUTHOR]

Background: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. Methods: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. Results: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. Conclusion: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells. [ABSTRACT FROM AUTHOR]

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects. Methods: Murine and in vitro ANXA1-/- models were used to study the generation of protective TH1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis. Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a well-documented bacterial contaminant in platelet concentrates (PCs), a blood component used to treat patients with platelet deficiencies. This bacterium can evade routine PC culture screening and cause septic transfusion reactions. Here, we investigated the gene expression modulation within the PC niche versus trypticase soy media (TSB) of S. aureus CBS2016-05, a strain isolated from a septic reaction, in comparison to PS/BAC/317/16/W, a strain identified during PC screening. RNA-seq analysis revealed upregulation of the capsule biosynthesis operon (capA-H), surface adhesion factors (sasADF), clumping factor A (clfA), protein A (spa), and anaerobic metabolism genes (pflAB, nrdDG) in CBS2016-05 when grown in PCs versus TSB, implying its enhanced pathogenicity in this milieu, in contrast to the PS/BAC/317/16/W strain. Furthermore, we investigated the impact of S. aureus CBS2016-05 on platelet functionality in spiked PCs versus non-spiked PC units. Flow cytometry analyses revealed a significant decrease in glycoprotein (GP) IIb (CD41) and GPIbα (CD42b) expression, alongside increased P-selectin (CD62P) and phosphatidylserine (annexin V) expression in spiked PCs compared to non-spiked PCs (p = 0.01). Moreover, spiked PCs exhibited a drastic reduction in MitoTrack Red FM and Calcein AM positive platelets (87.3% vs. 29.4%, p = 0.0001 and 95.4% vs. 24.7%, p = 0.0001) in a bacterial cell density manner. These results indicated that S. aureus CBS2016-05 triggers platelet activation and apoptosis, and compromises mitochondrial functionality and platelet viability, in contaminated PCs. Furthermore, this study enhanced our understanding of the effects of platelet-bacteria interactions in the unique PC niche, highlighting S. aureus increased pathogenicity and deleterious effect on platelet functionality in a strain specific manner. Our novel insights serve as a platform to improve PC transfusion safety. [ABSTRACT FROM AUTHOR]

Background. Still, cancer remains to be one of the main causes of death globally. Sesquiterpene lactones (SLs) appeared to have remarkable pharmacologic properties, particularly as anticancer. This study evaluated the cytotoxicity and apoptogenic potential of Janerin and its underlying mechanism in HL60, THP‐1, and Jurkat leukemia cell lines vs. normal peripheral blood mononuclear cells (PBMCs). Methods. The viability of leukemia and PBMCs following treatment with Janerin (2.5–20 μM) and doxorubicin (2 μM, as the positive control) for 48 h was determined via the resazurin assay. The apoptotic cells were determined by annexin V and propidium iodide test. Also, the genes expressions involved in apoptosis were detected by real‐time PCR. Results. Janerin reduced cell viability in leukemia cells in a dose‐dependent manner with no significant toxicity toward normal PBM cells. Janerin significantly increased apoptosis in leukemia cells after 48 h of treatment. In these cells, the expressions of p21, p53, CASP3, CASP9, and Bax/Bcl2 ratio were significantly elevated, whereas CASP8 remained unchanged (p < 0.01). It was suggested that the intrinsic pathway was the mechanism by which Janerin induced apoptosis in HL60, THP‐1, and Jurkat leukemia cells in a time‐ and dose‐dependent manner. Conclusion. The findings imply that Janerin might be a suitable substitute for doxorubicin in leukemia patients. [ABSTRACT FROM AUTHOR]

In recent years, in the development of emerging immunotherapy, B7‐H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)‐T target against glioma and other tumours, and aroused extensive attention. However, B7‐H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7‐H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7‐H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5‐methylcytosine reader Y‐box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7‐H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Glucotoxicity is a condition that leads to β-cell death, driving a decrease in insulin secretion and leading to hyperglycemia and, eventually, diabetes. Here, we investigate whether connexin Cx30.2 could protect against glucotoxicity-induced apoptosis in β cells. We found that RIN-m5F β cells exposed to high glucose exhibited increased Cx30.2 protein expression. Interestingly, decreased expression of Cx30.2 by specific siRNAs resulted in augmented β-cell death, suggesting that Cx30.2 has an important role in maintaining cell survival during glucotoxicity. Overall, Cx30.2 could be an attractive pharmacological target to avoid high glucose-induced β-cell apoptosis. Glucotoxicity may exert its deleterious effects on pancreatic β-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. β-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in β cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of β cells (RIN-m5F). Methods: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. Results: High glucose did not induce apoptosis in RIN-m5F β cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. Conclusion: The data suggest that the upregulation of Cx30.2 protects β cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders. [ABSTRACT FROM AUTHOR]

Objective and Design: Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. Methods: We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. Results: LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin − 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. Conclusion: ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Major facilitator superfamily domain containing 12 (MFSD12) regulates lysosomal cysteine import and promotes the proliferation and survival of melanoma cells. However, the expression and function of MFSD12 in other cancers, particularly in lung cancer, remain unclear. The expression of MFSD12 across various types of cancers and corresponding control tissues was examined using TIMER. MFSD12 expression in lung adenocarcinoma (LUAD) and its correlation with distinct clinicopathological features of LUAD patients were analyzed with UALCAN. The correlation between MFSD12 expression and survival of LUAD patients was assessed using the R package, survival, and the relationship between MFSD12 expression and immune infiltration status in LUAD was investigated using CIBERSORT. In addition, MFSD12 expression was knocked down in PC9 LUAD cells and their proliferation, capacity for expansion, cell cycle, apoptosis, and migration/invasion were evaluated through CCK-8 assays, colony formation assays, 7-AAD staining, Annexin V/PI staining, and Transwell assays, respectively. The stemness of these PC9 cells was determined through Western blotting, flow cytometry, and tumor sphere formation assays. MFSD12 mRNA levels were significantly elevated in multiple types of cancers, including LUAD. MFSD12 expression was also positively correlated with cancer stage, nodal metastasis, and infiltration of various immune cells in LUAD, and high MFSD12 levels predicted poor survival among LUAD patients. Knockdown of MFSD12 in PC9 cells resulted in decreased proliferation, attenuated colony formation capacity, cell cycle arrest, elevated apoptosis, impaired migration/invasion, and reduced stemness in PC9 cells. MFSD12 is an oncogene in LUAD. [ABSTRACT FROM AUTHOR]