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For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.
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Educación de Posgrado en Odontología/estadística & datos numéricos , Humanos , Facultades de Odontología/estadística & datos numéricos , Encuestas y Cuestionarios , Estados UnidosRESUMEN
We propose a framework for general Bayesian inference. We argue that a valid update of a prior belief distribution to a posterior can be made for parameters which are connected to observations through a loss function rather than the traditional likelihood function, which is recovered as a special case. Modern application areas make it increasingly challenging for Bayesians to attempt to model the true data-generating mechanism. For instance, when the object of interest is low dimensional, such as a mean or median, it is cumbersome to have to achieve this via a complete model for the whole data distribution. More importantly, there are settings where the parameter of interest does not directly index a family of density functions and thus the Bayesian approach to learning about such parameters is currently regarded as problematic. Our framework uses loss functions to connect information in the data to functionals of interest. The updating of beliefs then follows from a decision theoretic approach involving cumulative loss functions. Importantly, the procedure coincides with Bayesian updating when a true likelihood is known yet provides coherent subjective inference in much more general settings. Connections to other inference frameworks are highlighted.
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AIMS: To investigate the effects of anthrax lethal toxin (LeTx) on human primary keratinocytes. METHODS AND RESULTS: We show here that human primary keratinocytes are resistant to LeTx-triggered cytotoxicity. All but one of the MEKs (mitogen-activated protein kinase kinases) are cleaved within 3 h, and the cleavage of MEKs in keratinocytes leads to their subsequent proteasome-mediated degradation at different rates. Moreover, LeTx reduced the concentration of several cytokines except RANTES in culture. CONCLUSIONS: Our results indicate that primary keratinocytes are resistant to LeTx cytotoxicity, and MEK cleavage does not correlate with LeTx cytotoxicity. Although LeTx is considered as an anti-inflammatory agent, it upregulates RANTES. SIGNIFICANCE AND IMPACT OF THE STUDY: According to a current view, the action of LeTx results in downregulation of the inflammatory response, as evidenced by diminished expression of several inflammatory biomarkers. Paradoxically, LeTx has been reported to attract neutrophils to cutaneous infection sites. This paper, which shows that RANTES, a chemoattractant for immune cells, is upregulated after exposure of keratinocytes to LeTx, although a number of other markers of the inflammatory response are downregulated. Our results might explain why the exposure of keratinocytes to LeTx results in the recruitment of neutrophils to cutaneous infection sites, while the expression of several inflammatory biomarkers is diminished.
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Antígenos Bacterianos/farmacología , Antígenos Bacterianos/toxicidad , Bacillus anthracis , Toxinas Bacterianas/farmacología , Toxinas Bacterianas/toxicidad , Queratinocitos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Animales , Biomarcadores/metabolismo , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Prepucio , Glicoproteínas/farmacología , Humanos , Queratinocitos/enzimología , Queratinocitos/metabolismo , MasculinoRESUMEN
A national survey of 1023 people with epilepsy in the US assessed their attitudes about their therapies. Subjects were drawn from responders to a previous national survey of US households or from those who phoned the Epilepsy Foundation. Overall response rate was 49%. Approximately 90% of the respondents were taking medications for their epilepsy. Only 56% were on monotherapy, while 26% were taking two, 6% three, and 2% four medications. Only 68% of respondents were very satisfied with their current seizure medications. When asked to rank five areas of importance regarding their seizure medication, the rank order (highest to lowest) was seizure control, fewer side effects, convenient dosing regimens and cost. Adverse medication events were listed in descending rank order as problems with cognition, energy level, school performance, childbearing, coordination, and sexual function. Inter-individual differences in side effects of concern were listed, suggesting medication choices should be individualized according to potential side effects. Twenty percent of 920 respondents adjusted their medications on their own, by adjusting amount (62%), dosing schedule (31%), or both (3%). Eighty percent of respondents were satisfied with their medical care systems. In this group, 82% had health insurance that covered epilepsy. The large majority (94%) of respondents had seen a neurologist. Subjects expressed dissatisfaction about time limits and lack of accessible information about epilepsy. People with epilepsy are generally satisfied with efforts to treat their disorder, but adverse events are of concern. Many patients requested more information about epilepsy.
