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1.
Hu Li Za Zhi ; 67(1): 81-88, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-31960399

RESUMO

BACKGROUND & PROBLEMS: Early rehabilitation after stroke is important for the recovery of bodily functions in stroke patients. However, the percentage of completion of early limb rehabilitation among stroke patients is only 16%. PURPOSE: Raise the early rehabilitation intervention rate to 88% for patients with stroke within 24 hours of hospitalization. RESOLUTION: We developed an education course on post-stroke rehabilitation and a related e-Learning course as well as organized an 'alliance for recovery' team. In addition, we established a standard for post-stroke relay rehabilitation and designed rehabilitation relay cards, Xbox rehabilitation games, and nine squares challenge for brain stroke care. RESULTS: The accuracy of the knowledge of nursing staff related to physical rehabilitation improved from 72.4% to 100%; the accuracy of their perceptions regarding early limb rehabilitation increased from 16% to 100%; and patient satisfaction increased from 68% to 98%. CONCLUSIONS: We deployed diverse and innovative strategies to assist limb rehabilitation in patients with stroke. Patients and caregivers should be encouraged to participate in early rehabilitation and related programs and should apply the skills and rehabilitation activities learned to daily life.


Assuntos
Extremidades/fisiopatologia , Reabilitação do Acidente Vascular Cerebral/métodos , Reabilitação do Acidente Vascular Cerebral/enfermagem , Acidente Vascular Cerebral/fisiopatologia , Difusão de Inovações , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Pesquisa em Avaliação de Enfermagem , Recursos Humanos de Enfermagem no Hospital/educação , Recursos Humanos de Enfermagem no Hospital/psicologia , Satisfação do Paciente/estatística & dados numéricos , Desenvolvimento de Programas
2.
Infect Immun ; 86(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29891543

RESUMO

Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A (csrA of B. burgdorferi, or csrABb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C terminus), and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection.


Assuntos
Proteínas de Bactérias/genética , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidade , Ácidos Graxos Voláteis/farmacologia , Acetatos/farmacologia , Animais , Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Borrelia burgdorferi/efeitos dos fármacos , Butiratos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Lipoproteínas/genética , Doença de Lyme/microbiologia , Mutação , Propionatos/farmacologia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fator sigma/genética , Virulência
3.
Infect Immun ; 86(7)2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29685985

RESUMO

Borrelia burgdorferi, the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the B orrelia host adaptation regulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086, shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi, were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the B orrelia host adaptation protein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of B burgdorferi.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Doença de Lyme/fisiopatologia , Virulência/fisiologia , Animais , Doença de Lyme/epidemiologia , Camundongos , Camundongos Endogâmicos C3H/microbiologia , Camundongos SCID/microbiologia , Ratos , Estados Unidos/epidemiologia
4.
Methods Mol Biol ; 1690: 155-175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29032544

RESUMO

Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re.3gulator (BadR-a DNA binding protein) and Carbon storage regulators A of B. b urgdorferi (CsrABb-an RNA binding protein) as examples. Best laboratory practices associated with overexpression/purification of recombinant borrelial proteins, synthesis of target nucleic acid sequences, and electrophoretic mobility assays to assess the protein/nucleic acid interactions are described. The methods described are intended to facilitate empirical assessment of the binding affinity, co-factor requirements, quality of the interacting partners, and readily modifiable assay conditions to assess the binding properties to define known and unknown regulatory properties of nucleic acid binding proteins of B. burgdorferi.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Clonagem Molecular/métodos , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Doença de Lyme/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Infect Immun ; 85(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28052993

RESUMO

Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferirel (relBbu ; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Borrelia burgdorferi/fisiologia , Regulação Bacteriana da Expressão Gênica , Espermidina/metabolismo , Espermina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Poliaminas/metabolismo , Poliaminas/farmacologia , Espermidina/farmacologia , Espermina/farmacologia , Transcrição Genética , Fatores de Virulência/genética
6.
Infect Immun ; 81(8): 2972-85, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23753623

RESUMO

Carbon storage regulator A of Borrelia burgdorferi (CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABb are dependent on environmental signals and on select residues. We analyzed the phenotype of csrABb deletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABb in the csrABb mutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in the csrABb mutant to parental levels, indicating that both the levels of CsrABb and the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb (8S) with alanines resulted in increased levels of CsrABb and reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members of rpoS regulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb (7D) resulted in reduced csrABb transcripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream of pta compared to the parental and 7D truncated protein. Two CsrABb binding sites were also identified upstream of fliW within the flgK coding sequence. These observations reveal conserved and unique functions of CsrABb that regulate adaptive gene expression in B. burgdorferi.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidade , Interações Hospedeiro-Parasita/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Western Blotting , Borrelia burgdorferi/genética , Sequência Conservada , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Carrapatos/microbiologia
7.
PLoS One ; 7(5): e38171, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22675445

