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2.
Appl Microbiol Biotechnol ; 105(3): 1253-1268, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33475797

RESUMO

Ergosterol, a major lipid present in the fungal cell membrane, is considered as an effective antifungal drug target. A rational strategy for increasing drug reservoir relies on functionally validation of essential enzymes involved in fungal key biological pathway. Current knowledge regarding the essential genes in the ergosterol biosynthesis pathway is still limited in the opportunistic human pathogen Aspergillus fumigatus. In this study, we characterized two endoplasmic reticulum-localized sterol C-14 reductases encoded by both erg24A and erg24B homologs that are essential for the viability of A. fumigatus despite the fact that neither paralog is essential individually. Loss of one homolog of Erg24 impairs hyphal growth, conidiation, and virulence but has no effect on ergosterol biosynthesis. To investigate the functional significance of erg24, a conditional double mutant (Δerg24B niiA::erg24A) was constructed in the Δerg24B background. Strikingly, the conditional erg24 double mutant exhibited severe growth defects and accumulation of sterol intermediate. Moreover, the addition of metal ions and the overexpression of the corresponding ion transporters could rescue the growth defects of the erg24 double mutant in A. fumigatus, implying that the defective phenotype of the erg24 double mutant is tightly associated with dysregulation of ion homeostasis. Taken together, our results demonstrate the critical role of Erg24 in ergosterol biosynthesis and ion homeostasis in A. fumigatus, which may have important implications for antifungal discovery. KEY POINTS: • We characterized two endoplasmic reticulum-localized sterol C-14 reductases Erg24A and Erg24B in A. fumigatus. • Erg24A and Erg24B in combination, but not individually, are required for the viability of A. fumigatus. • Inactivation of Erg24 leads to the disruption of ion homeostasis and affects ergosterol biosynthesis.


Assuntos
Aspergillus fumigatus , Oxirredutases , Antifúngicos , Aspergillus fumigatus/genética , Ergosterol , Proteínas Fúngicas/genética , Homeostase , Humanos , Oxirredutases/genética
4.
Med Sci Monit ; 26: e926187, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33203828

RESUMO

BACKGROUND Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators in the signaling pathways of Toll-like receptors (TLRs)/IL1Rs. Targeting the IRAK4/IRAK1/TRAF6 axis and its associated pathway has therapeutic benefits in liver fibrosis. However, the function of IRAK1 itself in the development of liver fibrosis remains unknown. MATERIAL AND METHODS Irak1 global knockout (KO) mice were generated to study the functional role of Irak1 in liver fibrosis. Male Irak1 knockout and control mice were challenged with chronic carbon tetrachloride (CCl4) or fed a methionine- and choline-deficient diet (MCDD) to generate models of nonalcoholic steatohepatitis (NASH). Liver inflammation and collagen deposition were assessed by histological examination, quantitative real-time PCR (qRT-PCR), and western blotting of hepatic tissues. RESULTS The mRNA expression of the downstream inflammatory gene Il1b was significantly lower in Irak1-KO than in control mice. Irak1 ablation had little effect on inflammatory cell infiltration into livers of mice with NASH. Collagen deposition and the expression of genes related to fibrogenesis were similar in the livers of Irak1-KO and control mice exposed to CCl4 and MCDD. The loss of Irak1 did not affect lipid or glucose metabolism in these experimental models of steatohepatitis. CONCLUSIONS Irak1 knockout reduced the expression of inflammatory genes but had no effect on hepatic fibrogenesis. The Irak1-related pathway may regulate liver fibrosis via other pathways or be compensated for by other factors.


Assuntos
Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/deficiência , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Inflamação/complicações , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Metabolismo dos Lipídeos , Cirrose Hepática/complicações , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/complicações
5.
Orphanet J Rare Dis ; 15(1): 276, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028371

RESUMO

BACKGROUND: Propionic acidemia (PA) is a serious metabolic disorder, and different approaches have been applied to its prenatal diagnosis. To evaluate the reliability and validity of a biochemical strategy in the prenatal diagnosis of PA, we conducted a retrospective study of our 11-year experiences at a single center. METHODS: We accumulated data from 78 pregnancies from 58 families referred to our center and provided prenatal diagnosis by directed genetic analysis and/or metabolite measurement using tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) of amniotic fluid (AF) samples. RESULTS: Sixty-five unaffected fetuses (83.33%) and 13 affected fetuses (16.67%) were confirmed in our study. The characteristic metabolites including propionylcarnitine (C3) level, C3/acetylcarnitine (C2) ratio and 2-methylcitric acid (2MCA) level in unaffected and affected groups showed significant differences (P < 0.0001), while the level of 3-hydroxypropionic acid (3HPA) showed no significant difference between the two groups (P > 0.05).Of the 78 pregnancies, 24 fetuses were found to have either one causative pathogenic variant or were without genetic information in the proband. Three of these fetuses had elevated AF levels of C3, C3/C2 ratio, and 2MCA and, thus, were determined to be affected, while the remaining fetuses were determined to be unaffected based on a normal AF metabolite profile. Our genetic and biochemical results were highly consistent with postnatal follow-up results on all unaffected fetuses. CONCLUSIONS: We conclude that a biochemical approach can serve as a fast and convenient prenatal diagnostic method for pregnancies at an increased risk for PA, which could be used in conjunction with genetic testing for precise prenatal diagnosis of this disorder. In our analysis, the characteristic metabolites C3 level, C3/C2 ratio, and 2MCA level in AF supernatant were dependable biochemical markers for diagnosis, of which the C3/C2 ratio appears to be the most reliable biochemical marker for the prenatal diagnosis of PA.


