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1.
ACS Synth Biol ; 12(12): 3669-3679, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37963151

RESUMO

Exchanging the native iron of heme for other metals yields artificial metalloproteins with new properties for spectroscopic studies and biocatalysis. Recently, we reported a method for the biosynthesis and incorporation of a non-natural metallocofactor, cobalt protoporphyrin IX (CoPPIX), into hemoproteins using the common laboratory strain Escherichia coli BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in E. coli. Herein, we report detailed kinetic analysis of the ferrochelatase responsible for metal insertion, EcHemH (E. coli ferrochelatase). This enzyme exhibits a small, less than 2-fold preference for Fe2+ over the non-native Co2+ substrate in vitro. To test how mutations impact EcHemH, we used a surrogate metal specificity screen to identify variants with altered metal insertion preferences. This engineering process led to a variant with an ∼30-fold shift in specificity toward Co2+. When assayed in vivo, however, the impact of this mutation is small compared to the effects of alteration of the external metal concentrations. These data suggest that incorporation of cobalt into PPIX is enabled by the native promiscuity of EcHemH coupled with BL21's impaired ability to maintain transition-metal homeostasis. With this knowledge, we generated a method for CoPPIX production in rich media, which yields cobalt-substituted hemoproteins with >95% cofactor purity and yields comparable to standard expression protocols for the analogous native hemoproteins.


Assuntos
Cobalto , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Cinética , Metais/química
2.
J Bacteriol ; 205(5): e0033222, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37154694

RESUMO

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate. In this review, we examine two known heme-dependent transcription factors, CooA and RcoM, that regulate inducible CO metabolism pathways in anaerobic and aerobic microorganisms. We provide an analysis of the known physiological and genomic contexts of these sensors and employ this analysis to contextualize known biochemical properties. In addition, we describe a growing list of putative transcription factors associated with CO metabolism that potentially use cofactors other than heme to sense CO.


Assuntos
Monóxido de Carbono , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Monóxido de Carbono/metabolismo , Oxirredução , Heme/metabolismo , Expressão Gênica , Proteínas de Bactérias/metabolismo
3.
Phys Rev E ; 107(2-1): 024409, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36932546

RESUMO

Quorum sensing (QS) allows bacterial cells to sense changes in local cell density and, hence, to regulate multicellular processes, including biofilm formation, regulation of virulence, and horizontal gene transfer. While, traditionally, QS was thought to involve the exchange of extracellular signal molecules free in solution, recent experiments have shown that for some bacterial systems a substantial fraction of signal molecules are packaged and delivered in extracellular vesicles. How the packaging of signal molecules in extracellular vesicles influences the ability of cells to communicate and coordinate multicellular behaviors remains largely unknown. We present here a stochastic reaction-diffusion model of QS that accounts for the exchange of both freely diffusing and vesicle-associated signal molecules. We find that the delivery of signal molecules via extracellular vesicles amplifies local fluctuations in the signal concentration, which can strongly affect the dynamics and spatial range of bacterial communication. For systems with multiple bacterial colonies, extracellular vesicles provide an alternate pathway for signal transport between colonies, and may be crucial for long-distance signal exchange in environments with strong degradation of free signal molecules.


Assuntos
Bactérias , Vesículas Extracelulares , Percepção de Quorum/fisiologia , Comunicação
4.
Appl Environ Microbiol ; 88(23): e0134622, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36342184

RESUMO

The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. IMPORTANCE The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange.


Assuntos
Bactérias , Vesículas Extracelulares , Bactérias/genética , Bactérias/metabolismo , Transferência Genética Horizontal , Vesículas Extracelulares/metabolismo , Membranas , Eucariotos
5.
Microbiol Spectr ; 10(5): e0235322, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36106751

