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1.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 10): 483-487, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33006576

RESUMO

The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL Mpro.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/química , Betacoronavirus/química , Betacoronavirus/genética , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Humanos , Modelos Moleculares , Difração de Nêutrons , Pandemias , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura , Proteínas não Estruturais Virais/genética
2.
Nat Commun ; 11(1): 4916, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004803

RESUMO

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.


Assuntos
Brassica rapa/fisiologia , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Animais , Cristalografia , Flores/metabolismo , Haplótipos , Simulação de Dinâmica Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Pólen/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera
3.
Nat Commun ; 11(1): 4905, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999288

RESUMO

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/genética , Insetos , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/isolamento & purificação , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/isolamento & purificação , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
4.
Med Hypotheses ; 143: 110244, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33017910

RESUMO

Corona virus disease 2019 (Covid-19), a pandemia emerged recently, caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). The receptor for corona virus and influenza A is the mucosal cell membrane protein angiotensin converting enzyme 2 (ACE2), which is abundant on the membrane of alveolar cells and enterocytes. Viral spike protein 1 (S1) is the ligand, with an affinity of 14.7 nM to the receptor. The main port of entry for the virus is the upper respiratory tract, and the diagnosis is usually by PCR of the viral RNA with nasal and pharyngeal swab test. Human defensin 5 (HDEF5) is a protein encoded by the DEFA gene, secreted by Paneth cells in the small intestine and by granules of neutrophils. It has an affinity of 39.3 nM to ACE2, much higher than that of the corona S1. HDEF5 may also attach to glycosylated Corona S1 protein, make its efficiency even better. The issues to be investigated are the affinity of HDEF5 to S1 protein, the ability of recombinant HDEF5 function in attaching both ACE2 and S1, and the feasibility to perform aerosol spray of this protein. In addition, safety and efficiency should be studied in phases I, II and II clinical protocols. Thus, an aerosol spray of HDEF5 given through the nose and throat, once to several times a day, may be a very efficient approach to prevent infection with SARA-CoV-2 as well as influenza A.


Assuntos
Betacoronavirus , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , alfa-Defensinas/administração & dosagem , Aerossóis , Humanos , Ligantes , Peptidil Dipeptidase A/química , Proteínas Recombinantes/química , alfa-Defensinas/química
5.
Medicine (Baltimore) ; 99(35): e21825, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32871904

RESUMO

OBJECTIVE: To conduct a meta-analysis evaluating the effect of combining traditional Chinese medicine (TCM) with Western medicine in treating hepatitis C, and to provide an evidence-based medical strategy. METHODS: Randomized controlled trials (RCTs) comparing the effect of pegylated interferon (Peginterferon) combined with ribavirin (PR) alone and its combination with TCM were manually retrieved from the Weipu Information Resources System (VIP), Wan Fang Database, PubMed, and the Chinese Journal Full Text Database (CNKI). Studies meeting the inclusion criteria were selected and analyzed using the Review Manager 5.3 software. Suitable tests were also performed to determine the quality, heterogeneity, and sensitivity of the studies included in the meta-analysis. RESULTS: Twenty-eight RCTs met the inclusion criteria. The combination therapy or intervention group showed significantly greater HCV-RNA negative rate post-treatment compared to the monotherapy or the control group (P < .05). In addition, the serum levels of the liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) were significantly improved after the combination therapy compared to PR alone (P < .05), while total bilirubin (TB) and r-glutamyltransferase (GGT) levels were not affected by TCM (P > .05). Finally, the parameters of liver fibrosis were also reduced by the combination therapy more effectively than the monotherapy. CONCLUSION: The combination of TCM and PR can improve the Comprehensive Clinical Efficacy of hepatitis C and have a better negative rate of HCV-RNA with a better benefit in the liver function. The effect of TCM + PR is better than that of PR alone in treating hepatitis C.


Assuntos
Antivirais/uso terapêutico , Hepatite C/terapia , Medicina Tradicional Chinesa , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Terapia Combinada , Quimioterapia Combinada , Hepacivirus/genética , Humanos , Interferon-alfa/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Polietilenoglicóis/uso terapêutico , RNA Viral/sangue , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Albumina Sérica , gama-Glutamiltransferase/sangue
6.
Nat Commun ; 11(1): 4566, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917903

