RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Multifunctional chitosan-bimetallic nanocarrier deliver 5-fluorouracil for enhanced treatment of pancreatic and triple-negative breast cancer.

This work aimed to prepare multifunctional aptamer-conjugated, photothermally responsive 5-fluorouracil (5fu)-loaded chitosan-bimetallic (Au/Pd) nanoparticles (APT-CS-5fu-Au/Pd NPs) for improved cytotoxicity in two cancer cell lines (PANC-1 and MDA-MD 231). The CS-5fu-Au/Pd NPs were polydispersed with a size of 34.43 ± 1.59 nm. FTIR analysis indicated the presence of CS, 5fu in CS-5fu-Au/Pd NPs. The 2 theta degrees in CS-5fu-Au/Pd NPs accounted for CS and Au/Pd. Additionally, AGE revealed the conjugation of APT in CS-5fu-Au/Pd NPs. The APT-CS-5fu-Au/Pd NPs (180 µg/mL) with NIR treatment increased the temperature to >50 °C. The optimized 5fu input was 0.075 % in CS-5fu-Au/Pd NPs, exhibiting a hydrodynamic size of 112.96 ± 17.23 nm, DEE of 64.2 ± 3.77 %, and DLE of 11.1 ± 0.65 %. A higher level of 5fu release (69.8 ± 2.78 %) was observed under pH 5.4 at 74 h. In conclusion, NIR-APT-CS-5fu-Au/Pd NPs did not cause toxicity to RBC and Egg CAM, but increased cytotoxicity in MDA-MB 231 and PANC-1 cells by triggering oxidative stress-mediated cell death.

HDAC6-MYCN-CXCL3 axis mediates allergic inflammation and is necessary for allergic inflammation-promoted cellular interactions.

Histone deacetylase 6 (HDAC6) has been shown to play an important role in allergic inflammation. This study hypothesized that novel downstream targets of HDAC6 would mediate allergic inflammation. Experiments employing HDAC6 knock out C57BL/6 mice showed that HDAC6 mediated passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Antigen stimulation increased expression of N-myc (MYCN) and CXCL3 in an HDAC6-dependent manner in the bone marrow-derived mast cells. MYCN and CXCL3 were necessary for both PCA and PSA. The role of early growth response 3 (EGR3) in the regulation of HDAC6 expression has been reported. ChIP assays showed EGR3 as a direct regulator of MYCN. miR-34a-5p was predicted to be a negative regulator of MYCN. Luciferase activity assays showed miR-34a-5p as a direct regulator of MYCN. miR-34a-5p mimic negatively regulated PCA and PSA. MYCN decreased miR-34a-5p expression in antigen-stimulated rat basophilic leukemia cells (RBL2H3). MYCN was shown to bind to the promoter sequence of CXCL3. In an IgE-independent manner, recombinant CXCL3 protein increased expression of HDAC6, MYCN, and ß-hexosaminidase activity in RBL2H3 cells. Mouse recombinant CXCL3 protein enhanced the angiogenic potential of the culture medium of RBL2H3. CXCL3 was necessary for the enhanced angiogenic potential of the culture medium of antigen-stimulated RBL2H3. The culture medium of RBL2H3 was able to induce M2 macrophage polarization in a CXCL3-dependent manner. Recombinant CXCL3 protein also increased the expression of markers of M2 macrophage. Thus, the identification of the novel role of HDAC6-MYCN-CXCL3 axis can help better understand the pathogenesis of anaphylaxis.

Cancer/testis antigen CAGE mediates osimertinib resistance in non-small cell lung cancer cells and predicts poor prognosis in patients with pulmonary adenocarcinoma.

