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.

Feeding state regulates pheromone-mediated avoidance behavior via the insulin signaling pathway in Caenorhabditis elegans.

Animals change sensory responses and their eventual behaviors, depending on their internal metabolic status and external food availability. However, the mechanisms underlying feeding state-dependent behavioral changes remain undefined. Previous studies have shown that Caenorhabditis elegans hermaphrodite exhibits avoidance behaviors to acute exposure of a pheromone, ascr#3 (asc-ΔC9, C9). Here, we show that the ascr#3 avoidance behavior is modulated by feeding state via the insulin signaling pathway. Starvation increases ascr#3 avoidance behavior, and loss-of-function mutations in daf-2 insulin-like receptor gene dampen this starvation-induced ascr#3 avoidance behavior. DAF-2 and its downstream signaling molecules, including the DAF-16 FOXO transcription factor, act in the ascr#3-sensing ADL neurons to regulate synaptic transmission to downstream target neurons, including the AVA command interneurons. Moreover, we found that starvation decreases the secretion of INS-18 insulin-like peptides from the intestine, which antagonizes DAF-2 function in the ADL neurons. Altogether, this study provides insights about the molecular communication between intestine and sensory neurons delivering hunger message to sensory neurons, which regulates avoidance behavior from pheromones to facilitate survival chance.

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy.

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.

Haploinsufficiency of Cyfip2 Causes Lithium-Responsive Prefrontal Dysfunction.

OBJECTIVE: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy. METHODS: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. RESULTS: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. INTERPRETATION: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543.

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.

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.

Mitochondrial carnitine palmitoyltransferase 2 is involved in Nε-(carboxymethyl)-lysine-mediated diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in the world. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of DN via multifactorial mechanisms including the generation of oxidative stress and overproduction of various growth factors and cytokines. AGEs are heterogeneous cross-linked sugar-derived proteins, and Nε-(carboxymethyl)-lysine (CML)-conjugated BSA is a major component of AGEs. However, the proteins involved in DN induction by CML have never been reported. Herein, we investigated specific protein regulators of AGE-mediated DN via proteomic analysis of streptozotocin (STZ)-induced diabetic mice kidneys. We identified 937, 976, and 870 proteins in control, STZ, and STZ + CML-BSA samples, respectively. Bioinformatics analysis identified several CML-mediated proteins potentially involved in kidney damage, activation of fatty acid oxidation (FAO), and mitochondrial dysfunction. Furthermore, we identified the CML-specific differential protein carnitine palmitoyltransferase 2 (CPT2), related to FAO. To confirm the effect of CPT2 and the CML-mediated mechanism, human renal tubular HK-2 cells were treated with CML-BSA and cpt2 siRNA, and examined for FAO-mediated fibrosis and mitochondrial dysfunction. CML-BSA and CPT2 knockdown induced fibrosis-related gene expression and damage to mitochondrial membrane potential. Moreover, CPT2 overexpression recovered CML-induced fibrosis-related gene expression. Based on these results, a decrease in CML-induced CPT2 expression causes mitochondrial FAO damage, leading to renal fibrosis and DN.

Mitochondrial Damage and Necroptosis in Aging Cochlea.

Age-related hearing loss (ARHL) is an irreversible, progressive neurodegenerative disorder and is presently untreatable. Previous studies using animal models have suggested mitochondrial damage and programmed cell death to be involved with ARHL. Thus, we further investigated the pathophysiologic role of mitochondria and necroptosis in aged C57BL/6J male mice. Aged mice (20 months old) exhibited a significant loss of hearing, number of hair cells, neuronal fibers, and synaptic ribbons compared to young mice. Ultrastructural analysis of aged cochleae revealed damaged mitochondria with absent or disorganized cristae. Aged mice also showed significant decrease in cochlear blood flow, and exhibited increase in gene expression of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α), receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL). Immunofluorescence (IF) assays of cytochrome C oxidase I (COX1) confirmed mitochondrial dysfunction in aged cochleae, which correlated with the degree of mitochondrial morphological damage. IF assays also revealed localization and increased expression of RIPK3 in sensorineural tissues that underwent significant necroptosis (inner and outer hair cells and stria vascularis). Together, our data shows that the aging cochlea exhibits damaged mitochondria, enhanced synthesis of proinflammatory cytokines, and provides new evidence of necroptosis in the aging cochlea in in vivo.

Trafficking of synaptic vesicles is changed at the hypothalamus by exposure to an 835 MHz radiofrequency electromagnetic field.

