RESUMO
Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that neutrophils from patients with APS used glycolysis more avidly than neutrophils from people in the healthy control group, especially when the neutrophils were from patients with APS with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.
Assuntos
Síndrome Antifosfolipídica , Armadilhas Extracelulares , Glucose , Glicólise , Neutrófilos , Via de Pentose Fosfato , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Animais , Camundongos , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Síndrome Antifosfolipídica/tratamento farmacológico , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Masculino , Feminino , Glucose/metabolismo , Trombose/metabolismo , Trombose/imunologia , Trombose/patologia , Trombose/genética , Adulto , Espécies Reativas de Oxigênio/metabolismo , Pessoa de Meia-IdadeRESUMO
Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.
Assuntos
Neutrófilos , Infecções por Salmonella , Animais , Humanos , Camundongos , Caspases/metabolismo , Células Epiteliais , Mucosa Intestinal/microbiologia , Infecções por Salmonella/metabolismo , Salmonella typhimuriumRESUMO
Activation of the endoplasmic reticulum stress sensor, IRE1α, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases in which neutrophils are a key immune component. However, the specific role of IRE1α in regulating neutrophil effector function has not been studied. In this study, we show that infection-induced IRE1α activation licenses neutrophil antimicrobial capacity, including IL-1ß production, formation of neutrophil extracellular traps (NETs), and methicillin-resistant Staphylococcus aureus (MRSA) killing. Inhibition of IRE1α diminished production of mitochondrial reactive oxygen species and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in CASPASE-2 or neutrophil IRE1α were highly susceptible to MRSA infection and failed to effectively form NETs in the s.c. abscess. IRE1α activation enhanced calcium influx and citrullination of histone H3 independently of mitochondrial reactive oxygen species production, suggesting that IRE1α coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1α-CASPASE-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1α in neutrophil antibacterial function.
Assuntos
Endorribonucleases/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Neutrófilos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Voluntários Saudáveis , Humanos , Interleucina-1beta/biossíntese , Camundongos , Transdução de Sinais/imunologiaRESUMO
A good long-term outcome following a total knee arthroplasty relies on restoration of the mechanical axis and effective soft tissue balancing of the prosthetic knee. Arthroplasty surgery in patients with secondary osteoarthritis of the knee with an extra-articular tibial deformity is a complex and challenging procedure. The correction of mal-alignment of the mechanical axis is associated with unpredictable result and with higher revision rates. Single-staged deformity correction and replacement surgery often result in the use of constraint implants. We describe our experience with staged correction of deformity using a Taylor Spatial Frame (TSF) followed by total knee arthroplasty in these patients and highlight the advantage of staged approach. The use of TSF fixator for deformity correction prior to a primary total knee arthroplasty has not been described in the literature. We describe three cases of secondary osteoarthritis of the knee associated with multiplanar tibial deformity treated effectively with a total knee arthroplasty following deformity correction and union using a TSF. All patients had an improved Knee Society score and Oxford Knee score postoperatively and were satisfied with their replacement outcome. Staged deformity correction followed by arthroplasty allows the use of standard primary arthroplasty implants with predicable results and flexible aftercare. This approach may also provide significant improvement of patient symptoms following correction of deformity resulting in deferment of the arthroplasty surgery.
RESUMO
This article provides an overview of the recruitment strategies utilized in the Mumbai Worksites Tobacco Control Study, a cluster randomized trial testing the effectiveness of an integrated tobacco control and occupational safety and health program in Indian manufacturing worksites. From June 2012 to June 2013, 20 companies were recruited. Companies were identified using association lists, referrals, internet searches and visits to industrial areas. Four hundred eighty companies were contacted to validate information, introduce the study and seek an in-person meeting with a company representative. Eighty-three company representatives agreed to meet. Of those 83 companies, 55 agreed to a formal 'pitch meeting' with key decision makers at the company. Seventy-seven recruitment 'pitches' were given, including multiple meetings in the same companies. If the company was interested, we obtained a letter of participation and employee roster. Based on this experience, recommendations are made that can help inform future researchers and practitioners wishing to recruit Indian worksites. When compared with recruitment of US manufacturing worksites, recruitment of Indian worksites lacked current industrial lists of companies to serve as a sampling frame, and required more in-person visits, incentives for control companies and more assurances around confidentiality to allow occupational safety and health experts into their worksite.
