RESUMO
OBJECTIVE: Correlating mechanical forces with quantifiable physical changes in the dentoalveolar complex. SETTING AND SAMPLE POPULATION: Male 6-week C57BL/6 mice (N=3), micro X-ray-computed tomography; post-analysis software to extract physical changes in periodontal ligament (PDL)-space. MATERIALS AND METHODS: Silicone-elastic bands were placed between maxillary molars for 1 week, with the contralateral side as internal control. Average displacements between crowns and roots, and changes in PDL-spaces were evaluated by registering X-ray tomograms of experimental and control hemi-maxillae. Histology illustrated mineral formation and resorption-related events within narrowed and widened volumes of the PDL-space. RESULTS: 3D maps of changes in PDL-space between molars illustrated coronal and root displacements of 640 µm and 180 µm, respectively, compared to 70 µm in controls. Orthodontic tooth movement (OTM) specimens exhibited an average net change of -20 µm in narrowed and +30 µm in widened PDL-spaces. Bone and cementum were affected by the force on molars, and primary cementum was more affected than secondary cementum. CONCLUSIONS: This novel approach illustrates the importance of 3D-imaging and analysing 3D alveolar socket subjected to OTM otherwise omitted by 2D micrographs. A measured force on the crown elicits a response related to narrowed and widened regions in the 3D complex. OTM that exceeds PDL-space can illicit biological responses that attempt to restore physiologic PDL-space via remodelling of the periodontium. Regenerated weaker bone due to aseptic inflammation caused by orthodontics could leave patients at a higher risk of bone loss or root resorption if they later develop periodontitis, a form of septic inflammation.
Assuntos
Ligamento Periodontal/fisiologia , Coroa do Dente , Técnicas de Movimentação Dentária/métodos , Animais , Fenômenos Biomecânicos , Cemento Dentário/fisiologia , Análise do Estresse Dentário , Imageamento Tridimensional , Masculino , Maxila , Camundongos , Camundongos Endogâmicos C57BL , Ligamento Periodontal/diagnóstico por imagem , Interpretação de Imagem Radiográfica Assistida por Computador , Reabsorção da Raiz/fisiopatologia , Estresse Mecânico , Torque , Microtomografia por Raio-XRESUMO
BACKGROUND: Various minimally invasive approaches exist for the management of choledocholithiasis at the time of laparoscopic cholecystectomy. The aim of this study was to compare endoscopic retrograde cholangiopancreatography (ERCP) with laparoscopic bile duct exploration (LBDE) and test the hypothesis that intraoperative ERCP is no different to LBDE in terms of rate of bile duct clearance or retained stones. METHODS: Eligible patients with choledocholithiasis undergoing emergency laparoscopic cholecystectomy were randomized to intraoperative ERCP or LBDE in a 1 : 1 ratio. The primary outcomes were rates of bile duct clearance and retained stones. Secondary outcomes were postprocedure complication rate, mortality rate, postoperative length of hospital stay, conversion to open surgery rate, procedural time and total duration of surgery. RESULTS: Some 104 patients were randomized, and 52 patients in each group were included in an intention-to-treat analysis. Duct clearance rates were 87 per cent for patients who had intraoperative ERCP and 69 per cent for those in the LBDE group (P = 0·057). The rate of retained stones was lower in the ERCP group than in the LBDE group: 15 versus 42 per cent respectively (P = 0·004). Median postoperative length of stay was shorter with ERCP (2 days versus 3 days for LBDE; P = 0·015). CONCLUSION: Intraoperative ERCP is more effective than LBDE in terms of minimizing the rate of retained stones in patients with choledocholithiasis undergoing emergency laparoscopic cholecystectomy. REGISTRATION NUMBER: ACTRN12613000761763 (http://www.anzctr.org.au/).
Assuntos
Colangiopancreatografia Retrógrada Endoscópica , Colecistectomia Laparoscópica/métodos , Coledocolitíase/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Coledocolitíase/diagnóstico por imagem , Feminino , Seguimentos , Humanos , Análise de Intenção de Tratamento , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp-/-) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the "polymer-induced liquid-precursor" (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp-/- and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp-/- dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair.
RESUMO
BACKGROUND AND OBJECTIVE: The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses. MATERIAL AND METHODS: Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton. RESULTS: CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk. CONCLUSION: Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.
