RESUMEN
A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.
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COVID-19/inmunología , Megacariocitos/inmunología , Monocitos/inmunología , ARN Viral , SARS-CoV-2/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , China , Estudios de Cohortes , Citocinas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Análisis de la Célula Individual , Transcriptoma/inmunología , Adulto JovenRESUMEN
In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation1-3. However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4. Chimeric knockout of LKB1 kinase-an activator of AMPK-related protein kinase SIK35-7-in adult mouse brain markedly reduces the amount and delta power-a measure of sleep depth-of non-rapid eye movement sleep (NREMS). Downstream of the LKB1-SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increased sleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research.
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Transducción de Señal , Duración del Sueño , Transcripción Genética , Animales , Ratones , Regulación de la Expresión Génica , Fosforilación , Transducción de Señal/fisiología , Sueño de Onda Lenta/genética , Perfilación de la Expresión GénicaRESUMEN
CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.
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Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Sistemas CRISPR-Cas/fisiología , Proteínas Virales/metabolismo , ADP-Ribosilación/fisiología , Proteínas Bacterianas/genética , Bacteriófagos/genética , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Microscopía por Crioelectrón/métodos , ADN/metabolismo , Modelos Moleculares , ARN Bacteriano/metabolismo , Proteínas Virales/genéticaRESUMEN
The CRISPR-Cas system is a highly adaptive and RNA-guided immune system found in bacteria and archaea, which has applications as a genome editing tool and is a valuable system for studying the co-evolutionary dynamics of bacteriophage interactions. Here introduces CRISPRimmunity, a new web server designed for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of key CRISPR-associated molecular events. CRISPRimmunity is built on a suite of CRISPR-oriented databases providing a comprehensive co-evolutionary perspective of the CRISPR-Cas and anti-CRISPR systems. The platform achieved a high prediction accuracy of 0.997 for Acr prediction when tested on a dataset of 99 experimentally validated Acrs and 676 non-Acrs, outperforming other existing prediction tools. Some of the newly identified class 2 CRISPR-Cas loci using CRISPRimmunity have been experimentally validated for cleavage activity in vitro. CRISPRimmunity offers the catalogues of pre-identified CRISPR systems to browse and query, the collected resources or databases to download, a well-designed graphical interface, a detailed tutorial, multi-faceted information, and exportable results in machine-readable formats, making it easy to use and facilitating future experimental design and further data mining. The platform is available at http://www.microbiome-bigdata.com/CRISPRimmunity. Moreover, the source code for batch analysis are published on Github (https://github.com/HIT-ImmunologyLab/CRISPRimmunity).
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Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Bacterias/genética , Archaea/genética , ComputadoresRESUMEN
Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1ß, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.
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Enfermedades de los Peces , Animales , Línea Celular , Enfermedades de los Peces/inmunología , Aletas de Animales/inmunología , Poli I-C/farmacología , Inmunidad Innata , Perciformes/inmunología , Perciformes/genética , Peces/inmunologíaRESUMEN
The dedicator of cytokinesis 2(DOCK2) protein, an atypical guanine nucleotide exchange factor (GEFs), is a member of the DOCKA protein subfamily. DOCK2 protein deficiency is characterized by early-onset lymphopenia, recurrent infections, and lymphocyte dysfunction, which was classified as combined immune deficiency with neutrophil abnormalities as well. The only cure is hematopoietic stem cell transplantation. Here, we report two patients harboring four novel DOCK2 mutations associated with recurrent infections including live attenuated vaccine-related infections. The patient's condition was partially alleviated by symptomatic treatment or intravenous immunoglobulin. We also confirmed defects in thymic T cell output and T cell proliferation, as well as aberrant skewing of T/B cell subset TCR-Vß repertoires. In addition, we noted neutrophil defects, the weakening of actin polymerization, and BCR internalization under TCR/BCR activation. Finally, we found that the DOCK2 protein affected antibody affinity although with normal total serum immunoglobulin. The results reported herein expand the clinical phenotype, the pathogenic DOCK2 mutation database, and the immune characteristics of DOCK2-deficient patients.
