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In this study, the bacterial diversity of acquired middle ear cholesteatoma (MEC) was evaluated to reveal its pathogenesis and provides a guide for the use of antibiotics. Twenty-nine cases of acquired MEC and eight cases of healthy middle ears undergoing cochlear implantation (CI) were evaluated. Full-length 16S rRNA gene sequencing was performed to profile the bacterial communities in lesions and healthy tissues of the middle ear. ACE (P = 0.043) and Chao1 (P = 0.039) indices showed significant differences in alpha diversity (P < 0.05). Analysis of PERMANOVA/Anosim using the Bray-Curtis distance matrix results suggested that the between-group differences were greater than the within-group differences (R = 0.238, P < 0.05, R2 = 0.066, P < 0.05). Bacterial community analysis revealed that Alphaproteobacteria at the class level and Caulobacterales and Sphingomonadales at the order level were significantly different (P < 0.05). In the LefSe (Linear discriminant analysis effect size) analysis, Porphyromonas bennonis was elevated, and Bryum argenteum and unclassified Cyanobacteriales were reduced at the species level in MEC (P < 0.05). Fifteen metabolic pathways were found to be significantly different between the two groups by analysing the abundance of metabolic pathways in level 2 of the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Seven and eight metabolic pathways were significantly elevated in the MEC and control groups, respectively (P < 0.05). The role of bacteria in the pathogenesis of acquired MEC was further refined through analysis of metabolic pathways. These findings indicate that the acquired MEC and healthy middle ear contain more diverse microbial communities than previously thought.
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Colesteatoma da Orelha Média , Humanos , Colesteatoma da Orelha Média/genética , RNA Ribossômico 16S/genética , Genes de RNAr , Bactérias/genética , ChinaRESUMO
Mutations in RAS, a family of proteins found in all human cells, drive a third of cancers, including many pancreatic, colorectal, and lung cancers. However, there is a lack of clinical therapies that can effectively prevent RAS from causing tumor growth. Recently, a protease was engineered that specifically degrades active RAS, offering a promising new tool for treating these cancers. However, like many other intracellularly acting protein-based therapies, this protease requires a delivery vector to reach its site of action within the cell. In this study, we explored the incorporation of cationic lipids into ionizable lipid nanoparticles (LNPs) to develop a RAS protease delivery platform capable of inhibiting cancer cell proliferation in vitro and in vivo. A library of 13 LNPs encapsulating RAS protease was designed, and each formulation was evaluated for in vitro delivery efficiency and toxicity. A subset of four top-performing LNP formulations was identified and further evaluated for their impact on cancer cell proliferation in human colorectal cancer cells with mutated KRAS in vitro and in vivo, as well as their in vivo biodistribution and toxicity. In vivo, both the concentration of cationic lipid and type of cargo influenced LNP and cargo distribution. All lead candidate LNPs showed RAS protease functionality in vitro, and the top-performing formulation achieved effective intracellular RAS protease delivery in vivo, decreasing cancer cell proliferation in an in vivo xenograft model and significantly reducing tumor growth and size. Overall, this work demonstrates the use of LNPs as an effective delivery platform for RAS proteases, which could potentially be utilized for cancer therapies.
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Proliferação de Células , Lipídeos , Nanopartículas , Humanos , Animais , Proliferação de Células/efeitos dos fármacos , Nanopartículas/administração & dosagem , Nanopartículas/química , Lipídeos/química , Linhagem Celular Tumoral , Camundongos Nus , Feminino , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Distribuição Tecidual , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Camundongos , Sistemas de Liberação de MedicamentosRESUMO
Waardenburg syndrome is an autosomal dominant inherited syndromic hereditary hearing loss characterized by varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. The aim of this study was to analyze the clinical phenotypes and genetic variants of a Chinese boy with Waardenburg syndrome type 2 and to explore the possible molecular pathogenesis of Waardenburg syndrome type 2. Clinical, audiological, and ophthalmologic evaluations were performed on the proband. Clinical data from the principal members in the proband's family were collected through questionnaires. Genetic analysis was conducted, including targeted next-generation sequencing of 144 known deafness genes, Sanger sequencing, and bioinformatic analysis. Waardenburg syndrome type 2was diagnosed in a 4-year-old boy according to the Waardenburg Syndrome Consortium Criteria. The novel missense mutation c.426G>T (p.Trp142Cys) was identified in SOX10 in the proband but was absent in his parents and the controls. A de novo missense mutation in SOX10 was the genetic cause of Waardenburg syndrome type 2 in the proband, which was useful for the molecular diagnosis of Waardenburg syndrome type 2.