Assuntos
Osso e Ossos/fisiologia , Cemento Dentário/fisiologia , Perfilação da Expressão Gênica , Ligamento Periodontal/fisiologia , Animais , Antígenos/análise , Antígenos CD/análise , Fenômenos Biofísicos , Diferenciação Celular , Histocitoquímica , Camundongos Transgênicos , Ligamento Periodontal/citologia , Proteoglicanas/análise , Sialoglicoproteínas/análise , Fator de Transcrição Sp7 , Análise Espaço-Temporal , Fatores de Transcrição/análiseRESUMO
BACKGROUND AND OBJECTIVE: Adaptive properties of the bone-periodontal ligament-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models, such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed periodontal ligament space, and changes in the mineral level of the cortical bone and alveolar jaw bone and in the glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-periodontal ligament-cementum complex to prolonged reduced loads has not been fully explained to date, especially with regard to concurrent adaptations of bone, periodontal ligament and cementum. Therefore, in the present study, using a rat model, the temporal effect of reduced functional loads on physical characteristics, such as morphology and mechanical properties and the mineral profiles of the bone-periodontal ligament-cementum complex was investigated. MATERIAL AND METHODS: Two groups of 6-wk-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127-158 N/mm for hard pellet or 0.3-0.5 N/mm for soft powder forms. Spatio-temporal adaptation of the bone-periodontal ligament-cementum complex was identified by mapping changes in the following: (i) periodontal ligament collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro-X-ray computed tomography; (ii) mineral profiles of the periodontal ligament-cementum and periodontal ligament-bone interfaces by X-ray attenuation; and (iii) microhardness of bone and cementum by microindentation of specimens at ages 6, 8, 12 and 15 wk. RESULTS: Reduced functional loads over prolonged time resulted in the following adaptations: (i) altered periodontal ligament orientation and decreased periodontal ligament collagen birefringence, indicating decreased periodontal ligament turnover rate and decreased apical cementum resorption; (ii) a gradual increase in X-ray attenuation, owing to mineral differences, at the periodontal ligament-bone and periodontal ligament-cementum interfaces, without significant differences in the gradients for either group; (iii) significantly (p < 0.05) lower microhardness of alveolar bone (0.93 ± 0.16 GPa) and secondary cementum (0.803 ± 0.13 GPa) compared with the higher load group insert bone = (1.10 ± 0.17 and cementum = 0.940 ± 0.15 GPa, respectively) at 15 wk, indicating a temporal effect of loads on the local mineralization of bone and cementum. CONCLUSION: Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology, mechanical properties and mineral variations of the local load-bearing sites in the bone-periodontal ligament-cementum complex. These observed local changes in turn could help to explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease and/or orthodontic and prosthodontic treatments.
Assuntos
Adaptação Fisiológica , Processo Alveolar/fisiologia , Cemento Dentário/fisiologia , Análise do Estresse Dentário , Ligamento Periodontal/fisiologia , Processo Alveolar/anatomia & histologia , Processo Alveolar/química , Processo Alveolar/diagnóstico por imagem , Animais , Birrefringência , Densidade Óssea , Colágeno/ultraestrutura , Força Compressiva , Cemento Dentário/anatomia & histologia , Cemento Dentário/química , Cemento Dentário/diagnóstico por imagem , Alimentos , Dureza , Testes de Dureza , Masculino , Ligamento Periodontal/anatomia & histologia , Ligamento Periodontal/química , Ligamento Periodontal/diagnóstico por imagem , Ratos , Ratos Sprague-Dawley , Suporte de Carga , Microtomografia por Raio-XRESUMO
Silver diamine fluoride (SDF) is a dental biomaterial used to arrest dental caries. To better understand SDF's mechanism of action, we examined the localization of silver within the tissues of SDF-treated teeth. Carious primary teeth fixed within 2 min of SDF application (SDF-minutes, n = 3), at 3 wk after SDF application in vivo (SDF-weeks, n = 4), and at 2 y after multiple SDF applications in vivo (SDF-multiple, n = 1) were investigated in this study. Carious primary teeth without SDF application (no-SDF, n = 3) served as controls. Mineral density and structural analyses were performed via micro-X-ray computed tomography and scanning electron microscopy. Elemental analyses were performed through X-ray fluorescence microprobe and energy-dispersive X-ray spectroscopic techniques. SDF-treated teeth revealed higher X-ray-attenuated surface and subsurface regions within carious lesions, and similar regions were not present in no-SDF teeth. Regions of higher mineral density correlated with regions of silver abundance in SDF-treated teeth. The SDF penetration depth was approximated to 0.5 ± 0.02 mm and 0.6 ± 0.05 mm (mean ± SD) for SDF-minutes and SDF-weeks specimens, respectively. A higher percentage of dentin tubular occlusion by silver or calcium phosphate particles was observed in primary teeth treated with SDF-weeks as compared with SDF-minutes. Elemental analysis also revealed zinc abundance in carious lesions and around the pulp chamber. SDF-weeks teeth had significantly increased tertiary dentin than SDF-minutes and no-SDF teeth. These results suggest that SDF treatment on primary teeth affected by caries promotes pathologic biomineralization by altering their physicochemical properties, occluding dentin tubules, and increasing tertiary dentin volume. These seemingly serendipitous effects collectively contribute to the cariostatic activity of SDF.
