Establishing Three-Dimensional Explant Culture of Human Dental Pulp Tissue.

Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.

Mechanism of Methylene Blue Inducing the Disulfide Bond Formation of Tubulin-Associated Unit Proteins.

Methylene blue (MB) has recently completed a Phase-3 clinical trial as leuco-methylthioninium (LMT) bis(hydromethanesulfonate) for treating Alzheimer's disease. Herein, we investigated the mechanism underlying the MB inhibition of tubulin-associated unit (tau) aggregation by focusing on tau monomers. We found that MB causes disulfide bond formation, resulting in strong nuclear magnetic resonance chemical shift perturbations in a large area of tau proteins. The oxidized form of MB, namely methylthioninium (MT+), specifically catalyzed the oxidation of cysteine residues in tau proteins to form disulfide bonds directly using O2. This process is independent of the MT+-to-LMT redox cycle. Moreover, MT+ preferentially oxidized C291 and C322 in the lysine-rich R2 and R3 domains. Under in vivo brain physoxia conditions, LMT may convert to MT+, possibly interfering with tau fibrillation via disulfide bond formation.

Rotundifuran Induces Ferroptotic Cell Death and Mitochondria Permeability Transition in Lung Cancer Cells.

Rotundifuran (RF), a potent anti-inflammatory and anti-cancer compound, is a natural compound predominantly present in Vitex Rotundifolia. Herein, we investigated the effects of RF on the growth of lung cancer cells. Our findings suggested that RF inhibits cell growth, highlighting its potential as a therapeutic agent for cancer treatment. Interestingly, we observed that cell growth inhibition was not due to apoptosis, as caspases were not activated and DNA fragmentation did not occur. Furthermore, we found that intracellular vacuoles and autophagy were induced, but RF-induced cell death was not affected when autophagy was inhibited. This prompted us to investigate other possible mechanisms underlying cell growth inhibition. Through a cDNA chip analysis, we confirmed changes in the expression of ferroptosis-related genes and observed lipid peroxidation. We further examined the effect of ferroptosis inhibitors and found that they alleviated cell growth inhibition induced by RF. We also observed the involvement of calcium signaling, ROS accumulation, and JNK signaling in the induction of ferroptosis. Our findings suggested that RF is a potent anti-cancer drug and further studies are needed to validate its clinal use.

Crif1 deficiency in dopamine neurons triggers early-onset parkinsonism.

Mitochondrial dysfunction has been implicated in Parkinson's Disease (PD) progression; however, the mitochondrial factors underlying the development of PD symptoms remain unclear. One candidate is CR6-interacting factor1 (CRIF1), which controls translation and membrane insertion of 13 mitochondrial proteins involved in oxidative phosphorylation. Here, we found that CRIF1 mRNA and protein expression were significantly reduced in postmortem brains of elderly PD patients compared to normal controls. To evaluate the effect of Crif1 deficiency, we produced mice lacking the Crif1 gene in dopaminergic neurons (DAT-CRIF1-KO mice). From 5 weeks of age, DAT-CRIF1-KO mice began to show decreased dopamine production with progressive neuronal degeneration in the nigral area. At ~10 weeks of age, they developed PD-like behavioral deficits, including gait abnormalities, rigidity, and resting tremor. L-DOPA, a medication used to treat PD, ameliorated these defects at an early stage, although it was ineffective in older mice. Taken together, the observation that CRIF1 expression is reduced in human PD brains and deletion of CRIF1 in dopaminergic neurons leads to early-onset PD with stepwise PD progression support the conclusion that CRIF1-mediated mitochondrial function is important for the survival of dopaminergic neurons.

Diplacone Isolated from Paulownia tomentosa Mature Fruit Induces Ferroptosis-Mediated Cell Death through Mitochondrial Ca2+ Influx and Mitochondrial Permeability Transition.

