Formation Of Cell Research Articles

Liquid-liquid phase inclusion bodies in acute and persistent parainfluenaza virus type 5 infections.

Cytoplasmic inclusion bodies (IBs) are a common feature of single-stranded, non-segmented, negative-strand RNA virus (Mononegavirales) infections and are thought to be regions of active virus transcription and replication. Here we followed the dynamics of IB formation and maintenance in cells infected with persistent and lytic/acute variants of the paramyxovirus, parainfluenza virus type 5 (PIV5). We show that there is a rapid increase in the number of small inclusions bodies up until approximately 12 h post-infection. Thereafter the number of inclusion bodies decreases but they increase in size, presumably due to the fusion of these liquid organelles that can be disrupted by osmotically shocking cells. No obvious differences were observed at these times between inclusion body formation in cells infected with lytic/acute and persistent viruses. IBs are also readily detected in cells persistently infected with PIV5, including in cells in which there is little or no ongoing virus transcription or replication. In situ hybridization shows that genomic RNA is primarily located in IBs, whilst viral mRNA is more diffusely distributed throughout the cytoplasm. Some, but not all, IBs show incorporation of 5-ethynyl-uridine (5EU), which is integrated into newly synthesized RNA, at early times post-infection. These results strongly suggest that, although genomic RNA is present in all IBs, IBs are not continuously active sites of virus transcription and replication. Disruption of IBs by osmotically shocking persistently infected cells does not increase virus protein synthesis, suggesting that in persistently infected cells most of the virus genomes are in a repressed state. The role of IBs in PIV5 replication and the establishment and maintenance of persistence is discussed.

Abstract MP34: Muscle Sodium Content and Isolevuglandin Formation in Antigen Presenting Cells in Human Salt Sensitive Hypertension

Background: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular mortality. However, the mechanisms by which salt exposure affects blood pressure in SSBP are still unclear. We have shown that under high sodium (Na + ) concentrations, Na + enters antigen-presenting cells (APCs) via epithelial Na + channel (ENaC), which leads to isolevuglandin (IsoLG) production and hypertension. Baseline %IsoLG+ APC correlates with SSBP. While the exact location where APCs are activated by salt and trigger an inflammatory response remains to be elucidated, muscle and skin are proposed as potential sites. We hypothesize that muscle and skin Na + levels are associated with SSBP. Methods: We assessed SSBP in 30 hypertensive subjects using an inpatient salt loading and depletion protocol. Flow cytometry was used to identify IsoLG-containing APCs; dendritic cells (DCs), classic (CD14+16-), non-classic (CD14-16+), and intermediate (CD14+16+) monocytes. Salt loading was achieved through saline infusion and a high-salt diet (460mmol/24 hours), while salt depletion was achieved via diuretic administration (furosemide 40mg x3) and a low-salt diet (10mmol/24hours). Baseline muscle and skin Na + levels were assessed with the 23 Na MRI technique. Spearman's correlation was used to analyze the associations. Demographic data are presented with mean and standard error of the mean. Results: Participants’ age was 53 ± 1.34 years, 53% female, baseline blood pressure was 140.2 ± 2.62 / 87.30 ± 1.77 mmHg, and salt sensitivity index was 3.72 ± 1.4 mmHg. We found an inverse correlation between baseline muscle Na + levels and the decrease in IsoLGs from salt loading to depletion (ΔIsoLGs) in DCs (r=-0.50, p=0.007), intermediate (r=-0.42 p=0.0284) and non-classic monocytes (r=-0.39 p=0.0432), but not in classical monocytes. Skin Na + levels were not correlated with any of these measures. There was a decreasing trend of salt sensitivity index with increasing muscle Na + , although this correlation did not reach statistical significance (r=-0.35 p=0.0686) Conclusion: This highlights the potential importance of muscle Na + in SSBP, such that patients with lower muscle Na + exhibited a higher trend of salt sensitivity index and increased variability in IsoLG+ APCs. Moreover, baseline tissue Na + assessment may emerge as a potential tool for evaluating the degree of salt sensitivity and IsoLG activation in SSBP.

Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway

Background Macrophage-derived foam cells are essential in the progression of atherosclerosis (AS). Based on our previous study, the Huxin Formula (HXF), a traditional Chinese medicine formula, demonstrates potential in anti-atherosclerosis. Nevertheless, it is still unknown how HXF affects the formation of foam cells derived from THP-1 macrophages. Purpose This research aims to examine the preventive role of HXF in the development of foam cells and its underlying molecular mechanism. Methods THP-1 derived macrophages and THP-1 cells overexpressing LOX-1 (LV-OLR1) were exposed to ox-LDL to establish foam cell models, and then treated with HXF. Meantime, Oil red O staining was used to detect lipid droplet production. ELISA kit was performed to measure intracellular levels of IL-6 and TGF-β. RT-qPCR and Western Blot were then utilized to determine the LOX-1 and NF-κB mRNA/protein levels. Results The findings indicated that HXF treatment potently reduced the lipid accumulation, downregulated IL-6 levels and upregulated TGF-β levels. However, this impact was almost reversed when LOX-1 was overexpressed in THP-1 cells stimulated with ox-LDL. Moreover, THP-1 were treated with HXF markedly reduced the levels of LOX-1 and NF-κB mRNA/protein, whereas overexpressing LOX-1 significantly reversed this effect. Conclusion HXF reduced the formation of foam cell in ox-LDL-stimulated THP-1 macrophages via inhibiting lipid accumulation and inflammation through regulating the LOX-1/ NF-κB pathway. These present findings further indicate a potential beneficial role of HXF in ameliorating atherosclerosis and foam cell formation, while provide a novel potential therapeutic strategy for preventing atherosclerosis.

Targeting proliferating cell nuclear antigen enhances ionizing radiation-induced cytotoxicity in prostate cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation. The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated. We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S2056) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status. These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.

Abstract P233: Amyloid Beta Plaque Deposits Co-Localize with Endothelial Cells in the Hippocampus of Female APPswe/PSEN1dE9 Mice During the Onset of Amyloid Pathology

Increasing evidence shows that cardiovascular diseases are associated with an increased risk of cognitive impairment and Alzheimer’s diseases (AD). It is unknown whether systemic vascular dysfunction occurs prior to the development of AD, if this occurs in a sex-dependent manner, or whether endothelial cells play a role in the deposition of amyloid beta (Aβ) peptides. We hypothesized that female mice present with an exacerbated formation of Aβ peptides, and vascular dysfunction would occur during the onset of amyloid pathology, thus escalating its progression. To test this hypothesis, we used a double transgenic mouse model of early onset AD (APPswe/PSEN1dE9) prior to the onset of amyloid pathology (3 months of age) and during the onset of amyloid pathology (7 months of age). Immunofluorescence was performed in the hippocampus stained with Aβ 1-42 and CD31 antibodies. High resolution images were acquired on the confocal microscope. Acetylcholine-induced relaxation (1pM-30µM) was evaluated in mesenteric resistance arteries (MRA), and the data analyzed as non-linear curve regression and maximum effect (Emax). The statistical analysis was performed using Student’s t test, or two-way ANOVA followed by Tukey post-hoc ( p <0.05*). We observed that during the onset of amyloid pathology (7-months-old), there was elevated Aβ deposition which co-localized with endothelial cells in the hippocampus from female but not male APP/PS1 mice [females colocalization rate (%): control 12.9 ± 2.0 vs . APP/PS1 25.1 ± 7.8, n=4; males colocalization rate (%): control 23.2 ± 6.5 vs. APP/PS1 15.3 ± 3.3, n=4]. In addition, the 7-month-old female APP/PS1 mice showed an exacerbated endothelium-dependent relaxation compared to the 3-month-old group (female APP/PS1 7-month-old ACh Emax: 97.1 ± 1.6*%, n=4 vs. 3-month-old ACh Emax: 70.1 ± 8.3%, n=6). Furthermore, endothelial dysfunction occurred prior to the onset of amyloid pathology irrespective of sex. Overall, these data showed that the endothelial dysfunction prior to the onset of amyloid pathology may be an early marker of AD. Further, a greater burden of Aβ formation in hippocampal endothelial cells from female but not male APP/PS1 mice may be associated with vascular abnormalities and dysfunction.