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Proteínas Activadoras de GTPasa , Síndromes de Inmunodeficiencia , Humanos , Vacunas Atenuadas , Proteínas Activadoras de GTPasa/genética , Reinfección , Factores de Intercambio de Guanina Nucleótido/genética , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Mutación , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Aromatic ring fusion on BODIPY core can effectively tune its electronic property, and red-shift its absorption and emission wavelength. In this work, we report that a one-pot Pd(II) catalyzed multiple C-H activation to access acenaphtho[b]-fused BODIPYs though the reaction of α,ß-unsubstituted-BODIPYs and 1,8-dibromonaphthalenes. These newly synthesized acenaphtho[b]-fused BODIPYs revealed intensified deep red absorptions (639-669â nm) and emissions (643-683â nm), with high fluorescence quantum yields (0.53-0.84) in dichloromethane. Notably, these acenaphtho[b]-fused BODIPYs exhibited well-defined self-aggregation behavior in water/THF mixture, and for instance, the absorption of 3 a was red-shifted by 53â nm to 693â nm after forming aggregates.
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Colorantes Fluorescentes , Paladio , Paladio/química , Colorantes Fluorescentes/química , Compuestos de Boro/química , CatálisisRESUMEN
The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters' activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano.
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Peces , Inmunidad Innata , Animales , Inmunidad Innata/genética , Proteínas de Peces/química , Interferones , AntiviralesRESUMEN
Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic-pituitary-gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.
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Evolución Molecular , Proteína Forkhead Box L2/genética , Duplicación de Gen , Carpa Dorada/genética , Poliploidía , Animales , Femenino , Proteína Forkhead Box L2/metabolismo , Carpa Dorada/crecimiento & desarrollo , Carpa Dorada/metabolismo , Masculino , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismoRESUMEN
Tongue squamous cell carcinoma (TSCC) is one of the most common cancers in the oral cavity. Notch signaling is frequently dysregulated in cancer. However, the role of Notch2 in TSCC is not well understood. The aim of this study was to investigate the effect of abnormal expression of Notch2 in TSCC. The expression of Notch2 was tested in 47 pairs of tissues from tongue cancer and normal samples by using immunohistochemical staining. Tongue cancer cells were transfected with siRNA or plasmid. The proliferation of the cells was tested by the CCK8 assay and colony formation assay. Subcutaneous tumor model was established to observe tumor growth. Transwell assay was used to detect the changes of cell migration and invasion ability. A humanized anti-Notch2 antibody was used to TSCC cells. We found that Notch2 was upregulated in tongue carcinoma tissues. Knocking down the expression of Notch2 by siRNA in the TSCC cell lines decreased proliferation ability both in vitro and in vivo. In addition, migration and invasion abilities were inhibited by knockdown of Notch2 in the TSCC cells. However, overexpression of Notch2 increased tongue cancer cell proliferation, invasion and migration. The humanized anti-Notch2 antibody inhibited TSCC cell growth. The results indicated that Notch2 is an oncogene in tongue squamous cell carcinoma and may become the target of a new approach for treating TSCC.
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Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Receptor Notch2/genética , Neoplasias de la Lengua/genética , Animales , Carcinoma de Células Escamosas/patología , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Lengua/patología , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Matrix metalloproteinase 7 (MMP7), as the smallest member of the matrix metalloproteinase family, has been verified to be implicated in cancer progression, especially metastasis. However, its expression pattern and function in tongue cancer is not clear. METHODS: The expression of MMP7 in human tongue squamous cell carcinoma (TSCC) specimens compared with their respective paired nontumour tissues by real-time PCR and immunohistochemical staining. The effect of MMP7 on the proliferation, apoptosis, migration, invasion of tongue cancer cells was tested in appropriate ways after MMP7 siRNA knockdown or overexpression. The effect of MMP7 on lymph node metastasis in vivo was analyzed using a high-metastasis orthotopic nude mouse tongue transplanted tumour model. RESULTS: We found markedly elevated expression of MMP7 in human TSCC specimens compared with their respective paired nontumour tissues, and this high expression was correlated with the patients' lymph node metastasis. Furthermore, the results of molecular functional assays confirmed that MMP7 promotes cell proliferation, migration and invasion of TSCC cells. Knockdown of MMP7 inhibited lymph nodes metastasis in vivo. CONCLUSIONS: MMP7 plays an oncogenic role in carcinogenesis and metastasis of tongue cancer, and may serve as a potential therapeutic target for tongue cancer.
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Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 7 de la Matriz/genética , Neoplasias de la Lengua/genética , Adulto , Anciano , Animales , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 7 de la Matriz/metabolismo , Ratones , Persona de Mediana Edad , Neoplasias de la Lengua/metabolismo , Neoplasias de la Lengua/mortalidad , Neoplasias de la Lengua/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.
