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1.
Mol Microbiol ; 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33368680

RESUMO

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

3.
PLoS Negl Trop Dis ; 14(11): e0008840, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33137811

RESUMO

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

4.
NPJ Breast Cancer ; 6: 44, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32964118

RESUMO

Germline genetic variation has been suggested to influence the survival of breast cancer patients independently of tumor pathology. We have studied survival associations of genetic variants in two etiologically unique groups of breast cancer patients, the carriers of germline pathogenic variants in BRCA1 or BRCA2 genes. We found that rs57025206 was significantly associated with the overall survival, predicting higher mortality of BRCA1 carrier patients with estrogen receptor-negative breast cancer, with a hazard ratio 4.37 (95% confidence interval 3.03-6.30, P = 3.1 × 10-9). Multivariable analysis adjusted for tumor characteristics suggested that rs57025206 was an independent survival marker. In addition, our exploratory analyses suggest that the associations between genetic variants and breast cancer patient survival may depend on tumor biological subgroup and clinical patient characteristics.

5.
Euro Surveill ; 25(25)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32613938

RESUMO

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

6.
PLoS Negl Trop Dis ; 14(7): e0008452, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658917

RESUMO

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. METHODS AND PRINCIPAL FINDINGS: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. CONCLUSIONS: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Melioidose/sangue , Melioidose/diagnóstico , Fitas Reagentes , Testes Sorológicos/métodos , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias , Proteínas de Bactérias , Burkholderia pseudomallei/genética , Humanos , Melioidose/microbiologia , Sensibilidade e Especificidade
7.
Clin Infect Dis ; 71(7): e135-e140, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31677383

RESUMO

BACKGROUND: Technology has changed the way that men who have sex with men (MSM) seek sex. More than 60% of MSM in the United States use the internet and/or smartphone-based geospatial networking apps to find sex partners. We correlated use of the most popular app (Grindr) with sexual risk and prevention behavior among MSM. METHODS: A nested cohort study was conducted between September 2018 and June 2019 among MSM receiving community-based human immunodeficiency virus (HIV) and sexually transmitted infection (STI) screening in central San Diego. During the testing encounter, participants were surveyed for demographics, substance use, risk behavior (previous 3 months), HIV pre-exposure prophylaxis (PrEP) use, and Grindr usage. Participants who tested negative for HIV and who were not on PrEP were offered immediate PrEP. RESULTS: The study included 1256 MSM, 1090 of whom (86.8%) were not taking PrEP. Overall, 580 of 1256 (46%) participants indicated that they used Grindr in the previous 7 days. Grindr users reported significantly higher risk behavior (greater number of male partners and condomless sex) and were more likely to test positive for chlamydia or gonorrhea (8.6% vs 4.7% of nonusers; P = .005). Grindr users were also more likely to be on PrEP (18.7% vs 8.7% of nonusers; P < .001) and had fewer newly diagnosed HIV infections (9 vs 26 among nonusers; P = .014). Grindr users were also nearly twice as likely as nonusers to initiate PrEP (24.6% vs 14%; P < .001). CONCLUSIONS: Given the higher risk behavior and greater acceptance of PrEP among MSM who used Grindr, Grindr may provide a useful platform to promote HIV and STI testing and increase PrEP uptake.

8.
PLoS Negl Trop Dis ; 13(10): e0007821, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31634353

RESUMO

BACKGROUND: Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil. METHODOLOGY/PRINCIPAL FINDINGS: Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h. CONCLUSIONS/SIGNIFICANCE: In the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future.


Assuntos
Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/metabolismo , Meios de Cultura/química , Eritritol/metabolismo , Oryza/microbiologia , Microbiologia do Solo , Técnicas Bacteriológicas , Burkholderia pseudomallei/crescimento & desenvolvimento , Carbono/metabolismo , Melioidose/microbiologia , Solo , Vietnã
9.
Chem Commun (Camb) ; 55(83): 12575-12578, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31578536

RESUMO

NMR spectroscopy is generally used to investigate molecules under equilibrium conditions. Despite recent technological and methodogical developments to study on-going reactions, tracing the fate of individual atoms during an irreversible chemical reaction is still a challenging and elaborate task. Reaction-interrupted excitation transfer (ExTra) NMR provides a selective tracking of resonances from atoms, which undergo chemical conversion. We show that reactions triggered either by rapid mixing or by photo-excitation can be conveniently followed at a sub-second time scale using standard NMR equipment. In ExTra NMR we use the selectively inverted magnetization of a selected atom to follow its conversion in the course of a fast chemical reaction. The chemical reaction has to be started within the relaxation period of an initial inverting 180° pulse. The presented protocol provides a generally applicable NMR method for reaction monitoring.