Induction of phospholipase A2 group 4C by hepatitis C virus infection regulates lipid droplet formation

Background & AimsHepatic steatosis, characterized by lipid accumulation in hepatocytes, is a key diagnostic feature in patients with chronic hepatitis C virus (HCV) infection. This study aimed to clarify the involvement of phospholipid metabolic pathways in the pathogenesis of HCV-induced steatosis. MethodsThe expression and distribution of lipid species in the livers of human liver chimeric mice were analyzed using imaging mass spectrometry. Triglyceride accumulation and lipid droplet formation were studied in phospholipase A2 group 4C (PLA2G4C) knockout or overexpressing cells. ResultsImaging mass spectrometry of the infected mouse model revealed increased lysophosphatidylcholine levels and decreased phosphatidylcholine levels in HCV-positive regions of the livers. Among the transcripts associated with phosphatidylcholine biosynthesis, up-regulation of PLA2G4C mRNA was most pronounced following HCV infection. Activation of the transcription factor NF-κB and up-regulation of c-Myc were important for activation of PLA2G4C transcription by HCV infection and expression of the viral proteins Core-NS2. The amount and size of lipid droplets were reduced in PLA2G4C-knockout cells. Inhibition of NF-κB or c-Myc activity suppressed lipid droplet formation in HCV-infected cells. HCV infection promoted the stabilization of lipid droplets, but this stability was reduced in PLA2G4C-knockout cells. Overexpression of PLA2G4C decreased the levels of phosphatidylcholine species in the lipid droplet fraction and led to lower levels of key factors involved in lipolysis (breakdown of triglycerides into glycerol and free fatty acids), such as ATGL, PLIN1 and ABHD5 on the lipid droplets. ConclusionsHCV infection markedly increases PLA2G4C expression. This may alter the phospholipid composition of the lipid droplet membrane, leading to stabilization and enlargement of the droplets.

Study of the relationship among biomarkers, cell and tissue of glioma through Raman spectroscopy

Glioma is the most common brain tumors with high mortality and recurrence rates. Currently, the diagnosis methods for glioma are mainly based on tissue level, cellular level and biomarker level. In this paper, the characteristics of biomarkers (γ-aminobutyric acid and matrix mtalloproteinses-2), U87MG glioma cell and tissue were studied based on Raman spectroscopy, respectively. The results showed that the γ-aminobutyric acid concentration exhibited a linear relation with the intensity of characteristic peaks in 800–1600 cm−1 region, whereas the spectral baseline increased with the increasing of sample concentration in 200–700 cm−1 region. The Raman characteristics of matrix mtalloproteinses-2 in 20–1800 cm−1 region was investigated. Especially, it is demonstrated that the matrix mtalloproteinses-2 showed sixteen low-wavenumber Raman peaks in the range of 20–300 cm−1. Moreover, the U87MG glioma cell showed seven different Raman characteristic peaks in 600–1800 cm−1 region. Compared with the normal tissue, the Raman intensity of tumor tissue showed apparent intensity differences in 300–1800 cm−1, where the intensity changes of these Raman peaks were related to the reducing of the lipid metabolic pathways, and increase of the RNA in tumor tissue region. Furthermore, it is found that the Raman spectra of U87MG glioma cell and tumor tissue had corresponding peaks in the Raman spectra of the liquid γ-aminobutyric acid and matrix mtalloproteinses-2. It is suggested that the γ-aminobutyric acid and matrix mtalloproteinses-2 contributed to the formation and growth of glioma cell and tissue. Thus, Raman spectroscopy not only can diagnose glioma at the biomarkers, cellular and tissue level, but also analyze the relationship among the three. Furthermore, the results provided a physical marker for the detection of glioma in clinically.