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Perda Auditiva Neurossensorial , Fatores de Transcrição SOXE , Síndrome de Waardenburg , Humanos , População do Leste Asiático , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Mutação , Fenótipo , Fatores de Transcrição SOXE/genética , Síndrome de Waardenburg/diagnóstico , Síndrome de Waardenburg/genética , Masculino , Pré-EscolarRESUMO
Spontaneous cerebrospinal fluid (CSF) leak is a condition that commonly presents with unilateral watery drainage from the nose or ear, tinnitus, and stuffy ears or hearing loss. Spontaneous CSF rhinorrhea and otorrhea together are rare. A 64-year-old woman presented at our department with complaints of clear watery rhinorrhea and hearing loss on the right side persisting for 10 months. Imaging and surgery were used to diagnose the condition. Through surgical treatment, she was eventually cured. Review of the literature has shown that patients with both nasal and aural CSF leaks are rare. When a patient presents with both unilateral watery drainage from both the nose and ear, a diagnosis of CSF rhinorrhea and otorrhea should be considered. This case report will benefit clinicians by providing more information to assist with diagnosing the disease.
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Background: Rape pollen allergy is a common allergic reaction disorder that affects the health and life of patients seriously. The research on ceRNA regulatory network in rape pollen allergy is poor. Methods: High throughput whole-transcriptome sequencing was conducted on rape pollen allergic samples and non-allergic samples. Differentially expressed microRNAs (DEmiRNAs), circRNAs (DEcircRNAs), long non-coding RNA (DElncRNAs), mRNA (DEmRNAs) were identified and a ceRNA regulatory network was constructed by Cytoscape. Functional enrichment analyses were performed on DEmRNAs in the ceRNA network. Then, the least absolute shrinkage and selection operator (LASSO) regression model was used to identify characteristic genes for rape pollen allergy. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic ability of characteristic genes. Results: A total of 25 DEmiRNAs, 258 DEcircRNAs, 304 DElncRNAs, and 383 DEmRNAs in the allergic group compared with the non-allergic group were uncovered, respectively. A ceRNA network containing 21 miRNAs, 57 circRNAs, 28 lncRNAs, and 33 mRNAs was generated with 139 nodes and 160 edges. The signal transduction-related processes, immune-related processes, the ion, inorganic substance, and hormone regulation processes were associated with mRNAs in the ceRNA network. The results of pathway enrichment illustrated that mRNAs in the ceRNA were significantly linked to IL-17 signaling pathway, inflammatory mediator regulation of trp channels, GMP-PKG signaling pathway, signaling by GPCR, and GPCR downstream signaling pathway. Then, five characteristic genes (KCNQ3, CCR5, FOSB, CFAP43, and PRKG1) were defined by the LASSO algorithm. The AUC values of these genes indicated that these genes had a powerful discrimination ability in discriminating allergic samples from non-allergic samples. Conclusion: Taken together, we revealed the ceRNA regulatory network in rape pollen allergy and excavated five characteristic genes (KCNQ3, CCR5, FOSB, CFAP43, and PRKG1) with the diagnostic value that may be a potential target in diagnosis and treatment.
