LINC00511 accelerates the proliferation of cardiomyocytes after myocardial ischemia/reperfusion injury by absorbing miRNA-515-5p.

OBJECTIVE: The aim of this study was to clarify the role of LINC00511 in regulating the proliferative ability of cardiomyocytes undergoing ischemia/reperfusion (I/R) injury by absorbing miRNA-515-5p. MATERIALS AND METHODS: Adult male C57BL/6 mice were subjected to I/R injury, and I/R model was constructed in vivo. Primary cardiomyocytes were isolated from 1-2 days-old male mice and treated with H2O2 to establish the I/R model in vitro. The relative expression level of LINC00511 was determined after ligation of the anterior descending coronary artery (LAD) in mice or H2O2 induction in primary cardiomyocytes for different time points, respectively. The regulatory effect of LINC00511 on the viability of H2O2-treated cardiomyocytes was assessed. Subsequently, the interaction between LINC00511 and miRNA-515-5p was evaluated by Dual-Luciferase Reporter Gene Assay. Furthermore, the viability and 5-Ethynyl-2'-deoxyuridine (EdU)-positive rate influenced by LINC00511/miRNA-515-5p were examined. RESULTS: LINC00511 was gradually downregulated with the prolongation of I/R procedures in mice or H2O2 treatment in primary cardiomyocytes. The overexpression of LINC00511 significantly elevated the viability and EdU-positive rate in H2O2-treated cardiomyocytes. LINC00511 could bind to miRNA-515-5p. Meanwhile, there was a negative correlation between the levels of LINC00511 and miRNA-515-5p. In addition, the overexpression of miRNA-515-5p reversed the promoting effect of LINC00511 on the proliferative ability of H2O2-treated cardiomyocytes. CONCLUSIONS: LINC00511 accelerates the proliferation of cardiomyocytes after I/R by targeting miRNA-515-5p.

[Mutation analysis of GJB2 gene in 1 822 patients with nonsyndromic hearing loss in Zhejiang Province].

Objective:To analyze the genetic characteristics in nonsyndromic hearing impairment (NSHL) patients in Zhejiang province.Method:Peripheral blood samples were obtained from 1822 NSHL patients and 467 normal hearing controls in Zhejiang province. We carried out a systematic mutational screening of GJB2 gene in these subjects by amplifying the coding region of GJB2 gene and sequencing directly.Result:Thirty kinds of mutation were identified, including eleven pathogenic mutations, one hypomorphic allele, sixteen polymorphic mutations and two novel mutations. The c.235delC mutation was the most prevalent pathogenic mutation in this cohort (18.50%), and the rate of allele mutation was 12.16%. The frequency of c.299_300delAT,c.176_191del16,c.512_513insAACG,c.35delG,c.283G>A,c.427C>T,c.35insG,c.439G>A,c.571T>C,c.139G>T mutations were decreased in turn.Conclusion:c.235delC mutation is the hot spot of GJB2 gene mutation in NSHL patients in Zhejiang province and the most common mutational pattern is frame-shift mutation. The discovery of novel mutations enriches the spectrum and frequency of variants in GJB2 gene.

Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation.

Deafness or hearing loss is a major issue in human health. Inner ear hair cells are the main sensory receptors responsible for hearing. Defects in hair cells are one of the major causes of deafness. A combination of induced pluripotent stem cell (iPSC) technology with genome-editing technology may provide an attractive cell-based strategy to regenerate hair cells and treat hereditary deafness in humans. Here, we report the generation of iPSCs from members of a Chinese family carrying MYO15A c.4642G>A and c.8374G>A mutations and the induction of hair cell-like cells from those iPSCs. The compound heterozygous MYO15A mutations resulted in abnormal morphology and dysfunction of the derived hair cell-like cells. We used a CRISPR/Cas9 approach to genetically correct the MYO15A mutation in the iPSCs and rescued the morphology and function of the derived hair cell-like cells. Our data demonstrate the feasibility of generating inner ear hair cells from human iPSCs and the functional rescue of gene mutation-based deafness by using genetic correction.