RESUMO

Influenza virus exposures in childhood can establish long-lived memory B cell responses that can be recalled later in life. Here, we complete a large serological survey to elucidate the specificity of antibodies against contemporary H3N2 viruses in differently aged individuals who were likely primed with different H3N2 strains in childhood. We find that most humans who were first infected in childhood with H3N2 viral strains from the 1960s and 1970s possess non-neutralizing antibodies against contemporary 3c2.A H3N2 viruses. We find that 3c2.A H3N2 virus infections boost non-neutralizing H3N2 antibodies in middle-aged individuals, potentially leaving many of them in a perpetual state of 3c2.A H3N2 viral susceptibility.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Suscetibilidade a Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Philadelphia , Proteínas Recombinantes , Estações do Ano , Adulto Jovem
7.
Nat Commun ; 11(1): 4576, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917905

RESUMO

Endosome maturation depends on membrane contact sites (MCSs) formed between endoplasmic reticulum (ER) and endolysosomes (LyLEs). The mechanism underlying lipid supply for this process and its pathophysiological relevance remains unclear, however. Here, we identify PDZD8-the mammalian ortholog of a yeast ERMES subunit-as a protein that interacts with protrudin, which is located at ER-LyLE MCSs. Protrudin and PDZD8 promote the formation of ER-LyLE MCSs, and PDZD8 shows the ability to extract various lipids from the ER. Overexpression of both protrudin and PDZD8 in HeLa cells, as well as their depletion in mouse primary neurons, impairs endosomal homeostasis by inducing the formation of abnormal large vacuoles reminiscent of those apparent in spastin- or REEP1-deficient neurons. The protrudin-PDZD8 system is also essential for the establishment of neuronal polarity. Our results suggest that protrudin and PDZD8 cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, thereby maintaining neuronal polarity and integrity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/fisiologia , Metabolismo dos Lipídeos , Neurônios/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Lipídeos , Lipossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mitocôndrias , Domínios Proteicos , Proteômica , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética
8.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 355-360, 2020 Aug 24.
Artigo em Chinês | MEDLINE | ID: mdl-32935508

RESUMO

OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.


Assuntos
Proteínas de Helminto , Proteínas Recombinantes , Schistosoma japonicum , Animais , Anticorpos Anti-Helmínticos/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo
9.
Front Immunol ; 11: 2072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922409

RESUMO

A dysregulated immune response with hyperinflammation is observed in patients with severe coronavirus disease 2019 (COVID-19). The aim of the present study was to assess the safety and potential benefits of human recombinant C1 esterase inhibitor (conestat alfa), a complement, contact activation and kallikrein-kinin system regulator, in severe COVID-19. Patients with evidence of progressive disease after 24 h including an oxygen saturation <93% at rest in ambient air were included at the University Hospital Basel, Switzerland in April 2020. Conestat alfa was administered by intravenous injections of 8400 IU followed by 3 additional doses of 4200 IU in 12-h intervals. Five patients (age range, 53-85 years; one woman) with severe COVID-19 pneumonia (11-39% lung involvement on computed tomography scan of the chest) were treated a median of 1 day (range 1-7 days) after admission. Treatment was well-tolerated. Immediate defervescence occurred, and inflammatory markers and oxygen supplementation decreased or stabilized in 4 patients (e.g., median C-reactive protein 203 (range 31-235) mg/L before vs. 32 (12-72) mg/L on day 5). Only one patient required mechanical ventilation. All patients recovered. C1INH concentrations were elevated before conestat alfa treatment. Levels of complement activation products declined after treatment. Viral loads in nasopharyngeal swabs declined in 4 patients. In this uncontrolled case series, targeting multiple inflammatory cascades by conestat alfa was safe and associated with clinical improvements in the majority of severe COVID-19 patients. Controlled clinical trials are needed to assess its safety and efficacy in preventing disease progression.


Assuntos
Betacoronavirus/efeitos dos fármacos , Proteína Inibidora do Complemento C1/uso terapêutico , Complemento C1/antagonistas & inibidores , Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Sistema Calicreína-Cinina/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Proteína Inibidora do Complemento C1/análise , Fator XIa/antagonistas & inibidores , Feminino , Humanos , Calicreínas/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Pandemias , Proteínas Recombinantes/uso terapêutico , Carga Viral/efeitos dos fármacos
10.
Emerg Med Clin North Am ; 38(4): 871-889, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32981623

RESUMO

Massive gastrointestinal hemorrhage is a life-threatening condition that can result from numerous causes and requires skilled resuscitation to decrease patient morbidity and mortality. Successful resuscitation begins with placement of large-bore intravenous or intraosseous access; early blood product administration; and early consultation with a gastroenterologist, interventional radiologist, and/or surgeon. Activate a massive transfusion protocol when initial red blood cell transfusion does not restore effective perfusion or the patient's shock index is greater than 1.0. Promptly reverse coagulopathies secondary to oral anticoagulant or antiplatelet use. Use thromboelastography or rotational thromboelastometry to guide further transfusions. Secure a definitive airway and minimize aspiration.


