A biochemical and cell biological characterization of TAFAZZIN in a novel Barth syndrome cell model

Barth syndrome (BTHS) is an X-linked disease characterized by cardio- and skeletal myopathy, hypotonia, growth delay, neutropenia, and 3-methylglutaconic aciduria. Patients have mutations in the TAZ gene on chromosome Xq28 (G4.5) most of which are presumed to result in a loss-of-function of the protein product, tafazzin (TAZ). As TAZ is involved in the remodeling of the phospholipid, cardiolipin (CL), the resultant lipid profile in the absence of its function shows decreased CL levels, accumulated monolyso-CL, and the remaining CL contains an altered acyl chain composition. Unfortunately, the lack of an antibody against endogenous TAZ has prevented a detailed cell biologic and biochemical characterization of the endogenous protein. This in turn has impeded a molecular understanding of the protein’s role in BTHS pathogenesis. Here, we have developed three mouse monoclonal antibodies capable of detecting endogenous TAZ in mammals. A complicating aspect of mammalian TAZ research is the presence of many predicted alternatively spliced variants, of which only the hTAZ –exon5 was able to fully restore aberrant CL profile in the Saccharomyces cerevisiae Δtaz1 mutant. Importantly, each TAZ antibody has the ability to detect every predicted isoform as determined by epitope mapping. Our results also show that only one isoform of TAZ is normally expressed in human fibroblasts, HEK293 Flp-In cells, amd mouse heart and liver, despite the reported detection of mRNA corresponding to multiple splice variants. Using mouse tissues, 293 Flp-In cells, and immortalized fibroblasts derived from healthy and BTHS patients, we demonstrate that mammalian TAZ is a highly protease-resistant and localized to the mitochondria where it associates non-integrally with membranes and assembles in a range of complexes. Like its yeast counterpart, TAZ in humans and rodents associates with the IMS-facing leaflets of the inner and outer mitochondrial membrane, Finally, using a novel mammalian BTHS cell culture model established via TALEN-mediated genome editing, we demonstrate that the loss-of-function mechanisms for two pathogenic alleles, R57L and H69Q, when overexpressed in tazTALEN cells, are the same as originally modeled and defined in yeast. Thus, our results reveal that tazTALEN cells can serve as a convenient platform to systematically dissect the loss-of-function mechanisms that underlie other BTHS variants, especially those that cannot be modeled in yeast due to lack of conservation. Combined with the generation of antibodies against endogenous TAZ, this work provides a major leap forward in our ability to characterize this enigmatic protein, to assign and discern the functions of TAZ and its numerous variants, and to study its role in CL metabolism

Defining functional classes of Barth syndrome mutation in humans

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ

(S)-(+)-4-(Oxiran-2-ylmeth­oxy)-9H-carbazole

In the title compound, C15H13NO2, all atoms of the carbazole group are coplanar (r.m.s. deviation = 0.005 Å), and the dihedral angle between this plane and C—O—C plane of oxane group is 57.1 (4)°. The crystal packing is stabilized by an N—H⋯O hydrogen bond, resulting in infinite supra­molecular chains along [001]

Serotonin-mediated modulation of Na+/K+ pump current in rat hippocampal CA1 pyramidal neurons

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to investigate whether serotonin (5-hydroxytryptamine, 5-HT) can modulate Na⁺/K⁺pump in rat hippocampal CA1 pyramidal neurons.</p> <p>Results</p> <p>5-HT (0.1, 1 mM) showed Na⁺/K⁺pump current (Ip) densities of 0.40 ± 0.04, 0.34 ± 0.03 pA/pF contrast to 0.63 ± 0.04 pA/pF of the control of 0.5 mM strophanthidin (Str), demonstrating 5-HT-induced inhibition of Ip in a dose-dependent manner in hippocampal CA1 pyramidal neurons. The effect was partly attenuated by ondasetron, a 5-HT₃receptor (5-HT₃R) antagonist, not by WAY100635, a 5-HT_1AR antagonist, while 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG), a 5-HT₃R specific agonist, mimicked the effect of 5-HT on Ip.</p> <p>Conclusion</p> <p>5-HT inhibits neuronal Na⁺/K⁺pump activity via 5-HT₃R in rat hippocampal CA1 pyramidal neurons. This discloses novel mechanisms for the function of 5-HT in learning and memory, which may be a useful target to benefit these patients with cognitive disorder.</p

Clinical observation on treating evaporative dry eye with the tonifying kidney pill combining with mingmuwuzi

AIM: To observe the clinical effect of the tonifying kidney pills with mingmuwuzi treating evaporative dry eyes.METHODS: This study adopted the positive drug control, prospective study, random number remainder grouping method to 65 cases of outpatient patients diagnosed with evaporative dry eyes which were divided into the treatment group 32 cases(64 eyes)and the control group 33 cases(66 eyes). The treatment group took the decoction of kidney pills with mingmuwuzi, combined with sodium hyaluronate eye drops. The control group simply use sodium hyaluronate eye drops, both group were set to 4wk for a course of treatment. To observe the symptoms and signs of two groups before and after the treatment, the change of the evaluation index and curative effect were evaluated.RESULTS: The effectiveness of the treatment group was 87.5%, the control group was 78.8%, the difference was statistically significant(z=-3.149, PCONCLUSION: The treatment of the kidney pills with mingmuwuzi combined with sodium hyaluronate eye drops to evaporative dry eyes is more effective than the simple use of sodium hyaluronate eye drops

Reciprocal regulation of MicroRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells

BACKGROUND: MicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%. METHODS: The expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors. RESULTS: We observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3′UTR of IGF1R mRNA into the 3′UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3′UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling. CONCLUSION: Overall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation

Moir\'{e} Flat Bands of Twisted Few-layer Graphite

We report that the twisted few layer graphite (tFL-graphite) is a new family of moir\'{e} heterostructures (MHSs), which has richer and highly tunable moir\'{e} flat band structures entirely distinct from all the known MHSs. A tFL-graphite is composed of two few-layer graphite (Bernal stacked multilayer graphene), which are stacked on each other with a small twisted angle. The moir\'{e} band structure of the tFL-graphite strongly depends on the layer number of its composed two van der Waals layers. Near the magic angle, a tFL-graphite always has two nearly flat bands coexisting with a few pairs of narrowed dispersive (parabolic or linear) bands at the Fermi level, thus, enhances the DOS at $E_F$. This coexistence property may also enhance the possible superconductivity as been demonstrated in other multiband superconductivity systems. Therefore, we expect strong multiband correlation effects in tFL-graphite. Meanwhile, a proper perpendicular electric field can induce several isolated nearly flat bands with nonzero valley Chern number in some simple tFL-graphites, indicating that tFL-graphite is also a novel topological flat band system.Comment: Submitted version,supplementary materials are adde

catena-Poly[[tetra­aqua­(μ-4,4′-bipyridine-κ2 N:N′)zinc(II)] fumarate tetra­hydrate]

In the title compound, {[Zn(C10H8N2)(H2O)4](C4H2O4)·4H2O}n, the ZnII atom is coordinated by two N atoms from two μ-4,4′-bipyridine ligands and four water mol­ecules in a distorted octa­hedral geometry. The coordination unit is extended through the Zn—N bond, leading to a one-dimensional cationic chain. A twofold rotation axis passes through the Zn atom and along the axis of the 4,4′-bipyridine ligand. Each uncoordinated water mol­ecule acts as both hydrogen-bond donor and acceptor. A three-dimensional network is constructed through hydrogen bonds involving water mol­ecules and fumarate dianions