L-serine restored lysosomal failure in cells derived from patients with BPAN reducing iron accumulation with eliminating lipofuscin.

Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2'-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether L-serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. L-serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and L-serine treatment reduced the increase of lipofuscin. These data suggest that L-serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity.

Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

Alpha-2-macroglobulin as a novel diagnostic biomarker for human bladder cancer in urinary extracellular vesicles.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p<0.0001). There was no significant difference in whole urine a2M levels between patients with BC and controls (p=0.317). We observed that compared to classical differential centrifugation, ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method for uEV protein analysis. We expect that our approach for EV analysis will provide an efficient route for the identification of clinically meaningful uEV-based biomarkers for cancer diagnosis.

Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

Estrogen-Related Receptor γ Maintains Pancreatic Acinar Cell Function and Identity by Regulating Cellular Metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.

Photo-Cross-Linkable Human Albumin Colloidal Gels Facilitate In Vivo Vascular Integration for Regenerative Medicine.

Biodegradable cellular and acellular scaffolds have great potential to regenerate damaged tissues or organs by creating a proper extracellular matrix (ECM) capable of recruiting endogenous cells to support cellular ingrowth. However, since hydrogel-based scaffolds normally degrade through surface erosion, cell migration and ingrowth into scaffolds might be inhibited early in the implantation. This could result in insufficient de novo tissue formation in the injured area. To address these challenges, continuous and microsized strand-like networks could be incorporated into scaffolds to guide and recruit endogenous cells in rapid manner. Fabrication of such microarchitectures in scaffolds is often a laborious and time-consuming process and could compromise the structural integrity of the scaffold or impact cell viability. Here, we have developed a fast single-step approach to fabricate colloidal hydrogels, which are made up of randomly packed human serum albumin-based photo-cross-linkable microparticles with continuous internal networks of microscale voids. The human serum albumin conjugated with methacrylic groups were assembled to microsized aggregates for achieving unique porous structures inside the colloidal gels. The albumin hydrogels showed tunable mechanical properties such as elastic modulus, porosity, and biodegradability, providing a suitable ECM for various cells such as cardiomyoblasts and endothelial cells. In addition, the encapsulated cells within the hydrogel showed improved cell retention and increased survivability in vitro. Microporous structures of the colloidal gels can serve as a guide for the infiltration of host cells upon implantation, achieving rapid recruitment of hematopoietic cells and, ultimately, enhancing the tissue regeneration capacity of implanted scaffolds.

Iron Accumulation and Changes in Cellular Organelles in WDR45 Mutant Fibroblasts.

Iron overload in the brain, defined as excess stores of iron, is known to be associated with neurological disorders. In neurodegeneration accompanied by brain iron accumulation, we reported a specific point mutation, c.974-1G>A in WD Repeat Domain 45 (WDR45), showing iron accumulation in the brain, and autophagy defects in the fibroblasts. In this study, we investigated whether fibroblasts with mutated WDR45 accumulated iron, and other effects on cellular organelles. We first identified the main location of iron accumulation in the mutant fibroblasts and then investigated the effects of this accumulation on cellular organelles, including lipid droplets, mitochondria and lysosomes. Ultrastructure analysis using transmission electron microscopy (TEM) and confocal microscopy showed structural changes in the organelles. Increased numbers of lipid droplets, fragmented mitochondria and increased numbers of lysosomal vesicles with functional disorder due to WDR45 deficiency were observed. Based on correlative light and electron microscopy (CLEM) findings, most of the iron accumulation was noted in the lysosomal vesicles. These changes were associated with defects in autophagy and defective protein and organelle turnover. Gene expression profiling analysis also showed remarkable changes in lipid metabolism, mitochondrial function, and autophagy-related genes. These data suggested that functional and structural changes resulted in impaired lipid metabolism, mitochondrial disorder, and unbalanced autophagy fluxes, caused by iron overload.

Overexpressed p32 localized in the endoplasmic reticulum and mitochondria negatively regulates calcium­dependent endothelial nitric oxide synthase activit.