The recently defined type of cell death ferroptosis has garnered significant attention as a potential new approach to cancer treatment owing to its more immunogenic nature when compared with apoptosis. Ferroptosis is characterized by the depletion of glutathione (GSH)/glutathione peroxidase-4 (GPx4) and iron-dependent lipid peroxidation. Diplacone (DP), a geranylated flavonoid compound found in Paulownia tomentosa fruit, has been identified to have anti-inflammatory and anti-radical activity. In this study, the potential anticancer activity of DP was explored against A549 human lung cancer cells. It was found that DP induced a form of cytotoxicity distinct from apoptosis, which was accompanied by extensive mitochondrial-derived cytoplasmic vacuoles. DP was also shown to increase mitochondrial Ca2+ influx, reactive oxygen species (ROS) production, and mitochondrial permeability transition (MPT) pore-opening. These changes led to decreases in mitochondrial membrane potential and DP-induced cell death. DP also induced lipid peroxidation and ATF3 expression, which are hallmarks of ferroptosis. The ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 were effective in counteracting the DP-mediated ferroptosis-related features. Our results could contribute to the use of DP as a ferroptosis-inducing agent, enabling studies focusing on the relationship between ferroptosis and the immunogenic cell death of cancer cells.

Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease.

Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.

GPR143 controls ESCRT-dependent exosome biogenesis and promotes cancer metastasis.

Exosomes transport a variety of macromolecules and modulate intercellular communication in physiology and disease. However, the regulation mechanisms that determine exosome contents during exosome biogenesis remain poorly understood. Here, we find that GPR143, an atypical GPCR, controls the endosomal sorting complex required for the transport (ESCRT)-dependent exosome biogenesis pathway. GPR143 interacts with HRS (an ESCRT-0 Subunit) and promotes its association to cargo proteins, such as EGFR, which subsequently enables selective protein sorting into intraluminal vesicles (ILVs) in multivesicular bodies (MVBs). GPR143 is elevated in multiple cancers, and quantitative proteomic and RNA profiling of exosomes in human cancer cell lines showed that the GPR143-ESCRT pathway promotes secretion of exosomes that carry unique cargo, including integrins signaling proteins. Through gain- and loss-of-function studies in mice, we show that GPR143 promotes metastasis by secreting exosomes and increasing cancer cell motility/invasion through the integrin/FAK/Src pathway. These findings provide a mechanism for regulating the exosomal proteome and demonstrate its ability to promote cancer cell motility.

Suppression of Lung Cancer Malignancy by Micellized siRNA through Cell Cycle Arrest.

UBA6-specific E2 conjugation enzyme 1 (USE1) is frequently overexpressed in lung cancer patients. Moreover, the critical role of USE1 in the progression of human lung cancer is also indicated. As the next step, the authors aim to develop USE1-targeted therapeutic agents based on RNA interference (RNAi). In this study, a lipid-modified DNA carrier, namely U4T, which consists of four consecutive dodec-1-ynyluracil (U) nucleobases to increase the cell permeability of siRNA targeting of USE1 is introduced. The U4Ts aggregate to form micelles, and the USE1-silencing siRNA-incorporated soft spherical nucleic acid aggregate (siSNA) can be created simply through base-pairing with siRNA. Treatment with siSNA is effective in suppressing tumor growth in vivo as well as cell proliferation, migration, and invasion of lung cancer cells. Furthermore, siSNA inhibited tumor cell growth by inducing cell cycle arrest in the G1 phase and apoptosis. Thus, the anti-tumor efficacy of siSNA in lung cancer cell lines and that siSNA possesses effective cell-penetrating ability without using cationic transfection moieties are confirmed. Collectively, these results suggest that siSNA can be applied to the clinical application of RNAi-based therapeutics for lung cancer treatment.

Limpet teeth microstructure unites auxeticity with extreme strength and high stiffness.

Materials displaying negative Poisson's ratio, referred to as auxeticity, have been found in nature and created in engineering through various structural mechanisms. However, uniting auxeticity with high strength and high stiffness has been challenging. Here, combining in situ nanomechanical testing with microstructure-based modeling, we show that the leading part of limpet teeth successfully achieves this combination of properties through a unique microstructure consisting of an amorphous hydrated silica matrix embedded with bundles of single-crystal iron oxide hydroxide nanorods arranged in a pseudo-cholesteric pattern. During deformation, this microstructure allows local coordinated displacement and rotation of the nanorods, enabling auxetic behavior while maintaining one of the highest strengths among natural materials. These findings lay a foundation for designing biomimetic auxetic materials with extreme strength and high stiffness.

In situ synthesis of hierarchically-assembled three-dimensional ZnS nanostructures and 3D printed visualization.

Nanomaterials have gained enormous interest in improving the performance of energy harvest systems, biomedical devices, and high-strength composites. Many studies were performed fabricating more elaborate and heterogeneous nanostructures then the structures were characterized using TEM tomographic images, upgrading the fabrication technique. Despite the effort, intricate fabrication process, agglomeration characteristic, and non-uniform output were still limited to presenting the 3D panoramic views straightforwardly. Here we suggested in situ synthesis method to prepare complex and hierarchically-assembled nanostructures that consisted of ZnS nanowire core and nanoparticles under Ag2S catalyst. We demonstrated that the vaporized Zn and S were solidified in different shapes of nanostructures with the temperatures solely. To our knowledge, this is the first demonstration of synthesizing heterogeneous nanostructures, consisting of a nanowire from the vapor-liquid-solid and then nanoparticles from the vapor-solid grown mechanism by in situ temperature control. The obtained hierarchically-assembled ZnS nanostructures were characterized by various TEM technologies, verifying the crystal growth mechanism. Lastly, electron tomography and 3D printing enabled the nanoscale structures to visualize with centimeter scales. The 3D printing from randomly fabricated nanomaterials is rarely performed to date. The collaborating work could offer a better opportunity to fabricate advanced and sophisticated nanostructures.

Mesenchymal Stem Cells Potentiate the Vasculogenic Capacity of Endothelial Colony-Forming Cells under Hyperglycemic Conditions.

Many studies have demonstrated a reduced number and vasculogenic capacity of endothelial colony-forming cells (ECFCs) in diabetic patients. However, whether the vasculogenic capacity of ECFCs is recovered or not when combined with pericyte precursors, mesenchymal stem cells (MSCs), under hyperglycemic conditions has not been studied. Thus, we investigated the role of MSCs in ECFC-mediated vascular formation under high-glucose conditions. The ECFCs and MSCs were treated with normal glucose (5 mM; NG) or high glucose (30 mM; HG) for 7 days. The cell viability, proliferation, migration, and tube formation of ECFCs were reduced in HG compared to NG. Interestingly, the ECFC+MSC combination after HG treatment formed tubular structures similar to NG-treated ECFCs+MSCs. An in vivo study using a diabetic mouse model revealed that the number of perfused vessels formed by HG-treated ECFCs+MSCs in diabetic mice was comparable with that of NG-treated ECFCs+MSCs in normal mice. Electron microscopy revealed that the ECFCs+MSCs formed pericyte-covered perfused blood vessels, while the ECFCs alone did not form perfused vessels when injected into the mice. Taken together, MSCs potentiate the vasculogenic capacity of ECFCs under hyperglycemic conditions, suggesting that the combined delivery of ECFCs+MSCs can be a promising strategy to build a functional microvascular network to repair vascular defects in diabetic ischemic regions.

Mitochondrial dysfunction in skeletal muscle contributes to the development of acute insulin resistance in mice.