RESUMO

Phylogenetic and sequence similarity network analyses of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors indicate the presence of numerous subgroups, many of which have not been analyzed. Five homologs of the CRP/FNR family are present in the Rhodobacter capsulatus genome. One is a member of a broadly disseminated, previously uncharacterized CRP/FNR family subgroup encoded by the gene rcc01561. In this study, we utilize mutational disruption, transcriptome sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the role of RCC01561 in regulating R. capsulatus physiology. This analysis shows that a mutant strain disrupted for rcc01561 exhibits altered expression of 451 genes anaerobically. A detailed analysis of the affected loci shows that RCC01561 represses photosynthesis and favors catabolism over anabolism and the use of the Entner-Doudoroff shunt and glycolysis over that of the tricarboxylic acid (TCA) cycle to limit NADH and ATP formation. This newly characterized CRP/FNR family member with a predominant role in reducing the production of reducing potential and ATP is given the nomenclature RedB as it functions as an energy and redox brake. Beyond limiting energy production, RedB also represses the expression of numerous genes involved in protein synthesis, including those involved in translation initiation, tRNA synthesis and charging, and amino acid biosynthesis. IMPORTANCE CRP and FNR are well-characterized members of the CRP/FNR family of regulatory proteins that function to maximize cellular energy production. In this study, we identify several new subgroups of the CRP/FNR family, many of which have not yet been characterized. Using Rhodobacter capsulatus as a model, we have mutationally disrupted the gene rcc01561, which codes for a transcription factor that is a member of a unique subgroup of the CRP/FNR family. Transcriptomic analysis shows that the disruption of rcc01561 leads to the altered expression of 451 genes anaerobically. Analysis of these regulated genes indicates that RCC01561 has a novel role in limiting cellular energy production. To our knowledge, this is first example of a member of the CRP/FNR family that functions as a brake on cellular energy production.


Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Filogenia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , NAD/genética , NAD/metabolismo , Fatores de Transcrição/metabolismo , Oxirredução , Fumaratos , Ácidos Tricarboxílicos , Aminoácidos/metabolismo , RNA de Transferência/metabolismo , Trifosfato de Adenosina/metabolismo
6.
Microbiol Spectr ; 10(5): e0235422, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36106752

RESUMO

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicates that RedB and Fnr directly coregulate only 4 out of 199 genes. The primary mechanism for the divergent regulation of target genes thus involves indirect regulation by both RedB and FnrL (156 cases). Additional regulation involves direct binding by RedB and indirect regulation by FnrL (36 cases) or direct binding by FnrL and indirect regulation by RedB (3 cases). Analysis of physiological pathways under direct and indirect control by these global regulators demonstrates that RedB functions primarily to limit energy production, while FnrL functions to enhance energy production. This regulation includes glycolysis, gluconeogenesis, photosynthesis, hydrogen oxidation, electron transport, carbon fixation, lipid biosynthesis, and protein synthesis. Finally, we show that 75% of genomes from diverse species that code for RedB proteins also harbor genes coding for FNR homologs. This cooccurrence indicates that RedB likely has an important role in buffering FNR-mediated energy production in a broad range of species. IMPORTANCE The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in Rhodobacter capsulatus), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions. In a companion article (N. Ke, J. E. Kumka, M. Fang, B. Weaver, et al., Microbiol Spectr 10:e02353-22, 2022, https://doi.org/10.1128/Spectrum02353-22), we identified a new subgroup of the CRP/FNR family and demonstrated that a member of this new subgroup, called RedB, has a role in limiting cellular energy production. In this study, we show that numerous genes encompassing the RedB regulon significantly overlap genes that are members of the FnrL regulon. Furthermore, 97% of the genes that are members of both the RedB and FnrL regulons are divergently regulated by these two transcription factors. RedB thus functions as a buffer limiting the amount of energy production that is promoted by FnrL.


Assuntos
Rhodobacter capsulatus , Rhodobacter sphaeroides , Trifosfato de Adenosina/metabolismo , Anaerobiose , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrogênio/metabolismo , Lipídeos , NAD/genética , NAD/metabolismo , Oxirredução , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
World Neurosurg ; 163: e223-e229, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35367390