[Spectrum of GJB6 variants in 318 pedigrees with non-syndromic hearing loss:one deafness pedigree carrying both GJB6 and GJB2 deletion variant].

Objective:To investigate the mutation characteristics of GJB6 (gap juction bata 6) gene in 318 Han Chinese pedigrees with non-syndromic hearing loss.Method:Polymerase chain reaction was used to detect the coding region of GJB6 gene in 318 Han Chinese pedigrees with non-syndromic hearing loss.Gene arrays and second generation sequencing were used to detect 118 genes which had reported to be accosiated with deafness in members of pedigree which possibly carried pathogenic GJB6 gene mutation.Result:Here,we have screened the mutations of GJB6 gene in 318 Han Chinese pedigrees with non-syndromic hearing loss and found one pedigree carrying both GJB6 and GJB2 gene deletion.Clinical and molecular genetic evaluation revealed the variable phenotype of hearing impairments including age-at-onset,audiometric configuration and severity in these subjects.Mutational analysis of the GJB2 and GJB6 gene coding region showed a heterozygous 235 del C of GJB2 gene and a novel 228 del G of GJB6 gene.Conclusion:GJB6 gene 228 del G variant,which occurs at a highly evolutionarily conserved nucleotide,forward the stop codon to 81 position and result in the corresponding polypeptide 181 amino acids shorter than wildtype polypeptide.In addition,GJB6 gene 228 del G absent varies among 94 unrelated Chinese controls.Our finding suggest that GJB6 gene 228 del G maybe a novel pathogenic mutation associated with non-syndromic hearing loss.

Nuclear background determines biochemical phenotype in the deafness-associated mitochondrial 12S rRNA mutation.

The pathogenetic mechanism of the human mitochondrial 12S rRNA gene mutation at position 1555, associated with non-syndromic deafness and aminoglycoside-induced deafness, has been investigated in 33 transformants obtained by transferring mitochondria from lymphoblastoid cell lines into human mitochondrial DNA (mtDNA)-less (rho *206) cells. In this nearly constant nuclear background, 15 transformants derived from five symptomatic individuals from a large Arab-Israeli family, carrying this mutation in homoplasmic form, exhibited significant decreases compared with nine control transformants in the rate of growth in a medium containing galactose instead of glucose, as well as in the rates of mitochondrial protein synthesis and of substrate-dependent respiration. Most significantly, these decreases were very similar to those observed in nine transformants derived from three asymptomatic members of the family. This result in transmitochondrial cybrids is in contrast to the differences in the same parameters previously demonstrated between the original lymphoblastoid cell lines derived from the symptomatic and asymptomatic members of the Arab-Israeli family. In addition, the intragroup variability in biochemical dysfunction among the lymphoblastoid cell lines from different symptomatic or asymptomatic or control individuals was significantly reduced in the derived mitochondrial transformants carrying the same nuclear background. These observations provide strong genetic and biochemical evidence in support of the idea that the nuclear background plays a determinant role in the phenotypic manifestation of the non-syndromic deafness associated with the A1555G mutation.

A biochemical basis for the inherited susceptibility to aminoglycoside ototoxicity.

The A1555 G mutation in mitochondrial 12S rRNA has been found to be associated with non-syndromic deafness and aminoglycoside-induced deafness. The sensitivity to the aminoglycoside paromomycin has been analyzed in lymphoblastoid cell lines derived from five deaf individuals and five hearing individuals from an Arab-Israeli family carrying the A1555G mutation, and three married-in controls from the same family. Exposure to a high concentration of paromomycin (2 mg/ml), which caused an 8% average increase in doubling time (DT) in the control cell lines, produced higher average DT increases (49 and 47%) in the A1555G mutation-carrying cell lines derived from symptomatic and asymptomatic individuals, respectively. The ratios of translation rates in the presence and absence of paromomycin, which reflected the effect of the drug on mitochondrial protein synthesis, were significantly decreased in the cell lines derived from symptomatic and asymptomatic individuals (by 30 and 28% on average, respectively), compared with the ratios in the control cell lines. These ratios showed, in both groups of mutant cell lines, a significant negative correlation with the ratios of DTs in the presence and absence of the antibiotic. These results have provided the first direct evidence that the mitochondrial 12S rRNA carrying the A1555G mutation is the main target of aminoglycosides. They suggest that these antibiotics exert their detrimental effect through an alteration of mitochondrial protein synthesis, which exacerbates the inherent defect caused by the mutation, reducing the overall translation rate down to and below the minimal level required for normal cellular function (40-50%).

Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance.

Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.

The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of approximately 70% in the tRNASer(UCN) level and a decrease of approximately 45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to approximately 40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located approximately 7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

Infection of T cell subsets by HIV-1 and the effects of interleukin-12.

CD4+ lymphocytes constitute one of the major cell targets for human immunodeficiency virus type 1 (HIV-1) infection. The eventual loss of CD4+ lymphocytes contributes substantially to the pathogenesis of HIV-1 and development of acquired immunodeficiency syndrome (AIDS). CD4+ lymphocytes consist of the subgroups Th1, Th2, and Th0, which differ in their cytokine profile. Th1 cells produce cytokines that favor cell-mediated immune responses, whereas Th2 cells produce cytokines that favor humoral immunity. Th0 cells are precursors to the Th1 and Th2 subsets. A shift from a Th1 to a Th2 response has been reported for HIV-1-infected patients (Kannagi et al. 1990. J. Virol. 64, 3399-3406; Walker et al. 1986. Science 234, 1563-1566; Walker et al. 1991. J. Virol. 65, 5921-5927). For this reason, the potential role of cytokines in the development of AIDS has received a great deal of attention. Interleukin (IL)-12 is a disulfide-linked, 70-kDa heterodimeric cytokine produced by antigen-presenting cells. IL-12 has a central role in the development of the Th1-type immune responses. Therefore, we investigated the ability of T-tropic HIV-1 IIIB to replicate in Th1, Th2, and Th0 T cell clones and studied the effects of IL-12 on HIV-1 replication in these cells types. These studies demonstrate several points. (1) Th1, Th2, and Th0 T cell clones support HIV-1 IIIB replication nearly equally well, and it is, therefore, unlikely that differences in ability to support HIV-1 replication can explain changes in Th1, Th2, or Th0 subtype 1 following HIV-1 infection. (2) Using this model, we show that IL-12 can inhibit HIV-1 replication, consistent with a role for IL-12 in HIV-1 replication in T cells. (3) HIV-1 can form a persistent infection in T cell clones, providing a reservoir model for study of viral sanctuary and persistence in a system closely approximating the in vivo situation.

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.

Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae.

The yeast nuclear mutation mgm104-1, which leads to slow growth on glucose medium and temperature-sensitive (ts) loss of mitochondrial DNA (mtDNA), has been identified by screening a collection of temperature-sensitive mutants on glycerol medium. A nuclear gene was isolated from a genomic DNA library by complementation of the mgm104-1 allele and was found to be identical to TTS1, which encodes the cytoplasmic tyrosyl-tRNA synthetase required for cytoplasmic protein synthesis. A gene disruption in a diploid strain demonstrated that the TTS1 gene is essential for cell viability. The lack of mutations in TTS1 in the mgm104-1 mutant indicates that TTS1 and MGM104 are different genes. The ability to rescue the mgm104-1 phenotype with a single additional copy of TTS1 suggests that TTS1 has an additional function that is directly or indirectly involved in the maintenance of the mitochondrial genome.

A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Biochemical evidence for nuclear gene involvement in phenotype of non-syndromic deafness associated with mitochondrial 12S rRNA mutation.

The phenotypic effects of the human mitochondrial 12S rRNA gene mutation at position 1555 associated with maternally inherited non-syndromic deafness and sensitivity to aminoglycoside-induced deafness have been analyzed in 25 lymphoblastoid cell lines derived from members of a large family carrying this mutation in homoplasmic form and from control individuals. A clear decrease in the rates of growth in galactose medium, mitochondrial protein synthesis, total oxygen consumption, and complex I-, complex III- and complex IV-dependent respiration was observed in two groups of nine and 10 mutant cell lines derived, respectively, from symptomatic and asymptomatic members of the family, as compared with six control cell lines. The severity of mitochondrial dysfunction in the mutant cell lines was correlated with the presence or absence of hearing loss in the donor individuals. These observations strongly suggest a role of a nuclear factor(s) in the phenotypic manifestation of the mutation. The approach used here provides a paradigm for the analysis of the nuclear background involvement in other mtDNA-linked disorders, including the putative ones associated with neurodegenerative diseases. Exposure of the cell lines derived from several symptomatic or asymptomatic individuals from the same family to high concentrations of neomycin or paromomycin decreased to a significant, nearly identical extent their rate of growth in glucose-containing medium, as contrasted with the unchanged growth rate of control cell lines or of mtDNA-less cells. These results support the hypothesis that the main target of the antibiotics is the mitochondrial 12S rRNA carrying the 1555 mutation, without any apparent role of the nuclear background.

Susceptibility testing of Dientamoeba fragilis ATCC 30948 with iodoquinol, paromomycin, tetracycline, and metronidazole.

Susceptibility testing was performed on Dientamoeba fragilis ATCC 30948 in a dixenic culture with Klebsiella pneumoniae and Bacteroides vulgatus. D. fragilis was cocultured with the bacteria in TYGM-9 medium (ATCC medium 1171). The activities of antiparasitic drugs were assessed by counting viable D. fragilis trophozoites with a hemacytometer by trypan blue exclusion. The minimal amebicidal concentrations of the following four drugs were determined: iodoquinol at 128 micrograms/ml, paromomycin at 16 micrograms/ml, tetracycline (questionably) at 32 micrograms/ml, and metronidazole at 32 micrograms/ml.

MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.

A nuclear mutation, mgm101, results in temperature sensitive loss of mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae. The corresponding gene, MGM101, was isolated from a genomic DNA library by complementation. Sequence analysis shows that MGM101 encodes a positively charged protein of 269 amino acids with a calculated molecular weight of 30 kDa. This analysis also reveals that MGM101 is adjacent to the ribosomal protein gene RPS7A on chromosome X and hybridization indicates it occurs in single copy. Creation of a null mutant by targeted disruption showed that the gene has no essential cellular function, aside from its participation in mitochondrial genome maintenance. As no counterpart has been identified in databases it is a novel protein whose role has yet to be determined. Expression of MGM101 is low on glucose medium but on galactose there is a two-fold increase in the level of the transcript.

Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens.

An indirect fluorescent-antibody (IFA) assay was carried out to examine for the presence of Dientamoeba fragilis trophozoites in preserved fecal specimens. Antiserum to D. fragilis trophozoites was raised in a rabbit with a dixenic culture of D. fragilis (ATCC 30948) from the American Type Culture Collection. After absorption with Klebsiella pneumoniae and Bacteroides vulgatus, the immune rabbit serum was used for examination by the IFA assay. A total of 155 clinical samples were tested; 42 with no parasites, 9 with D. fragilis, and 104 with other parasites. The IFA assay identified seven D. fragilis organisms. Two specimens with doubtful IFA assay readings showed very scanty amounts of D. fragilis trophozoites on stained smears. There were no false-positive IFA assay readings. The IFA assay appeared to be a promising method because of its speed in screening. The specificity of the IFA assay indicates that other diagnostic tests such as an enzyme-linked immunosorbent assay could be developed to identify D. fragilis antigens in fecal specimens.

Large-scale screening of human sera with cytomegalovirus recombinant antigens.

One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulins G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.

A simple and rapid procedure for the direct detection of cytomegalovirus in urine samples.

We compared cytomegalovirus (CMV) isolation in tissue culture and viral DNA detection by DNA:DNA hybridization in 60 clinical urine samples concentrated by different procedures. Our results showed that urine concentration by filtration is the easiest and quickest procedure allowing further detection of CMV. With optimized working conditions, the CMV detection in urine can be done in 5 hr without requiring cell cultures or costly instruments.