The p32 protein plays a crucial role in the regulation of cytosolic Ca2+ concentrations ([Ca2+]c) that contributes to the Ca2+­dependent signaling cascade. Using an adenovirus and plasmid p32­overexpression system, the aim of the study was to evaluate the role of p32 in the regulation of [Ca2+] and its potential associated with Ca2+­dependent endothelial nitric oxide synthase (eNOS) activation in endothelial cells. Using electron and confocal microscopic analysis, p32 overexpression was observed to be localized to mitochondria and the endoplasmic reticulum and played an important role in Ca2+ translocation, resulting in increased [Ca2+] in these organelles and reducing cytosolic [Ca2+] ([Ca2+]c). This decreased [Ca2+]c following p32 overexpression attenuated the Ca2+­dependent signaling cascade of calcium/calmodulin dependent protein kinase II (CaMKII)/AKT/eNOS phosphorylation. Moreover, in aortic endothelia of wild­type mice intravenously administered adenovirus encoding the p32 gene, increased p32 levels reduced NO production and accelerated reactive oxygen species (ROS) generation. In a vascular tension assay, p32 overexpression decreased acetylcholine (Ach)­induced vasorelaxation and augmented phenylephrine (PE)­dependent vasoconstriction. Notably, decreased levels of arginase II (ArgII) protein using siArgII were associated with downregulation of overexpressed p32 protein, which contributed to CaMKII­dependent eNOS phosphorylation at Ser1177. These results indicated that increased protein levels of p32 caused endothelial dysfunction through attenuation of the Ca2+­dependent signaling cascade and that ArgII protein participated in the stability of p32. Therefore, p32 may be a novel target for the treatment of vascular diseases associated with endothelial disorders.

Autophagic death of neural stem cells mediates chronic stress-induced decline of adult hippocampal neurogenesis and cognitive deficits.

Macroautophagy/autophagy is generally regarded as a cytoprotective mechanism, and it remains a matter of controversy whether autophagy can cause cell death in mammals. Here, we show that chronic restraint stress suppresses adult hippocampal neurogenesis in mice by inducing autophagic cell death (ACD) of hippocampal neural stem cells (NSCs). We generated NSC-specific, inducible Atg7 conditional knockout mice and found that they had an intact number of NSCs and neurogenesis level under chronic restraint stress and were resilient to stress- or corticosterone-induced cognitive and mood deficits. Corticosterone treatment of adult hippocampal NSC cultures induced ACD via SGK3 (serum/glucocorticoid regulated kinase 3) without signs of apoptosis. Our results demonstrate that ACD is biologically important in a mammalian system in vivo and would be an attractive target for therapeutic intervention for psychological stress-induced disorders.Abbreviations: AAV: adeno-associated virus; ACD: autophagic cell death; ACTB: actin, beta; Atg: autophagy-related; ASCL1/MASH1: achaete-scute family bHLH transcription factor 1; BafA1: bafilomycin A1; BrdU: Bromodeoxyuridine/5-bromo-2'-deoxyuridine; CASP3: caspase 3; cKO: conditional knockout; CLEM: correlative light and electron microscopy; CORT: corticosterone; CRS: chronic restraint stress; DAB: 3,3'-diaminobenzidine; DCX: doublecortin; DG: dentate gyrus; GC: glucocorticoid; GFAP: glial fibrillary acidic protein; HCN: hippocampal neural stem; i.p.: intraperitoneal; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MKI67/Ki67: antigen identified by monoclonal antibody Ki 67; MWM: Morris water maze; Nec-1: necrostatin-1; NES: nestin; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NSC: neural stem cell; PCD: programmed cell death; PFA: paraformaldehyde; PX: Phox homology; PtdIns3P: phosphatidylinositol-3-phosphate; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; SGK: serum/glucocorticoid-regulated kinases; SGZ: subgranular zone; SOX2: SRY (sex determining region Y)-box 2; SQSTM1: sequestosome 1; STS: staurosporine; TAM: tamoxifen; Ulk1: unc-51 like kinase 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VIM: vimentin; WT: wild type; ZFYVE1: zinc finger, FYVE domain containing 1; Z-VAD/Z-VAD-FMK: pan-caspase inhibitor.

Heterogeneity in liver histopathology is associated with GSK-3ß activity and mitochondrial dysfunction in end-stage diabetic rats on differential diets.