With the rapidly increasing use of mobile phones and their close-contact usage to the brain, there are some concerns about the possible neuronal effects induced by exposure to excessive electromagnetic radiation. Exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate (SAR) 5 h/day for 12 weeks) may affect hypothalamic presynaptic neurons in C57BL/6 mice. The number and size of the synaptic vesicles (SVs) in the hypothalamic presynaptic terminals were significantly decreased after RF-EMF exposure. Further, the density (SVs numbers/µm) of docking and fusing SVs in the active zones of the presynaptic terminal membrane was significantly decreased in hypothalamic neurons. The expression levels of synapsin I/II and synaptotagmin 1, two regulators of SV trafficking in neurons, were also significantly decreased in the hypothalamus. In parallel, the expression of calcium channel was significantly decreased. These changes in SVs in the active zones may directly decrease the release of neurotransmitters in hypothalamic presynaptic terminals. Therefore, we further studied the possible changes in hypothalamic function by testing the core body temperature and body weight and performed the buried pellet test. The trafficking of SVs was changed by RF-EMF; however, we could not find any significant phenotypical changes in our experimental condition.

Autophagy Activation by Crepidiastrum Denticulatum Extract Attenuates Environmental Pollutant-Induced Damage in Dermal Fibroblasts.

Pollution-induced skin damage results in oxidative stress; cellular toxicity; inflammation; and, ultimately, premature skin aging. Previous studies suggest that the activation of autophagy can protect oxidation-induced cellular damage and aging-like changes in skin. In order to develop new anti-pollution ingredients, this study screened various kinds of natural extracts to measure their autophagy activation efficacy in cultured dermal fibroblast. The stimulation of autophagy flux by the selected extracts was further confirmed both by the expression of proteins associated with the autophagy signals and by electron microscope. Crepidiastrum denticulatum (CD) extract treated cells showed the highest autophagic vacuole formation in the non-cytotoxic range. The phosphorylation of adenosine monophosphate kinase (AMPK), but not the inhibition of mammalian target of rapamycin (mTOR), was observed by CD-extract treatment. Its anti-pollution effects were further evaluated with model compounds, benzo[a]pyrene (BaP) and cadmium chloride (CdCl2), and a CD extract treatment resulted in both the protection of cytotoxicity and a reduction of proinflammatory cytokines. These results suggest that the autophagy activators can be a new protection regimen for anti-pollution. Therefore, CD extract can be used for anti-inflammatory and anti-pollution cosmetic ingredients.

Mutations in SEC24D, encoding a component of the COPII machinery, cause a syndromic form of osteogenesis imperfecta.

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205(∗)) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).

Intracellular formyl peptide receptor regulates naïve CD4 T cell migration.

We found that formyl peptide receptor (FPR) 1 and FPR3 were expressed intracellularly and/or the nucleus of naïve CD4 T cell. Activation of naïve CD4 T cells with synthetic intracellular agonists dTAT-WKYMVm and CTP-WKYMVm for FPR members stimulated CD4 T cell migration via pertussis toxin-sensitive manner. Knockdown of FPR1, but not knockdown of FPR3, blocked dTAT-WKYMVm-induced naïve CD4 T cell migration. Stimulation of naïve CD4 T cells with dTAT-WKYMVm elicited the activation of ERK, p38 MAPK, and Akt. Activation of CD4 T cells with anti-CD3 and anti-CD28 antibodies caused surface expression of FPR1 and FPR3, but not FPR2. CD4 T cells isolated from sepsis patients expressed the three members of FPR family on their cell surface. Taken together, our results suggest that intracellular FPR in naïve CD4 T cells and surface FPRs in activated CD4 T cells might regulate immune responses by regulating CD4 T cell activity.

The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

Exposure to 835 MHz radiofrequency electromagnetic field induces autophagy in hippocampus but not in brain stem of mice.

The exploding popularity of mobile phones and their close proximity to the brain when in use has raised public concern regarding possible adverse effects from exposure to radiofrequency electromagnetic fields (RF-EMF) on the central nervous system. Numerous studies have suggested that RF-EMF emitted by mobile phones can influence neuronal functions in the brain. Currently, there is still very limited information on what biological mechanisms influence neuronal cells of the brain. In the present study, we explored whether autophagy is triggered in the hippocampus or brain stem after RF-EMF exposure. C57BL/6 mice were exposed to 835 MHz RF-EMF with specific absorption rates (SAR) of 4.0 W/kg for 12 weeks; afterward, the hippocampus and brain stem of mice were dissected and analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that several autophagic genes, which play key roles in autophagy regulation, were significantly upregulated only in the hippocampus and not in the brain stem. Expression levels of LC3B-II protein and p62, crucial autophagic regulatory proteins, were significantly changed only in the hippocampus. In parallel, transmission electron microscopy (TEM) revealed an increase in the number of autophagosomes and autolysosomes in the hippocampal neurons of RF-EMF-exposed mice. The present study revealed that autophagy was induced in the hippocampus, not in the brain stem, in 835 MHz RF-EMF with an SAR of 4.0 W/kg for 12 weeks. These results could suggest that among the various adaptation processes to the RF-EMF exposure environment, autophagic degradation is one possible mechanism in specific brain regions.

Pyruvate dehydrogenase kinase 4 deficiency attenuates cisplatin-induced acute kidney injury.

Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Since details of the underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury.

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

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.

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.