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Objective:To analyze the clinical characteristics, treatment and prognosis of the otolaryngology head and neck malignant tumors in children, in order to improve the diagnosis and treatment of the diseases. Methods:The patients of otorhinolaryngology head and neck malignant solid tumors under 14 years old hospitalized in Kunming Children's Hospital and the First Affiliated Hospital of Kunming Medical University from 2014 to 2020 were retrospectively analyzed. All cases were statistically analyzed according to gender, age, location, pathological type and treatment method. Results:The main clinical manifestations of 91 children were mainly facial and neck masses, including nasal congestion, swallowing discomfort, and continuous intermittent fever. CT and MRI examination showed that the diameter of the tumor was 1.2 cm ×2.0 cm to 5.0 cm×12.0 cm, with a mean of 2.8 cm×3.2 cm, and 19 cases had distant metastasis. The main tissue sources were soft tissue ï¼56 casesï¼ and epithelial tissue ï¼35 casesï¼. There were 6 pathological types, the most common was sarcoma ï¼41 casesï¼, followed by neuroblastoma ï¼15 casesï¼, papillary carcinoma ï¼14 casesï¼, squamous cell carcinoma ï¼10 casesï¼, mucoepidermoid carcinoma ï¼8 casesï¼, and adenocarcinoma ï¼3 casesï¼. According to the classification of tissue origin, the statistical analysis of gender and pathological type showed statistically significant differences in both gender and pathological typesï¼P<0.01ï¼. Conclusion:The age of onset, primary site, tissue origin and pathological type of otolaryngology head and neck malignancy in children have their own characteristics, which should be comprehensively evaluated and treated with multidisciplinary treatment.
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Adenocarcinoma , Neoplasias de Cabeça e Pescoço , Sarcoma , Humanos , Criança , Adolescente , Estudos Retrospectivos , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/terapia , PrognósticoRESUMO
Objective:To analyze the consistency of pepsin assay kit, pepsin IHC, reflux symptom indexï¼RSIï¼ and reflux finding scoreï¼RFSï¼ in the diagnosis of laryngopharyngeal reflux diseaseï¼LPRDï¼. Methods:The clinical data of 61 inpatients with laryngeal diseases who were admitted to the Department of Otolaryngology, the First Affiliated Hospital of Kunming Medical University from May 2020 to December 2021 were retrospectively analyzed. The RSI and RFS scores, the Formwitz score of pepsin immunohistochemistry, and the results of pepsin detection kit were recorded. ICC group correlation coefficient and Kappa consistency analysis was used for three detection methods. Results:Among 61 patients, 30 cases were positive and 31 cases were negative for the pepsin test kit, with a positive rate of 49.18%. The positive rate of pepsin immunohistochemistry was 45.90%ï¼28/61ï¼, and the diagnostic agreement rate between the two was 70.49%. The consistency between them was highï¼κ=0.409ï¼. The positive rate of RSI and RFS in diagnosing LPRD was 62.30%ï¼38/61ï¼, and the consistency rate was 73.77% with pepsin detection kit. The consistency between them was highï¼κ=0.486ï¼. Taking pepsin IHC as the reference standard, the sensitivity, specificity, positive predictive value and negative predictive value of pepsin detection kit were 71.43%ï¼20/28ï¼, 69.70%ï¼23/33ï¼, 66.67%ï¼20/30ï¼ and 74.19%ï¼23/31ï¼, respectively. Using RSI and RFS scales as reference criteria, the sensitivity, specificity, positive predictive value and negative predictive value of pepsin detection kit were 89.29%ï¼25/28ï¼, 60.61%ï¼20/33ï¼, 65.79%ï¼25/38ï¼ and 86.96%ï¼20/23ï¼, respectively. Analysis of correlation coefficient within ICC group: ICC value was 0.628, 95% confidence intervalï¼0.497-0.741ï¼, the three methods have good consistency. Conclusion:The RSI and RFS scale scores were in good agreement with the pepsin test kit, and the pepsin test kit was also in good agreement with pepsin immunohistochemistry. As a non-invasive diagnostic technique, the pepsin test kit can be widely used in the diagnosis of pharyngeal reflux in combination with pepsin immunohistochemistry and RSI and RFS scale.
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Refluxo Laringofaríngeo , Humanos , Refluxo Laringofaríngeo/diagnóstico , Pepsina A/análise , Estudos Retrospectivos , Imuno-Histoquímica , FaringeRESUMO
To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, ß-grasp, and α/ß-plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or ß-grasp fold in the smaller form but an α/ß-plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.