Assuntos
Hemorragia Gastrointestinal/terapia , Manuseio das Vias Aéreas , Antibacterianos/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticoagulantes/efeitos adversos , Antifibrinolíticos/uso terapêutico , Oclusão com Balão , Fatores de Coagulação Sanguínea/administração & dosagem , Transfusão de Sangue/métodos , Cateteres , Serviço Hospitalar de Emergência , Fator Xa/administração & dosagem , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/etiologia , Humanos , Infusões Intraósseas , Infusões Intravenosas , Anamnese , Exame Físico , Inibidores da Bomba de Prótons/uso terapêutico , Proteínas Recombinantes/administração & dosagem , Ressuscitação , Tromboelastografia , Vasoconstritores/uso terapêutico
11.
Open Biol ; 10(9): 200209, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898469

RESUMO

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.


Assuntos
Betacoronavirus/metabolismo , Eucariotos/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Membrana Celular/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Eucariotos/citologia , Humanos , Microssomos/metabolismo , Mutação , Pandemias , Filogenia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética
12.
Trials ; 21(1): 769, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895056

RESUMO

OBJECTIVES: To assess the effect of anticoagulation with bivalirudin administered intravenously on gas-exchange in patients with COVID-19 and respiratory failure using invasive mechanical ventilation. TRIAL DESIGN: This is a single centre parallel group, superiority, randomized (1:1 allocation ratio) controlled trial. PARTICIPANTS: All patients admitted to the Hamad Medical Corporation -ICU in Qatar for COVID-19 associated respiratory distress and in need of mechanical ventilation are screened for eligibility. INCLUSION CRITERIA: all adult patients admitted to the ICU who test positive for COVID-19 by PCR-test and in need for mechanical ventilation are eligible for inclusion. Upon crossing the limit of D-dimers (1.2 mg/L) these patients are routinely treated with an increased dose of anticoagulant according to our local protocol. This will be the start of randomization. EXCLUSION CRITERIA: pregnancy, allergic to the drug, inherited coagulation abnormalities, no informed consent. INTERVENTION AND COMPARATOR: The intervention group will receive the anticoagulant bivalirudin intravenously with a target aPTT of 45-70 sec for three days while the control group will stay on the standard treatment with low-molecular-weight heparins /unfractionated heparin subcutaneously (see scheme in Additional file 1). All other treatment will be unchanged and left to the attending physicians. MAIN OUTCOMES: As a surrogate parameter for clinical improvement and primary outcome we will use the PaO2/FiO2 (P/F) ratio. RANDOMISATION: After inclusion, the patients will be randomized using a closed envelope method into the conventional treatment group, which uses the standard strategy and the experimental group which receives anticoagulation treatment with bivalirudin using an allocation ratio of 1:1. BLINDING (MASKING): Due to logistical and safety reasons (assessment of aPTT to titrate the study drug) only the data-analyst will be blinded to the groups. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): We performed a sample size calculation and assumed the data for P/F ratio (according to literature) is normally distributed and used the mean which would be: 160 and SD is 80. We expect the treatment will improve this by 30%. In order to reach a power of 80% we would need 44 patients per group (in total 88 patients). Taking approximately 10% of dropout into account we will include 100 patients (50 in each group). TRIAL STATUS: The local registration number is MRC-05-082 with the protocol version number 2. The date of approval is 18th June 2020. Recruitment started on 28th June and is expected to end in November 2020. TRIAL REGISTRATION: The protocol is registered before starting subject recruitment under the title: "Anticoagulation in patients suffering from COVID-19 disease. The ANTI-CO Trial" in ClinicalTrials.org with the registration number: NCT04445935 . Registered on 24 June 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 2). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.


Assuntos
Antitrombinas/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Respiração Artificial , Síndrome do Desconforto Respiratório do Adulto/terapia , Anticoagulantes/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/sangue , Estado Terminal , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Heparina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Hirudinas , Humanos , Pandemias , Tempo de Tromboplastina Parcial , Pneumonia Viral/sangue , Catar , Proteínas Recombinantes/uso terapêutico
13.
Nat Commun ; 11(1): 4458, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895383

RESUMO

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.