While liver histopathology is heterogeneous in diabetes, the underlying mechanisms remain unclear. We investigated whether glycemic variation resulting from differential diets can induce heterogeneity in diabetic liver and the underlying molecular mechanisms. We generated end-stage non-obese diabetic model rats by subtotal-pancreatectomy in male Sprague- Dawley rats and ad libitum diet for 7 weeks (n = 33). The rats were then divided into three groups, and fed a standard- or a low-protein diet (18 or 6 kcal%, respectively), for another 7 weeks: to maintain hyperglycemia, 11 rats were fed ad libitum (18AL group); to achieve euglycemia, 11 were calorierestricted (18R group), and 11 were both calorie- and proteinrestricted with the low-protein diet (6R group). Overnightfasted liver samples were collected after the differential diets together with sham-control (18S group), and histology and molecular changes were compared. Hyperglycemic-18AL showed glycogenic hepatopathy (GH) without steatosis, with the highest GSK-3ß inactivation because of Akt activation during hyperglycemia; mitochondrial function was not impaired, compared to the 18S group. Euglycemic-18R showed neither GH nor steatosis, with intermediate GSK-3ß activation and mitochondrial dysfunction. However, euglycemic-6R showed both GH and steatosis despite the highest GSK-3ß activity and no molecular evidence of increased lipogenesis or decreased ApoB expression, where mitochondrial dysfunction was highest among the groups. In conclusion, heterogeneous liver histopathology developed in end-stage non-obese diabetic rats as the glycemic levels varied with differential diets, in which protein content in the diets as well as glycemic levels differentially influenced GSK-3ß activity and mitochondrial function in insulin-deficient state. [BMB Reports 2020; 53(2): 100-105].

Propionic acid induces mitochondrial dysfunction and affects gene expression for mitochondria biogenesis and neuronal differentiation in SH-SY5Y cell line.

Studies in animal models have shown that the short-chain fatty acid, propionic acid (PPA), interferes with mitochondrial metabolism leading to mitochondrial dysfunction and behavioral abnormalities. The aim of this study was to investigate the effects of PPA on mitochondrial function and gene expression in neuronal cells. SH-SY5Y cells and normal human neural progenitor (NHNP) cells were exposed to 1, 5 mM PPA for 4 or 24 h and we found that the mitochondrial potential measured in SH-SY5Y cells decreased in a dose-dependent manner after PPA treatment. Electron microscopy analysis revealed that the size of the mitochondria was significantly reduced following PPA treatment. A dose-dependent increase in the mitochondrial DNA copy number was observed in the PPA-treated cells. The expression of the mitochondrial biogenesis-related proteins PGC-1α, TFAM, SIRT3, and COX4 was significantly increased after PPA treatment. Transcriptome analysis revealed that mRNA expression in the notch signaling-related genes ASCL1 and LFNG changed after PPA treatment and the positive correlated protein expression changes were also observed. These results revealed that PPA treatment may affect neurodevelopment by altering mitochondrial function and notch signaling-related gene expression.

Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

Atopic dermatitis (AD) is an inflammatory skin disorder caused by immunological dysregulation and genetic factors. Whether the expression levels of cytokine and skin barrier protein were altered by S100 calcium binding protein A8 (S100A8) and S100A9 in human keratinocytic HaCaT cells was examined in the present study. Alterations of cytokine expression were examined by ELISA following treatment with S100A8/9 and various signal protein­specific inhibitors. Activation of the mitogen activated protein kinase (MAPK) pathway and nuclear factor (NF)­κB was evaluated by using western blotting and an NF­κB activity test, respectively. The expression levels of interleukin (IL)­6, IL­8 and monocyte chemoattractant protein­1 increased following treatment with S100A8 and S100A9, and the increase was significantly blocked by specific signaling pathway inhibitors, including toll­like receptor 4 inhibitor (TLR4i), rottlerin, PD98059, SB203580 and BAY­11­7085. Extracellular signal­regulated kinase (ERK) and p38 MAPK pathways were activated in a time­dependent manner following treatment with S100A8 and S100A9. Phosphorylation of ERK and p38 MAPK were blocked by TLR4i and rottlerin. S100A8 and S100A9 induced translocation of NF­κB in a time­dependent manner, and the activation of NF­κB was inhibited by TLR4i, rottlerin, PD98059 and SB203580. In addition, S100A8 and S100A9 decreased the expression of skin barrier proteins, filaggrin and loricrin. These results may help to elucidate the pathogenic mechanisms of AD and develop clinical strategies for controlling AD.