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Dobramento de Proteína , Proteínas , Proteínas/metabolismo , Sequência de Aminoácidos , Proteína Estafilocócica A , MutaçãoRESUMO
Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains.
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Plasmodium falciparum/química , Subtilisinas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Terciária de Proteína , Alinhamento de Sequência , SoluçõesRESUMO
Gout nodules (tophi) are formed by a chronic inflammatory reaction in tissues resulting in deposition of urate crystals. They are commonly seen in joints and surrounding tissues, subcutaneous tissues, as well as the pinna and kidney, and are characteristic manifestations of gout. Vocal cord tophi are rarely reported in the literature, and patients often present with hoarseness, progressive dysphagia, and other symptoms. We report a case of a vocal cord mass found by gastroscopy in a patient with a history of gout for more than 20 years. Postoperative pathological findings were vocal cord tophi. Tophi can have serious consequences and should be included in the differential diagnosis of laryngeal masses in patients with a history of gout. Therapy should involve a combination of systemic uric acid-lowering treatment and surgery to improve symptoms and reduce recurrence.
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Objective:To evaluate the preliminary value of the cross-sectional area and morphological changes of the external ear canal opening after the two-flap auriculoplasty through the I shaped posterior incision. Methods:One hundred and thirty-seven patientsï¼a total of 155 earsï¼ who received open radical mastoidectomy in the department of otolaryngology in the First Affiliated Hospital of Kunming Medical University were treated with I shaped incision and two-flap auriculoplasty. Vertical diameterï¼D1ï¼ and horizontal diameterï¼D2ï¼ of the external ear canal were measured at the completion of surgery, 1 month and 6 months post-operation, respectively. The cross-sectional areaï¼S=1/4πD1×D2ï¼ of the external ear canal was calculated according to the two diameters. The dry ear time and intraoperative lumen epithelialization time were observed after operation. At 6 months after operation, the morphology of the external ear canal opening was analyzed. Results:The postoperative dry ear duration was 18-61 daysï¼27.32±7.52ï¼ days. The time to complete epithelialization of the operative cavity was 24-70 daysï¼32.18±10.36ï¼ days. Six months after the operation, the morphological classification of 155 outer ear meatal openings was as follows: 117 earsï¼ 75.48%ï¼ were roundï¼the difference between vertical diameter and horizontal diameter was within 2 mmï¼; Ovalï¼oval appearance, difference between vertical diameter and horizontal diameter greater than 2 mmï¼ 35 earsï¼22.58%ï¼, triangle 3 earsï¼1.94%ï¼; Irregular ear canal orifice was not observed in all cases. During the operation, and at 1 month and 6 months after the operation, the cross-sectional area of the external ear canal wasï¼2.51±0.48ï¼ cm², ï¼2.45±0.35ï¼ cm², ï¼2.41±0.43ï¼ cm², respectively. And no significant differences were observed. ï¼P>0.05ï¼. Conclusion:The I shaped posterior auricular incision and two-flap auricular lumenoplasty is not compex and easy to perform. The morphology of the external ear opening is regular after the operation, which can effectively match the ventilation of the operative cavity.
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Pavilhão Auricular , Meato Acústico Externo , Pavilhão Auricular/cirurgia , Meato Acústico Externo/cirurgia , Humanos , Processo Mastoide/cirurgia , Mastoidectomia , TimpanoplastiaRESUMO
OBJECTIVES: To determine the clinical characteristics and genetic causes of Waardenburg syndrome type 1 (WS1) present in a Chinese Han family. METHODS: Evaluations, including the familial history, clinical features and audiological tests, were performed on the proband and her parents. Genetic analyses were conducted using targeted next-generation sequencing of 144 known deafness genes, and confirmed by Sanger sequencing. Bioinformatics analyses of the candidate variant were performed. RESULTS: The proband suffered from moderate hearing loss of the right ear, and her mother suffered from profound congenital bilateral hearing loss. The proband exhibited a left blue iris. The calculated W index of the proband was 2.61, while her mother's W index was 2.12. The proband and her mother were diagnosed with WS1 according to the Waardenburg Syndrome Consortium criteria. A novel missense variant NM_181457.3: c.127G > T; p.(Gly43Cys) in exon 2 in Paired Box 3 (PAX3) was identified in the proband and her mother, but this variant was not detected in the father and the controls. This variant was not reported in the HGMD, ClinVar, 1000G and ESP6500 databases. CONCLUSION: We identified a novel missense variant in exon 2 of PAX3 as the genetic cause of WS1 in this two-generation family, which broadened the genetic spectrum of WS1.