CAGE, a cancer/testis antigen, was originally isolated from the sera of patients with gastric cancers. Previously, we have shown the role of CAGE in resistance to chemotherapy and target therapy. The aim of this study was to investigate the role of CAGE in osimertinib resistance and determine the prognostic value of CAGE in patients with pulmonary adenocarcinomas. The clinicopathological correlation with CAGE and autophagy flux in patients was examined using immunohistochemistry and in situ hybridization. The possible role of autophagy in osimertinib resistance was analyzed using immune blot, immune fluorescence staining and immunohistochemistry. This study found that immunohistochemical staining (IHC) showed CAGE expression in more than 50% of patients with pulmonary adenocarcinomas (pADCs). CAGE expression was increased in pADCs after the acquisition of EGFR-TKIs resistance. High expression of CAGE was correlated with shorter overall survival and progression free survival in patients with pADCs. Thus, CAGE mediates osimertinib resistance and predicts poor prognosis in patients with pADCs. Osimertinib-resistant non-small cell lung cancer cells (PC-9/OSI) were established and mechanistic studies of CAGE-mediated osimertinib resistance were performed. PC-9/OSI cells showed increased autophagic flux and CAGE expression compared with parental sensitive PC-9 cells. PC-9/OSI cells showed higher tumorigenic, metastatic, and angiogenic potential compared with parental PC-9 cells. CAGE CRISPR-Cas9 cell lines showed decreased autophagic flux, invasion, migration potential, and tumorigenic potential compared with PC-9/OSI cells in vitro and in vivo. CAGE plays a crucial role in the cancer progression by modulating autophagy and can predict the poor prognosis of patients with pulmonary adenocarcinomas. Our findings propose CAGE as a potential therapeutic target for developing anticancer drugs that can overcome osimertinib resistance.

Autophagy and mitophagy-related extracellular mitochondrial dysfunction of cerebrospinal fluid cells in patients with hemorrhagic moyamoya disease.

We aimed to investigate whether mitochondrial dysfunction in extracellular cerebrospinal fluid (CSF), which is associated with autophagy and mitophagy, might be involved in neurological outcomes in adult patients with hemorrhagic moyamoya disease (MMD) whose pathogenesis related to poor outcomes is not well-known. CSF samples were collected from 43 adult MMD patients and analyzed according to outcomes at 3 months. Fluorescence-activated cell sorter analysis (FACS) and the JC-1 red/green ratio were used to assess mitochondrial cells and intact mitochondrial membrane potential (MMP). We performed quantitative real-time polymerase chain reaction and Western blotting analyses of autophagy and mitophagy-related markers, including HIF1α, ATG5, pBECN1, BECN1, BAX, BNIP3L, DAPK1, and PINK1. Finally, FACS analysis with specific fluorescence-conjugated antibodies was performed to evaluate the potential cellular origin of CSF mitochondrial cells. Twenty-seven females (62.8%) with a mean age of 47.4 ± 9.7 years were included in the study. Among 43 patients with hemorrhagic MMD, 23 (53.5%) had poor outcomes. The difference in MMP was evident between the two groups (2.4 ± 0.2 in patients with poor outcome vs. 3.5 ± 0.4 in patients with good outcome; p = 0.02). A significantly higher expression (2-ΔCt) of HIF1α, ATG5, DAPK1 followed by BAX and BNIP3L mRNA and protein was also observed in poor-outcome patients compared to those with good outcomes. Higher percentage of vWF-positive mitochondria, suggesting endothelial cell origins, was observed in patients with good outcome compared with those with poor outcome (25.0 ± 1.4% in patients with good outcome vs. 17.5 ± 1.5% in those with poor outcome; p < 0.01). We observed the association between increased mitochondrial dysfunction concomitant with autophagy and mitophagy in CSF cells and neurological outcomes in adult patients with hemorrhagic MMD. Further prospective multicenter studies are needed to determine whether it has a diagnostic value for risk prediction.

Real-Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label-Free Imaging with Optical Diffraction Tomography.

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug-resistant bacteria. As a mimic of AMPs, peptoids with N-substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure-activity relationship (SAR) of a library of indole side chain-containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label-free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real-time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance-breaking novel antibiotic drug.

Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors.

Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen over 4 months, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted during the same period. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging.

Photothermally responsive chitosan-coated iron oxide nanoparticles for enhanced eradication of bacterial biofilms.

This work developed a pH/NIR responsive antibacterial agent (CS-FeNPs) composed of chitosan (CS) and Fe3O4 nanoparticles (FeNPs). CS triggers bacterial attraction through surface charge, while Fe acts as a photothermal agent (PTA). The CS-Fe NPs exhibited antibacterial and antibiofilm activity against both bacteria (G+/G-). However, higher activity was observed against bacteria (G-) due to electrostatic interactions. The CS-FeNPs bind with the bacterial membrane through electrostatic interactions and disturb bacterial cells. Later, in an acidic environment, CS-FeNPs bind with bacterial membrane, and NIR irradiation leads the antibacterial activity. CS-FeNPs exhibited a potential photothermal conversion efficiency (η) of 21.53 %. Thus, it converts NIR irradiation into heat to kill the bacterial pathogen. The CS-FeNPs were found to be less cytotoxic with great antibacterial efficiency on planktonic bacteria and their biofilm, which indicates that they deserve to develop potential and safe treatment strategies for the treatment of bacterial infections.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Esculetin and Fucoidan Attenuate Autophagy and Apoptosis Induced by Zinc Oxide Nanoparticles through Modulating Reactive Astrocyte and Proinflammatory Cytokines in the Rat Brain.