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Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Fluorescentes Verdes/biosíntesis , Vibrio/química , Sistema Libre de Células , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Plásmidos , Vibrio/metabolismoRESUMEN
Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.
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Escherichia coli/genética , Ingeniería Genética , Saccharomyces cerevisiae/genética , Streptomyces/genética , Aminoglicósidos/biosíntesis , Aminoglicósidos/química , Carbazoles/química , Carbazoles/metabolismo , Biología Computacional , Monoterpenos Ciclohexánicos , Enediinos/química , Escherichia coli/metabolismo , Alcoholes Grasos/química , Alcoholes Grasos/metabolismo , Furanos/química , Furanos/metabolismo , Lactonas/química , Lactonas/metabolismo , Estructura Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Péptidos/química , Presión , Nucleósidos de Pirimidina/biosíntesis , Nucleósidos de Pirimidina/química , Pirrolnitrina/biosíntesis , Pirrolnitrina/química , Saccharomyces cerevisiae/metabolismo , Streptomyces/metabolismo , Tiazoles/química , Tiazoles/metabolismo , Factores de Tiempo , Vincristina/biosíntesis , Vincristina/químicaRESUMEN
BACKGROUND: Our previous study demonstrated a close relationship between NOTCH signaling pathway and salivary adenoid cystic carcinoma (SACC). HES1 is a well-known target gene of NOTCH signaling pathway. The purpose of the present study was to further explore the molecular mechanism of HES1 in SACC. METHODS: Comparative transcriptome analyses by RNA-Sequencing (RNA-Seq) were employed to reveal NOTCH1 downstream gene in SACC cells. Immunohistochemical staining was used to detect the expression of HES1 in clinical samples. After HES1-siRNA transfected into SACC LM cells, the cell proliferation and cell apoptosis were tested by suitable methods; animal model was established to detect the change of growth ability of tumor. Transwell and wound healing assays were used to evaluate cell metastasis and invasion. RESULTS: We found that HES1 was strongly linked to NOTCH signaling pathway in SACC cells. The immunohistochemical results implied the high expression of HES1 in cancerous tissues. The growth of SACC LM cells transfected with HES1-siRNAs was significantly suppressed in vitro and tumorigenicity in vivo by inducing cell apoptosis. After HES1 expression was silenced, the SACC LM cell metastasis and invasion ability was suppressed. CONCLUSIONS: The results of this study demonstrate that HES1 is a specific downstream gene of NOTCH1 and that it contributes to SACC proliferation, apoptosis and metastasis. Our findings serve as evidence indicating that HES1 may be useful as a clinical target in the treatment of SACC.
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Carcinoma Adenoide Quístico/genética , Oncogenes , Neoplasias de las Glándulas Salivales/genética , Factor de Transcripción HES-1/genética , Adulto , Anciano , Animales , Apoptosis/genética , Carcinoma Adenoide Quístico/patología , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , ARN Interferente Pequeño/genética , Receptor Notch1/genética , Recurrencia , Neoplasias de las Glándulas Salivales/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.
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Aminoácidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , Aminoacil-ARNt Sintetasas/metabolismo , Modelos Moleculares , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Proteínas/genética , Proteínas/metabolismoRESUMEN
BACKGROUND: Brain-derived neurotrophic factor (BDNF) has been implicated in cognitive performance and the modulation of several metabolic parameters in some disease models, but its potential roles in successful aging remain unclear. We herein sought to define the putative correlation between BDNF Val66Met and several metabolic risk factors including BMI, blood pressure, fasting plasma glucose (FPG) and lipid levels in a long-lived population inhabiting Hongshui River Basin in Guangxi. METHODS: BDNF Val66Met was typed by ARMS-PCR for 487 Zhuang long-lived individuals (age ≥ 90, long-lived group, LG), 593 of their offspring (age 60-77, offspring group, OG) and 582 ethnic-matched healthy controls (aged 60-75, control group, CG) from Hongshui River Basin. The correlations of genotypes with metabolic risks were then determined. RESULTS: As a result, no statistical difference was observed on the distribution of allelic and genotypic frequencies of BDNF Val66Met among the three groups (all P > 0.05) except that AA genotype was dramatically higher in females than in males of CG. The HDL-C level of A allele (GA/AA genotype) carriers was profoundly lower than was non-A (GG genotype) carriers in the total population and the CG (P = 0.009 and 0.006, respectively), which maintained in females, hyperglycemic and normolipidemic subgroup of CG after stratification by gender, BMI, glucose and lipid status. Furthermore, allele A carriers, with a higher systolic blood pressure, exhibited 1.63 folds higher risk than non-A carriers to be overweight in CG (OR = 1.63, 95% CI: 1.05 - 2.55, P = 0.012). Multiple regression analysis displayed that the TC level of LG reversely associated with BDNF Val66Met genotype. CONCLUSIONS: These data suggested that BDNF 66Met may play unfavorable roles in blood pressure and lipid profiles in the general population in Hongshui River area which might in part underscore their poorer survivorship versus the successful aging individuals and their offspring.