RESUMO

Borrelia burgdorferi, the agent of Lyme disease, is a spirochetal pathogen with limited metabolic capabilities that survives under highly disparate host-specific conditions. However, the borrelial genome encodes several proteins of the mevalonate pathway (MP) that utilizes acetyl-CoA as a substrate leading to intermediate metabolites critical for biogenesis of peptidoglycan and post-translational modifications of proteins. In this study, we analyzed the MP and contributions of acetate in modulation of adaptive responses in B. burgdorferi. Reverse-transcription PCR revealed that components of the MP are transcribed as individual open reading frames. Immunoblot analysis using monospecific sera confirmed synthesis of members of the MP in B. burgdorferi. The rate-limiting step of the MP is mediated by HMG-CoA reductase (HMGR) via conversion of HMG-CoA to mevalonate. Recombinant borrelial HMGR exhibited a K(m) value of 132 µM with a V(max) of 1.94 µmol NADPH oxidized minute(-1) (mg protein)(-1) and was inhibited by statins. Total protein lysates from two different infectious, clonal isolates of B. burgdorferi grown under conditions that mimicked fed-ticks (pH 6.8/37°C) exhibited increased levels of HMGR while other members of the MP were elevated under unfed-tick (pH 7.6/23°C) conditions. Increased extra-cellular acetate gave rise to elevated levels of MP proteins along with RpoS, CsrA(Bb) and their respective regulons responsible for mediating vertebrate host-specific adaptation. Both lactone and acid forms of two different statins inhibited growth of B. burgdorferi strain B31, while overexpression of HMGR was able to partially overcome that inhibition. In summary, these studies on MP and contributions of acetate to host-specific adaptation have helped identify potential metabolic targets that can be manipulated to reduce the incidence of Lyme disease.


Assuntos
Acetatos/farmacologia , Borrelia burgdorferi/metabolismo , Ácido Mevalônico/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/genética , Ativação Enzimática/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Dados de Sequência Molecular , Fases de Leitura Aberta , Alinhamento de Sequência , Transcriptoma
8.
Phytochemistry ; 78: 7-19, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22513011

RESUMO

AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 were identified using LC-MS/MS. Analysis of trypsin-released signature peptides showed that this E3-independent ubiquitination of AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 was dominated by mono-ubiquitination at multiple sites. Unlike AtUBC8-type E2s, AtUBC36 was not able to transfer ubiquitin to AtMAPR2. The truncated mutants AtMAPR2Δ1-10, AtMAPR2Δ1-30, and AtMAPR2_1-73 could also be ubiquitinated. The presence of a ubiquitin-binding domain (UBD) allows proteins to be ubiquitinated independently of E3 ligases. However, AtMAPRs do not contain any known UBD. In vitro ubiquitination of AtMAPR2 observed in this study will be further studied in biochemical and physiological aspects.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Transporte/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Arabidopsis/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos
9.
Invest Ophthalmol Vis Sci ; 51(12): 6277-85, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20435600

RESUMO

PURPOSE: To compare the effects of early, mid, and late subconjunctival injection of bevacizumab on corneal neovascularization (NV) and conjunctivalization in a rabbit limbal insufficiency model. METHODS: Limbal insufficiency was created surgically, and subconjunctival injections of 2.5 mg bevacizumab twice weekly for 1 month were started immediately (early group), 1 week (mid group), and 1 month after injury (late group). The corneal epithelial alterations, stromal opacity, centricity, extent, and PICS (percentage of involved corneal surface) of the corneal NV were evaluated. The expression of cytokeratins K3, K4, K12, K13, and MUC5 by the corneal surface cells was examined by immunohistochemistry. RESULTS: Bevacizumab significantly inhibited corneal NV in the early and mid, but not the late, treatment groups at 4 weeks after treatment (PICS: P < 0.01 in the early group, P < 0.01 in the mid group, and P > 0.05 in the late group). Early and mid treatment produced significant inhibition of corneal alteration (P < 0.01 in the early group and P = 0.03 in the mid group) and stromal opacity (P < 0.01 in the early group and P = 0.02 in the mid group) at 4 months after treatment but not in the late group. The immunohistochemistry of cytokeratin on the corneal surface cells at 1 month after treatment was K3(+), K4(-), K12(+), K13(-), and MUC5(-) in the early group; K3(+), K4(+), K12(+), K13(+), and MUC5(-) in the mid group; and K3(+), K4(+), K12(-), K13(+), and MUC5(+) in the late treatment group. CONCLUSIONS: Early and mid bevacizumab injection inhibited corneal NV, epithelial alteration, and stromal opacity in limbal insufficiency, but late treatment did not. Early treatment with bevacizumab seems to be clinically beneficial in the management of limbal injury such as chemical burn.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Túnica Conjuntiva/efeitos dos fármacos , Neovascularização da Córnea/tratamento farmacológico , Limbo da Córnea/patologia , Células 3T3 , Animais , Anticorpos Monoclonais Humanizados , Bevacizumab , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Túnica Conjuntiva/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Substância Própria/metabolismo , Substância Própria/patologia , Modelos Animais de Doenças , Epitélio Anterior/metabolismo , Epitélio Anterior/patologia , Técnica Indireta de Fluorescência para Anticorpo , Injeções , Queratinas/metabolismo , Camundongos , Microscopia de Fluorescência , Mucina-5B/metabolismo , Fenótipo , Coelhos , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Cicatrização
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