10.
Sci Rep ; 9(1): 14479, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597939

RESUMO

Universal HIV and HCV screening in emergency departments (ED) can reach populations who are less likely to get tested otherwise. The objective of this analysis was to evaluate universal opt-out HIV and HCV screening in two EDs in San Diego. HIV screening for persons aged 13-64 years (excluding persons known to be HIV+ or reporting HIV testing within last 12 months) was implemented using a 4th generation HIV antigen/antibody assay; HCV screening was offered to persons born between 1945 and 1965. Over a period of 16 months, 12,575 individuals were tested for HIV, resulting in 33 (0.26%) new HIV diagnoses, of whom 30 (90%) were successfully linked to care. Universal screening also identified 74 out-of-care for >12-months HIV+ individuals of whom 50 (68%) were successfully relinked to care. Over a one-month period, HCV antibody tests were conducted in 905 individuals with a seropositivity rate of 9.9% (90/905); 61 seropositives who were newly identified or never treated for HCV had HCV RNA testing, of which 31 (51%) resulted positive (3.4% of all participants, including 18 newly identified RNA positives representing 2% of all participants), and 13/31 individuals (42%) were linked to care. The rate of newly diagnosed HCV infections exceeded the rate of newly diagnosed HIV infections by >7-fold, underlining the importance of HCV screening in EDs.


Assuntos
Serviço Hospitalar de Emergência , Infecções por HIV/diagnóstico , Hepatite C/diagnóstico , Programas de Rastreamento/métodos , Sorodiagnóstico da AIDS , Adolescente , Adulto , Idoso , Algoritmos , California/epidemiologia , Estudos de Coortes , Infecções por HIV/epidemiologia , Soroprevalência de HIV , Hepatite C/epidemiologia , Humanos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Adulto Jovem
11.
Circ Res ; 125(1): 43-52, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31219742

RESUMO

RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.


Assuntos
Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Interferon Tipo I/biossíntese , Mitocôndrias/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Adulto Jovem
12.
Biomol NMR Assign ; 13(1): 121-125, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30617945

RESUMO

Transfer of genetic material is the main mechanism underlying the spread of antibiotic resistance and virulence factors within the bacterial community. Conjugation is one such process by which the genetic material is shared from one bacterium to another. The DNA substrate is processed and prepared for transfer by a multi-protein complex called the relaxosome .The relaxosome of plasmid R1 possesses the most crucial enzyme TraI which, both nicks and unwinds the dsDNA substrate. TraI comprises 1765 residues and multiple functional domains, including those catalyzing the DNA trans-esterase (relaxase) on the dsDNA designated for a conjugative transfer and DNA helicase activities. Structural and functional studies have been reported for most of the TraI except the C-terminal domain spanning from residue 1630 to 1765. This region is the least understood part of TraI and is thought to be highly disordered and flexible. This region, being intrinsically disordered, is hypothesized to be serving as an interacting platform for other proteins involved in this DNA transfer initiation mechanism. In this work, we report the 1H, 13C, 15N resonance assignment of this region as well as the secondary structure information based on the backbone chemical shifts.


Assuntos
Proteínas de Bactérias/química , Ressonância Magnética Nuclear Biomolecular , Plasmídeos/genética , Isótopos de Carbono , Isótopos de Nitrogênio , Domínios Proteicos , Estrutura Secundária de Proteína , Prótons
13.
Trop Med Infect Dis ; 3(2)2018 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30274435

RESUMO

The first cases of human melioidosis were described in Vietnam in the 1920s, almost a century ago. It was in Vietnam in the thirties that the saprophytic nature of B. pseudomallei was first recognized. Although a significant number of French and U.S. soldiers acquired the disease during the Vietnam wars, indigenous cases in the Vietnamese population were only sporadically reported over many decades. After reunification in 1975, only two retrospective studies reported relatively small numbers of indigenous cases from single tertiary care hospitals located in the biggest cities in the South and the North, respectively. Studies from provincial hospitals throughout the country were missing until the Research Network on Melioidosis and Burkholderia pseudomallei (RENOMAB) project started in 2014. From then on seminars, workshops, and national scientific conferences on melioidosis have been conducted to raise awareness among physicians and clinical laboratory staff. This led to the recognition of a significant number of cases in at least 36 hospitals in 26 provinces and cities throughout Vietnam. Although a widespread distribution of melioidosis has now been documented, there are still challenges to understand the true epidemiology of the disease. Establishment of national guidelines for diagnosis, management, and reporting of the disease together with more investigations on animal melioidosis, genomic diversity of B. pseudomallei and its environmental distribution are required.