Assuntos
Cárie Dentária , Biomineralização , Cariostáticos , Cárie Dentária/tratamento farmacológico , Dentina , Fluoretos Tópicos , Humanos , Compostos de Amônio Quaternário/farmacologia , Compostos de PrataRESUMO
Spatiotemporal implant-bone biomechanics and mechanoadaptive strains in peri-implant tissue are poorly understood. Physical and chemical characteristics of an implant-bone complex (IBC) were correlated in three-dimensional space (along the length and around a dental implant) to gather insights into time related integration of the implant with the cortical portion of a jaw bone in a rat. Rats (N = 9) were divided into three experimental groups with three rats per time point; 3-, 11-, and 24-day. All rats were fed crumbled hard pellets mixed with water (soft-food diet) for the first 3 days followed by a hard-food diet with intact hard-food pellets (groups of 11- and 24-day only). Biomechanics of the IBCs harvested from rats at each time point was evaluated by performing mechanical testing in situ in tandem with X-ray imaging. The effect of physical association (contact area) of a loaded implant with adapting peri-implant tissue, and resulting strain within was mapped by using digital volume correlation (DVC) technique. The IBC stiffness at respective time points was correlated with mechanical strain in peri-implant tissue. Results illustrated that IBC stiffness at 11-day was lower than that observed at 3-day. However, at 24-day, IBC stiffness recovered to that which was observed at 3-day. Correlative microscopy and spectroscopy illustrated that the lower IBC stiffness was constituted by softer and less mineralized peri-implant tissue that contained varying expressions of osteoconductive elements. Lower IBC stiffness observed at 11-day was constituted by less mineralized peri-implant tissue with osteoconductive elements that included phosphorus (P) which was co-localized with higher expression of zinc (Zn), and lower expression of calcium (Ca). Higher IBC stiffness at 24-day was constituted by mineralized peri-implant tissue with higher expressions of osteoconductive elements including Ca and P, and lower expressions of Zn. These spatiotemporal correlative maps of peri-implant tissue architecture, heterogeneous distribution of mineral density, and elemental colocalization underscore mechanoadaptive physicochemical properties of peri-implant tissue that facilitate functional osseointegration of an implant. These results provided insights into 1) plausible "prescription" of mechanical loads as an osteoinductive "therapeutic dose" to encourage osteoconductive elements in the peri-implant tissue that would facilitate functional osseointegration of the implant; 2) a "critical temporal window" between 3 and 11 days, and perhaps it is this acute phase during which key candidate regenerative molecules can be harnessed to accelerate osseointegration of an implant under load.
Assuntos
Implantes Dentários , Osseointegração , Animais , Fenômenos Biomecânicos , Ratos , TitânioRESUMO
Implant-bone biomechanics and mechanoadaptation of periimplant tissue in space (around and along the length of an implant) and time (3-, 11-, and 24-day following implantation) are important for functional osseointegration of dental implants. Spatiotemporal shifts in biomechanics of implant-bone complex in rat maxillae were correlated with maximum (tensile) and minimum (compressive) principal strain profiles in periimplant tissue using a hybrid model; biomechanics in situ paired with digital volume correlation. Spatiotemporal changes in elemental counts and their association with mineral density of the periimplant tissue were mapped using electron dispersive X-ray and X-ray fluorescence microprobe techniques. Data provided within are related to biomechanical testing of an implant-bone complex in situ. Data also highlight the power of correlating elemental colocalization with tension and compression regions of the periimplant tissues to explain spatiotemporal mechanoadaptation of implant-bone complexes. Further interpretation of data is provided in "Mechanoadaptive Strain and Functional Osseointegration of Dental Implants in Rats [1]."