RESUMO

BACKGROUND: Shaken baby syndrome occurs following inertial loading of the pediatric head, resulting in retinal hemorrhaging, subdural hematoma, and encephalopathy. However, the anatomically vulnerable cervical spine receives little attention. Automotive safety literature is replete with biomechanical data involving forward-facing pediatric surrogates in frontal collisions, an environment analogous to shaking. Publicly available data involving child occupants were utilized to study pediatric neck and head injury potential. We hypothesized that inertial loading provides a greater risk of injury to the cervical spine than to the head. METHODS: Full-scale automotive crash tests (n = 131) and deceleration sled tests (n = 32) utilizing forward-facing 3-year-old surrogates with head accelerometers and cervical force sensors were analyzed. One hundred sixty-seven full-scale vehicle and 33 sled test runs were assessed in the context of published injury assessment reference values (IARVs) for closed head injury (head injury criterion 15 [HIC15]) and cervical tensile strength in the 3-year-old model. RESULTS: One hundred sixty-one (96%) child surrogates in full-scale crash tests exceeded the cervical peak tension IARV, while only 37 (22%) surpassed the HIC15 IARV. Similarly, in sled testing runs, 27 (82%) pediatric surrogates exceeded cervical tension IARVs, while 1 (3%) surpassed the HIC15 IARV. In both full-scale and sled tests, all surrogates surpassing the HIC15 IARV also exceeded the cervical tension IARV. Positive linear correlations were observed between HIC15 and cervical tensile forces in both full-scale vehicle (R2 = 0.15) and sled testing runs (R2 = 0.54). CONCLUSIONS: These data support the hypothesis that inertial loading of the head provides a greater injury risk to the cervical spine than to closed-head injury.


Assuntos
Traumatismos Craniocerebrais , Síndrome do Bebê Sacudido , Aceleração , Acidentes de Trânsito , Fenômenos Biomecânicos , Vértebras Cervicais , Criança , Pré-Escolar , Humanos , Lactente , Pescoço , Síndrome do Bebê Sacudido/diagnóstico
8.
Biochemistry ; 61(8): 678-688, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35394749

RESUMO

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.


Assuntos
Heme , Hemeproteínas , Proteínas de Bactérias/química , Monóxido de Carbono/metabolismo , DNA/metabolismo , Heme/química , Hemeproteínas/metabolismo , Ligação Proteica , Fatores de Transcrição/química
9.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33850014

RESUMO

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.


Assuntos
Metaloporfirinas/química , Porfirinas/biossíntese , Porfirinas/química , Biocatálise , Cobalto/química , Cobalto/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Ferro , Metais/química , Mioglobina/química , Protoporfirinas/biossíntese , Protoporfirinas/química
10.
Sports Biomech ; 15(3): 370-84, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27240101

RESUMO

The goal of the current study was to expand on previous work to validate the use of pressure insole technology in conjunction with linear regression models to predict the free torque at the shoe-surface interface that is generated while wearing different athletic shoes. Three distinctly different shoe designs were utilised. The stiffness of each shoe was determined with a material's testing machine. Six participants wore each shoe that was fitted with an insole pressure measurement device and performed rotation trials on an embedded force plate. A pressure sensor mask was constructed from those sensors having a high linear correlation with free torque values. Linear regression models were developed to predict free torques from these pressure sensor data. The models were able to accurately predict their own free torque well (RMS error 3.72 ± 0.74 Nm), but not that of the other shoes (RMS error 10.43 ± 3.79 Nm). Models performing self-prediction were also able to measure differences in shoe stiffness. The results of the current study showed the need for participant-shoe specific linear regression models to insure high prediction accuracy of free torques from pressure sensor data during isolated internal and external rotations of the body with respect to a planted foot.


Assuntos
Desenho de Equipamento , Pé/fisiologia , Teste de Materiais , Corrida/fisiologia , Sapatos , Adolescente , Adulto , Fenômenos Biomecânicos , Humanos , Modelos Lineares , Pressão , Rotação , Torque , Adulto Jovem
11.
J Biomech Eng ; 138(6): 061007, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27109294

RESUMO

A direct method to express the center of pressure (CoP) measured by an insole pressure sensor system (IPSS) into a known coordinate system measured by motion tracking equipment is presented. A custom probe was constructed with reflective markers to allow its tip to be precisely tracked with motion tracking equipment. This probe was utilized to activate individual sensors on an IPSS that was placed in a shoe fitted with reflective markers used to establish a local shoe coordinate system. When pressed onto the IPSS the location of the probe's tip was coincident with the CoP measured by the IPSS (IPSS-CoP). Two separate pushes (i.e., data points) were used to develop vectors in each respective coordinate system. Simple vector mathematics determined the rotational and translational components of the transformation matrix needed to express the IPSS-CoP into the local shoe coordinate system. Validation was performed by comparing IPSS-CoP with an embedded force plate measured CoP (FP-CoP) from data gathered during kinematic trials. Six male subjects stood on an embedded FP and performed anterior/posterior (AP) sway, internal rotation, and external rotation of the body relative to a firmly planted foot. The IPSS-CoP was highly correlated with the FP-CoP for all motions, root mean square errors (RMSRRs) were comparable to other research, and there were no statistical differences between the displacement of the IPSS-CoP and FP-CoP for both the AP and medial/lateral (ML) axes, respectively. The results demonstrated that this methodology could be utilized to determine the transformation variables need to express IPSS-CoP into a known coordinate system measured by motion tracking equipment and that these variables can be determined outside the laboratory anywhere motion tracking equipment is available.