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Síndrome de Waardenburg , China , Feminino , Humanos , Mutação , Fator de Transcrição PAX3/genética , Linhagem , Síndrome de Waardenburg/genéticaRESUMO
Matrix metalloproteinase (MMP)9 is a key enzyme responsible for extracellular matrix degradation and contributes to the progressive histological changes observed in lower respiratory tract infections. Integrin ß1 and αtubulin are potential MMP9interacting proteins, and microRNA (miR)29b3p can regulate MMP9 expression. MMP9 is highly expressed in chronic rhinosinusitis with nasal polyps (CRSwNPs), regardless of its effects on miR29b3p, integrin ß1 and αtubulin expression. In the present study, samples from 100 patients with CRSwNPs were examined via reverse transcriptionquantitative PCR to assess the mRNA expression of miR29b3p, and western blotting was performed to assess the protein expression of MMP2, MMP9, acetylαtubulin, integrin ß1 and tissue inhibitor of metalloproteinase 1 (TIMP1). A dualluciferase reporter assay was used to verify the direct binding of miR29b3p and MMP2/MMP9. Coimmunoprecipitation (CoIP) and GST pulldown assays showed that integrin ß1 and αtubulin were MMP9interacting proteins. Cell viability, apoptosis and inflammatory cytokine levels were determined via a Cell Counting Kit8 assay, flow cytometry and ELISA, respectively. miR29b3p expression was found to be positively correlated with MMP2 and MMP9 expression. Whereas, TIMP1 expression was negatively correlated with MMP2 and MMP9 expression. The dualluciferase assay revealed that miR29b3p targeted the 3' untranslated region of MMP2/MMP9. The CoIP and GST pulldown assays showed that MMP9 could directly bind to integrin ß1 and indirectly bind to αtubulin. Finally, the overexpression of miR29b3p decreased the expression of MMP9 and increased the levels of acetylαtubulin. By contrast, the knockdown of miR29b3p increased the expression of MMP9 and decreased the levels of acetylαtubulin. Additionally, MMP9 expression was found to be negatively correlated with acetylαtubulin expression. Of note, the expression of integrin ß1 did not change following the overexpression and knockdown of MMP9. Finally, the overexpression of miR29b3p not only decreased MMP9 expression, but also alleviated lipopolysaccharideinduced inflammation in NP69 cells. The results showed that the downregulation of miR29b3p promoted αtubulin deacetylation by increasing the number of MMP9integrin ß1 complexes in CRSwNPs, thus targeting miR29b3p/MMP9 may be a potential novel strategy for the clinical treatment of CRSwNPs.