Assuntos
Expressão Gênica , Interleucina-8/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/genética , Infecções por Pseudomonas/genética , RNA Mensageiro/metabolismo , Antracenos/farmacologia , Brônquios , Células Cultivadas , Células Epiteliais , Flavonoides/farmacologia , Humanos , Interleucina-8/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Pirrolidinas/farmacologia , Tiocarbamatos/farmacologiaRESUMO
AIMS: The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. METHODS AND RESULTS: Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60 degrees C and pH 6.5. Addition of 1 mmol l(-1) of Ca(2+) markedly enhanced both CMCase and avicelase activities of the enzyme. CONCLUSIONS: We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.
Assuntos
Bacillaceae/isolamento & purificação , Bacillaceae/metabolismo , Celulases/biossíntese , Celulose/metabolismo , Bacillaceae/enzimologia , Celulase/biossíntese , Celulases/classificação , Ácidos Graxos/metabolismo , RNA Ribossômico 16S/genética , Especificidade por Substrato , TemperaturaRESUMO
The load-bearing dentoalveolar fibrous joint is composed of biomechanically active periodontal ligament (PDL), bone, cementum, and the synergistic entheses of PDL-bone and PDL-cementum. Physiologic and pathologic loads on the dentoalveolar fibrous joint prompt natural shifts in strain gradients within mineralized and fibrous tissues and trigger a cascade of biochemical events within the widened and narrowed sites of the periodontal complex. This review highlights data from in situ biomechanical simulations that provide tooth movements relative to the alveolar socket. The methods and subsequent results provide a reasonable approximation of strain-regulated biochemical events resulting in mesial mineral formation and distal resorption events within microanatomical regions at the ligament-tethered/enthesial ends. These biochemical events, including expressions of biglycan, decorin, chondroitin sulfated neuroglial 2, osteopontin, and bone sialoprotein and localization of various hypertrophic progenitors, are observed at the alkaline phosphatase-positive widened site, resulting in mineral formation and osteoid/cementoid layers. On the narrowed side, tartrate-resistant acid phosphatase regions can lead to a sequence of clastic activities resulting in resorption pits in bone and cementum. These strain-regulated biochemical and subsequently biomineralization events in the load-bearing periodontal complex are critical for maintenance of the periodontal space and overall macroscale joint biomechanics.
Assuntos
Fenômenos Biomecânicos/fisiologia , Mastigação/fisiologia , Doenças da Boca/fisiopatologia , Ligamento Periodontal/fisiologia , Coroa do Dente/fisiologia , Adaptação Fisiológica , Animais , Osso e Ossos/fisiologia , Cemento Dentário/fisiologia , Análise do Estresse Dentário , HumanosRESUMO
OBJECTIVE: To determine the role of polymorphisms of genes regulating glutathione S-transferase (GST) and its plasma GST activity in the pathogenesis of chronic obstructive pulmonary disease (COPD). DESIGN: Case-control study. METHODS: One hundred and sixty-three patients with stable COPD from several community or regional hospitals were matched for age and pack-years smoked with the same number of health controls from the general population. Each participant underwent an interview-based respiratory and smoking questionnaire, lung function testing and gave a blood sample. Genotyping was carried out using a polymerase chain reaction-based method for polymorphisms of glutathione S-transferase theta 1 (GSTT1), glutathione S-transferase mu 1 (GSTM1) and glutathione S-transferase P 1 (GSTP1) genes. Plasma GST activity was measured using the spectrofluorometric method. RESULTS: There were no significant differences in the distribution of various genotypes of polymorphisms of GSTT1, GSTM1 and GSTP1 between COPD patients and healthy controls. GST activity was significantly higher in patients compared with controls, irrespective of their different genotypes, and was not different between patients with different levels of airflow obstruction. CONCLUSION: Polymorphisms of GSTT1, GSTM1 and GSTP1 genes are unlikely to be involved in the pathogenesis of COPD in Chinese in Hong Kong and Southern China.
Assuntos
Glutationa Transferase/fisiologia , Polimorfismo Genético/genética , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/genética , Fumar/metabolismo , Idoso , Povo Asiático , Estudos de Casos e Controles , Feminino , Volume Expiratório Forçado , Hong Kong , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/etnologia , Capacidade VitalRESUMO
Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.