We examined the protective effects of esculetin and fucoidan against the neurotoxicity of ZnO NPs in rats. Ninety rats were divided into nine groups and pre-treated with esculetin or fucoidan 1 h before ZnO NP administration on a daily basis for 2 weeks. Serum and brain homogenates were examined by enzyme-linked immunosorbent assay (ELISA), and neurons, microglia, and astrocytes in the hippocampal region were examined with immunohistochemical analysis. The serum levels of interleukin-1-beta (IL-1ß), 3-nitrotyrosine (3-NT), superoxide dismutase (SOD), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were altered in the ZnO NP treatment groups. Brain IL-1ß and TNF-α levels were elevated after ZnO NP administration, and these effects were inhibited by esculetin and fucoidan. SOD, 8-OHdG, and acetylcholinesterase (AChE) levels in the brain were decreased after ZnO NP administration. The brain levels of beclin-1 and caspase-3 were elevated after ZnO NP treatment, and these effects were significantly ameliorated by esculetin and fucoidan. The number of reactive astrocytes measured by counting glial fibrillary acidic protein (GFAP)-positive cells, but not microglia, increased following ZnO NP treatment, and esculetin and fucoidan ameliorated the changes. Esculetin and fucoidan may be beneficial for preventing ZnO NP-mediated autophagy and apoptosis by the modulation of reactive astrocyte and proinflammatory cytokines in the rat brain.

Combination of Paraconiothyrium brasiliense fabricated titanium dioxide nanoparticle and antibiotics enhanced antibacterial and antibiofilm properties: A toxicity evaluation.

Antimicrobial resistance (AMR) causes global consequences through increased mortality and economic loss. Antimicrobial drugs including nanomaterials are an emerging environmental impact. Hence, this work aimed to synthesize and characterize the titanium dioxide nanoparticles (TiO2 NPs) using the aqueous extract of endophytic fungus Paraconiothyrium brasiliense (Pb) for enhancing the antibacterial efficiency of existing standard antibiotics at minimum concentration. The FTIR and XRD results confirmed the capping of functional molecules and the crystalline nature of Pb-TiO2 NPs. The spherical-shaped TiO2 NPs with the size of 57.39 ± 13.65 nm were found in TEM analysis. The average hydrodynamic size (68.43 ± 1.49 d. nm) and the zeta potential (-19.6 ± 1.49 mV) was confirmed the stability of Pb-TiO2 NPs. Antibacterial studies revealed that bare Pb-TiO2 NPs (20 µg/mL) did not exhibit significant antibacterial activity while combination of TCH + Pb-TiO2 NPs considerably increased the inhibition of E. coli biofilm evidenced by CLSM and SEM analysis. Further, Pb-TiO2 NPs (100 µg/mL) were found to be moderately toxic to cell line (NIH3T3), red blood cells (RBC), and egg embryos. Hence, this study concluded that <50 µg/mL of TiO2 NPs can be mixed with antibiotics for enhanced antibacterial application thereby minimizing the AMR and the environmental toxicity.

Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule.

SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.

Folic acid conjugated chitosan encapsulated palladium nanoclusters for NIR triggered photothermal breast cancer treatment.

This study developed folic acid (FA) conjugated chitosan (CS) encapsulated rutin (R) synthesized palladium nanoclusters (Pd NCs) for NIR triggered and folate receptor (FR) targeted triple-negative breast cancer (MDA-MB 231 cells) treatment. R-Pd NCs exhibited flower-shaped particles with an average size of <100 nm. FA-CS encapsulation concealed the flower shape of R-Pd NCs with a positive charge. The XRD spectrum confirmed the cubic crystalline structure of Pd. The FA conjugation on CS improved the cellular uptake of R-Pd NCs in MDA-MB 231 cells was confirmed by TEM. FA-CS-R-Pd NCs (+NIR) treatment was considerably inhibited the MDA-MB 231 cells proliferation evidenced by cell viability, fluorescent staining, and flow cytometry analysis. Further, in vitro hemolysis assay and in Ovo model confirmed the non-toxic nature of FA-CS-R-Pd-NCs with or without NIR radiation. Hence, this study concluded that FA-CS-R-Pd NCs can be applied for the treatment of drug-resistant breast cancer.

Different Inhibitory Effects of Erythromycin and Chlortetracycline on Early Growth of Brassica campestris Seedlings.

