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1.
Life Sci ; 264: 118662, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33127517

RESUMEN

The core machinery for vesicular membrane trafficking broadly comprises of coat proteins, RABs, tethering complexes and SNAREs. As cellular membrane traffic modulates key processes of mitogenic signaling, cell migration, cell death and autophagy, its dysregulation could potentially results in increased cell proliferation and survival, or enhanced migration and invasion. Changes in the levels of some components of the core machinery of vesicular membrane trafficking, likely due to gene amplifications and/or alterations in epigenetic factors (such as DNA methylation and micro RNA) have been extensively associated with human cancers. Here, we provide an overview of association of membrane trafficking with cancer, with a focus on mutations and variants of coat proteins, RABs, tethering complex components and SNAREs that have been uncovered in human cancer cells/tissues. The major cellular and molecular cancer-driving or suppression mechanisms associated with these components of the core membrane trafficking machinery shall be discussed.


Asunto(s)
Membrana Celular/metabolismo , Mutación/genética , Neoplasias/genética , Exocitosis , Humanos , Modelos Biológicos , Transporte de Proteínas
2.
Life Sci ; 264: 118655, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33141042

RESUMEN

AIM: Large tumor suppressor 1 (LATS1) is a Ser/Thr kinase to mediate Hippo signaling pathway and plays a pivotal role in tumor suppression. By searching the COSMIC database, we found a somatic missense mutation (NM_004690.4:c.2552C>T) of human LATS1 (NP_004681.1:p.851T>I) in two colorectal cancer cell lines, and investigated the role and underlying mechanism of this mutation in the colorectal tumorigenesis. MAIN METHODS: We performed structural and biochemistry analyses to investigate the role of LATS1 T851I mutation in Hippo signaling activation and used the mouse xenograft model to assess the role of this mutation in the colorectal tumorigenesis. KEY FINDINGS: By structural and biochemistry approaches, we propose that T851 is an active residue other than Ser909 on the activation loop and is essential for LATS1 phosphorylation and kinase activity. We then reveal that T851I mutation in LATS1 not only destabilizes the phospho-Thr1079-LATS1, a prerequisite of LATS1 kinase activity, but also reduces its binding to the downstream effectors, YAP and TAZ. As a result, T851I mutation in LATS1 attenuates Hippo signaling and decreases its tumor-suppressor functions in the colorectal cancer. SIGNIFICANCE: The present study identifies the T851 as an essential residue for LATS1 kinase activity and uncovers the T851I mutation of LATS1 and consequent Hippo signaling suppression as a hitherto uncharacterized mechanism controlling colorectal tumorigenesis.


Asunto(s)
Mutación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Masculino , Ratones Desnudos , Modelos Moleculares , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Gene ; 765: 145045, 2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-32777524

RESUMEN

To find the variant spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and evaluate its frequent variants in Chinese congenital absence of vas deferens (CAVD) patients. A total of 276 patients with azoospermia and CAVD (aged from 21 to 44 years old) were investigated from May 2013 to September 2019 in the Third Affiliated Hospital of Sun Yat-sen University. Additionally, 50 healthy, unrelated volunteers were recruited as controls (aged from 21 to 46 years old). The 5'-UTR, exons and their flanking side of the CFTR gene were sequenced by high-throughput sequencing technology. The results were compared with those retrieved from the Ensembl Genome Browser. In addition, all 13 novel variants were further confirmed independently by Sanger sequencing and evaluated in the bioinformatics web servers. A schematic of the variant spectrum of the CFTR gene, including 13 novel variants (12 in CAVD patients, one in the control group), is shown, and the frequent variants in Chinese CAVD patients were 5 T (27.54%), c.-8G > C (7.25%), p.Q1352H (5.98%), and p.I556V (3.08%). 5 T was found to be the most frequent variant. p.Q1352H had a significantly high allelic frequency in CAVD patients (P < 0.05). c.-8G > C and p.I556V had high allelic frequencies but showed no difference between patients and controls (P > 0.05). p.Q1352H is the most common and important missense variant in Chinese patients with CAVD, while the pathological effects of C.-8G > C and p.I556V may be weak after evaluation.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Enfermedades Urogenitales Masculinas/genética , Conducto Deferente/anomalías , Adulto , Alelos , Grupo de Ascendencia Continental Asiática/genética , Azoospermia/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Análisis Mutacional de ADN/métodos , Exones/genética , Frecuencia de los Genes/genética , Humanos , Infertilidad Masculina/genética , Masculino , Enfermedades Urogenitales Masculinas/metabolismo , Mutación/genética , Conducto Deferente/metabolismo
4.
J Vis Exp ; (165)2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-33283789

RESUMEN

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Embrión no Mamífero/metabolismo , Microinyecciones , Mutagénesis/genética , Phlebotomus/embriología , Animales , Femenino , Masculino , Ratones , Microtecnología , Mutación/genética , Agujas , Phlebotomus/genética , Phlebotomus/inmunología , Phlebotomus/parasitología
5.
Nat Commun ; 11(1): 6331, 2020 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-33303757

RESUMEN

In modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.


Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrocarburos Aromáticos/metabolismo , Proteínas Bacterianas/química , Benceno/metabolismo , Sitios de Unión , Biodegradación Ambiental , Transporte Biológico , Difusión , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Mutación/genética
6.
Nat Commun ; 11(1): 6402, 2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33328475

RESUMEN

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and  alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2.


Asunto(s)
/inmunología , /prevención & control , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Peso Corporal , Línea Celular , Cricetinae , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Genoma Viral , Pulmón/patología , Pulmón/virología , Ratones Endogámicos C57BL , Mutación/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Vacunación , Carga Viral
7.
Sci Rep ; 10(1): 22214, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33335187

RESUMEN

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.


Asunto(s)
/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /genética , Australia , Genes Virales/genética , Humanos , Mutación/genética , ARN Viral/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(12): 1360-1363, 2020 Dec 10.
Artículo en Chino | MEDLINE | ID: mdl-33306822

RESUMEN

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with N-acetylglutamate synthase deficiency. METHODS: Trio whole exome sequencing (WES) was carried out for the pedigree. Pathogenicity of the identified variant was predicted based on the latest recommendation of the American College of Medical Genetics and Genomics (ACMG). Prenatal diagnosis was provided for subsequent pregnancy through Sanger sequencing. RESULTS: Trio WES showed that the proband has carried compound heterozygous c.68delG and c.796G>C variants of NAGS gene, for which the mother and father were respectively heterozygous carriers. Neither variant was reported previously. Based on the ACMG guidelines, the c.68delG variant was classified as "likely pathogenic" (PVS1+PM2), while the c.796G>C variant was classified as with "uncertain significance" (PM2+BP4). Sanger sequencing validated the above findings, and only detected the heterozygous c.796G>C variant in the amniotic fluid sample. The fetus was followed up till 6 month after birth with no obvious abnormality. CONCLUSION: The compound heterozygous c.68delG and c.796G>C variants of the NAGS gene probably underlay the disorder in this pedigree, and the resulth asenabled genetic counseling and prenatal diagnosis for this pedigree.


Asunto(s)
Pruebas Genéticas , Diagnóstico Prenatal , Trastornos Innatos del Ciclo de la Urea , N-Acetiltransferasa de Aminoácidos/genética , China , Femenino , Humanos , Masculino , Mutación/genética , Linaje , Embarazo , Trastornos Innatos del Ciclo de la Urea/diagnóstico , Trastornos Innatos del Ciclo de la Urea/genética , Secuenciación del Exoma Completo
9.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(12): 1368-1370, 2020 Dec 10.
Artículo en Chino | MEDLINE | ID: mdl-33306824

RESUMEN

OBJECTIVE: To detect pathological variants of the SLC12A3 gene in a Chinese pedigree affected with Gitelman syndrome (GS). METHODS: Clinical data and peripheral blood samples of the proband and his family members were collected. All exons of the SLC12A3 gene were amplified by PCR and subjected to Sanger sequencing. RESULTS: Sanger sequencing has revealed that the proband has carried a c.486_489 delTACG (p.Ile162Met fs*8) deletion and a heterozygous c.2890C>T (p.Arg964Trp) missense variant in the SLC12A3 gene. Neither variant was reported previously and was not found among healthy controls. CONCLUSION: The c.486_489delTACG (p.Ile162Met fs*8) and c.2890C>T (p.Arg964Trp) variants of the SLC12A3 gene probably underlay the GS in the proband. Above discovery has enriched the variant spectrum of GS.