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Actitud Frente a la Salud , Atención a la Salud , Epilepsia/psicología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Terapias Complementarias , Epilepsia/fisiopatología , Epilepsia/terapia , Femenino , Estado de Salud , Humanos , Seguro de Salud/estadística & datos numéricos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Estados UnidosRESUMEN
The partition behaviour of nanoparticulate inclusion bodies (IBs) in various states of purity were evaluated as surrogate mimics for adenovirus and retrovirus products in method scouting experiments with aqueous two-phase systems (ATPSs). Such systems are proposed for effective, high capacity downstream processing (DSP) of viral gene therapy vectors. Studies with mimics provided simple descriptions of particle partition which may benefit the field of vector DSP, where experimental material is rarely available for research and development in quantities and concentrations representative of clinical manufacture. Polyethylene glycol (PEG)-salt and PEG-dextran ATPSs were screened in respect of the partition recovery of IBs from crude feedstocks. Select candidate systems were similarly evaluated with limited preparations of adenovirus and retrovirus with respect to fractional recoveries of infectivity and particle number. Maintenance of the former was good, whilst comparison of the latter with quantitation of unwashed and washed IBs indicated poor utilisation of the inherent high capacities of ATPSs in viral experiments. This is discussed in the context of the volumetric throughput and capacities reported in the literature for the recovery of infective viruses in ultracentrifugation and chromatographic processes.
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Terapia Genética , Vectores Genéticos , Adenoviridae/genética , Adenoviridae/aislamiento & purificación , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Tamaño de la Partícula , Retroviridae/genética , Retroviridae/aislamiento & purificación , Agua/químicaRESUMEN
Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.
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Lipopolisacáridos/química , Treponema/química , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/análisis , Hexosas/análisis , Lipopolisacáridos/aislamiento & purificación , Azúcares Ácidos/análisisRESUMEN
We developed a radioassay to assess the adherence of the oral treponemes Treponema denticola and Treponema pectinovorum to live HEp-2 epithelial cells. T. pectinovorum bound firmly to the epithelial cell monolayer in a concentration-dependent manner. The results indicated that a subpopulation of T. pectinovorum appeared to bind and that the binding could be influenced by environmental factors. Increasing concentrations of fetal bovine serum inhibited binding, whereas T. pectinovorum membrane vesicles and co-incubation with T. denticola ATCC 35404 increased the number of cells bound to the monolayer. Treatment of T. pectinovorum with periodic acid, but not trypsin or proteinase K, decreased the binding suggesting that a cell surface carbohydrate, such as the O-antigenic component of the lipopolysaccharide, mediates attachment of the bacteria to the epithelial cells. Co-infection of the HEp-2 cells with both T. denticola and T. pectinovorum did not interfere with each other in attachment to the epithelial cell suggesting that they do not compete for the same cellular receptor on the host cell surface. This study demonstrates that T. pectinovorum is capable, in vitro, of forming a tight association with host cells and that this binding could represent an initial step in the pathogenesis of T. pectinovorum.
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Adhesión Bacteriana/fisiología , Células Epiteliales/microbiología , Treponema/fisiología , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Línea Celular , Endopeptidasa K/farmacología , Células Epiteliales/efectos de los fármacos , Lipopolisacáridos , Ácido Peryódico/farmacología , Inhibidores de Proteasas/farmacología , Treponema/patogenicidadRESUMEN
UNLABELLED: To assess the efficacy of the analgesic technique and the incidence of complications, we prospectively evaluated patients who received intrathecal opioid analgesia (ITOA) to manage postsurgical pain. Daily quality assurance data were collected on the first postoperative day and tabulated for 5969 adult patients who had received ITOA for major urologic, orthopedic, general/ vascular, thoracic, and nonobstetrical gynecologic surgery. A scale of 1-10 was used to quantify each patient's satisfaction with analgesia. The incidence of side effects, complications, and naloxone usage was also recorded and tabulated. The mean satisfaction score using a 10-point numeric rating scale was 8.51, with a score of 1 connoting "complete dissatisfaction" and 10 connoting "complete satisfaction." Side effects were minor and easily managed. Pruritus was the most common (37%). Respiratory depression was the least common (3%), easily detected by nursing observation, never life-threatening, and always responsive to treatment with naloxone. There were no deaths, nerve injuries, central nervous system infections, or naloxone-related complications. Postdural puncture headaches were rare (0.54%), as was the need for epidural blood patch (0.37%). IMPLICATIONS: Over a 7-yr period, intrathecal opioid analgesia was used to control acute postoperative pain on nearly 6000 patients, resulting in a high degree of patient satisfaction and a low incidence of side effects and complications.