Veterinary antibiotics, including erythromycin (Ery) and chlortetracycline (CTC), are often detected in agricultural land. Although these contaminants affect plant growth and development, their effects on crops remain elusive. In this study, the effects of Ery and CTC on plant growth were investigated and compared by analyzing transcript abundance in Brassica campestris seedlings. Treatment with Ery and/or CTC reduced chlorophyll content in leaves and photosynthetic efficiency. Examination of the chloroplast ultrastructure revealed the presence of abnormally shaped plastids in response to Ery and CTC treatments. The antibiotics produced similar phenotypes of lower accumulation of photosynthetic genes, including RBCL and LHCB1.1. Analysis of the transcript levels revealed that Ery and CTC differentially down-regulated genes involved in the tetrapyrrole biosynthetic pathway and primary root growth. In the presence of Ery and CTC, chloroplasts were undeveloped and photosynthesis efficiency was reduced. These results suggest that both Ery and CTC individually affect gene expression and influence plant physiological activity, independently of one another.

HDAC6 and CXCL13 Mediate Atopic Dermatitis by Regulating Cellular Interactions and Expression Levels of miR-9 and SIRT1.

Histone deacetylase 6 (HDAC6) has been known to regulate inflammatory diseases. The role of HDAC6 in allergic skin inflammation has not been studied. We studied the role of HDAC6 in atopic dermatitis (AD) and the mechanisms associated with it. The decreased expression or chemical inhibition of HDAC6 suppressed AD by decreasing autophagic flux and cellular features of AD. AD increased expression levels of the Th1 and Th2 cytokines, but decreased expression levels of forkhead box P3 (FoxP3) and interleukin-10 (IL-10) in an HDAC6-dependent manner. CXC chemokine ligand 13 (CXCL13), which was increased in an HDAC6-depenednt manner, mediated AD. MiR-9, negatively regulated by HDAC6, suppressed AD by directly regulating the expression of sirtuin 1 (SIRT1). The downregulation or inhibition of SIRT1 suppressed AD. Experiments employing culture medium and transwell suggested that cellular interactions involving mast cells, keratinocytes, and dermal fibroblast cells could promote AD; HDAC6 and CXCL13 were found to be necessary for these cellular interactions. Mouse recombinant CXCL13 protein increased HDAC6 expression in skin mast cells and dermal fibroblast cells. CXCL13 protein was found to be present in the exosomes of DNCB-treated skin mast cells. Exosomes of DNCB-treated skin mast cells enhanced invasion potentials of keratinocytes and dermal fibroblast cells and increased expression levels of HDAC6, SIRT1 and CXCL13 in keratinocytes and dermal fibroblast cells. These results indicate that HDAC6 and CXCL13 may serve as targets for the developing anti-atopic drugs.

Inhibitors of Lipoxygenase and Cyclooxygenase-2 Attenuate Trimethyltin-Induced Neurotoxicity through Regulating Oxidative Stress and Pro-Inflammatory Cytokines in Human Neuroblastoma SH-SY5Y Cells.

Trimethyltin (TMT) is an environmental neurotoxin that mediates dopaminergic neuronal injury in the brain. In this study, we characterized the toxic mechanism and possible protective compounds against TMT-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. Antioxidants such as melatonin, N-acetylcysteine (NAC), α-tocopherol, and allopurinol alleviated TMT toxicity. Apoptosis induced by TMT was identified by altered expression of cleaved caspase-3, Bax, Bcl-2, and Bcl-xL through Western blot analysis. The iron chelator deferoxamine ameliorated the alteration of apoptosis-related proteins through TMT exposure. TMT also induced delayed ultrastructural necrotic features such as mitochondrial swelling and cytoplasmic membrane rupture; NAC reduced these necrotic injuries. Esculetin, meloxicam, celecoxib, and phenidone decreased TMT toxicity. Elevation of the pro-inflammatory cytokines IL-1ß, TNF-α, and NF-ĸB and reduction of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) were induced by TMT and ameliorated by inhibitors of LOX and COX-2 enzymes. Both NMDA and non-NMDA antagonists attenuated TMT toxicity. The free calcium ion modulators nimodipine and BAPTA/AM contributed to neuronal survival against TMT toxicity. Inhibitors of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, an autophagy regulator, decreased TMT toxicity. These results imply that TMT neurotoxicity is the chief participant in LOX- and COX-2-mediated apoptosis, partly via necrosis and autophagy in SH-SY5Y cells.