Assuntos
, Teste de Materiais , Pressão , Sapatos , Adulto , Fenômenos Biomecânicos , Humanos , Masculino , Rotação , Adulto Jovem
12.
J Pediatr ; 161(1): 120-4, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22364851

RESUMO

OBJECTIVE: To develop a statewide school-based program of measuring and reporting cardiovascular fitness levels in children, and to create age- and sex-specific cardiovascular fitness percentile-based distribution curves. STUDY DESIGN: A pilot study validated cardiovascular fitness assessment with Progressive Aerobic Cardiovascular Endurance Run (PACER) testing as an accurate predictor of cardiovascular fitness measured by maximal oxygen consumption treadmill testing. Schools throughout the state were then recruited to perform PACER and body mass index (BMI) measurement and report de-identified data to a centralized database. RESULTS: Data on 20 631 individual students with a mean age 12.1 ± 2.0 years, BMI of 21.4 ± 5.1, and a cardiovascular fitness measured with PACER of 29.7 ± 18.2 laps (estimated maximal oxygen consumption of 36.5 mL/kg/min) were submitted for analysis. Standardized fitness percentiles were calculated for age and sex. CONCLUSIONS: This study demonstrates the feasibility of performing, reporting, and recording annual school-based assessments of cardiovascular fitness to develop standardized childhood fitness percentiles on the basis of age and sex. Such data can be useful in comparing populations and assessing initiatives that aim to improve childhood fitness. Because health consequences of obesity result from both adiposity and physical inactivity, supplementation of BMI measurement with tracking of cardiovascular fitness adds a valuable tool for large-scale health assessment.


Assuntos
Fenômenos Fisiológicos Cardiovasculares , Aptidão Física , Adolescente , Criança , Feminino , Humanos , Masculino , Projetos Piloto , Valores de Referência , Instituições Acadêmicas
13.
Can J Anaesth ; 58(12): 1110-4, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21971741

RESUMO

INTRODUCTION: Extensive evidence has established a link between sympathetic nervous system hyperactivity, ventricular arrhythmias, and sudden cardiac death. For this reason, cardiac sympathectomy is often beneficial in the treatment of patients at high risk for ventricular ectopy, although it involves an invasive procedure associated with potential morbidity. We report a case in which we used guided lytic thoracic sympathetic block in a patient with underlying cardiomyopathy and refractory polymorphic ventricular tachycardia. CLINICAL FEATURES: A 74-yr-old African American male with ischemic cardiomyopathy presented with refractory episodes of ventricular tachycardia despite maximal medical therapy involving antiarrhythmic drugs and previous interventions, including endovascular epicardial ablation and open cryoablation via sternotomy. During his inpatient admission, the patient developed sustained ventricular tachycardia associated with cardiac depression requiring vasopressors. An open thoracoscopic sympathectomy was considered as a possible treatment, but in our view, the patient would not tolerate this procedure. As an alternative, the pain medicine team successfully performed a lytic thoracic sympathetic block. Subsequently, the patient demonstrated a period of clinical improvement with no apparent morbidity related to the procedure. CONCLUSION: Lytic thoracic sympathetic blockade is a novel technique for the treatment of sympathetically mediated ventricular tachycardia, and it is less invasive than other types of cardiac sympathectomy. Additional studies are required to evaluate this treatment as a viable alternative in patients at high risk for ventricular ectopy. This report suggests the feasibility of this approach and the potential for minimal morbidity in cases of refractory ventricular arrhythmias.