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Integrina beta1/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pólipos Nasais/etiologia , Sinusite/etiologia , Tubulina (Proteína)/metabolismo , Regiões 3' não Traduzidas/genética , Acetilação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Regulação para Baixo , Feminino , Humanos , Inflamação/induzido quimicamente , Integrina beta1/genética , Lipopolissacarídeos/efeitos adversos , Masculino , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Tubulina (Proteína)/genética , Adulto JovemRESUMO
Acute otitis media (AOM) is a common infectious disease in children that is accompanied by signs and symptoms of middle ear inflammation and infection. Previous studies have shown that the long non-coding (lnc)RNA nuclear-enriched abundant transcript 1(NEAT1) participates in various inflammatory conditions and plays an important regulatory role. The focus of the present study was the biological function of NEAT1 and underlying molecular mechanism in lipopolysaccharide (LPS)-induced human middle ear epithelial cells (HMEECs). The expression of NEAT1, miR-301b-3p and toll-like receptor 4 (TLR4) protein were determined by reverse transcription-quantitative PCR and western blot assays, respectively. Dual-luciferase reporter assay was performed to investigate the combination of miR-301b-3p and NEAT1 or TLR4. In addition, cell viability, apoptosis and the levels of pro-inflammatory factors (IL-1ß, TNF-α and IL-6) were measured by Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. Cell viability was significantly decreased, whereas apoptosis and inflammation were increased in LPS-stimulated HMEECs. Functional analyses demonstrated that NEAT1 was upregulated following LPS treatment, whereas knockdown of NEAT1 significantly increased cell viability and alleviated apoptosis and inflammation. Mechanistically, NEAT1 directly bound to and negatively regulated miR-301b-3p expression, whereas miR-301b-3p inhibitors abolished the inhibitory effect of NEAT1 knockdown on cell apoptosis and inflammation. As a target of miR-301b-3p, TLR4 was regulated by NEAT1 and miR-301b-3p. TLR4 overexpression alleviated NEAT1 silencing-induced inflammatory suppression. Rescue experiments demonstrated that NEAT1 promoted TLR4 expression by inhibiting miR-301b-3p. Collectively, the results of the present study suggested that NEAT1 may attenuate LPS-induced inflammation and apoptosis in HMEECs by modulating the miR-301b-3p/TLR4 axis, and may provide a new therapeutic target for the clinical treatment of AOM.
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Waardenburg syndrome (WS) is a congenital hereditary disease, attributed to the most common symptoms of sensorineural deafness and iris hypopigmentation. It is also known as the hearing-pigmentation deficient syndrome. Mutations on SOXl0 gene often lead to congenital deafness and has been shown to play an important role in the pathogenesis of WS. We investigated one family of five members, with four patients exhibiting the classic form of WS2, whose DNA samples were analyzed by the technique of Whole-exome sequencing (WES). From analysis of WES data, we found that both the mother and all three children in the family have a heterozygous mutation on the Sex Determining Region Y - Box 10 (SOX10) gene. The mutation was c.298_300delinsGG in exon 2 of SOX10 (NM_006941), which leads to a frameshift of nine nucleotides, hence the amino acids (p. S100Rfs*9) are altered and the protein translation may be terminated prematurely. Further flow cytometry confirmed significant down-regulation of SOX10 protein, which indicated the SOX10 gene mutation was responsible for the pathogenesis of WS2 patients. In addition, we speculated that some other mutated genes might be related to disease phenotype in this family, which might also participate in promoting the progression of WS2.
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Mutação da Fase de Leitura , Fatores de Transcrição SOXE/genética , Síndrome de Waardenburg/genética , Povo Asiático/genética , China , Análise Mutacional de DNA , Progressão da Doença , Heterogeneidade Genética , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Linhagem , Fenótipo , Fatores de Transcrição SOXE/sangue , Síndrome de Waardenburg/sangue , Síndrome de Waardenburg/diagnóstico , Síndrome de Waardenburg/etnologia , Sequenciamento do ExomaRESUMO
We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.
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Engenharia de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Subtilisina/metabolismo , Células HEK293 , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Especificidade por Substrato , Subtilisina/genéticaRESUMO
Aims: To determine the clinical characteristics and genetic cause of Waardenburg syndrome type 1 (WS1) in a Chinese family. Materials and Methods: Evaluations, including history, clinical features, and audiological tests, were performed on the proband and her parents. Genetic analyses were performed targeting 144 known deafness genes using a next-generation sequencing panel. Bioinformatic analyses were used to analyze the candidate mutation. Results: The proband and her parents suffered from congenital bilateral profound hearing loss. Her mother exhibited bilateral blue irides. WS1 was diagnosed in the proband and her mother according to the Waardenburg syndrome consortium criteria: the calculated W index of the proband was 2.39 and that of her mother was 2.31. A novel mutation c.1076_1077del (p.Thr359fs) in exon 7 of the PAX3 gene (paired box 3) was identified in the proband and her mother that was absent in the father and controls. Conclusion: Mutations in exon 7 of the PAX3 gene are rare. We identified a novel frameshift mutation in exon 7 of the PAX3 gene that we determined was responsible for WS1 in this family.