BACKGROUND: Although mounting evidence indicates that insulin resistance (IR) co-occurs with mitochondrial dysfunction in skeletal muscle, there is no clear causal link between mitochondrial dysfunction and IR pathogenesis. In this study, the exact role of mitochondria in IR development was determined. METHODS: Six-week-old C57BL/6 mice were fed a high-fat diet for 2 weeks to induce acute IR or for 24 weeks to induce chronic IR (n = 8 per group). To characterize mitochondrial function, we measured citrate synthase activity, ATP content, mitochondrial DNA (mtDNA) content, and oxygen consumption rate in gastrocnemius and liver tissues. We intraperitoneally administered mitochondrial division inhibitor 1 (mdivi-1) to mice with acute IR and measured mitochondrial adaptive responses such as mitophagy, mitochondrial unfolded protein response (UPRmt), and oxidative stress (n = 6 per group). RESULTS: Acute IR occurred coincidently with impaired mitochondrial function, including reduced citrate synthase activity (-37.8%, P < 0.01), ATP production (-88.0%, P < 0.01), mtDNA (-53.1%, P < 0.01), and mitochondrial respiration (-52.2% for maximal respiration, P < 0.05) in skeletal muscle but not in liver. Administration of mdivi-1 attenuated IR development by increasing mitochondrial function (+58.5% for mtDNA content, P < 0.01; 4.06 ± 0.69 to 5.84 ± 0.95 pmol/min/mg for citrate synthase activity, P < 0.05; 13.06 ± 0.70 to 34.87 ± 0.70 pmol/min/g for maximal respiration, P < 0.001). Western blot analysis showed acute IR resulted in increased autophagy (mitophagy) and UPRmt induction in muscle tissue. This adaptive response was inhibited by mdivi-1, which reduced the mitochondrial oxidative stress of skeletal muscle during acute IR. CONCLUSIONS: Acute IR induced mitochondrial oxidative stress that impaired mitochondrial function in skeletal muscle. Improving mitochondrial function has important potential for treating acute IR.

Junctional Modulation of Round Window Membrane Enhances Dexamethasone Uptake into the Inner Ear and Recovery after NIHL.

Delivery of substances into the inner ear via local routes is increasingly being used in clinical treatment. Studies have focused on methods to increase permeability through the round window membrane (RWM) and enhance drug diffusion into the inner ear. However, the clinical applications of those methods have been unclear and few studies have investigated the efficacy of methods in an inner ear injury model. Here, we employed the medium chain fatty acid caprate, a biologically safe, clinically applicable substance, to modulate tight junctions of the RWM. Intratympanic treatment of sodium caprate (SC) induced transient, but wider, gaps in intercellular spaces of the RWM epithelial layer and enhanced the perilymph and cochlear concentrations/uptake of dexamethasone. Importantly, dexamethasone co-administered with SC led to significantly more rapid recovery from noise-induced hearing loss at 4 and 8 kHz, compared with the dexamethasone-only group. Taken together, our data indicate that junctional modulation of the RWM by SC enhances dexamethasone uptake into the inner ear, thereby hastening the recovery of hearing sensitivity after noise trauma.

Carrier-free micellar CpG interacting with cell membrane for enhanced immunological treatment of HIV-1.

Unmethylated CpG motifs activate toll-like receptor 9 (TLR9), leading to sequence- and species-specific immune stimulation. Here, we engineered a CpG oligodeoxyribonucleotide (ODN) with multiple hydrophobic moieties, so-called lipid-modified uracil, which resulted in a facile micelle formation of the stimulant. The self-assembled CpG nanostructure (U4CpG) containing the ODN 2216 sequence was characterized by various spectroscopic and microscopic methods together with molecular dynamics simulations. Next, we evaluated the nano-immunostimulant for enhancement of anti-HIV immunity. U4CpG treatment induced activation of plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells in healthy human peripheral blood, which produced type I interferons (IFNs) and IFN-γ in human peripheral blood mononuclear cells (PBMCs). Moreover, we validated the activation and promotion efficacy of U4CpG in patient-derived blood cells, and HIV-1 spread was significantly suppressed by a low dosage of the immunostimulant. Furthermore, U4CpG-treated PBMC cultured medium elicited transcription of latent HIV-1 in U1 cells indicating that U4CpG reversed HIV-1 latency. Thus, the functions of U4CpG in eradicating HIV-1 by enhancing immunity and reversing latency make the material a potential candidate for clinical studies dealing with viral infection.