Asunto(s)
Síndrome de Gitelman , China , Síndrome de Gitelman/genética , Heterocigoto , Humanos , Mutación/genética , Linaje , Miembro 3 de la Familia de Transportadores de Soluto 12/genética
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(12): 1395-1398, 2020 Dec 10.
Artículo en Chino | MEDLINE | ID: mdl-33306831

RESUMEN

OBJECTIVE: To trace a rare case of chronic myeloid leukemia (CML) with a four-way Philadelphia chromosome variant by cytogenetic analysis in order to provide a basis for the selection of treatment. METHODS: Bone marrow morphology, chromosomal karyotyping, fluorescence in situ hybridization (FISH) and real-time quantitative PCR (RQ-PCR) were used for the diagnosis and staging of the disease. Point mutations in the tyrosine kinase domain of ABL1 gene were detected by Sanger sequencing. RESULTS: The patient was initially diagnosed as CML in chronic phase (CML-CP) with a chromosomal karyotype of 46,XX,t(5;9;22;6)(q13;q34;q11;q25), while FISH revealed presence of a variant Philadelphia chromosome translocation. Clonal evolution has occurred after 38 months of tyrosine kinase inhibitor (TKI) treatment, when cytogenetic analysis revealed coexisting t(5;9;22;6)(q13;q34;q11;q25) and t(5;9;22;6;17)(q13;q34;q11;q25;q11). After 57 months of TKIs treatment, only the t(5;9;22;6;17) clone was detected. Three months later, hyperdiploidy with additional abnormalities were detected in addition to t(5;9;22;6;17). Three mutations, including p.Tyr253Phe, p.Thr315Ile and p.Gly250Glu, were identified in the tyrosine kinase domain of the ABL1 gene during the course of disease. The patient did not attain cytogenetic and molecular response to TKIs. CONCLUSION: The four-way variant translocation may be genetically unstable. Clonal evolution and genetic mutations are likely to occur during TKIs treatment, resulting in poor response to drug therapy. This observation, however, needs to be confirmed by large-scale studies.


Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Cromosoma Filadelfia , Translocación Genética , Inhibidores Enzimáticos/uso terapéutico , Evolución Molecular , Femenino , Humanos , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación/genética
11.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(12): 1403-1406, 2020 Dec 10.
Artículo en Chino | MEDLINE | ID: mdl-33306833

RESUMEN

OBJECTIVE: To explore the pathogenesis for a SRY-negative male with 46,XX disorder of sex development (DSD). METHODS: Peripheral blood samples of the patient and his family members were subjected to chromosomal karyotyping, routine PCR, real-time fluorescence quantitative PCR, whole exome sequencing and whole genome sequencing. The data was analyzed with NextGENe software. RESULTS: Both the proband and his brother presented a 46,XX karyotype with negative SRY gene, while their father presented normal phenotype and karyotype with positive SRY gene. No pathogenic variant associated with sex development was detected by whole exome sequencing, while a 243 kb duplication was detected by whole genome sequencing in the 5' upstream region of the SOX9 gene in the proband, his brother and father. The same duplication was not found in his sister and mother. CONCLUSION: The 243 kb duplication at the 5' upstream of the SOX9 gene may predispose to the 46,XX DSD in this family. It is speculated that there exist an unknown core regulatory element in the upstream of the SOX9, and its duplication may trigger expression of SOX9 and initiate testicular differentiation in the absence of SRY gene.


Asunto(s)
Trastornos del Desarrollo Sexual , Proteína de la Región Y Determinante del Sexo , Trastornos del Desarrollo Sexual/genética , Femenino , Humanos , Masculino , Mutación/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteína de la Región Y Determinante del Sexo/genética , Testículo , Secuenciación del Exoma Completo
12.
Yakugaku Zasshi ; 140(12): 1433-1439, 2020.
Artículo en Japonés | MEDLINE | ID: mdl-33268685

RESUMEN

DNA preserves and inherits genetic information. 7,8-dihydro-8-oxoguanine (GO) and abasic sites are some of the most common DNA lesions generated endogenously in living organisms and they induce mutations. The resultant mutations in our DNA cause diseases such as cancers. GO and abasic sites are known to induce mutations at the positions of the lesions. We revealed GO induced mutations at points distant from a lesion besides mutations at the lesion site in human cells when WRN helicase or DNA polymerase λ was knocked down. In addition, an abasic site analog, tetrahydrofuran, also induced the same type of mutations and large deletions. Thus, these endogenous DNA damages could induce more diverse mutations than previously thought. Recently, much research toward the development of gene therapy approaches has been carried out to apply gene therapy in a clinical setting. In this study, we found that the usual plasmid DNA with suitable transcription regulatory sequences achieved durably expressed transgenes in mouse liver. In addition, we successfully improved gene-correction efficiency with tailed duplex DNA fragments by introducing a second mismatch. These results give us important information to apply a transgene expression approach and tailed duplexes in a clinical setting.


Asunto(s)
ADN/genética , Terapia Genética/métodos , Mutagénesis/genética , Mutación/genética , Animales , Daño del ADN/genética , Fragmentación del ADN , ADN Polimerasa beta , Furanos , Expresión Génica , Guanina/análogos & derivados , Humanos , Ratones , Transgenes
13.
PLoS Comput Biol ; 16(12): e1008449, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33270653

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 31 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic and confirmed cases of cross-species transmission, the question of the extent to which this transmission is possible emerges, as well as what molecular features distinguish susceptible from non-susceptible animal species. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also allow us to predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.