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Analgesia/métodos , Analgésicos Opioides/administración & dosificación , Dolor Postoperatorio/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Analgesia/efectos adversos , Analgésicos Opioides/efectos adversos , Femenino , Humanos , Inyecciones Espinales , Masculino , Estudios Prospectivos , Prurito/inducido químicamente , Respiración/efectos de los fármacosRESUMEN
Nurses are often the primary source of donor referrals to an organ procurement organization. Early exposure to donation information during nursing school may enhance nurses' ability to identify and refer potential donors once they are in nursing practice. Although organ procurement organizations strive to provide donation education to nursing students, this process can drain an organization's resources. Therefore, to facilitate the educational process with a minimum of disruption to the function of the organ procurement organization, a standardized approach to donation was developed. After the program had been in place for more than a year, students and organ procurement organization staff alike reported great satisfaction with this standardized approach.
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Curriculum , Bachillerato en Enfermería/organización & administración , Bachillerato en Enfermería/normas , Estudiantes de Enfermería , Materiales de Enseñanza/normas , Donantes de Tejidos/educación , HumanosRESUMEN
Aqueous two-phase protocols have been established which successfully generate highly purified preparations of small inclusion bodies (IBs) from whole cell homogenates. Particle size analysis of disruptates confirmed that intense disruption (concomitant with maximal product release) was compromised by the corelease of contaminating solutes and the micronisation of cell debris yielding a similar particle size range to the IBs (100-200 nm). PEG 300-phosphate systems enabled partial recovery of IBs in the top phase of ATPS. In contrast, PEG 8000-phosphate systems partitioned IBs more efficiently as a discrete sediment within the lower phase, whilst the majority of micronised debris remained in the interphase. The alpha-glucosidase IB yield and purity in ATPS was bettered only by analytical sucrose density gradient centrifugation, which is not readily scaleable for application in process operations. The successful recovery of such small IBs from complex homogenates highlights a generic role that ATPS techniques might play in the recovery and purification of new bioparticulate products (viral and plasmid gene therapy vectors, particulate protein vaccines etc.).
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Fraccionamiento Celular/métodos , Técnicas de Química Analítica/métodos , Cuerpos de Inclusión , Centrifugación , Centrifugación por Gradiente de Densidad , Escherichia coli/química , Fermentación , FiltraciónRESUMEN
A great deal of discussion has been generated regarding the effects of donor hypernatremia on recipient liver graft function. Much of this discussion is anecdotal and focuses on the detrimental effects of hypernatremia in the organ donor. The treatment of donor hypernatremia often leads to changes in donor management protocols. A retrospective study was conducted to determine if donor hypernatremia measurably alters the liver graft function of recipients. Results indicated that donor sodium values were unrelated to any recipient parameter assessed. Although further studies are pending, these findings suggest that a moderate rather than a severe approach to the management of donor hypernatremia may be preferred.
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Supervivencia de Injerto/fisiología , Hipernatremia/sangre , Hipernatremia/complicaciones , Trasplante de Hígado/fisiología , Trasplante de Hígado/estadística & datos numéricos , Sodio/sangre , Humanos , Hipernatremia/prevención & control , Pruebas de Función Hepática , Análisis de Regresión , Estudios RetrospectivosRESUMEN
This study was designed to investigate the virulence characteristics of Treponema denticola, T. socranskii, T. pectinovorum, and T. vincentii following challenge infection of mice. These microorganisms induced well-demarcated, dose-dependent, raised subcutaneous (s.c.) abscesses which were similar in time of onset, lesion progression, and duration of healing. Only viable cells were capable of inducing these characteristic s.c. abscesses. Histological examination of the skin lesion 3 and 5 days postinfection revealed abscess formation in the s.c. tissues, and abundant spiral organisms were demonstrated to be present in the abscess. Host resistance modulation by dexamethasone (neutrophil alteration) and cyclophosphamide (neutrophil depletion) pretreatment had a minimal effect on the virulence expression by any of these treponemes. The T. denticola isolates demonstrated significant trypsin-like protease (TLPase) activity, while both T. socranskii and T. vincentii were devoid of this activity. Interestingly, T. pectinovorum strains were heterogeneous with respect to TLPase as high producers, low producers, and nonproducers. However, no differences in lesion formation were noted regardless of whether the species expressed this proteolytic activity or whether treatment with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and dithiothreitol was performed. These results showed that (i) a murine model may be used to evaluate virulence expression by oral treponemes; (ii) while TLPase activity varies among the oral treponemes, this protease does not appear to participate in abscess induction in the mouse model; and (iii) T. pectinovorum strains show variation in TLPase activity.