Assuntos
Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Hipotálamo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteína Relacionada com Agouti/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Glicemia/análise , Comunicação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Humanos , Hipotálamo/citologia , Hipotálamo/patologia , Injeções Intraventriculares , Leptina/genética , Masculino , Melanocortinas/metabolismo , Hormônios Estimuladores de Melanócitos/administração & dosagem , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA-Seq , Receptor Tipo 4 de Melanocortina/genética , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismo , Indução de Remissão/métodos , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Técnicas Estereotáxicas , Transcriptoma/efeitos dos fármacos
14.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
15.
Nat Commun ; 11(1): 4515, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908127

RESUMO

The discovery of ancestral RAG transposons in early deuterostomia reveals the origin of vertebrate V(D)J recombination. Here, we analyze the functional regulation of a RAG transposon, ProtoRAG, in lancelet. We find that a specific interaction between the cis-acting element within the TIR sequences of ProtoRAG and a trans-acting factor, lancelet YY1-like (bbYY1), is important for the transcriptional regulation of lancelet RAG-like genes (bbRAG1L and bbRAG2L). Mechanistically, bbYY1 suppresses the transposition of ProtoRAG; meanwhile, bbYY1 promotes host DNA rejoins (HDJ) and TIR-TIR joints (TTJ) after TIR-dependent excision by facilitating the binding of bbRAG1L/2 L to TIR-containing DNA, and by interacting with the bbRAG1L/2 L complex. Our data thus suggest that bbYY1 has dual functions in fine-tuning the activity of ProtoRAG and maintaining the genome stability of the host.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/metabolismo , Anfioxos/genética , Recombinação V(D)J , Fator de Transcrição YY1/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes RAG-1 , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/isolamento & purificação
16.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
17.
Nat Commun ; 11(1): 4529, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913184

RESUMO

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.


Assuntos
Metilação de DNA , Epigênese Genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Ilhas de CpG/genética , Estudos Transversais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Técnicas de Introdução de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Carga Global da Doença , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteínas Recombinantes/genética , Sistema de Registros/estatística & dados numéricos , Índice de Gravidade de Doença , Ovinos , Adulto Jovem
18.
Nat Commun ; 11(1): 4414, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887877

RESUMO

CD4+ helper T cells contribute important functions to the immune response during pathogen infection and tumor formation by recognizing antigenic peptides presented by class II major histocompatibility complexes (MHC-II). While many computational algorithms for predicting peptide binding to MHC-II proteins have been reported, their performance varies greatly. Here we present a yeast-display-based platform that allows the identification of over an order of magnitude more unique MHC-II binders than comparable approaches. These peptides contain previously identified motifs, but also reveal new motifs that are validated by in vitro binding assays. Training of prediction algorithms with yeast-display library data improves the prediction of peptide-binding affinity and the identification of pathogen-associated and tumor-associated peptides. In summary, our yeast-display-based platform yields high-quality MHC-II-binding peptide datasets that can be used to improve the accuracy of MHC-II binding prediction algorithms, and potentially enhance our understanding of CD4+ T cell recognition.


Assuntos
Epitopos de Linfócito T/genética , Oligopeptídeos , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Técnicas de Visualização da Superfície Celular , Bases de Dados de Proteínas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo
19.
Pestic Biochem Physiol ; 170: 104704, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980065

RESUMO

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s-1 mM-1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL-1) could hydrolyze 79.89% chlorpyrifos (5 mg L-1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.


Assuntos
Carboxilesterase/genética , Clorpirifos , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Clonagem Molecular , Concentração de Íons de Hidrogênio , Filogenia , Proteínas Recombinantes/genética , Especificidade por Substrato , Temperatura
20.
Anticancer Res ; 40(9): 4869-4874, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878774

RESUMO

BACKGROUND/AIM: In the present study, we evaluated the efficacy of adjuvant administration of oral recombinant methioninase (o-rMETase) against recurrence and metastasis in a 4T1 murine breast-cancer syngeneic model. MATERIALS AND METHODS: 4T1 cells were orthotopically implanted into the 2nd mammary fat pad of BALB/c mice. The 4T1 orthotopic syngeneic models were randomized into 2 groups after primary tumor resection: untreated control and o-rMETase (100 units, oral, daily, 2 weeks). RESULTS: The frequency and extent of local recurrence were reduced by o-rMETase. The number of individual cancer cells and metastatic nodules on the lung surface was significantly lower in the o-rMETase-treated mice than the untreated control mice. CONCLUSION: Adjuvant o-rMETase inhibited local recurrence and lung metastasis after primary tumor resection.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Liases de Carbono-Enxofre/administração & dosagem , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Administração Oral , Animais , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Esquema de Medicação , Feminino , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Recidiva Local de Neoplasia/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
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