Assuntos
Acidemia Propiônica , Líquido Amniótico , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal , Acidemia Propiônica/diagnóstico , Acidemia Propiônica/genética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Espectrometria de Massas em Tandem
6.
Metallomics ; 12(9): 1370-1379, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32608423

RESUMO

Aluminum (Al) toxicity is one of the most important limiting factors for crop yield in acidic soils. Bound Al gets converted into a toxic ionic state (Al3+) in acidic soil. Recent studies have shown that Al can act on the cell walls, cell membranes, organelles, and nuclei of microorganisms and affect substance and energy metabolism. To explore the gene expression at the transcriptional level under Al stress, we sequenced the transcriptome of Cryptococcus humicola, which is a highly Al-resistant yeast strain isolated from acidic soil and tolerates up to 200 mM Al3+. The screening conditions for genes from the control and experimental group were a false discovery rate (FDR) <0.05 and log 2|FC| > 1. A total of 4760 genes were differentially expressed, among which 3066 were upregulated and 1694 were downregulated. These genes control glycometabolism, protein synthesis, lipid metabolism and signalling pathways. Eleven selected differentially expressed genes were further validated using qRT-PCR. The results suggested that Al stress leads to complex responses in C. humicola. The effects of Al on the ß-d-glucan and mannose contents and Al accumulation in the cell wall were determined. With an increase in the Al treatment time and concentration, the contents of ß-d-glucan and mannose showed a trend of first increasing and then decreasing. Under Al treatment, the Al content of the cell wall also showed a trend of first increasing and then decreasing. These results suggested that Al accumulates in the cell wall and the cell wall plays a vital role in the Al resistance of C. humicola. The differentially expressed genes provide a foundation for the further study of Al tolerance in C. humicola.

7.
Neuromuscul Disord ; 30(3): 219-226, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32169315

RESUMO

Spinal muscular atrophy (SMA) is caused by homozygous deletions of the SMN1 gene in approximately 95% of patients. The remaining 5% of patients with SMA retain at least one copy of the SMN1 gene carrying insertions, deletions, or point mutations. Although molecular genetic testing for most SMA patients is quite easy, diagnosing "nondeletion" SMA patients is still compromised by the presence of a highly homologous SMN2 gene. In this study, we analyzed the SMN1/SMN2 copy number by quantitative PCR and multiplex ligation-dependent probe amplification (MLPA). Further, common primers for both SMN1 and SMN2 sequences were used to screen DNA intragenic mutations. To confirm whether the identified mutations occurred in SMN1 or SMN2, we improved the traditional RT-PCR method by only amplifying SMN1 transcripts using an allelic-specific PCR (AS-RT-PCR) strategy. We identified six SMN1 point mutations and small indels in 8 families, which included c.683T>A, c.22dupA, c.815A>G, c.19delG, c.551_552insA and c.401_402delAG. To the best of our knowledge, the latter three have never been previously reported. The most common mutation in Chinese patients is c.22dupA, which was identified in three families. In this work, we demonstrated AS-RT-PCR to be reliable for identifying SMN1 subtle mutations, especially the prevalent mutation c.22dupA in Chinese SMA patients. By reviewing published papers and summarizing reported SMN1 mutations, a distinct ethnic specificity was found in SMA patients from China. Our research extends the SMN1 mutation spectrum.


Assuntos
Atrofia Muscular Espinal/genética , Mutação/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , China , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Masculino , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 2 de Sobrevivência do Neurônio Motor/genética
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 686-689, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302911

RESUMO

OBJECTIVE: To explore the pathogenesis of two fetuses from one family affected with Joubert syndrome (JS). METHODS: Whole exome sequencing was employed to screen potential mutations in both fetuses. Suspected mutations were verified by Sanger sequencing. Impact of intronic mutations on DNA transcription was validated by cDNA analysis. RESULTS: Two novel TCTN1 mutations, c.342-8A>G and c.1494+1G>A, were identified in exons 2 and 12, respectively.cDNA analysis confirmed the pathogenic nature of both mutations with interference of normal splicing resulting in production of truncated proteins. CONCLUSION: The genetic etiology of the family affected with JS has been identified.Above findings have enriched the mutation spectrum of TCTN1gene and facilitated understanding of the genotype-phenotype correlation of JS.