Characterisation and modelling of potassium-ion batteries

Potassium-ion batteries (KIBs) are emerging as a promising alternative technology to lithium-ion batteries (LIBs) due to their significantly reduced dependency on critical minerals. KIBs may also present an opportunity for superior fast-charging compared to LIBs, with significantly faster K-ion electrolyte transport properties already demonstrated. In the absence of a viable K-ion electrolyte, a full-cell KIB rate model in commercial cell formats is required to determine the fast-charging potential for KIBs. However, a thorough and accurate characterisation of the critical electrode material properties determining rate performance—the solid state diffusivity and exchange current density—has not yet been conducted for the leading KIB electrode materials. Here, we accurately characterise the effective solid state diffusivities and exchange current densities of the graphite negative electrode and potassium manganese hexacyanoferrate K2Mn[Fe(CN)6]\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${{{{\\rm{K}}}}}_{2}{{{\\rm{Mn}}}}[{{{\\rm{Fe}}}}{({{{\\rm{CN}}}})}_{6}]$$\\end{document} (KMF) positive electrode, through a combination of optimised material design and state-of-the-art analysis. Finally, we present a Doyle-Fuller-Newman model of a KIB full cell with realistic geometry and loadings, identifying the critical materials properties that limit their rate capability.

Distinct functional domains of Dystroglycan regulate inhibitory synapse formation and maintenance in cerebellar Purkinje cells.

Dystroglycan is a cell adhesion molecule that localizes to synapses throughout the nervous system. While Dystroglycan is required to maintain inhibitory synapses from cerebellar molecular layer interneurons (MLIs) onto Purkinje cells (PCs) whether initial synaptogenesis during development is dependent on Dystroglycan has not been examined. We show that conditional deletion of Dystroglycan from Purkinje cells prior to synaptogenesis results in impaired MLI:PC synapse formation and function due to reduced presynaptic inputs and abnormal postsynaptic GABAA receptor clustering. Using genetic manipulations that disrupt glycosylation of Dystroglycan or truncate its cytoplasmic domain, we show that Dystroglycan's role in synapse function requires both extracellular and intracellular interactions, whereas synapse formation requires only extracellular interactions. Together, these findings provide molecular insight into the mechanism of inhibitory synapse formation and maintenance in cerebellar cortex.

Comparison of Free‐Hydroxy With 3′,5′‐Di‐O‐Acetyl Ribose of 5‐Alkynyl Dicobalt Hexacarbonyl 2′‐Deoxy‐Furopyrimidines Hybrids: Synthesis, Antiproliferative Activity, and ROS Determination

ABSTRACTSynthesis of 5‐alkynyl furopyrimidine nucleoside analogs, with free‐ribose groups, was carried out, and their biological activity was evaluated. The substituents introduced at the C‐5 included propargyl and homopropargyl alcohols, 4‐methylphenyl (p‐tolyl), and 4‐pentylphenyl substituted alkynyl groups (56%–88%). Subsequently, alkyne function was coordinated to dicobalt hexacarbonyl unit, yielding the corresponding dicobalt hexacarbonyl 5‐alkynyl furopyrimidine nucleosides (51%–75%). All compounds contained 4‐pentylphenyl group attached at the C‐6 position of the bicyclic base, in line with most active antiviral structures. The antiproliferative effect of these modified nucleosides on cancer cells of different phenotypes was determined using in vitro studies. The tested compounds show antiproliferative effects with median inhibitory concentration (IC50) values of 16–23 and 9–15 μM for HeLa and K562 cells, respectively. The determination of reactive oxygen species (ROS) formation in K562 cells in the presence of modified nucleosides may suggest that the mode of action of the examined compounds may be attributed to the induction of oxidative stress.

Treatment of tumor-associated macrophages with PD-1 monoclonal antibodies affects vascular generation in cervical cancer via the PD-1/IRE1α/SHP2/HIF1α signaling pathway.