Assuntos
Promoção da Saúde/organização & administração , Saúde Ocupacional , Seleção de Pacientes , Local de Trabalho , Humanos , Índia , Prevenção do Hábito de FumarRESUMO
Recently, we reported that extract of Dalbergia sissoo made from leaves and pods have antiresorptive and bone-forming effects. The positive skeletal effect attributed because of active molecules present in the extract of Dalbergia sissoo. Caviunin 7-O-[ß-D-apiofuranosyl-(1-6)-ß-D-glucopyranoside] (CAFG), a novel isoflavonoid show higher percentage present in the extract. Here, we show the osteogenic potential of CAFG as an alternative for anabolic therapy for the treatment of osteoporosis by stimulating bone morphogenetic protein 2 (BMP2) and Wnt/ß-catenin mechanism. CAFG supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur and decreased bone turnover markers better than genistein. Oral administration of CAFG to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased the expression of osteogenic genes in femur and show new bone formation without uterine hyperplasia. CAFG increased mRNA expression of osteoprotegerin in bone and inhibited osteoclast activation by inhibiting the expression of skeletal osteoclastogenic genes. CAFG is also an effective accelerant for chondrogenesis and has stimulatory effect on the repair of cortical bone after drill-hole injury at the tissue, cell and gene level in mouse femur. At cellular levels, CAFG stimulated osteoblast proliferation, survival and differentiation. Signal transduction inhibitors in osteoblast demonstrated involvement of p-38 mitogen-activated protein kinase pathway stimulated by BMP2 to initiate Wnt/ß-catenin signaling to reduce phosphorylation of GSK3-ß and subsequent nuclear accumulation of ß-catenin. Osteogenic effects were abrogated by Dkk1, Wnt-receptor blocker and FH535, inhibitor of TCF-complex by reduction in ß-catenin levels. CAFG modulated MSC responsiveness to BMP2, which promoted osteoblast differentiation via Wnt/ß-catenin mechanism. CAFG at 1 mg/kg(/)day dose in ovariectomy mice (human dose â¼0.081 mg/kg) led to enhanced bone formation, reduced bone resorption and bone turnover better than well-known phytoestrogen genistein. Owing to CAFG's inherent properties for bone, it could be positioned as a potential drug, food supplement, for postmenopausal osteoporosis and fracture repair.
Assuntos
Doenças Ósseas Metabólicas/tratamento farmacológico , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Osso e Ossos/efeitos dos fármacos , Glicosídeos/farmacologia , Isoflavonas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Animais Recém-Nascidos , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Doenças Ósseas Metabólicas/fisiopatologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteína Morfogenética Óssea 2/genética , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica , Genisteína/farmacologia , Células HEK293 , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos Endogâmicos BALB C , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Ovariectomia , Fatores de Tempo , Transfecção , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic ß subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.
Assuntos
Anabolizantes/farmacologia , Osso e Ossos/patologia , Osteoporose/tratamento farmacológico , Inibidores de Proteassoma/química , Vitanolídeos/farmacologia , Cicatrização , Anabolizantes/química , Anabolizantes/farmacocinética , Anabolizantes/uso terapêutico , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiopatologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fêmur/fisiopatologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteoporose/genética , Osteoporose/patologia , Osteoporose/fisiopatologia , Ovariectomia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacocinética , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Proteólise/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vitanolídeos/química , Vitanolídeos/farmacocinética , Vitanolídeos/uso terapêutico , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H-N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.
Assuntos
Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Bactérias/química , Deutério , Proteínas de Escherichia coli/química , Isomerases de Dissulfetos de Proteínas/química , Prótons , alfa-Sinucleína/químicaRESUMO
Psoas syndrome is an easily missed diagnosis. However, it is important to consider this condition as part of the differential diagnosis for patients presenting with low back pain--particularly for osteopathic physicians, because patients may view these practitioners as experts in musculoskeletal conditions. The authors describe the case of a 48-year-old man with a 6-month history of low back pain that had been attributed to "weak core muscles." The diagnosis of psoas syndrome was initially overlooked in this patient. After the correct diagnosis was made, he was treated by an osteopathic physician using osteopathic manipulative treatment, in conjunction with at-home stretches between office treatments. At his 1-month follow-up appointment, he demonstrated continued improvement of symptoms and a desire for further osteopathic manipulative treatment.
Assuntos
Dor Lombar/terapia , Osteopatia/métodos , Dor Intratável/terapia , Músculos Psoas/patologia , Espasmo/diagnóstico , Diagnóstico Diferencial , Terapia por Exercício , Humanos , Dor Lombar/etiologia , Dor Lombar/patologia , Masculino , Pessoa de Meia-Idade , Médicos Osteopáticos , Dor Intratável/etiologia , Dor Intratável/patologia , Espasmo/patologia , Espasmo/terapia , SíndromeRESUMO
Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble ß1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.
RESUMO
We show that quantitative internuclear (15)N-(13)C distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of (13)C sites in the beta1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-(13)C or 2-(13)C-glycerol as the (13)C source. Quantitative dipolar trajectories are extracted from two-dimensional (15)N-(13)C planes, in which approximately 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one (13)C and several (15)N each), yielding quantitative precision as good as 0.1 A for approximately 350 sites, better than 0.3 A for another 150, and approximately 1.0 A for 150 distances in the range of 5-8 A. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25+/-0.09 A), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79+/-0.03 A). Moreover, side chain heavy atoms are well restrained (0.76+/-0.06 A total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.