Assuntos
Anormalidades Múltiplas/genética , Cerebelo/anormalidades , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico , Anormalidades do Olho/diagnóstico , Humanos , Doenças Renais Císticas/diagnóstico , Mutação , Linhagem , Sequenciamento Completo do Exoma
9.
Prenat Diagn ; 39(11): 993-997, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31278756

RESUMO

OBJECTIVES: This study reported the clinical prenatal diagnosis experience of families affected by methylmalonic acidemia (MMA) evaluated at a single prenatal diagnosis center over 8 years, and the reliability of a biochemical approach for prenatal diagnosis was analyzed. METHODS: Prenatal diagnosis data for 187 MMA families referred to our center from 2009 to 2016 were reviewed retrospectively. The results of the genetic analysis and biochemical approach were compared. RESULTS: A total of 41 MMA-affected pregnancies (21%) were identified. The biochemical analysis could identify the true status of 99.5% of fetuses. The diagnostic sensitivities of the propionylcarnitine (C3) level, the C3 to acetylcarnitine (C2) ratio (C3/C2), the methylmalonic acid, and methylcitrate levels in the amniotic fluid were 95.1%, 100%, 100%, and 82.9%, respectively, and the specificities were 98.7%, 99.3%, 97.4%, and 96.7%, respectively. CONCLUSIONS: The biochemical analysis could be optionally used in the prenatal diagnosis of MMA, especially in cases where the genetic results are inconclusive. Among the four tested biochemical markers, C3/C2 appeared to be the most reliable.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Líquido Amniótico/química , Biomarcadores/análise , Erros Inatos do Metabolismo dos Aminoácidos/genética , Amniocentese , Líquido Amniótico/citologia , Feminino , Testes Genéticos , Humanos , Metilmalonil-CoA Mutase/genética , Oxirredutases/genética , Gravidez , Estudos Retrospectivos
10.
PLoS One ; 9(8): e106100, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25166052

RESUMO

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5' end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.


Assuntos
Quirópteros/anatomia & histologia , Extremidades/crescimento & desenvolvimento , Proteínas do Olho/genética , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Sequência de Bases , Quirópteros/embriologia , Quirópteros/genética , Sequência Conservada , Embrião de Mamíferos/anatomia & histologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Dados de Sequência Molecular , Análise de Sequência de RNA
11.
Proc Biol Sci ; 281(1783): 20133133, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24695426

RESUMO

Bats are the only mammals capable of true flight. Critical adaptations for flight include a pair of dramatically elongated hands with broad wing membranes. To study the molecular mechanisms of bat wing evolution, we perform genomewide mRNA sequencing and in situ hybridization for embryonic bat limbs. We identify seven key genes that display unique expression patterns in embryonic bat wings and feet, compared with mouse fore- and hindlimbs. The expression of all 5'HoxD genes (Hoxd9-13) and Tbx3, six known crucial transcription factors for limb and digit development, is extremely high and prolonged in the elongating wing area. The expression of Fam5c, a tumour suppressor, in bat limbs is bat-specific and significantly high in all short digit regions (the thumb and foot digits). These results suggest multiple genetic changes occurred independently during the evolution of bat wings to elongate the hand digits, promote membrane growth and keep other digits short. Our findings also indicate that the evolution of limb morphology depends on the complex integration of multiple gene regulatory networks and biological processes that control digit formation and identity, chondrogenesis, and interdigital regression or retention.


Assuntos
Evolução Biológica , Quirópteros/anatomia & histologia , Quirópteros/fisiologia , Voo Animal , Regulação da Expressão Gênica , Asas de Animais/anatomia & histologia , Animais , Quirópteros/genética , Quirópteros/crescimento & desenvolvimento , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Estudo de Associação Genômica Ampla , Hibridização In Situ , Camundongos , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
12.
PLoS One ; 8(7): e68867, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23874796

RESUMO

A trade-off between the sensory modalities of vision and hearing is likely to have occurred in echolocating bats as the sophisticated mechanism of laryngeal echolocation requires considerable neural processing and has reduced the reliance of echolocating bats on vision for perceiving the environment. If such a trade-off exists, it is reasonable to hypothesize that some genes involved in visual function may have undergone relaxed selection or even functional loss in echolocating bats. The Gap junction protein, alpha 10 (Gja10, encoded by Gja10 gene) is expressed abundantly in mammal retinal horizontal cells and plays an important role in horizontal cell coupling. The interphotoreceptor retinoid-binding protein (Irbp, encoded by the Rbp3 gene) is mainly expressed in interphotoreceptor matrix and is known to be critical for normal functioning of the visual cycle. We sequenced Gja10 and Rbp3 genes in a taxonomically wide range of bats with divergent auditory characteristics (35 and 18 species for Gja10 and Rbp3, respectively). Both genes have became pseudogenes in species from the families Hipposideridae and Rhinolophidae that emit constant frequency echolocation calls with Doppler shift compensation at high-duty-cycles (the most sophisticated form of biosonar known), and in some bat species that emit echolocation calls at low-duty-cycles. Our study thus provides further evidence for the hypothesis that a trade-off occurs at the genetic level between vision and echolocation in bats.


Assuntos
Adaptação Biológica/genética , Quirópteros/genética , Conexinas/genética , Evolução Molecular , Proteínas do Olho/genética , Filogenia , Proteínas de Ligação ao Retinol/genética , Percepção Visual/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Ecolocação/fisiologia , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Pseudogenes/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie
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