Asunto(s)
Interacciones Huésped-Patógeno/genética , /química , /metabolismo , Animales , Sitios de Unión/genética , /transmisión , /virología , Biología Computacional , Secuencia Conservada/genética , Predisposición Genética a la Enfermedad , Humanos , Simulación de Dinámica Molecular , Mutación/genética , /metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
14.
Anticancer Res ; 40(12): 6781-6789, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33288571

RESUMEN

BACKGROUND/AIM: To examine the dynamics of circulating tumour cells (CTCs) in pancreatic cancer (PC), new mouse CTC models from human PC xenografts were developed. MATERIALS AND METHODS: Orthotopic (pancreas) and heterotopic (subcutaneous) transplantation models using GFP-tagged SUIT-2 PC cells were prepared. Using a cytology-based CTC detection platform, CTCs and metastasis were compared. RESULTS: The two types of orthotopic models, including the surgical transplantation model and the intraperitoneal injection model, showed a similar pattern of initial pancreatic tumour formation and subsequent development of peritoneal and hematogenous lung metastases. In the heterotopic model, only hematogenous lung metastasis was observed, and the number of CTCs and lung metastases was higher than that of the orthotopic model. Furthermore, KRAS mutation (G12D) was detected in CTCs. CONCLUSION: These orthotopic and heterotopic models clearly differ in terms of the pattern of metastasis and CTCs and therefore, would be useful PC models to investigate the effect of drug-therapy on CTCs and the role of KRAS mutation.


Asunto(s)
Citodiagnóstico , Mutación/genética , Células Neoplásicas Circulantes/patología , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Línea Celular Tumoral , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Sci Transl Med ; 12(573)2020 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-33229462

RESUMEN

Superspreading events shaped the coronavirus disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here, we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria's well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of low-frequency mutations that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2 and to gain fundamental insights into mutational dynamics and transmission properties.


Asunto(s)
/epidemiología , Mutación/genética , /genética , Austria/epidemiología , Secuencia de Bases , /virología , Interacciones Huésped-Patógeno/genética , Humanos , Tasa de Mutación , Filogenia
16.
Nat Metab ; 2(11): 1305-1315, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33139959

RESUMEN

A long-standing model holds that stochastic aberrations of transcriptional regulation play a key role in the process of ageing. While transcriptional dysregulation is observed in many cell types in the form of increased cell-to-cell variability, its generality to all cell types remains doubted. Here, we propose a new approach for analysing transcriptional regulation in single-cell RNA sequencing data by focusing on the global coordination between the genes rather than the variability of individual genes or correlations between pairs of genes. Consistently, across very different organisms and cell types, we find a decrease in the gene-to-gene transcriptional coordination in ageing cells. In addition, we find that loss of gene-to-gene transcriptional coordination is associated with high mutational load of a specific, age-related signature and with radiation-induced DNA damage. These observations suggest a general, potentially universal, stochastic attribute of transcriptional dysregulation in ageing.


Asunto(s)
Envejecimiento/genética , Transcripción Genética/genética , Animales , Daño del ADN , Drosophila , Redes Reguladoras de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Mutación/genética , Análisis de Secuencia de ARN , Procesos Estocásticos , Transcripción Genética/efectos de la radiación
17.
J Ayub Med Coll Abbottabad ; 32(4): 523-526, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33225656

RESUMEN

BACKGROUND: Chronic lymphocytic leukaemia (CLL), an indolent but malignant lymphoproliferative disorder, is characterized by unregulated and uninhibited growth of mature monoclonal lymphocytes, with deletion of 17p containing TP53 gene being the most important prognostic factor. TP53 mutations, reported in 10% of CLL cases, seem to have a direct correlation to a more advanced stage and aggressive transformation of CLL. METHODS: This was a retrospective cross-sectional descriptive study limited to a period from 1st June 2013 to 30th June 2016, conducted at Section of haematology, Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi. One thirty-nine cases of CLL received for TP53 mutation analysis at the Aga Khan University hospital clinical Laboratory were included in the study. Five ml of whole blood or one ml of bone marrow aspirate sample in EDTA tube was collected for the detection of TP53 mutation by the FISH technique. Statistical package for social sciences 21 was used for data entry and analysis. RESULTS: Of the 139 chronic lymphocytic leukaemia patients, 43 (31%) were females and 96 (69%) were males. The mean age of all patients was 56.3±10.84 years. Tp53 gene mutation in patients with chronic lymphocytic leukaemia was found only in 19(13.7%) patients. Among these patients 15 (10.9%) were male and 04(2.9%) were females. Age and gender were not statistically significant with Tp53 mutation with a p-value > 0.05 at a 95% confidence interval. CONCLUSIONS: In a cohort of Pakistani patients with Chronic lymphocytic leukaemia, TP53 gene mutation was found in 19 (13.7%).