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Presión Sanguínea/genética , Factor Neurotrófico Derivado del Encéfalo/genética , Metabolismo de los Lípidos/genética , Longevidad , Enfermedades Metabólicas , Anciano , Anciano de 80 o más Años , China/epidemiología , Etnicidad , Femenino , Genotipo , Humanos , Longevidad/genética , Longevidad/fisiología , Masculino , Enfermedades Metabólicas/epidemiología , Enfermedades Metabólicas/genética , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
We report a novel in vitro yeast ribosome display method based on cell-free protein synthesis (CFPS) using linear DNA templates. We demonstrate that our platform can enrich a target gene from a model library by 100-fold per round of selection. We demonstrate the utility of our approach by evolving cap-independent translation initiation (CITI) sequences, which result in a 13-fold increase in CFPS yields after four rounds of selection, and a threefold further increase by placing the beneficial short sequences in tandem. We also show that 12 of the selected CITI sequences permit precise control of gene expression in vitro over a range of up to 80-fold by enhancing translation (and not as cryptic promoters). These 12 sequences are then shown to tune protein expression in vivo, though likely due to a different mechanism. Looking forward, yeast ribosome display holds promise for evolving libraries of proteins and DNA regulatory parts for protein engineering and synthetic biology. Biotechnol. Bioeng. 2016;113: 1777-1786. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Evolución Molecular Dirigida/métodos , Biosíntesis de Proteínas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Técnicas de Visualización de Superficie Celular , Sistema Libre de Células , Ribosomas/genética , Saccharomyces cerevisiae/metabolismo , Biología SintéticaRESUMEN
Many studies have explored whether the Notch signaling pathway has a tumor-suppressive or an oncogenic role in various tumors; however, the role of the Notch signaling pathway in salivary adenoid cystic carcinoma (SACC) is still unknown. In this study, we attempt to define the role of Notch2 signaling in cell growth, invasion, and migration in SACC. We compared Notch2 expression in clinical SACC samples with that of normal samples by using immunohistochemical staining. Then, we down-regulated Notch2 expression to observe the effect of Notch2 on proliferation, invasion, migration, and the expression of known target genes of Notch signal pathway. According to our results, Notch2 expression was higher in SACC tissues compared with normal tissues. Knockdown of Notch2 inhibited cell proliferation, invasion, and migration in vitro and down-regulated the expression of HEY2 and CCND1. The results of this study suggest that Notch2 has an essential role in the cell growth, invasion, and migration of SACC. Notch2 may therefore be a potential target gene for the treatment of SACC by interfering with cell growth and metastasis.
Asunto(s)
Carcinoma Adenoide Quístico/patología , Proliferación Celular , Invasividad Neoplásica , Metástasis de la Neoplasia , Receptor Notch2/metabolismo , Neoplasias de las Glándulas Salivales/patología , Transducción de Señal , Carcinoma Adenoide Quístico/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Receptor Notch2/genética , Neoplasias de las Glándulas Salivales/metabolismoRESUMEN
In order to solve the problems of slow detection speed, large number of parameters and large computational volume of deep learning based gangue target detection method, we propose an improved algorithm for gangue target detection based on Yolov5s. First, the lightweight network EfficientVIT is used as the backbone network to increase the target detection speed. Second, C3_Faster replaces the C3 part in the HEAD module, which reduces the model complexity. once again, the 20 × 20 feature map branch in the Neck region is deleted, which reduces the model complexity; thirdly, the CIOU loss function is replaced by the Mpdiou loss function. The introduction of the SE attention mechanism makes the model pay more attention to critical features to improve detection performance. Experimental results show that the improved model size of the coal gang detection algorithm reduces the compression by 77.8%, the number of parameters by 78.3% the computational cost is reduced by 77.8% and the number of frames is reduced by 30.6%, which can be used as a reference for intelligent coal gangue classification.