Assuntos
Bloqueio Nervoso Autônomo/métodos , Cardiomiopatias/fisiopatologia , Taquicardia Ventricular/terapia , Idoso , Estudos de Viabilidade , Humanos , Masculino , Isquemia Miocárdica/fisiopatologia , Taquicardia Ventricular/fisiopatologia , Vértebras Torácicas , Resultado do Tratamento , Vasoconstritores/uso terapêutico
14.
J Biomech ; 44(15): 2636-41, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21889148

RESUMO

External rotation of the foot has been implicated in high ankle sprains. Recent studies by this laboratory, and others, have suggested that torsional traction characteristics of the shoe-surface interface may play a role in ankle injury. While ankle injuries most often involve damage to ligaments due to excessive strains, the studies conducted by this laboratory and others have largely used surrogate models of the lower extremity to determine shoe-surface interface characteristics based on torque measures alone. The objective of this study was to develop a methodology that would integrate a motion analysis-based kinematic foot model with a computational model of the ankle to determine dynamic ankle ligament strains during external foot rotation. Six subjects performed single-legged, internal rotation of the body with a planted foot while a marker-based motion analysis was conducted to track the hindfoot motion relative to the tibia. These kinematic data were used to drive an established computational ankle model. Ankle ligament strains, as a function of time, were determined. The anterior tibiofibular ligament (ATiFL) experienced the highest strain at 9.2±1.1%, followed by the anterior deltoid ligament (ADL) at 7.8±0.7%, averaged over the six subjects. The peak ATiFL strain occurred prior to peak strain in the ADL in all subjects. This novel methodology may provide new insights into mechanisms of high ankle sprains and offer a basis for future evaluations of shoe-surface interface characteristics using human subjects rather than mechanical surrogate devices.


Assuntos
Traumatismos do Tornozelo/fisiopatologia , Tornozelo/fisiopatologia , Simulação por Computador , Ligamentos Articulares/fisiopatologia , Modelos Biológicos , Caminhada , Adulto , Fenômenos Biomecânicos , Humanos , Masculino , Sapatos
15.
Methods Mol Biol ; 722: 167-78, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21590420

RESUMO

Deep transcriptome profiling of pathogen-infected tissues enhances the understanding of molecular mechanisms underlying host-pathogen interactions. Illumina's next generation sequencing technology sequencing-by-synthesis (SBS) is a powerful tool to rapidly sequence genomes and transcriptomes at an affordable rate. We modified the procedure for SBS library construction to significantly increase the efficiency of library construction. Using our improved method, two Sclerotinia homoeocarpa libraries were constructed from mycelia grown in potato dextrose broth (PDB) or potato dextrose agar (PDA) for 96 h, respectively, and two creeping bentgrass libraries were constructed from leaves 96 h after inoculation with S. homoeocarpa or water sprayed, respectively. About 4-7 million mRNA signatures were sequenced from each library. Sequence analysis using BLAST was performed against sequenced fungal genomes and rice genomic sequence to identify the expressed genes in both S. homoeocarpa mycelia and creeping bentgrass. Bioinformatic analysis identified many expressed genes in the pathogen and host. A public database to access the sequence data was developed at http://www.dstidb.org . Our results demonstrate how SBS technology can unravel transcriptome complexity during the creeping bentgrass-S. homoeocarpa interaction.


Assuntos
Agrostis/microbiologia , Ascomicetos/fisiologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Análise de Sequência de DNA/métodos , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Biologia Computacional/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Biblioteca Gênica , Genoma Fúngico , Genoma de Planta , Interações Hospedeiro-Patógeno/fisiologia , Micélio/genética , Micélio/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Nature ; 456(7223): 819-23, 2008 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-18849970

RESUMO

DNA double-strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiological processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell-cycle checkpoints and cell survival. DNA double-strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we show that in murine lymphocytes these physiological DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double-strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiological DNA double-strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.