Asunto(s)
Leucemia Linfocítica Crónica de Células B/epidemiología , Leucemia Linfocítica Crónica de Células B/genética , Mutación/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pakistán , Estudios Retrospectivos
18.
PLoS One ; 15(11): e0242812, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33253281

RESUMEN

Path testing is the basic approach of white box testing and the main approach to solve it by discovering the particular input data of the searching space to encompass the paths in the software under test. Due to the increasing software complexity, exhaustive testing is impossible and computationally not feasible. The ultimate challenge is to generate suitable test data that maximize the coverage; many approaches have been developed by researchers to accomplish path coverage. The paper suggested a hybrid method (NSA-GA) based on Negative Selection Algorithm (NSA) and Genetic Algorithm (GA) to generate an optimal test data avoiding replication to cover all possible paths. The proposed method modifies the generation of detectors in the generation phase of NSA using GA, as well as, develops a fitness function based on the paths' prioritization. Different benchmark programs with different data types have been used. The results show that the hybrid method improved the coverage percentage of the programs' paths, even for complicated paths and its ability to minimize the generated number of test data and enhance the efficiency even with the increased input range of different data types used. This method improves the effectiveness and efficiency of test data generation and maximizes search space area, increasing percentage of path coverage while preventing redundant data.


Asunto(s)
Automatización , Control de Calidad , Programas Informáticos , Algoritmos , Humanos , Mutación/genética , Selección Genética/genética
19.
Nat Med ; 26(11): 1788-1800, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33188278

RESUMEN

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.


Asunto(s)
Cardiomiopatía Dilatada/genética , Miocardio/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Alelos , Animales , Cardiomiopatía Dilatada/fisiopatología , Reprogramación Celular , Modelos Animales de Enfermedad , Femenino , Edición Génica , Humanos , Masculino , Mutación/genética , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Mensajero/genética , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Vesículas Secretoras/genética , Vesículas Secretoras/metabolismo , Porcinos
20.
PLoS One ; 15(11): e0242806, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33237975

RESUMEN

PURPOSE: To investigate whether a computer-aided diagnosis (CAD) program developed using the deep learning convolutional neural network (CNN) on neck US images can predict the BRAFV600E mutation in thyroid cancer. METHODS: 469 thyroid cancers in 469 patients were included in this retrospective study. A CAD program recently developed using the deep CNN provided risks of malignancy (0-100%) as well as binary results (cancer or not). Using the CAD program, we calculated the risk of malignancy based on a US image of each thyroid nodule (CAD value). Univariate and multivariate logistic regression analyses were performed including patient demographics, the American College of Radiology (ACR) Thyroid Imaging, Reporting and Data System (TIRADS) categories and risks of malignancy calculated through CAD to identify independent predictive factors for the BRAFV600E mutation in thyroid cancer. The predictive power of the CAD value and final multivariable model for the BRAFV600E mutation in thyroid cancer were measured using the area under the receiver operating characteristic (ROC) curves. RESULTS: In this study, 380 (81%) patients were positive and 89 (19%) patients were negative for the BRAFV600E mutation. On multivariate analysis, older age (OR = 1.025, p = 0.018), smaller size (OR = 0.963, p = 0.006), and higher CAD value (OR = 1.016, p = 0.004) were significantly associated with the BRAFV600E mutation. The CAD value yielded an AUC of 0.646 (95% CI: 0.576, 0.716) for predicting the BRAFV600E mutation, while the multivariable model yielded an AUC of 0.706 (95% CI: 0.576, 0.716). The multivariable model showed significantly better performance than the CAD value alone (p = 0.004). CONCLUSION: Deep learning-based CAD for thyroid US can help us predict the BRAFV600E mutation in thyroid cancer. More multi-center studies with more cases are needed to further validate our study results.


Asunto(s)
Inteligencia Artificial , Carcinoma Papilar/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Adulto , Anciano , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/epidemiología , Carcinoma Papilar/patología , Diagnóstico por Computador , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Glándula Tiroides/diagnóstico por imagen , Glándula Tiroides/patología , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/epidemiología , Neoplasias de la Tiroides/patología , Nódulo Tiroideo , Tomografía Computarizada por Rayos X
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