Assuntos
Isótopos de Carbono/química , Isótopos de Nitrogênio/química , Proteínas/química , Metilação , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação ProteicaRESUMO
Delayed presentation of a laryngeal foreign body in an adult is unusual. Symptoms of minimal hoarseness with no signs of odynophagia, infection or difficulty in swallowing in a fit young patient endow no clues to suspect a swallowed denture in a busy emergency department and can be easily missed by a junior medical staff. In this report, we present a case of a broken partial denture plate stuck in the supraglottic region of an adult male patient diagnosed after 10 days despite initial presentation to the emergency department.
Assuntos
Serviço Hospitalar de Emergência/normas , Migração de Corpo Estranho/complicações , Hemoptise/etiologia , Rouquidão/etiologia , Lesões do Pescoço/complicações , Sons Respiratórios/etiologia , Traqueia/diagnóstico por imagem , Adulto , Dentaduras , Migração de Corpo Estranho/diagnóstico por imagem , Humanos , Masculino , RadiografiaRESUMO
CONTEXT: Since the 1990s, there has been a heightened awareness of the value of teaching medical students about how aspects of spirituality and religion may affect patient care. OBJECTIVE: To determine the prevalence of spirituality-in-medicine instruction at colleges of osteopathic medicine (COMs) in the United States. METHODS: Prescreened subjects at 20 COMs were contacted by electronic mail and asked to complete a 25-item Web-based survey. The survey instrument consisted of questions about spirituality-in-medicine instruction at their institutions. If an institution was not represented in our survey results through subject response, we reviewed that institution's Web site to locate material suggestive of an extant spirituality-in-medicine curricula (eg, prospective student information). RESULTS: Surveys were submitted to investigators by representatives of 12 COMs for a response rate of 60%. Subjects from 8 COMs reported a structured spirituality-in-medicine curriculum currently in place at their institutions. Osteopathic medical students generally receive a total of 2 to 20 hours of instruction on spirituality and religion. Of the 10 unrepresented institutions, 4 COMs had material available on their Web sites that suggested spirituality-in-medicine topics were embedded in their curricula. Therefore, approximately 55% of all COMs have some form of spirituality-in-medicine program in place. CONCLUSION: Some form of spirituality-in-medicine instruction is available at slightly more than half the COMs in the United States. As the need for spirituality-in-medicine curricula is increasingly recognized, improved methods of documenting ongoing curricular development and student competency will be required.
Assuntos
Medicina Osteopática/educação , Faculdades de Medicina , Espiritualidade , Currículo , Humanos , Inquéritos e Questionários , Estados UnidosRESUMO
In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxina da Cólera/genética , Toxina da Cólera/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas/métodos , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodosRESUMO
Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of approximately 1 A, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 +/- 0.13 A (1.52 +/- 0.12 A for all heavy atoms), which improved to 0.49 +/- 0.05 A (1.19 +/- 0.07 A) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 +/- 0.06 A bbRMSD (1.06 +/- 0.07 A); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 +/- 0.09 A; 1.29 +/- 0.07 A) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.
Assuntos
Nanopartículas/química , Proteínas do Tecido Nervoso/química , Ressonância Magnética Nuclear Biomolecular , Bases de Dados de Proteínas , Marcação por Isótopo , Proteínas do Tecido Nervoso/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , TermodinâmicaRESUMO
INTRODUCTION: Complementary and alternative medicine (CAM) education has created a focus that not only brings awareness of various CAM therapies to conventional medical education but also highlights how these therapies can bring insight toward improved health care. METHODS: A survey of CAM educational leaders at institutions awarded grants for incorporating CAM education into medical curricula was performed to address how CAM education can improve health care delivery in America. RESULTS: Five (5) core themes were noted to be successful in achieving this goal. These included (1) education on the importance of relationship-centered care, (2) understanding holism, (3) the promotion of self-reflection and self-care, (4) collaboration with CAM providers to enhance communication, and (5) the need for faculty development in CAM. CONCLUSIONS: In discussing these themes, this paper explores how a shift in medical education toward a focus of understanding what is needed for the creation of health (salutogenesis) can bring balance to a curriculum that is currently weighted in teaching about the creation of disease (pathogenesis). Potential benefits, including reduced health care costs and improved quality of life for learners, are discussed.
Assuntos
Terapias Complementares/educação , Terapias Complementares/organização & administração , Educação Médica/organização & administração , Medicina Baseada em Evidências , Docentes de Medicina/organização & administração , Competência Clínica , Currículo , Educação de Pós-Graduação em Medicina/organização & administração , Educação de Graduação em Medicina/organização & administração , Educação em Enfermagem/organização & administração , Humanos , Comunicação Interdisciplinar , Liderança , National Institutes of Health (U.S.) , Garantia da Qualidade dos Cuidados de Saúde , Estados UnidosRESUMO
For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.