To investigate the effect of PD-1 monoclonal antibodies in tumor-associated macrophages on angiogenesis in cervical cancer and its mechanism of action. The effect of PD-1 monoclonal antibodies on the progression of cervical cancer was assessed using the nude mouse xenograft model and HE staining; the impact of PD-1 monoclonal antibodies on cervical cancer cell migration was evaluated using wound healing assay and Transwell assay; the effect on vascular formation in cervical cancer cells was examined using an angiogenesis assay; the impact on the expression of related proteins was tested using Western blotting. PD-1 monoclonal antibodies in tumor-associated macrophages can regulate and thus inhibit the progression of cervical cancer while promoting the expression of SHP2. Additionally, Sindilizumab inhibited the expression of tissue-type fibrinogen activator K and HIF1α through the PD-1/IRE1α/SHP2 signaling pathway, which inhibited the migration and neovascularization of cervical cancer cells. This study discovered that PD-1 monoclonal antibodies in tumor-associated macrophages inhibit vascular generation inside cervical cancer by affecting the PD-1/IRE1α/SHP2/HIF1α signaling pathway, providing a new therapeutic target for the treatment of cervical cancer.

Jiedu Xiaozheng Yin extract targets cancer stem cells by Wnt signaling pathway in colorectal cancer

Ethnopharmacological relevanceThe clinical application of the traditional Chinese medicinal formula Jiedu Xiaozheng Yin (JXY) for gastrointestinal tumors, particularly colorectal cancer (CRC), is well-established, yet the precise biological mechanism underlying its efficacy in CRC treatment remains elusive. Aims of the studyThis study endeavors to unravel the intricate mechanism through which JXY modulates colorectal cancer stem cells, thus elucidating the pathways by which it exerts its potent anti-tumor effects. Materials and methodsIn this study, the regulatory impact of JXY on the signaling pathway and function of CRC cells was analyzed through Network pharmacology. The ethyl acetate extract of JXY was detected the major compounds using HPLC and then treated the HCT-116 cells for RNA-Sequencing (RNA-Seq). Protein expression and stemness of HCT-15 and HCT-116 cells following JXY extract treatment were assessed using Western blot analysis and matrigel spheroid assays. Additionally, the β-catenin transcriptional activity was evaluated using a TOPflash reporter assay with or without Lithium chloride (LiCl) stimulation. Patient-derived organoids of CRC (CRC PDOs) were cultured using a stemness maintenance medium, and their viability was measured using ATP assays after treatment of JXY extract. Furthermore, the anti-tumor efficacy of JXY extract was assessed using a xenograft mice model derived from HCT-15 cells. ResultsNetwork pharmacology emphasized the influence of JXY on cancer stem cells and the Wnt signaling pathway. HPLC analysis confirmed that the JXY extract contained the three most prevalent pharmaceutical compounds among the four herbs documented in the Chinese Pharmacopoeia (rosmarinic acid, quercetin, and kaempferol). RNA-Seq results further elucidated the effect of JXY extract, particularly targeting cancer stem cells and the Wnt signaling pathway. Furthermore, JXY extract inhibited spheroid formation in CRC cells and downregulated CRC CSC markers (CD133, DCLK1, and C-MYC). Additionally, JXY extract suppressed the β-catenin expression and transcriptional activity as well as the Wnt pathway target proteins, including C-MYC and Cyclin D1. Consistent with findings from cell lines, JXY extract suppressed the growth of CRC PDOs exhibiting stemness characteristics. And JXY extract demonstrated a significant inhibitory effect on tumor growth, C-MYC, and β-catenin protein levels in xenograft tumors. ConclusionsThese results highlight the novel function of JXY extract in targeting CRC CSCs by regulating Wnt signaling pathway, underscoring its potential as a therapeutic agent for treating CRC.

FOSL1 promotes stem cell‑like characteristics and anoikis resistance to facilitate tumorigenesis and metastasis in osteosarcoma by targeting SOX2.