Assuntos
Sondas de Oligonucleotídeos/genética , RNA Mensageiro/genética , Receptores de Angiotensina/genética , Ribonuclease H/genética , Animais , Sequência de Bases , Células CHO , Quimera , Mapeamento Cromossômico , Cricetinae , Biblioteca Gênica , Masculino , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Transcrição Gênica/genética , TransfecçãoRESUMO
Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.
Assuntos
Nanopartículas Calcificantes/urina , Alendronato/química , Animais , Nanopartículas Calcificantes/química , Nanopartículas Calcificantes/metabolismo , Gatos , Ácido Cítrico/química , Durapatita/química , Feminino , Corantes Fluorescentes/química , Masculino , Microscopia Eletrônica de Transmissão , Osteopontina/química , Tamanho da Partícula , Urolitíase/diagnóstico , Urolitíase/veterináriaRESUMO
We present an evaluation of systematic effects associated with a continuously rotating, ambient-temperature half-wave plate (HWP) based on two seasons of data from the Atacama B-Mode Search (ABS) experiment located in the Atacama Desert of Chile. The ABS experiment is a microwave telescope sensitive at 145 GHz. Here we present our in-field evaluation of celestial (Cosmic Microwave Background (CMB) plus galactic foreground) temperature-to-polarization leakage. We decompose the leakage into scalar, dipole, and quadrupole leakage terms. We report a scalar leakage of â¼0.01%, consistent with model expectations and an order of magnitude smaller than other CMB experiments have been reported. No significant dipole or quadrupole terms are detected; we constrain each to be <0.07% (95% confidence), limited by statistical uncertainty in our measurement. Dipole and quadrupole leakage at this level lead to systematic error on r â² 0.01 before any mitigation due to scan cross-linking or boresight rotation. The measured scalar leakage and the theoretical level of dipole and quadrupole leakage produce systematic error of r < 0.001 for the ABS survey and focal-plane layout before any data correction such as so-called deprojection. This demonstrates that ABS achieves significant beam systematic error mitigation from its HWP and shows the promise of continuously rotating HWPs for future experiments.
RESUMO
OBJECTIVES: We studied artificial dentin lesions in human teeth generated by lactate and acetate buffers (pH 5.0), the two most abundant acids in caries. The objective of this study was to determine differences in mechanical properties, mineral density profiles and ultrastructural variations of two different artificial lesions with the same approximate depth. METHODS: 0.05M (pH 5.0) acetate or lactate buffer was used to create 1) 180µm-deep lesions in non-carious human dentin blocks (acetate 130h; lactate 14days); (2) demineralized, â¼180µm-thick non-carious dentin discs (3 weeks). We performed nanoindentation to determine mechanical properties across the hydrated lesions, and micro X-ray computed tomography (MicroXCT) to determine mineral profiles. Ultrastructure in lesions was analyzed by TEM/selected area electron diffraction (SAED). Demineralized dentin discs were analyzed by small angle X-ray scattering (SAXS). RESULTS: Diffusion-dominated demineralization was shown based on the linearity between lesion depths versus the square root of exposure time in either solution, with faster kinetics in acetate buffer. Nanoindentation revealed lactate induced a significantly sharper transition in reduced elastic modulus across the lesions. MicroXCT showed lactate demineralized lesions had swelling and more disorganized matrix structure, whereas acetate lesions had abrupt X-ray absorption near the margin. At the ultrastructural level, TEM showed lactate was more effective in removing minerals from the collagenous matrix, which was confirmed by SAXS analysis. CONCLUSIONS: These findings indicated the different acids yielded lesions with different characteristics that could influence lesion formation resulting in their distinct predominance in different caries activities, and these differences may impact strategies for dentin caries remineralization.
Assuntos
Acetatos/farmacocinética , Dentina/ultraestrutura , Ácido Láctico/farmacocinética , Desmineralização do Dente , Acetatos/química , Fenômenos Biomecânicos , Módulo de Elasticidade , Dureza , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ácido Láctico/química , Microscopia Eletrônica de Transmissão , Dente Serotino , Espalhamento a Baixo Ângulo , Microtomografia por Raio-XRESUMO
The importance of finding good antisense sequences cannot be underestimated. Poor inhibition of the targeted protein can compromise the final outcome of an antisense experiment, making it difficult to arrive at a definitive understanding of the function of the protein of interest. In antisense therapeutics, identification of potent sequences becomes even more important. RNA mapping greatly increases the odds of finding active sequences. When antisense sequences are selected randomly or by gene walking, a substantial number of the oligonucleotides have little to no activity. In contrast, oligonucleotides selected by RNA mapping typically produce an antisense inhibition of greater than 50%. Oligonucleotides targeted to 60% of the accessible sites in the 5' portion of the multidrug resistance transcript inhibited P-glycoprotein function with high potency. In the angiotensin type 1 receptor system, oligonucleotides to the eight accessible sites examined inhibited AT1 receptor binding by at least 50%, with oligonucleotides to four of the sites producing at least 70% inhibition. The RNA mapping assay, which is based on standard molecular techniques, therefore provides an easy and reliable method for potent antisense sequence selection.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Sequência de BasesRESUMO
Multiple, diverse sites in the coding region of the angiotensin type-1 receptor mRNA were targeted with 2'-deoxyribonucleotide antisense oligonucleotides (ONs). The uptake of 1 microM concentration of these ONs into Chinese hamster ovary cells was facilitated by the use of cationic liposomes. The antisense sequences reduced binding of 125I-angiotensin II by 57-73%, while mismatch ONs and reverse sequence ONs produced little reduction in receptor binding. These reductions in AT1 receptor binding were accompanied by comparable decreases in AT1 receptor mRNA levels. Furthermore, mRNA cleavage fragments corresponding in size to 3'-cleavage fragments were observed with two of the antisense ONs, consistent with the involvement of an RNase H-type enzyme. When 2'-methoxyribonucleotide analogs of these same sequences were tested, AT1 receptor mRNA levels were unchanged even though small reductions in AngII binding were observed. Antisense effects seen with these 2'-methoxyribonucleotide sequences may have arisen through a translational arrest mechanism. Direct comparisons between 2'-deoxyribonucleotide analogs and their 2'-methoxyribonucleotide counterparts show that antisense effects are significantly larger when they are mediated through an RNase H-type mechanism. 2'-methoxyribonucleotide sequences were most effective when they were directed against the translation initiation codon.
Assuntos
Angiotensina I/metabolismo , Elementos Antissenso (Genética)/genética , Regulação da Expressão Gênica/fisiologia , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Ribonuclease H/metabolismo , Animais , Northern Blotting , Células CHO/química , Células CHO/enzimologia , Cricetinae , Citometria de Fluxo , Fluoresceína , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/análise , TransfecçãoRESUMO
We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.
Assuntos
Corpo Estriado/fisiologia , Técnicas de Transferência de Genes , Oligodesoxirribonucleotídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Animais , Elementos Antissenso (Genética)/farmacologia , Biotina , Química Encefálica/fisiologia , Corpo Estriado/química , Corpo Estriado/citologia , Imunofluorescência , Expressão Gênica/fisiologia , Injeções Intraventriculares , Masculino , Microscopia Confocal , Neurônios/química , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Corticotropin releasing factor (CRF) is an important regulator of the endocrine, behavioral, autonomic and immune responses to stress. Two high affinity CRF receptors have been identified, which are distributed in distinct anatomical regions. CRF(1) receptors have been relatively well characterized and antagonists to this receptor effectively block stress-induced behaviors in rodents. The function of CRF(2) receptors, which are highly expressed in limbic brain regions, is less well understood. Therefore, an antisense oligonucleotide approach was used to study the role of CRF(2) receptors in the lateral septum in rats. An antisense oligonucleotide directed against the CRF(2) receptor mRNA reduced expression of CRF(2) receptors by 60--80%. In shock-induced freezing tests, animals administered the antisense oligonucleotide exhibited a significant reduction in freezing duration. However, pain sensitivity and locomotor activity were unaltered. A four-base mismatch of the antisense sequence had no significant effects on CRF(2) receptor density and on freezing behavior. These data support the involvement of CRF(2) receptors in fear conditioning. CRF(1) receptor antagonists also reduce freezing in this test. Additional studies to determine the effects of simultaneous inhibition of both receptor subtypes show that rats receiving both CRF(2) receptor antisense oligonucleotide and CRF(1) receptor antagonist froze significantly less than animals treated with either agent alone. These results provide additional evidence for the role of CRF(2) receptors in mediating the stress-induced actions of endogenous CRF.