The Role of ZNF143 in Breast Cancer Cell Survival Through the NAD(P)H Quinone Dehydrogenase 1⁻p53⁻Beclin1 Axis Under Metabolic Stress.

Autophagy is a cellular process that disrupts and uses unnecessary or malfunctioning components for cellular homeostasis. Evidence has shown a role for autophagy in tumor cell survival, but the molecular determinants that define sensitivity against autophagic regulation in cancers are not clear. Importantly, we found that breast cancer cells with low expression levels of a zinc-finger protein, ZNF143 (MCF7 sh-ZNF143), showed better survival than control cells (MCF7 sh-Control) under starvation, which was compromised with chloroquine, an autophagy inhibitor. In addition, there were more autophagic vesicles in MCF7 sh-ZNF143 cells than in MCF7 sh-Control cells, and proteins related with the autophagic process, such as Beclin1, p62, and ATGs, were altered in cells with less ZNF143. ZNF143 knockdown affected the stability of p53, which showed a dependence on MG132, a proteasome inhibitor. Data from proteome profiling in breast cancer cells with less ZNF143 suggest a role of NAD(P)H quinone dehydrogenase 1(NQO1) for p53 stability. Taken together, we showed that a subset of breast cancer cells with low expression of ZNF143 might exhibit better survival via an autophagic process by regulating the p53⁻Beclin1 axis, corroborating the necessity of blocking autophagy for the best therapy.

MiR-135-5p-p62 Axis Regulates Autophagic Flux, Tumorigenic Potential, and Cellular Interactions Mediated by Extracellular Vesicles During Allergic Inflammation.

The objective of this study was to investigate the relationship between autophagy and allergic inflammation. In vitro allergic inflammation was accompanied by an increased autophagic flux in rat basophilic leukemia (RBL2H3) cells. 3-MA, an inhibitor of autophagic processes, negatively regulated allergic inflammation both in vitro and in vivo. The role of p62, a selective receptor of autophagy, in allergic inflammation was investigated. P62, increased by antigen stimulation, mediated in vitro allergic inflammation, passive cutaneous anaphylaxis (PCA), and passive systemic anaphylaxis (PSA). P62 mediated cellular interactions during allergic inflammation. It also mediated tumorigenic and metastatic potential of cancer cells enhanced by PSA. TargetScan analysis predicted that miR-135-5p was a negative regulator of p62. Luciferase activity assay showed that miR-135-5p directly regulated p62. MiR-135-5p mimic negatively regulated features of allergic inflammation and inhibited tumorigenic and metastatic potential of cancer cells enhanced by PSA. MiR-135-5p mimic also inhibited cellular interactions during allergic inflammation. Extracellular vesicles mediated allergic inflammation both in vitro and in vivo. Extracellular vesicles were also necessary for cellular interactions during allergic inflammation. Transmission electron microscopy showed p62 within extracellular vesicles of antigen-stimulated rat basophilic leukemia cells (RBL2H3). Extracellular vesicles isolated from antigen-stimulated RBL2H3 cells induced activation of macrophages and enhanced invasion and migration potential of B16F1 mouse melanoma cells in a p62-dependent manner. Extracellular vesicles isolated from PSA-activated BALB/C mouse enhanced invasion and migration potential of B16F1 cells, and induced features of allergic inflammation in RBL2H3 cells. Thus, miR-135-5p-p62 axis might serve as a target for developing anti-allergy drugs.

Study of hyperpigmentation in human skin disorder using different electron microscopy techniques.

There is a global trend of increase in the demand for three-dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block-face scanning electron microscopy and focused ion beam-scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis-endocytosis theory of pigment in human skin.

Acute toxic effects of zinc oxide nanoparticles on Hydra magnipapillata.