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Fator de Transcrição PAX3/genética , Síndrome de Waardenburg/genética , Povo Asiático/genética , China , Éxons/genética , Família , Feminino , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Mutação , Fator de Transcrição PAX3/metabolismo , Linhagem , Síndrome de Waardenburg/metabolismoRESUMO
An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme's activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression-purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 A resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin's substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.
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Azidas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Subtilisinas/química , Subtilisinas/metabolismo , Ânions/química , Ânions/metabolismo , Azidas/química , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Fluoretos/química , Fluoretos/metabolismo , Cinética , Modelos Biológicos , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Subtilisinas/genéticaRESUMO
BACKGROUND: To analyze the clinical phenotypes and genetic variants of a Chinese family with Waardenburg syndrome (WS) and to explore the possible molecular pathogenesis of WS. METHODS: The clinical data from a patient and his family were collected. The genomic DNA of the patient and his family was purified from their peripheral blood. All exons and flanking sequences of the MITF, PAX3, SOX10, SNAI2, END3, and EDNRB genes were investigated through high-throughput sequencing. Based on the results of high-throughput sequencing, genetic variants in the patient and his family were verified and analyzed by Sanger sequencing. RESULTS: The patient was diagnosed with typical WS1 that manifested in hearing impairment, inner canthus ectopia and heterochromic iris. Sanger sequencing revealed the pathogenic heterozygous c.420-424de1CGCGGinsTTAC mutation in the PAX3 gene in the proband, which is a frameshift mutation that changed the amino acid sequence of the PAX3 protein from AVCDRNTVPSV to YSVIETPCRQ* (* refers to a stop codon) from amino acids 141-151. The stop codon induced by this mutation resulted in the truncation of the PAX3 protein. The same mutation sites were also found in the mother and younger sister of the proband. No previous report of this mutation was found in the Human Gene Mutation Database. CONCLUSION: The novel heterozygous c.420-424de1CGCGGinsTTAC mutation is the molecular pathological cause for WS1 in our patient. The clinical and genetic characterization of this family with WS1 elucidated the genetic heterogeneity of PAX3 in WS1. Moreover, the mutation detected in this case has expanded the database of PAX3 mutations.
Assuntos
Deleção de Genes , Fator de Transcrição PAX3/genética , Síndrome de Waardenburg/genética , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Síndrome de Waardenburg/patologiaRESUMO
Waardenburg syndrome type 2 (WS2) is a rare genetic disorder, characterized by bright blue eyes, moderate to profound hearing loss and pigmental abnormalities of the hair and skin. Between 10 and 20 mutations in the SRYbox 10 (SOX10) gene were previously identified to be associated with WS2. The present study aimed to identify the genetic causes of WS2 in a Chinese family. Clinical and molecular analyses were performed to genetically characterize a Chinese family with two cases of WS2. The clinical data of the proband were collected using a questionnaire. The genomic DNA was extracted from peripheral blood samples of each individual in the family, and 168 candidate genes associated with hearing loss were sequenced using the Illumina HiSeq 2000 and confirmed by Sanger sequencing. A heterozygous nonsense mutation [substitution; position 127; cytosine to thymine (c.127C>T)] was identified in exon 2 of SOX10 (transcript ID: NM_006941.3) in the proband and the mother; however, not in other family members or healthy controls. The novel nonsense heterozygous mutation may cause the replacement of codon 43 [arginine (Arg)] with a stop codon (Arg43stop), leading to premature termination of protein translation. The novel nonsense heterozygous mutation c.127C>T in the SOX10 gene was considered to be the cause of WS2 in the family. This mutation has not been identified in any databases, to the best of the authors' knowledge, including The Single Nucleotide Polymorphism Database, The Human Gene Mutation Database, 1000 Genomes Project and ClinVar and Exome Sequencing Project v. 6500.