Mitochondrial dysfunction associated with autophagy and mitophagy in cerebrospinal fluid cells of patients with delayed cerebral ischemia following subarachnoid hemorrhage.

Decreased mitochondrial membrane potential in cerebrospinal fluid (CSF) was observed in patients with subarachnoid hemorrhage (SAH) accompanied by delayed cerebral ischemia (DCI). However, whether abnormal mechanisms of mitochondria are associated with the development of DCI has not been reported yet. Under cerebral ischemia, mitochondria can transfer into the extracellular space. Mitochondrial dysfunction can aggravate neurologic complications. The objective of this study was to evaluate whether mitochondrial dysfunction might be associated with autophagy and mitophagy in CSF cells to provide possible insight into DCI pathogenesis. CSF samples were collected from 56 SAH patients (DCI, n = 21; and non-DCI, n = 35). We analyzed CSF cells using autophagy and mitophagy markers (DAPK1, BNIP3L, BAX, PINK1, ULK1, and NDP52) via qRT-PCR and western blotting of proteins (BECN1, LC3, and p62). Confocal microscopy and immunogold staining were performed to demonstrate the differentially expression of markers within dysfunctional mitochondria. Significant induction of autophagic flux with accumulation of autophagic vacuoles, increased expression of BECN1, LC3-II, and p62 degradation were observed during DCI. Compared to non-DCI patients, DCI patients showed significantly increased mRNA expression levels (2-ΔCt) of DAPK1, BNIP3L, and PINK1, but not BAX, ULK1, or NDP52. Multivariable logistic regression analysis revealed that Hunt and Hess grade ≥ IV (p = 0.023), DAPK1 (p = 0.003), and BNIP3L (p = 0.039) were related to DCI. Increased mitochondrial dysfunction associated with autophagy and mitophagy could play an important role in DCI pathogenesis.

Antiviral Activity of Chrysin against Influenza Virus Replication via Inhibition of Autophagy.

Influenza viruses cause respiratory infections in humans and animals, which have high morbidity and mortality rates. Although several drugs that inhibit viral neuraminidase are used to treat influenza infections, the emergence of resistant viruses necessitates the urgent development of new antiviral drugs. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid that exhibits antiviral activity against enterovirus 71 (EV71) by inhibiting viral 3C protease activity. In this study, we evaluated the antiviral activity of chrysin against influenza A/Puerto Rico/8/34 (A/PR/8). Chrysin significantly inhibited A/PR/8-mediated cell death and the replication of A/PR/8 at concentrations up to 2 µM. Viral hemagglutinin expression was also markedly decreased by the chrysin treatment in A/PR/8-infected cells. Through the time course experiment and time-of-addition assay, we found that chrysin inhibited viral infection at the early stages of the replication cycle. Additionally, the nucleoprotein expression of A/PR/8 in A549 cells was reduced upon treatment with chrysin. Regarding the mechanism of action, we found that chrysin inhibited autophagy activation by increasing the phosphorylation of mammalian target of rapamycin (mTOR). We also confirmed a decrease in LC3B expression and LC3-positive puncta levels in A/PR/8-infected cells. These results suggest that chrysin exhibits antiviral activity by activating mTOR and inhibiting autophagy to inhibit the replication of A/PR/8 in the early stages of infection.

Anti-Inflammatory Activity of 4-((1R,2R)-3-Hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol Isolated from Juglans mandshurica Maxim. in LPS-Stimulated RAW 264.7 Macrophages and Zebrafish Larvae Model.

Juglans mandshurica Maxim., a traditional folk medicinal plant, is widely distributed in Korea and China. In our previous study, we isolated a new phenylpropanoid compound, 4-((1R,2R)-3-hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol (HHMP), from J. mandshurica. In the present study, we evaluated the anti-inflammatory activity of HHMP on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and zebrafish larvae. HHMP significantly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 production in a dose-dependent manner. Moreover, HHMP treatment considerably suppressed LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. We also demonstrated the mechanisms of HHMP inhibition of inflammatory responses in LPS-stimulated RAW 264.7 cells via Western blot analysis and immunofluorescence staining. Furthermore, HHMP significantly inhibited NO production in LPS-stimulated zebrafish larvae. Consequently, we established that HHMP significantly inhibited the LPS-induced activation of NF-κB and MAPK and the nuclear translocation of p65 in RAW 264.7 cells. Taken together, our findings demonstrate the effect of HHMP on LPS-induced inflammatory responses in vitro and in vivo, suggesting its potential to be used as a natural anti-inflammatory agent.