Zinc oxide nanoparticles (ZnO NPs) are increasingly used in various products as coating and additive materials for household goods, personal-care products, and drug delivery systems. Because of their broad applications, the potential risks to nontarget organisms associated with their input into aquatic environments have generated much concern. We investigated the acute toxicity, morphological responses, and potential impact on physiology and metabolism in polyps exposed to spherical ZnO NPs of either 20 nm (ZnO NP20) or 100 nm (ZnO NP100). The median lethal concentrations (LC50) of ZnO NP20 were 55.3, 8.7, and 7.0 µg/mL after exposure for 48, 72, and 96 h, respectively; and those of ZnO NP100 were 262.0, 14.9, and 9.9 µg/mL, respectively. The morphological responses of the hydra polyps to a range of ZnO NP concentrations suggest that ZnO NPs may negatively affect neurotransmission in Hydra. ZnO NPs may also induce abnormal regeneration in the polyps by affecting the expression of several genes related to the Wnt signaling pathway. The presence of ZnO NP20 in the hydra tissue was confirmed with electron microscopy. A Gene Ontology analysis of the genes differentially expressed in hydra polyps after exposure to ZnO NP20 for 12 or 24 h revealed changes in various processes, including cellular and metabolic process, stress response, developmental process, and signaling. A KEGG pathway analysis of hydra polyps after exposure of ZnO NP20 or ZnO NP100 for 12 or 24 h demonstrated various changes, including in the DNA replication and repair, endocytosis, lysosomes, Wnt signaling, and natural killer-cell-mediated cytotoxicity pathways, suggesting the mechanisms that maintain cellular homeostasis in response to ZnO NPs. Progesterone-mediated oocyte maturation was also affected by the ZnO NPs nanoparticles, suggesting that they are potential endocrine disruptors. This study should increase our concern regarding the dispersal of ZnO NPs in aquatic environments.

Dual Function of USP14 Deubiquitinase in Cellular Proteasomal Activity and Autophagic Flux.

The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell.

TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions.

TAGLN is an actin-binding protein family that comprises three isoforms with theorized roles in smooth muscle differentiation, tumour development, lymphocyte activation, and brain chemistry. However, their fundamental characteristics in regulation of the actin-based cytoskeleton are not fully understood. Here we show that TAGLN2 (including TAGLN1 and TAGLN3) extensively nucleates G-actin polymerization under low-salt conditions, where polymerization would be completely suppressed. The calponin homology domain and actin-binding loop are essential to mechanically connect two adjacent G-actins, thereby mediating multimeric interactions. However, TAGLN2 blocked the Arp2/3 complex binding to actin filaments under physiological salt conditions, thereby inhibiting branched actin nucleation. In HeLa and T cells, TAGLN2 enhanced filopodium-like membrane protrusion. Collectively, the dual functional nature of TAGLN2-G-actin polymerization and Arp2/3 complex inhibition-may account for the mechanisms of filopodia development at the edge of Arp2/3-rich lamellipodia in various cell types.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.

Dequalinium-based functional nanosomes show increased mitochondria targeting and anticancer effect.

Mitochondria are targets with great potential for therapeutics for many human disorders. However, drug delivery systems for such therapeutics remain in need of more efficient mitochondrial-targeting carriers. In this study, we report that nanosomes composed of Dequalinium/DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)/DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), called DQA80s, can act in the dual role of mitochondrial-targeting carrier and anticancer agent for therapeutic interventions against mitochondrial diseases. In cytotoxicity assays, DQA80s were shown to be more toxic than DQAsomes. The DQA80s showed significantly increased cellular uptake as compared to that of DQAsomes, and DQA80s also showed more efficient escape from the endolysosome to the cytosol. We observed the efficient targeting of DQA80s to mitochondria in living cells using flow cytometry, confocal microscopy, and TEM imaging. We also found evidence of anticancer potential that mitochondrial-targeted DQA80s induced apoptosis by production of reactive oxygen species (ROS) via MAPK signaling pathways, loss of mitochondrial membrane potential, and the caspase-3 activation. The present study demonstrates that DQA80s have excellent dual potential both as a carrier and as an anticancer therapeutic for mitochondria-related disease therapy in vivo.