14.
Trop Med Infect Dis ; 3(2)2018 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30274458

RESUMO

Melioidosis is an often fatal infectious disease with a protean clinical spectrum, caused by the environmental bacterial pathogen Burkholderia pseudomallei. Although the disease has been reported from some African countries in the past, the present epidemiology of melioidosis in Africa is almost entirely unknown. Therefore, the common view that melioidosis is rare in Africa is not evidence-based. A recent study concludes that large parts of Africa are environmentally suitable for B. pseudomallei. Twenty-four African countries and three countries in the Middle East were predicted to be endemic, but no cases of melioidosis have been reported yet. In this study, we summarize the present fragmentary knowledge on human and animal melioidosis and environmental B. pseudomallei in Africa and the Middle East. We propose that systematic serological studies in man and animals together with environmental investigations on potential B. pseudomallei habitats are needed to identify risk areas for melioidosis. This information can subsequently be used to target raising clinical awareness and the implementation of simple laboratory algorithms for the isolation of B. pseudomallei from clinical specimens. B. pseudomallei was most likely transferred from Asia to the Americas via Africa, which is shown by phylogenetic analyses. More data on the virulence and genomic characteristics of African B. pseudomallei isolates will contribute to a better understanding of the global evolution of the pathogen and will also help to assess potential differences in disease prevalence and outcome.

15.
J Biol Chem ; 293(49): 18977-18988, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30209131

RESUMO

Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg735) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-π interaction in TM7 between Arg735 and two highly conserved nearby aromatic residues, Tyr733 and Trp737 We propose a mechanism by which the transient, membrane-embedded cation-π complex provides the necessary energy to keep the charged side chain of Arg735 within the hydrophobic membrane. Such cation-π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.


Assuntos
Arginina/química , Prótons , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Técnicas de Inativação de Genes , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eletricidade Estática , Triptofano/química , Triptofano/genética , Tirosina/química , Tirosina/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética
16.
Front Microbiol ; 9: 935, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867844

RESUMO

Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

17.
J Biomol NMR ; 70(4): 211-218, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29564580

RESUMO

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.


Assuntos
Sondas Moleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Silanos/química , Humanos , Ligantes , Peptídeo Hidrolases/química , Ligação Proteica , Proteínas/metabolismo , Proteínas de Ligação a Tacrolimo , Proteínas Virais/química , Zika virus/química
18.
Sci Rep ; 8(1): 3420, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467422

RESUMO

Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2 nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2 NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+ on the surface of the nanoparticle. We conclude that CaF2 NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology.


Assuntos
Fluoreto de Cálcio/metabolismo , Calmodulina/metabolismo , Nanopartículas/metabolismo , Fluoreto de Cálcio/química , Calmodulina/química , Dicroísmo Circular , Motivos EF Hand , Humanos , Luminescência , Modelos Moleculares , Nanopartículas/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Ressonância de Plasmônio de Superfície
19.
Genes Dev ; 31(20): 2099-2112, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29118048

RESUMO

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.


Assuntos
Neoplasias Pulmonares/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/fisiologia , Células Epiteliais Alveolares/metabolismo , Animais , Respiração Celular , Células Cultivadas , Metabolismo Energético , Feminino , Hormônios Esteroides Gonadais/fisiologia , Homeostase , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Mucosa Respiratória/metabolismo
20.
J Virol ; 91(17)2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28615205

RESUMO

Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART)-naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to 6 months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against tier 1 subtype B viruses (P = 0.003 for SF-162 and P = 0.017 for NL4-3) and marginally against autologous virus (P = 0.054). Lower presuperinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between 2 and 3 years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by presuperinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary infecting and tier 1 subtype B viruses are more susceptible to superinfection.IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some superinfected individuals. These findings will inform HIV vaccine design by providing testable correlates of protection from initial HIV infection.


Assuntos
Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/classificação , Superinfecção/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , California , Estudos de Casos e Controles , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/virologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Testes de Neutralização , Superinfecção/virologia , Adulto Jovem
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