Assuntos
Linfócitos B/metabolismo , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Camundongos SCID , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos
17.
Mol Cell Biol ; 27(13): 4603-16, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17485448

RESUMO

Whereas interleukin-10 (IL-10) is an anti-inflammatory cytokine known to regulate macrophage activation, its full mechanism of action remains incompletely defined. In a screen to identify novel IL-10-induced genes, we cloned the mouse ortholog of human ABIN-3 (also termed LIND). ABIN-3 expression was induced selectively by IL-10 in both mouse and human mononuclear phagocytes coordinately undergoing proinflammatory responses. In contrast to the previously characterized ABINs, mouse ABIN-3 was incapable of inhibiting NF-kappaB activation by proinflammatory stimuli. Generation and analysis of ABIN-3-null mice demonstrated that ABIN-3 is unnecessary for the anti-inflammatory effects of IL-10 as well as for proper negative regulation of NF-kappaB. Conversely, human ABIN-3 was capable of inhibiting NF-kappaB activation in response to signaling from Toll-like receptor, IL-1, and tumor necrosis factor. Enforced expression of human ABIN-3 in human monocytic cells suppressed the cytoplasmic degradation of IkappaBalpha, the activation of NF-kappaB, and the induction of proinflammatory genes. Comparative sequence analyses revealed that mouse ABIN-3 lacks a complete ABIN homology domain, which was required for the functional activity of human ABIN-3. ABIN-3 is, thus, an IL-10-induced gene product capable of attenuating NF-kappaB in human macrophages yet is inoperative in mice and represents a basis for species-specific differences in IL-10 actions.


Assuntos
Inflamação/patologia , Interleucina-10/farmacologia , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Ativação Enzimática/efeitos dos fármacos , Variação Genética , Humanos , Quinase I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Células U937
18.
Am J Phys Med Rehabil ; 86(5): 339-45 quiz 346-8, 379, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17449977

RESUMO

OBJECTIVES: To report long-term survival of spinal muscular atrophy type 1 (SMA 1) and consequences on speech and ventilator dependence as a function of mode of ventilator use. DESIGN: A retrospective chart review of 106 consecutively referred SMA 1 patients, the 92 most severe of which were considered in three groups: untreated (group 1), tracheostomy managed (group 2), and noninvasively managed (group 3). RESULTS: The untreated patients died at 9.6 +/- 4.0 mos of age. The mean age of the 22 patients referred with tracheostomy tubes (group 2) was 70.5 +/- 43.3 mos (range 2-159 mos); five died at 66.2 +/- 114.2 mos (range 8-270 mos) of age. Six had comprehendible speech at the time of tracheotomy and retained some ability to vocalize afterward. None of the 21 patients who had not developed the ability to speak did so after tracheotomy. Twenty-five of the 27 total lost all autonomous breathing ability immediately, and definitively, after tracheotomy. The 47 patients who used noninvasive mechanical ventilation (NIV) (group 3) were extubated to it during episodes of acute respiratory failure. Thirty-nine of these were 65.2 +/- 45.8 mos (range 11-153 mos) of age, and eight died at 60.9 +/- 26.1 mos (range 36-111 mos) of age. There was no significant difference in longevity with or without tracheostomy, but the NIV patients had significantly fewer (P = 0.04) hospitalizations per year after age 5; 39 of the 47 could communicate verbally, and only nine were continuously dependent on NIV. CONCLUSIONS: NIV and tracheostomy can both prolong survival for SMA 1 patients, but the latter results in continuous ventilator dependence and speech does not develop.


Assuntos
Atrofias Musculares Espinais da Infância/mortalidade , Adolescente , Criança , Pré-Escolar , Hospitalização/estatística & dados numéricos , Humanos , Lactente , Recém-Nascido , Prontuários Médicos , Qualidade de Vida , Respiração Artificial , Estudos Retrospectivos , Atrofias Musculares Espinais da Infância/fisiopatologia , Atrofias Musculares Espinais da Infância/terapia , Traqueotomia
19.
Blood ; 109(8): 3360-8, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17158234

RESUMO

The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.


Assuntos
Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas Proto-Oncogênicas c-vav/imunologia , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória/imunologia , Transdução de Sinais/imunologia , Animais , Células Cultivadas , Ciclo-Oxigenase 2/imunologia , Citocinas/imunologia , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/imunologia , Humanos , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/imunologia , Macrófagos/enzimologia , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/imunologia , Fator 88 de Diferenciação Mieloide/deficiência , NADH NADPH Oxirredutases/deficiência , NADH NADPH Oxirredutases/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Proteínas Proto-Oncogênicas c-vav/deficiência , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/imunologia , Proteína RAC2 de Ligação ao GTP
20.
Mech Dev ; 122(7-8): 939-48, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15925495

RESUMO

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal , Linhagem Celular , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Janus Quinases , Ligantes , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética
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