Metastasis is the leading cause of cancer‑related death in osteosarcoma (OS). OS stem cells (OSCs) and anoikis resistance are considered to be essential for tumor metastasis formation. However, the underlying mechanisms involved in the maintenance of a stem‑cell phenotype and anoikis resistance in OS are mostly unknown. Fos‑like antigen 1 (FOSL1) is important in maintaining a stem‑like phenotype in various cancers; however, its role in OSCs and anoikis resistance remains unclear. In the present study, the dynamic expression patterns of FOSL1 were investigated during the acquisition of cancer stem‑like properties using RNA sequencing, PCR, western blotting and immunofluorescence. Flow cytometry, tumor‑sphere formation, clone formation assays, anoikis assays, western blotting and in vivo xenograft and metastasis models were used to further investigate the responses of the stem‑cell phenotype and anoikis resistance to FOSL1 overexpression or silencing in OS cell lines. The underlying molecular mechanisms were evaluated, focusing on whether SOX2 is crucially involved in FOSL1‑mediated stemness and anoikis in OS. FOSL1 expression was observed to be upregulated in OSCs and promoted tumor‑sphere formation, clone formation and tumorigenesis in OS cells. FOSL1 expression correlated positively with the expression of stemness‑related factors (SOX2, NANOG, CD117 and Stro1). Moreover, FOSL1 facilitated OS cell anoikis resistance and promoted metastases by regulating the expression of apoptosis related proteins BCL2 and BAX. Mechanistically, FOSL1 upregulated SOX2 expression by interacting with the SOX2 promoter and activating its transcription. The results also showed that SOX2 is critical for FOSL1‑mediated stem‑like properties and anoikis resistance. The current findings indicated that FOSL1 is an important regulator that promotes a stem cell‑like phenotype and anoikis resistance to facilitate tumorigenesis and metastasis in OS by regulating the transcription of SOX2. Thus, FOSL1 might represent an attractive target for therapeutic interventions in OS.

Pericyte-to-Endothelial Cell Communication via Tunneling Nanotubes Is Disrupted by a Diol of Docosahexaenoic Acid.

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.

Aplastic anemia in association with multiple myeloma: clinical and pathophysiological insights

We investigated immune cytopenia in multiple myeloma (MM) patients with concurrent acquired aplastic anemia (AA), focusing on three clinical cases treated with plasma cell-directed therapy. All three patients achieved partial response in MM and one patient experienced complete resolution of AA. Two patients had partial improvement in transfusion requirement but continued to suffer from severe AA, leading to immunosuppressive therapy (IST) with improvement of transfusion requirement in both patients. In vitro serum testing of these patients demonstrated platelet mitochondrial dysfunction and platelet apoptosis but did not show sera-specific inhibition of erythroid colony formation in progenitor cells. The levels of IL8 and IL15 were elevated in MM patients with AA, implicating their potential roles in this co-occurrence. Response to IST points to the possibility of myeloma-dysregulated immune system leading to autoreactive T-cell destruction of hematopoietic stem and progenitor cells, offering insights for developing new treatment for cytopenia in MM.

RNA-binding motif protein 28 enhances angiogenesis by improving STAT3 translation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor characterized by extensive angiogenesis. However, the underlying mechanisms of HCC pathogenesis remain unclear. Previous studies have shown that RNA-binding proteins (RBPs) are implicated in HCC pathogenesis. In this study, we observed that increased RBM28 expression in HCC tissues was positively correlated with tumor microvascular density and negatively correlated with patient prognosis. Overexpression of RBM28 in HCC cells promoted tubule formation in human umbilical vein endothelial cells, whereas inhibition of RBM28 had the opposite effect, furthermore, the role of RBM28 in the progression of HCC was assessed using transgenic mouse models and chemically induced HCC models. We used various molecular assays and high-throughput detection methods to evaluate the role of RBM28 in promoting angiogenesis in HCC. Increased RBM28 expression in HCC directly binds to STAT3 mRNA, recruiting EIF4E to increase STAT3 expression and enhancing the secretion and expression of vascular endothelial growth factor A; consequently, promoting neovascularization in HCC. The potential of RBM28 as a viable diagnostic and therapeutic target for HCC was assessed using multi-cohort clinical samples and animal models. In summary, our results provide insights into the pathogenesis, clinical diagnosis, and treatment of HCC.

Small Molecule Induces Time-Dependent Inhibition of Stat3 Dimerization and DNA-Binding Activity and Regresses Human Breast Tumor Xenografts.

Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 μM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 μM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, and Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1, pErk1/2MAPK, pShc, pJAK2, or pSrc induction, or on cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.