Hearing Impairment in a Mouse Model of Diabetes Is Associated with Mitochondrial Dysfunction, Synaptopathy, and Activation of the Intrinsic Apoptosis Pathway.

Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.

Ultrastructure of phytoplasma-infected jujube leaves with witches' broom disease.

The subcellular characteristics of phytoplasma-infected jujube (Ziziphus jujuba) leaves were investigated using transmission electron microscopy. Midrib fragments of witches' broom-diseased jujube leaves were collected from abnormally small leaves at an early stage of branch clustering. The diseased jujube leaves showed multivesicular bodies (MVBs) with vesicles and tubules in the phloem parenchyma cells and sieve elements. The MVBs were connected to the plasma membrane appressed to the cell wall. There were increased callose collars at the pore-plasmodesma unit ends of the sieve elements in the diseased leaves than in control leaves. The proliferation of MVBs in the diseased jujube leaves could be associated with endoplasmic reticulum stress-dependent exosome release. The phytoplasma produced pleomorphic cells in sieve elements. Several types of putative extracellular structures were observed on the phytoplasma cells: (i) fimbriae-like threads, (ii) pili-like projections, (iii) flagella-like appendages, and (iv) tube-like structures. This study provides novel insights into intracellular obligate cell wall-less prokaryotes and host phloem structures.

Super-resolution imaging reveals cytoskeleton-dependent organelle rearrangement within platelets at intermediate stages of maturation.

A steady supply of platelets maintains their levels in the blood, and this is achieved by the generation of progeny from platelet intermediates. Using systematic super-resolution microscopy, we examine the ultrastructural organization of various organelles in different platelet intermediates to understand the mechanism of organelle redistribution and sorting in platelet intermediate maturation as the early step of platelet progeny production. We observe the dynamic interconversion between the intermediates and find that microtubules are responsible for controlling the overall shape of platelet intermediates. Super-resolution images show that most of the organelles are located near the cell periphery in oval preplatelets and confined to the bulbous tips in proplatelets. We also find that the distribution of the dense tubular system and α granules is regulated by actin, whereas that of mitochondria and dense granules is governed by microtubules. Altogether, our results call for a reassessment of organelle redistribution in platelet intermediates.

Super-resolution imaging of platelet-activation process and its quantitative analysis.

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.

Mitofusin-2 boosts innate immunity through the maintenance of aerobic glycolysis and activation of xenophagy in mice.

Mitochondrial function and innate immunity are intimately linked; however, the mechanisms how mitochondrion-shaping proteins regulate innate host defense remains largely unknown. Herein we show that mitofusin-2 (MFN2), a mitochondrial fusion protein, promotes innate host defense through the maintenance of aerobic glycolysis and xenophagy via hypoxia-inducible factor (HIF)-1α during intracellular bacterial infection. Myeloid-specific MFN2 deficiency in mice impaired the antimicrobial and inflammatory responses against mycobacterial and listerial infection. Mechanistically, MFN2 was required for the enhancement of inflammatory signaling through optimal induction of aerobic glycolysis via HIF-1α, which is activated by mitochondrial respiratory chain complex I and reactive oxygen species, in macrophages. MFN2 did not impact mitophagy during infection; however, it promoted xenophagy activation through HIF-1α. In addition, MFN2 interacted with the late endosomal protein Rab7, to facilitate xenophagy during mycobacterial infection. Our findings reveal the mechanistic regulations by which MFN2 tailors the innate host defense through coordinated control of immunometabolism and xenophagy via HIF-1α during bacterial infection.