Inhibition of the mitochondria-shaping protein Opa1 restores sensitivity to Gefitinib in a lung adenocarcinomaresistant cell line

Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it

Novel SPEF2 Variant in a Japanese Patient with Primary Ciliary Dyskinesia: A Case Report and Literature Review

Primary ciliary dyskinesia (PCD) is a genetic and congenital disease associated with an abnormal ciliary ultrastructure and function and is estimated to affect 1 in 15,000–20,000 individuals. A PCD diagnosis can be achieved by genotyping. Here, we performed whole-exome analysis for the diagnosis of PCD and described the detailed clinical characteristics of the case. A 39-year-old Japanese woman with sinusitis and bronchiectasis without situs inversus had had upper and lower respiratory symptoms since childhood and had received long-term macrolide therapy without an accurate diagnosis. A moderate deterioration of cilia function was observed by high-speed video microscopy analysis; additionally, the number of cells with moving cilia was fewer than that in patients without PCD. Electron microscopy revealed no apparent structural abnormalities. We performedwhole-exome analysis and identified novel biallelic variants of SPEF2 in the homozygous state (c.1860_1861insCT).We confirmed the absence of SPEF2 protein expression in the cilia of the nasal mucosa using fluorescent immunostaining. Accordingly, she was diagnosed as having PCD with the SPEF2 variant. The present case suggests that the deterioration of cilia function is moderate, the number of respiratory cells with moving cilia might be reduced, and the respiratory condition could be severe in patients with PCD with the SPEF2 variant

Infantile Pain Episodes Associated with Novel Nav1.9 Mutations in Familial Episodic Pain Syndrome in Japanese Families

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8-9 weeks old; n = 10-12 for each group) and mature (36-38 weeks old; n = 5-6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8-9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations

The DBCLS BioHackathon: standardization and interoperability for bioinformatics web services and workflows. The DBCLS BioHackathon Consortium*

Web services have become a key technology for bioinformatics, since life science databases are globally decentralized and the exponential increase in the amount of available data demands for efficient systems without the need to transfer entire databases for every step of an analysis. However, various incompatibilities among database resources and analysis services make it difficult to connect and integrate these into interoperable workflows. To resolve this situation, we invited domain specialists from web service providers, client software developers, Open Bio* projects, the BioMoby project and researchers of emerging areas where a standard exchange data format is not well established, for an intensive collaboration entitled the BioHackathon 2008. The meeting was hosted by the Database Center for Life Science (DBCLS) and Computational Biology Research Center (CBRC) and was held in Tokyo from February 11th to 15th, 2008. In this report we highlight the work accomplished and the common issues arisen from this event, including the standardization of data exchange formats and services in the emerging fields of glycoinformatics, biological interaction networks, text mining, and phyloinformatics. In addition, common shared object development based on BioSQL, as well as technical challenges in large data management, asynchronous services, and security are discussed. Consequently, we improved interoperability of web services in several fields, however, further cooperation among major database centers and continued collaborative efforts between service providers and software developers are still necessary for an effective advance in bioinformatics web service technologies

AN INVESTIGATION OF MOLECULAR LESIONS IN TWO JAPANESE FAMILIES WITH FAMILIAL PAROXYSMAL KINESIGENIC DYSKINESIA

Familial paroxysmal kinesigenic dyskinesia (PKD) is an episodic involuntary movement disorder characterized by recurrent and brief attacks induced by sudden voluntary movement.　Prolinerich transmembrane protein 2 (PRRT2) has been identified as a gene responsible for PKD and its related disorders.　Recently, the protein encoded by PRRT2 was identified as a synaptic protein with a regulatory role in neurotransmitter release, which indicated that PKD may be a synaptopathy.　At present, more than 50 PRRT2 mutations have been identified, but the molecular mechanisms underlying the heterozygous mutations that cause the disorder remain unclear.　A novel PRRT2 mutation, c.649delC (p.R217Efs*12), was identified as a heterozygous allele in one of two Japanese families with PKD.　The mutation encodes a truncated PRRT2 protein, which consists of 216 amino acid residues compared to the full length protein of 429 amino acid residues.　To examine the subcellular localization of the wild and mutant PRRT2 proteins, we induced the transient expression of the PRRT2 protein fused with fluorescent proteins, pAcGFP1-C1 and pDsRed-monomer-C1, in COS7 cells.　Although the transient intracellular expression of wild PRRT2 protein fused with pAcGFP1-C1 confirmed its subcellular localization at the cell membrane, the mutant p.R217Efs*12 PRRT2 protein fused with pDsRed-monomer-C1 was detected in the cytosol and nucleus of COS7 cells.　In the co-transfection experiment, the mutant truncated PRRT2 protein did not inhibit the subcellular localization of the wild-type PRRT2 protein.　The results suggested that a heterozygous PRRT2 mutation might cause the disorder through a reduction in the amount of the prot ein encoded by the PRRT2 gene

BUTYRATE REDUCES FREE CHOLESTEROL ACCUMULATION IN NIEMANN-PICK DISEASE TYPE C1 CELLS (NOVA SCOTIA FORM) THROUGH THE INDUCTION OF ACID SPHINGOMYELINASE

Niemann-Pick disease type C (NPC) is an inherited disorder caused by mutations in either the NPC1 or the NPC2 gene that affect intracellular free cholesterol trafficking. Most cases of NPC have mutations in the NPC1 gene. Acid sphingomyelinase (ASM) is a lysosomal enzyme in which an inherited deficiency leads to Niemann-Pick disease types A and B (NPDA/NPDB). Despite having a normal ASM gene, NPC cells have a secondary defect in ASM activity ; the mechanism remains to be fully elucidated. Butyrate, one of the short chain fatty acids, is known as an inducer of ASM. We investigated the effects of butyrate on ASM activity and mRNA expression in normal and NPC1 mutant (Nova Scotia form ; NPC1^) lymphoblasts. After incubation with 10 mM butyric acid for 24 h, ASM activity was significantly increased by 3.3- and 4.6-fold in normal and NPC1^ cells, respectively (p cells was restored to normal levels by treatment of 10 mM butyric acid. In quantitative RT-PCR analysis, butyric acid significantly increased ASM mRNA levels in both types of cells (p lymphoblasts. Free cholesterol levels in cells treated with or without 10 mM butyric acid were 0.019±0.002 and 0.026±0.006 μg/μg protein, respectively, demonstrating that butyric acid significantly decreased free cholesterol levels in NPC1^ cells (p cells, NPDB and NPC1^ fibroblasts were treated with 10 mM butyric acid and stained with filipin. We found that butyric acid dramatically reduced the accumulation of intracellular free cholesterol in NPC1^ cells, but it did not affect NPDB cells. These data suggest that ASM induced by butyrate is related to intracellular cholesterol trafficking and metabolism in NPC1^ cells. ASM inducers, such as butyrate, may reduce the accumulation of intracellular free cholesterol in NPC1^ cells