Phillyrin promotes autophagosome formation in A53T-αSyn-induced Parkinson's disease model via modulation of REEP1

BackgroundThe preservation of autophagosome formation presents a promising strategy for tackling neurological disorders, such as Parkinson's disease (PD). Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) serve not only as a focal point linked to various neurological disorders but also play a crucial role in supporting the biogenesis of autophagosomes. PurposeThis investigation aimed to elucidate the neuroprotective properties of phillyrin against PD and its underlying mechanisms in promoting autophagosome formation. MethodsER and mitochondria co-localization was assessed via fluorescent staining. Annexin V-fluorescein isothiocyanate (FITC) fluorescence was employed to quantify accessible cardiolipin (CL) on mitochondrial surfaces. The levels of CL within the MAM fraction of SH-SY5Y cells were evaluated using a CL probe assay kit. Monodansylcadaverine staining was utilized to detect autophagosome formation in SH-SY5Y cells. In an A53T-alpha-synuclein (αSyn)-induced PD mouse model, the anti-PD properties of phillyrin were assessed using open field, pole climbing, and rotarod tests, as well as immunohistochemistry staining of TH+ neurons in the brain sections. ResultsIn A53T-αSyn-treated SH-SY5Y cells, phillyrin facilitated autophagosome formation by suppressing CL externalization and restoring MAM integrity. Phillyrin enhanced the localization of receptor expression-enhancing protein 1 (REEP1) within MAM and mitochondria, bolstering MAM formation. Increased REEP1 levels in mitochondria, attributed to phillyrin, enhanced the interaction between REEP1 and NDPK-D, thereby reducing CL externalization. Furthermore, phillyrin exhibited a dose-dependent enhancement of motor function in mice, accompanied by an increase in the abundance of dopaminergic neurons within the substantia nigra. ConclusionsThese findings illuminate phillyrin's ability to enhance MAM formation through upregulation of REEP1 expression within MAM, while concurrently attenuating CL externalization via the REEP1-NDPK-D interaction. These mechanisms bolster autophagosome biogenesis, offering resilience against A53T-αSyn-induced PD. Thus, our study advances the understanding of phillyrin's complex mechanisms and underscores its potential as a therapeutic approach for PD, opening new avenues in natural product pharmacology.

Inhibition of XPR1-dependent phosphate efflux induces mitochondrial dysfunction: A potential molecular target therapy for hepatocellular carcinoma?

Xenotropic and polytropic retrovirus receptor 1 (XPR1) is the only known transporter associated with Pi efflux in mammals, and its impact on tumor progression is gradually being revealed. However, the role of XPR1 in hepatocellular carcinoma (HCC) is unknown. A bioinformatics screen for the phosphate exporter XPR1 was performed in HCC patients. The expression of XPR1 in clinical specimens was analyzed using quantitative real-time PCR, Western blot analysis, and immunohistochemical assays. Knockdown of the phosphate exporter XPR1 was performed by shRNA transfection to investigate the cellular phenotype and phosphate-related cytotoxicity of the Huh7 and HLF cell lines. In vivo tests were conducted to investigate the tumorigenicity of HCC cells xenografted into immunocompromised mice after silencing XPR1. Compared with that in paracancerous tissue, XPR1 expression in HCC tissues was markedly upregulated. High XPR1 expression significantly correlated with poor patient survival. Silencing of XPR1 leads to decreased proliferation, migration, invasion, and colony formation in HCC cells. Mechanistically, knockdown of XPR1 causes an increase in intracellular phosphate levels; mitochondrial dysfunction characterized by reduced mitochondrial membrane potential and adenosine triphosphate levels; increased reactive oxygen species levels; abnormal mitochondrial morphology; and downregulation of key mitochondrial fusion, fission, and inner membrane genes. This ultimately results in mitochondria-dependent apoptosis. These findings reveal the prognostic value of XPR1 in HCC progression and, more importantly, suggest that XPR1 might be a potential therapeutic target.

R-loops and impaired autophagy trigger cGAS-dependent inflammation via micronuclei formation in Senataxin-deficient cells

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.