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Treponema/patogenicidad , Infecciones por Treponema/microbiología , Animales , Modelos Animales de Enfermedad , Ratones , VirulenciaRESUMEN
The major protein present in the isolated outer membrane of Treponema pectinovorum ATCC 33768, MompA, was identified, purified, and characterized. Immuno-gold electron microscopy, using anti-MompA serum, and cell fractionation experiments confirmed the localization of MompA to the outer membrane. MompA was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 42 kDa when heat denatured, whereas native MompA formed a number of detergent-stable forms with molecular masses of 71, 76, and 83 kDa. A temperature of 60 degrees C was required to convert the native protein to the 42-kDa form. A number of detergents and chemical agents that are capable of breaking ionic and hydrogen bonds of proteins did not convert native MompA to the 42-kDa species. The native forms of the protein were resistant to the combined action of proteinase K, trypsin, and chymotrypsin, whereas the 42-kDa form of MompA was not. The N-terminal amino acid sequence of MompA was determined to be DVTVNINSRVRPVLYTT, and database searches did not identify any homology with known protein sequences. Amino acid compositional analysis showed the protein to be rich in proline and glycine, with these amino acids accounting for 28 and 13%, respectively, of the total amino acids. Antiserum raised against the major outer membrane protein of T. denticola GM-1 and ATCC 35405 did not cross-react with MompA, and antiserum raised against MompA did not react with any cellular components of Treponema denticola, Treponema vincentii, or Treponema socranskii. A major outer membrane protein similar in molecular mass to MompA was identified in eight clinical isolates of T. pectinovorum. The major outer membrane protein produced by four of the clinical isolates reacted strongly, by Western blotting, with anti-MompA serum, whereas proteins of the other strains did not.
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Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Treponema/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Reacciones Cruzadas , Detergentes/farmacología , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Temperatura , Treponema/ultraestructuraRESUMEN
Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.
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This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).
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Tronco Encefálico/química , Fosfatos/química , Polietilenglicoles/química , Priones/aislamiento & purificación , Extractos de Tejidos/química , Agua/química , Animales , Bovinos , Cromatografía en Agarosa , Electroforesis en Gel de Poliacrilamida , Ferritinas/química , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Péptidos/inmunología , Enfermedades por Prión/fisiopatología , Priones/inmunología , Espectrofotometría UltravioletaRESUMEN
Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis (beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an O polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the O polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.
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Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/genética , Calcio/farmacología , Caulobacter crescentus/genética , Membrana Celular/química , Lipopolisacáridos/química , Glicoproteínas de Membrana , Amino Azúcares/análisis , Proteínas Bacterianas/biosíntesis , Caulobacter crescentus/efectos de los fármacos , Caulobacter crescentus/ultraestructura , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Clonación Molecular , Desoxiazúcares/análisis , Genes Bacterianos , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/aislamiento & purificación , Modelos Estructurales , Monosacáridos/análisis , Mutación , Fenotipo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Evidence indicates that osseous healing of acute spondylolysis can occur without surgery, although no existing data supports a particular regimen that optimizes healing. This article presents a case study of a 17-year-old athletic male student who presented with acute spondylolysis and who was treated with intermittent bracing and daily external electric stimulation. The patient was treated with a thoracic lumbar sacral orthosis to which an external bone growth stimulator was added. Computer tomography scans performed throughout the treatment process and described in this report illustrate the progressive healing of the right and left pars fractures.
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Tirantes , Terapia por Estimulación Eléctrica , Vértebras Lumbares , Espondilólisis/terapia , Adolescente , Estudios de Seguimiento , Humanos , Vértebras Lumbares/diagnóstico por imagen , Masculino , Cintigrafía , Columna Vertebral/diagnóstico por imagen , Espondilólisis/diagnóstico por imagen , Factores de Tiempo , Tomografía Computarizada por Rayos XRESUMEN
The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.
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Antígenos Bacterianos/química , Caulobacter crescentus/química , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Membrana Celular/química , Galactosa/análisis , Variación Genética , Glucosa/análisis , Heptosas/análisis , Ácidos Láuricos/análisis , Lípido A/análisis , Lípidos/análisis , Manosa/análisis , Oligosacáridos/química , Azúcares Ácidos/análisisRESUMEN
Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa. All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface.