In the present study we investigated the changes in miRNA levels inhuman rhinovirus 16 (HRV16)-infected cells. A small RNA deep sequencing experiment was performed through next-generation sequencing. In total, 53 differentially expressed miRNAs were confirmed by RT-qPCR, including 37 known miRNAs and 16 novel miRNAs. Interaction networks between differentially expressed miRNAs and their targets were established by mirDIP and Navigator. The prediction results showed that QKI, NFAT5, BNC2, CELF2, LCOR, MBNL2, MTMR3, NFIB, PPARGC1A, RSBN1, TRPS1, WDR26, and ZNF148, which are associated with cellular differentiation and transcriptional regulation, were recognized by 12, 11, or 9 miRNAs. Many correlations were observed between transcriptional or post-transcriptional regulation of an miRNA and the expression levels of its target genes in HRV16-infected H1-HeLa cells.
CELF Proteins/metabolism* ; DNA-Binding Proteins/genetics* ; Gene Expression Profiling ; Gene Expression Regulation ; HeLa Cells ; High-Throughput Nucleotide Sequencing ; Humans ; MicroRNAs/metabolism* ; Nerve Tissue Proteins/genetics* ; Protein Tyrosine Phosphatases, Non-Receptor ; Repressor Proteins/metabolism* ; Sequence Analysis, RNA ; Transcription Factors/metabolism*

BACKGROUND: Invasive nonfunctioning pituitary adenomas (NFPAs) remain challenging due to their high complication rate and poor prognosis. We aimed to identify the distinctive molecular signatures of invasive NFPAs, compared with noninvasive NFPAs, using gene expression profiling by RNA sequencing. METHODS: We obtained frozen fresh tissue samples from 14 patients with NFPAs who underwent primary transsphenoidal surgery. Three non-invasive and 11 invasive NFPAs were used for RNA sequencing. The bioinformatics analysis included differential gene expression, gene ontology analysis, and pathway analysis. RESULTS: A total of 700 genes were differentially expressed (59 up-regulated and 641 down-regulated genes) between invasive and non-invasive NFPAs (false discovery rate <0.1, and |fold change| ≥2). Using the down-regulated genes in invasive NFPAs, gene ontology enrichment analyses and pathway analyses demonstrated that the local immune response was attenuated and that transforming growth factor-β (TGF-β) RII-initiated TGF-β signaling was down-regulated in invasive NFPAs. The overexpression of claudin-9 (CLDN9) and the down-regulation of insulin-like growth factor-binding protein 5 (IGFBP5), death-associated protein kinase 1 (DAPK1), and tissue inhibitor of metalloproteinase-3 (TIMP3) may be related with invasiveness in NFPAs. CONCLUSION: Invasive NFPAs harbor different gene expression profiles relative to noninvasive NFPAs. In particular, local suppression of the immune response and TGF-β signaling can make PAs prone to invasiveness.
Computational Biology ; Death-Associated Protein Kinases ; Down-Regulation ; Gene Expression ; Gene Expression Profiling ; Gene Ontology ; Humans ; Pituitary Neoplasms ; Prognosis ; Sequence Analysis, RNA ; Tissue Inhibitor of Metalloproteinase-3 ; Transcriptome

OBJECTIVE: To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T. METHODS: The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp. RESULTS: Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the of voltage-dependence of fast inactivation of I1363T mutant[(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type[(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type. CONCLUSIONS I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.
Gene Expression Profiling ; Humans ; Ion Channel Gating ; genetics ; Muscle, Skeletal ; physiopathology ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Sequence Analysis, Protein

PURPOSE: Molecular testing in non-small cell lung cancer (NSCLC) aids in identifying oncogenic alterations. The aim of this study was to compare the rates of detection of oncogenic alterations and responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) according to EGFR mutation status as determined by peptide nucleic acid (PNA) clamping or direct sequencing (DS). MATERIALS AND METHODS: We performed a systematic literature search using MEDLINE, EMBASE, and the Cochrane Central Register. Data from included studies were pooled to yield summary sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and receiver operating characteristic curves. A meta-regression analysis was conducted to identify potential sources of heterogeneity between selected studies. RESULTS: We identified 10 studies comprising 924 patients. Oncogenic alterations were detected in 340 of 924 cases (36.8%) with PNA clamping and in 250 of 924 (27.1%) with DS. The pooled sensitivities of PNA clamping and DS were 0.93 [95% confidence interval (CI): 0.90−0.95] and 0.69 (95% CI: 0.64−0.73), respectively. According to meta-regression analysis, none of the covariates were found to be significant sources of heterogeneity. With respect to treatment responses to EGFR-TKIs, there was no significant difference therein between EGFR mutations detected by PNA clamping and DS (53.4% vs. 50.8%; risk ratio, 0.99; 95% CI 0.83−1.19; p=0.874). CONCLUSION: We demonstrated that PNA clamping has a higher sensitivity than DS for detecting oncogenic alterations in NSCLC. Our findings suggest that PNA clamping is a more useful method for clinical practice.
Antineoplastic Agents/therapeutic use ; Carcinoma, Non-Small-Cell Lung/drug therapy/*genetics ; Constriction ; Humans ; Lung Neoplasms/*genetics ; Molecular Diagnostic Techniques ; Mutation ; Peptide Nucleic Acids/*genetics ; Protein Kinase Inhibitors/*therapeutic use ; Receptor Protein-Tyrosine Kinases/*genetics ; Receptor, Epidermal Growth Factor/*genetics ; Sensitivity and Specificity ; Sequence Analysis ; Sequence Analysis, DNA ; Translocation, Genetic

PURPOSE: Refractory Mycoplasma pneumonia (RMP) has been increasing not only in Korea but worldwide. We investigated the incidence of M. pneumonia resistant to macrolides and risk factors for RMP. METHODS: From October 2015 to May 2016, 62 pediatric patients who were admitted due to pneumonia diagnosed on the basis of chest x-ray with respiratory symptoms and positive for M. pneumoniae in polymerase chain reaction with no evidence of other bacterial or viral infections were included. Sequence analysis of the 23S rRNA gene in M. pneumoniae was performed to identify macrolide resistance. Patients with congenital anomalies, history of pulmonary disease, and unclear information on antibiotic use were excluded. RESULTS: Mutations in the 23S rRNA gene were detected in 50 of 62 patients (80.6%). Risk factors were analyzed in only 45 patients. The RMP group consisted of 26 patients (57.8%) who had fever lasting more than 5 days and deteriorating chest x-ray findings. The lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels were significantly higher in the RMP group than in the non-RMP group (LDH: 300±79 U/L vs. 469±206 U/L, CRP: 4.9±4.3 mg/dL vs. 2.5±1.7 mg/dL; P = 0.04 vs. P = 0.026). In univariate analysis, the RMP group was significantly associated with 23S rRNA gene mutation, lobar pneumonia, and pleural effusion (odds ration [OR]: 10.8, 4.1, 5.3; P = 0.004, P = 0.036, P = 0.046). The presence of macrolide resistance was found to be only a significant risk factor in logistic regression (OR; 8.827; 95% confidence interval, 1.376–56.622; P = 0.022). CONCLUSION: Macrolide resistance was a significant risk factor in patients with RMP and identification of macrolide resistance might be helpful in predicting RMP and establishing target therapy for RMP.
C-Reactive Protein ; Child ; Fever ; Genes, rRNA ; Humans ; Incidence ; Korea ; L-Lactate Dehydrogenase ; Logistic Models ; Lung Diseases ; Macrolides ; Mycoplasma ; Pleural Effusion ; Pneumonia ; Pneumonia, Mycoplasma ; Polymerase Chain Reaction ; Risk Factors ; Sequence Analysis ; Thorax

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.
Animals ; Classification ; Clone Cells ; Enterotoxins ; Epitopes ; Genetic Variation ; Genotype ; Humans ; Protein Folding ; Protein Processing, Post-Translational ; Rotavirus ; Sequence Analysis ; Viral Nonstructural Proteins ; Virulence

Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonoses. Pathogenesis of the B. abortus infection is complicated, and several researchers have attempted to elucidate the infection mechanism of B. abortus. While several proteins have been revealed as pathogenic factors by previous researchers, the underlying mechanism of B. abortus infection is unresolved. In this study, we identified proteins showing different expression levels in B. abortus mutants with different biological characteristics that were generated by random insertion of a transposon. Five mutants were selected based on biological characteristics, in particular, their growth features. Total proteins of mutant and wild-type B. abortus were purified and subjected to two-dimensional gel electrophoresis. Thirty protein spots of each mutant with expression increases or decreases were selected; those with a change of more than 2-fold were compared with the wild-type. Selected spots underwent liquid chromatography tandem mass spectrometry for peptide analysis. DnaK and ClpB, involved in protein aggregation, increased. SecA and GAPDH, associated with energy metabolism, decreased in some mutants with a growth rate slower than that of the wild-type. Mutants with slower growth showed a decrease in energy metabolism-related proteins, while mutants with faster growth showed an increase in pathogenicity-related proteins.
Brucella abortus ; Brucella ; Brucellosis ; Chromatography, Liquid ; Electrophoresis, Gel, Two-Dimensional ; Energy Metabolism ; Population Characteristics ; Sequence Analysis, Protein ; Tandem Mass Spectrometry ; Zoonoses

Hereditary spherocytosis (HS) is caused by mutations in the SPTA1, SPTB, ANK1, SLC4A1, and EPB42 genes, all of which encode erythrocyte membrane proteins. Mutations in SLC4A1, which encodes band 3 protein, have rarely been reported as the causative factor among Korean patients with HS. Here, we report two Korean patients with HS carrying mutations in SLC4A1. Patient 1 was a 3-year-old girl with unremarkable past and family histories and was evaluated for anemia that was detected after a complete blood count. She was suspected of having HS considering the spherocytosis of her peripheral blood smear, increased osmotic fragility, hemolytic features in blood chemistry tests, and splenomegaly. Sequence analysis revealed that the patient harbored a single heterozygous missense mutation, c.2278C>T (p.Arg760Trp) in exon 17 of SLC4A1. Patient 2 was a 23-year-old man who had a prior history of intermittent jaundice. Although the patient did not have anemia, a genetic test for HS was performed due to evidence of hemolytic features in the blood chemistry test, splenomegaly, and a family history of HS. The test confirmed a single heterozygous missense mutation, c.2423G>T (p.Arg808Leu) in exon 18 of SLC4A1.
Anemia ; Anion Exchange Protein 1, Erythrocyte ; Blood Cell Count ; Chemistry ; Child, Preschool ; Erythrocyte Membrane ; Exons ; Female ; Humans ; Jaundice ; Mutation, Missense ; Osmotic Fragility ; Sequence Analysis ; Splenomegaly ; Young Adult

The NAC family is an important transcription factor which regulate plant growth and development， signal transduction， and stress response.In this study， the protein identification， subfamily classification， the determination of physical and chemical properties， protein structure， and expression pattern of NAC family were performed using bioinformatic methods based on the RNA-seq data of ginger. The results showed that a total of 72 NAC transcription factors were identified in 271.1 Mb total nucleotides， and they could be clustered into 13 subfamilies according to the phylogenetic tree.The physical and chemical properties， structure analysis revealed that the amino acid number and isoelectric point were different among 13 NAC subfamilies; the secondary structure of NACs transcription factors mainly consist of random coil， and the tertiary structure is similar.In addition，the expression patterns of genes under different soil moisture and Ralstonia solanacearum infection showed that 23 NACs were differentially expressed， which were mainly distributed in Ⅷ，Ⅶ， and ⅩⅤ subfamilies related to plant senescence， hormone metabolism and cell wall metabolism.The results provide some valuable information for the research and development of NAC transcription factors in ginger.
Gene Expression Regulation, Plant ; Ginger ; genetics ; Multigene Family ; Phylogeny ; Plant Proteins ; genetics ; Protein Structure, Tertiary ; RNA, Plant ; genetics ; Sequence Analysis, RNA ; Transcription Factors ; genetics

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.
Animals ; Axons ; physiology ; Brain ; anatomy & histology ; Brain Mapping ; Brain Stem ; cytology ; GABAergic Neurons ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Pathways ; physiology ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Transduction, Genetic ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism