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Find video protocols related to scientific articles indexed in Pubmed.
Additively manufactured 3D porous Ti-6Al-4V constructs mimic trabecular bone structure and regulate osteoblast proliferation, differentiation and local factor production in a porosity and surface roughness dependent manner.
Biofabrication
PUBLISHED: 10-08-2014
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Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15-70% with compressive moduli of 2579-3693 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity, an early differentiation marker, decreased as porosity increased, while osteocalcin, a late differentiation marker, as well as osteoprotegerin, vascular endothelial growth factor and bone morphogenetic proteins 2 and 4 increased with increasing porosity. Three-dimensional (3D) constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo.
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Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination.
J Vis Exp
PUBLISHED: 10-07-2014
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Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the 'gold standard' Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. This method of production and quality control may also be suitable for other testing purposes.
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Toxic epidermal necrolysis due to voriconazole: case report and review.
Dermatol. Online J.
PUBLISHED: 09-15-2014
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Toxic epidermal necrolysis is an uncommon but potentially life-threatening adverse cutaneous drug reaction characterized by variable degrees of epidermal necrosis and detachment leading to morbidity and risk of mortality. We describe a 67-year-old woman who underwent allogeneic peripheral blood stem cell transplantation as treatment for chronic lymphocytic leukemia. She developed toxic epidermal necrolysis after she was transitioned to voriconazole, which was a component of her post-transplant regimen. The diagnosis of toxic epidermal necrolysis in our patient was made clinically and confirmed histologically. Based on the temporal initiation of voriconazole therapy and the development of her adverse cutaneous reaction, we concluded that voriconazole was the offending agent. There are limited reported cases of voriconazole-induced toxic epidermal necrolysis; we report this case to increase awareness of this potential life-threatening complication.
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Predictors of Immune Reconstitution Syndrome in Organ Transplant Recipients With Cryptococcosis: Implications for the Management of Immunosuppression.
Clin. Infect. Dis.
PUBLISHED: 09-09-2014
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?Risk factors including how changes in immunosuppression influence the occurrence of immune reconstitution syndrome (IRS) in solid organ transplant (SOT) recipients with cryptococcosis have not been fully defined.
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Donor artery aneurysm formation following the ligation of haemodialysis arteriovenous fistula: a systematic review and case reports.
J Vasc Access
PUBLISHED: 09-02-2014
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The purpose of this study is to investigate the pathogenesis, presentation and diagnosis of donor artery aneurysm formation following arteriovenous fistula (AVF) ligation and reach a consensus on their management.
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Rapidly polymerizing injectable click hydrogel therapy to delay bone growth in a murine re-synostosis model.
Biomaterials
PUBLISHED: 08-28-2014
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Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter.
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Superposition of nanostructures on microrough titanium-aluminum-vanadium alloy surfaces results in an altered integrin expression profile in osteoblasts.
Connect. Tissue Res.
PUBLISHED: 08-27-2014
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Recent studies of new surface modifications that superimpose well-defined nanostructures on microrough implants, thereby mimicking the hierarchical complexity of native bone, report synergistically enhanced osteoblast maturation and local factor production at the protein level compared to growth on surfaces that are smooth, nanorough, or microrough. Whether the complex micro/nanorough surfaces enhance the osteogenic response by triggering similar patterns of integrin receptors and their associated signaling pathways as with well-established microrough surfaces, is not well understood. Human osteoblasts (hOBs) were cultured until confluent for gene expression studies on tissue culture polystyrene (TCPS) or on titanium alloy (Ti6Al4V) disks with different surface topographies: smooth, nanorough, microrough, and micro/nanorough surfaces. mRNA expression of osteogenesis-related markers such as osteocalcin (BGLAP) and bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2), BMP4, noggin (NOG) and gremlin 1 (GREM1) were all higher on microrough and micro/nanorough surfaces, with few differences between them, compared to smooth and nanorough groups. Interestingly, expression of integrins ?1 and ?2, which interact primarily with collagens and laminin and have been commonly associated with osteoblast differentiation on microrough Ti and Ti6Al4V, were expressed at lower levels on micro/nanorough surfaces compared to microrough ones. Conversely, the ?v subunit, which binds ligands such as vitronectin, osteopontin, and bone sialoprotein among others, had higher expression on micro/nanorough surfaces concomitantly with regulation of the ?3 mRNA levels on nanomodified surfaces. These results suggest that the maturation of osteoblasts on micro/nanorough surfaces may be occurring through different integrin engagement than those established for microrough-only surfaces.
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Rapid 1?,25(OH)?D ? membrane-mediated activation of Ca²?/calmodulin-dependent protein kinase II in growth plate chondrocytes requires Pdia3, PLAA and caveolae.
Connect. Tissue Res.
PUBLISHED: 08-27-2014
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1?,25-Dihydroxy vitamin D3 [1?,25(OH)2D3] regulates growth zone chondrocytes (GC) via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. 1?,25(OH)2D3 initiates its membrane effects via its specific membrane-associated receptor (Pdia3) in caveolae. 1?,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating PLA2, resulting in prostaglandin E2 (PGE2) release and protein kinase C activation. Recently, we reported that 1?,25(OH)2D3 rapidly activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in GC cells. However, the roles of Pdia3, PLAA and caveolae in 1?,25(OH)2D3-dependent rapid activation of CaMKII are not clear. The aim of the present study was to evaluate the roles of Pdia3, PLAA and caveolae in 1?,25(OH)2D3 membrane-stimulated CaMKII activation. Pre-treating chondrocytes from the growth zone of the rat costochondral cartilage with antibodies against PLAA or Pdia3 blocked activation of CaMKII by 1?,25(OH)2D3. PLAA peptide rapidly activated CaMKII in GC cells. Caveolae disruption abolished CaMKII activation in response to 1?,25(OH)2D3 or PLAA peptide treatment. Immunoprecipitation studies showed increased CaM binding to PLAA in response to 1?,25(OH)2D3. The results indicated that Pdia3, PLAA and caveolae are required for rapid 1?,25(OH)2D3 membrane-mediated activation of CaMKII. 1?,25(OH)2D3 signaling via Pdia3 receptor triggered the interaction between PLAA and CaM suggesting that CaM may play a major role linking PLAA to CaMKII in membrane-mediated actions of 1?,25(OH)2D3.
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Anti-nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells.
Breast Cancer Res. Treat.
PUBLISHED: 08-26-2014
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The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.
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Decreasing trends in hospitalizations during anti-TNF therapy are associated with time to anti-TNF therapy: Results from two referral centres.
Dig Liver Dis
PUBLISHED: 08-22-2014
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Hospitalization is an important outcome measure and a major driver of costs in patients with inflammatory bowel disease. We analysed medical and surgical hospitalization rates and predictors of hospitalization before and during anti-TNF therapy.
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Osteoblast maturation on microtextured titanium involves paracrine regulation of bone morphogenetic protein signaling.
J Biomed Mater Res A
PUBLISHED: 08-11-2014
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Osteoblasts are sensitive to surface microtopography and chemistry. Osteoblast differentiation and maturation are higher in vitro and bone formation and osseointegration enhanced in vivo on microstructured titanium (Ti) compared to smooth surfaces. Cells increased BMP2 expression on microtextured Ti alloy, suggesting a paracrine role in regulating osteoblast maturation. However, recent studies show that exogenous BMP2 inhibits osteoblast production of anti-inflammatory cytokines and osteocalcin, indicating that control of BMP-signaling may be involved. This study examined whether cells modulate BMP ligands, receptors, and inhibitors during osteoblast maturation on Ti, specifically focusing on the roles of BMP2 and Noggin (NOG). mRNA and protein for BMP2, BMP4, and BMP7 and receptors BMPR1A, BMPR1B, and BMPR2, and BMP inhibitors were upregulated on microtextured surfaces in comparison to smooth surfaces. Maturation on microstructured Ti was slightly enhanced with exogenous BMP2 while NOG addition inhibited osteoblast maturation. Cells with NOG knocked down significantly increased osteoblast maturation. These results demonstrate that BMP-related molecules are controlled during osteoblast maturation on microstructured Ti surfaces and that endogenous NOG is an important regulator of the process. Modifying paracrine BMP signaling may yield more robust bone formation than application of exogenous BMPs. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.
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Estrogen receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast cancer cells via a membrane-associated mechanism.
Biochim. Biophys. Acta
PUBLISHED: 08-07-2014
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17?-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ER?36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17?-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ER?36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17?-estradiol pre-treatment was determined. Antibodies to ER?36, signal pathway inhibitors, ER?36 deletion mutants, and ER?36-silencing were used prior to these treatments to determine the role of ER?36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17?-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ER?36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; G?s and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17?-estradiol. Exon 9 of ER?36, a unique exon to ER?36 not found in other identified splice variants of ER? with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ER?36, suggesting that ER?36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer.
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Accuracy of computer-guided implantation in a human cadaver model.
Clin Oral Implants Res
PUBLISHED: 08-06-2014
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To examine the accuracy of computer-guided implantation using a human cadaver model with reduced experimental variability.
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Characterization of distinct classes of differential gene expression in osteoblast cultures from non-syndromic craniosynostosis bone.
J Genomics
PUBLISHED: 08-01-2014
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Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention.
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Exploring representations of protein structure for automated remote homology detection and mapping of protein structure space.
BMC Bioinformatics
PUBLISHED: 07-14-2014
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Due to rapid sequencing of genomes, there are now millions of deposited protein sequences with no known function. Fast sequence-based comparisons allow detecting close homologs for a protein of interest to transfer functional information from the homologs to the given protein. Sequence-based comparison cannot detect remote homologs, in which evolution has adjusted the sequence while largely preserving structure. Structure-based comparisons can detect remote homologs but most methods for doing so are too expensive to apply at a large scale over structural databases of proteins. Recently, fragment-based structural representations have been proposed that allow fast detection of remote homologs with reasonable accuracy. These representations have also been used to obtain linearly-reducible maps of protein structure space. It has been shown, as additionally supported from analysis in this paper that such maps preserve functional co-localization of the protein structure space.
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Effect of Cell Origin and Timing of Delivery for Stem Cell-Based Bone Tissue Engineering Using Biologically Functionalized Hydrogels.
Tissue Eng Part A
PUBLISHED: 07-11-2014
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Despite progress in bone tissue engineering, the healing of critically sized diaphyseal defects remains a clinical challenge. A stem cell-based approach is an attractive alternative to current treatment techniques. The objective of this study was to examine the ability of adult stem cells to enhance bone formation when co-delivered with the osteoinductive factor bone morphogenetic protein-2 (BMP-2) in a biologically functionalized hydrogel. First, adipose and bone marrow-derived mesenchymal stem cells (ADSCs and BMMSCs) were screened for their potential to form bone when delivered in an RGD functionalized alginate hydrogel using a subcutaneous implant model. BMMSCs co-delivered with BMP-2 produced significantly more mineralized tissue compared with either ADSCs co-delivered with BMP-2 or acellular hydrogels containing BMP-2. Next, the ability of BMMSCs to heal a critically sized diaphyseal defect with a nonhealing dose of BMP-2 was tested using the alginate hydrogel as an injectable cell carrier. The effect of timing of therapeutic delivery on bone regeneration was also tested in the diaphyseal model. A 7 day delayed injection of the hydrogel into the defect site resulted in less mineralized tissue formation than immediate delivery of the hydrogel. By 12 weeks, BMMSC-loaded hydrogels produced significantly more bone than acellular constructs regardless of immediate or delayed treatment. For immediate delivery, bridging of defects treated with BMMSC-loaded hydrogels occurred at a rate of 75% compared with a 33% bridging rate for acellular-treated defects. No bridging was observed in any of the delayed delivery samples for any of the groups. Therefore, for this cell-based bone tissue engineering approach, immediate delivery of constructs leads to an overall enhanced healing response compared with delayed delivery techniques. Further, these studies demonstrate that co-delivery of adult stem cells, specifically BMMSCs, with BMP-2 enhances bone regeneration in a critically sized femoral segmental defect compared with acellular hydrogels containing BMP-2.
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Biological control agents: from field to market, problems, and challenges.
Trends Biotechnol.
PUBLISHED: 06-27-2014
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Global food security is vulnerable due to massive growth of the human population, changes in global climate, the emergence of novel/more virulent pathogens, and demands from increasingly discerning consumers for chemical-free, sustainably produced food products. Bacterium-based biological control agents (BCAs), if used as part of an integrated management system, may satisfy the above demands. We focus on the advantages, limitations, problems, and challenges involved in such strategies.
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Membrane actions of 1?,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.
J. Steroid Biochem. Mol. Biol.
PUBLISHED: 06-24-2014
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1?,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1?,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKC? activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1?,25(OH)2D3-dependent signaling. The aim of the present study was to evaluate the roles of CaM and CaMKII as mediators of 1?,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKC?, and downstream biological effects. The results indicated that 1?,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9minutes. Silencing Cav1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against PLAA or Pdia3 blocked 1?,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1?,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1?,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1?,25(OH)2D3. The results indicate that membrane actions of 1?,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1?,25(OH)2D3.
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Osteoblast Lineage Cells Can Discriminate Microscale Topographic Features on Titanium-Aluminum-Vanadium Surfaces.
Ann Biomed Eng
PUBLISHED: 06-19-2014
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Titanium (Ti) and Ti alloys are used in orthopaedic/spine applications where biological implant fixation, or osseointegration, is required for long-term stability. These implants employ macro-scale features to provide mechanical stability until arthrodesis, features that are too large to influence healing at the cellular level. Micron-scale rough Ti alloy (Ti-6Al-4V) increases osteoblastic differentiation and osteogenic factor production in vitro and increases in vivo bone formation; however, effects of overall topography, including sub-micron scale and nanoscale features, on osteoblast lineage cells are less well appreciated. To address this, Ti6Al4V surfaces with macro/micro/nano-textures were generated using sand blasting and acid etching that had comparable average roughness values but differed in other roughness parameters (total roughness, profile roughness, maximum peak height, maximum valley depth, root-mean-squared roughness, kurtosis, skewness) (#5, #9, and #12). Human mesenchymal stem cells (HMSCs) and normal human osteoblasts (NHOst) were cultured for 7 days on the substrates and then analyzed for alkaline phosphatase activity and osteocalcin content, production of osteogenic local factors, and integrin subunit expression. All three surfaces supported osteoblastic differentiation of HMSCs and further maturation of NHOst cells, but the greatest response was seen on the #9 substrate, which had the lowest skewness and kurtosis. The #9 surface also induced highest expression of ?2 and ?1 integrin mRNA. HMSCs produced highest levels of ITGAV on #9, suggesting this integrin may play a role for early lineage cells. These results indicate that osteoblast lineage cells are sensitive to specific micro/nanostructures, even when overall macro roughness is comparable and suggest that skewness and kurtosis are important variables.
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Involvement of users and relatives in mental health service evaluation.
J. Nerv. Ment. Dis.
PUBLISHED: 06-03-2014
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Although Italian mental health (MH) services are community based, user and relative participation in service evaluation lagged behind until lately. We here review three recent studies involving stakeholder participation in service evaluation: two were quantitative studies, one on 204 users in an MH service in Pistoia (Central Italy) and the other on 2259 relatives, conducted with the National Union of Associations for Mental Health. The third (supported by The Centro per il Controllo delle Malattie, the ministerial Center for Disease Control) was a qualitative study in seven MH services, involving users, relatives, and professionals together, which collected interviews from 136 users, 119 relatives, and 79 professionals. In the quantitative studies, positive evaluations outnumbered negative ones. The qualitative study explored negative aspects in greater depth. Common findings were insufficient information, underinvolvement of users-relatives in planning, no choice of clinician, psychiatrist domination, and limited helpfulness of interventions. With stakeholder participation in service evaluation, the present medical framework will need reshaping.
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Nonmolecular methods for the diagnosis of respiratory fungal infections.
Clin. Lab. Med.
PUBLISHED: 04-12-2014
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Diagnosis of invasive fungal pneumonias by conventional culture methods is difficult to assess and often delayed. Nonmolecular fungal markers have emerged as an important adjunctive tool to support their diagnosis in combination with other clinical, radiologic, and microbiological criteria of invasive fungal diseases. Concerns about the sensitivity and specificity of some tests in different patient populations should lead to warnings about their widespread use. None can identify the emerging and particularly deadly fungal pathogens responsible for mucormycosis. The role of nonmolecular fungal markers should be better defined in combination with other microbiological and radiologic tools in preemptive antifungal strategies.
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Role of integrin ?2 ?1 in mediating osteoblastic differentiation on three-dimensional titanium scaffolds with submicron-scale texture.
J Biomed Mater Res A
PUBLISHED: 04-09-2014
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Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin ?2?1 was examined. The results showed ?1 and ?2?1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin ?2?1. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.
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Long-term stability at -20 °C of Aspergillus galactomannan in serum and bronchoalveolar lavage specimens.
J. Clin. Microbiol.
PUBLISHED: 04-09-2014
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Research to develop and validate novel methods for diagnosis of aspergillosis based on detection of galactomannan requires the use of clinical specimens that have been stored frozen. Data indicating that galactomannan remains stable when frozen are scant. The objective of this study was to determine the stability of galactomannan in clinical specimens stored at -20 °C that were positive in the Platelia Aspergillus enzyme immunoassay when initially tested. Prospective real-time testing of serum and bronchoalveolar lavage (BAL) fluid pools from positive and negative patient specimens showed no decline in galactomannan index (GMI) over 11 months at -20 °C and no development of positive reactions in the negative-control pool. Retrospective testing of positive specimens that had been stored at -20 °C for 5 years showed that 28 of 30 serum (n = 15) or BAL (n = 15) specimens remained positive. These findings support the use of frozen serum or BAL specimens stored for at least 5 years in evaluation of diagnostic tests based on detection of galactomannan.
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Role of autophagy genetic variants for the risk of Candida infections.
Med. Mycol.
PUBLISHED: 04-08-2014
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Candida albicans can cause candidemia in neutropenic and critically ill patients and oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients with low CD4(+) counts. Because all patients at risk do not develop Candida infections, it is possible that a patient's genetic background might play a role in his or her susceptibility to infection. Autophagy mediates pathogen clearance and modulation of inflammation. Our aim was to assess the effect of genetic variations in the ATG16L1 and IRGM autophagy genes on the susceptibility of patients with candidemia and oropharyngeal candidiasis. We assessed genetic variations in the ATG16L1 and IRGM genes in a cohort of candidemia patients of both African and European origin. In addition, we evaluated the effect of these polymorphisms on the susceptibility to oropharyngeal candidiasis of an HIV-positive cohort from Tanzania. Functional studies have been performed to assess the effect of the ATG16L1 and IRGM genetic variants on both in vitro and in vivo cytokine production. The results indicate that ATG16L1 variants modulate production of tumor necrosis factor-alpha, but not other cytokines, while no effects were seen in the presence of IRGM polymorphisms. In addition, no significant associations between the single-nucleotide polymorphisms in the ATG16L1 and IRGM genetic variants and the incidence of candidemia or oropharyngeal candidiasis were identified. Despite moderate effects on the modulation of proinflammatory cytokine production, genetic variation in the autophagy genes ATG16L1 and IRGM has a minor impact on the susceptibility to both mucosal and systemic Candida infections.
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Complexities associated with the molecular and proteomic identification of Paecilomyces species in the clinical mycology laboratory.
Med. Mycol.
PUBLISHED: 03-30-2014
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Paecilomyces species are emerging fungal pathogens. Morphological identifications are complicated by similarities among the members of the P. variotii complex as well as to some Rasamsonia and Hamigera species. The purpose of this study was to compare matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) with molecular diagnostic standards (i.e., multilocus DNA sequencing of the internal transcribed spacer regions 1 and 2, D1/D2 regions, and part of the ?-tubulin gene) for the identification of Paecilomyces spp. encountered in two clinical mycology laboratories. A total of 77 clinical isolates identified morphologically as P. variotii (n = 21), P. lilacinus (n = 52), and Paecilomyces spp. not otherwise specified (n = 4) were included. In accord with the most recent taxonomy, all P. lilacinus isolates were confirmed as Purpureocillium lilacinum by both sequencing and MALDI-TOF MS. Fungi phenotypically resembling P. variotii or Paecilomyces spp. were identified by molecular techniques as P. variotii sensu stricto (n = 12), P. formosus (n = 3), P. dactylethromorphus (n = 3), Rasamsonia argillacea (n = 4), or R. piperina (n = 1) and at the genus level as an isolate of a Hamigera sp. and a Paecilomyces sp. There was 92.2% (71/77) agreement between the molecular and proteomic methods only after supplementation of the MALDI-TOF MS database with type strains. Paecilomyces variotii-like organisms required multilocus DNA interrogations for differentiation and account for all of the fungi whose identification was missed by MALDI-TOF MS. Overall, MALDI-TOF MS was a rapid and reliable alternative to multilocus sequencing. However, significant augmentation of the commercially available database was required to reproducibly identify this group of important human pathogens.
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A review on the wettability of dental implant surfaces II: Biological and clinical aspects.
Acta Biomater
PUBLISHED: 03-26-2014
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Dental and orthopedic implants have been under continuous advancement to improve their interactions with bone and ensure a successful outcome for patients. Surface characteristics such as surface topography and surface chemistry can serve as design tools to enhance the biological response around the implant, with in vitro, in vivo and clinical studies confirming their effects. However, the comprehensive design of implants to promote early and long-term osseointegration requires a better understanding of the role of surface wettability and the mechanisms by which it affects the surrounding biological environment. This review provides a general overview of the available information about the contact angle values of experimental and of marketed implant surfaces, some of the techniques used to modify surface wettability of implants, and results from in vitro and clinical studies. We aim to expand the current understanding on the role of wettability of metallic implants at their interface with blood and the biological milieu, as well as with bacteria, and hard and soft tissues.
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Cordotomy in mesothelioma-related pain: a systematic review.
BMJ Support Palliat Care
PUBLISHED: 03-20-2014
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Cordotomy can be effective in relieving pain for patients with mesothelioma, but the evidence to support continued provision is limited. This review forms part of the Invasive Neurodestructive Procedures in Cancer Pain pilot study: The role of cordotomy in mesothelioma-related pain in the UK.
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Capsular serotyping of Streptococcus pneumoniae using the Quellung reaction.
J Vis Exp
PUBLISHED: 03-19-2014
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There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies. The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.
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Signaling components of the 1?,25(OH)2D3-dependent Pdia3 receptor complex are required for Wnt5a calcium-dependent signaling.
Biochim. Biophys. Acta
PUBLISHED: 03-03-2014
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Wnt5a and 1?,25(OH)2D3 are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1?,25(OH)2D3 initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A2 (PLA2)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA2, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA2 inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1?,25(OH)2D3 reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1?,25(OH)2D3-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1?,25(OH)2D3-stimulated PKC but higher Wnt5a concentrations reduced 1?,25(OH)2D3-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1?,25(OH)2D3 on PKC and CaMKII. 1?,25(OH)2D3 membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1?,25(OH)2D3 or Wnt5a treatment. This study demonstrates that 1?,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.
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Role of ?2?1 integrins in mediating cell shape on microtextured titanium surfaces.
J Biomed Mater Res A
PUBLISHED: 02-24-2014
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Surface microroughness plays an important role in determining osteoblast behavior on titanium. Previous studies have shown that osteoblast differentiation on microtextured titanium substrates is dependent on alpha-2 beta-1 (?2?1) integrin signaling. This study used focused ion beam milling and scanning electron microscopy, combined with three-dimensional image reconstruction, to investigate early interactions of individual cells with their substrate and the role of integrin ?2?1 in determining cell shape. MG63 osteoblast-like cells on sand blasted/acid etched (SLA) Ti surfaces after 3 days of culturing indicated decreased cell number, increased cell differentiation, and increased expression of mRNA levels for ?1, ?2, ?V, and ?1 integrin subunits compared to cells on smooth Ti (PT) surfaces. ?2 or ?1 silenced cells exhibited increased cell number and decreased differentiation on SLA compared to wild-type cells. Wild-type cells on SLA possessed an elongated morphology with reduced cell area, increased cell thickness, and more apparent contact points. Cells on PT exhibited greater spreading and were relatively flat. Silenced cells possessed a morphology and phenotype similar to wild-type cells grown on PT. These observations indicate that surface microroughness affects cell response via ?2?1 integrin signaling, resulting in a cell shape that promotes osteoblastic differentiation. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.
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A review on the wettability of dental implant surfaces I: theoretical and experimental aspects.
Acta Biomater
PUBLISHED: 02-14-2014
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The surface wettability of biomaterials determines the biological cascade of events at the biomaterial/host interface. Wettability is modulated by surface characteristics, such as surface chemistry and surface topography. However, the design of current implant surfaces focuses mainly on specific micro- and nanotopographical features, and is still far from predicting the concomitant wetting behavior. There is an increasing interest in understanding the wetting mechanisms of implant surfaces and the role of wettability in the biological response at the implant/bone or implant/soft tissue interface. Fundamental knowledge related to the influence of surface roughness (i.e. a quantification of surface topography) on titanium and titanium alloy surface wettability, and the different associated wetting regimes, can improve our understanding of the role of wettability of rough implant surfaces on the biological outcome. Such an approach has been applied to biomaterial surfaces only in a limited way. Focusing on titanium dental and orthopaedic implants, the present study reviews the current knowledge on the wettability of biomaterial surfaces, encompassing basic and applied aspects that include measurement techniques, thermodynamic aspects of wetting and models predicting topographical and roughness effects on the wetting behavior.
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Work disability and productivity loss in patients with inflammatory bowel diseases in Hungary in the era of biologics.
Eur J Health Econ
PUBLISHED: 01-31-2014
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To assess work disability (WD) rates in an inflammatory bowel disease (IBD) cohort involving patients with Crohn's disease (CD) or ulcerative colitis (UC) cohort and to identify possible clinical or demographic factors associated with WD. To our knowledge, this is the first study from Eastern Europe that has estimated indirect costs in IBD.
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Extracellular matrix synthesis in vascular disease: hypertension, and atherosclerosis.
J Biomed Res
PUBLISHED: 01-30-2014
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Extracellular matrix (ECM) within the vascular network provides both a structural and regulatory role. The ECM is a dynamic composite of multiple proteins that form structures connecting cells within the network. Blood vessels are distended by blood pressure and, therefore, require ECM components with elasticity yet with enough tensile strength to resist rupture. The ECM is involved in conducting mechanical signals to cells. Most importantly, ECM regulates cellular function through chemical signaling by controlling activation and bioavailability of the growth factors. Cells respond to ECM by remodeling their microenvironment which becomes dysregulated in vascular diseases such hypertension, restenosis and atherosclerosis. This review examines the cellular and ECM components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
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Evaluation of Potential Exposure to Metals in Laundered Shop Towels.
Hum Ecol Risk Assess
PUBLISHED: 01-24-2014
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We reported in 2003 that exposure to metals on laundered shop towels (LSTs) could exceed toxicity criteria. New data from LSTs used by workers in North America document the continued presence of metals in freshly laundered towels. We assessed potential exposure to metals based on concentrations of metals on the LSTs, estimates of LST usage by employees, and the transfer of metals from LST-to-hand, hand-to-mouth, and LST-to-lip, under average- or high-exposure scenarios. Exposure estimates were compared to toxicity criteria. Under an average-exposure scenario (excluding metals' data outliers), exceedances of the California Environmental Protection Agency, U.S. Environmental Protection Agency, and the Agency for Toxic Substances and Disease Registry toxicity criteria may occur for aluminum, cadmium, cobalt, copper, iron, and lead. Calculated intakes for these metals were up to more than 400-fold higher (lead) than their respective toxicity criterion. For the high-exposure scenario, additional exceedances may occur, and high-exposure intakes were up to 1,170-fold higher (lead) than their respective toxicity criterion. A sensitivity analysis indicated that alternate plausible assumptions could increase or decrease the magnitude of exceedances, but were unlikely to eliminate certain exceedances, particularly for lead.
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Implant osseointegration and the role of microroughness and nanostructures: lessons for spine implants.
Acta Biomater
PUBLISHED: 01-14-2014
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The use of spinal implants for spine fusion has been steadily increasing to avoid the risks of complications and donor site morbidity involved when using autologous bone. A variety of fusion cages are clinically available, with different shapes and chemical compositions. However, detailed information about their surface properties and the effects of such properties on osteogenesis is lacking in the literature. Here we evaluate the role of surface properties for spinal implant applications, covering some of the key biological processes that occur around an implant and focusing on the role of surface properties, specifically the surface structure, on osseointegration, drawing examples from other implantology fields when required. Our findings revealed that surface properties such as microroughness and nanostructures can directly affect early cell behavior and long-term osseointegration. Microroughness has been well established in the literature to have a beneficial effect on osseointegration of implants. In the case of the role of nanostructures, the number of reports is increasing and most studies reveal a positive effect from the nanostructures alone and a synergistic effect when combined with microrough surfaces. Long-term clinical results are nevertheless necessary to establish the full implications of surface nanomodifications.
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Impaired Bone Formation in Pdia3 Deficient Mice.
PLoS ONE
PUBLISHED: 01-01-2014
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1?,25-dihydroxyvitamin D3 [1?,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1?,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/- heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/- mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/- mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/- mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/- heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1?,25(OH)2D3's actions in regulating skeletal development.
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Aortic Carboxypeptidase-like Protein Enhances Lung Myofibroblast Differentiation Through Transforming Growth Factor ? Receptor Dependent and Independent Pathways.
J. Biol. Chem.
PUBLISHED: 12-16-2013
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Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix (ECM) remodeling and accumulation of active transforming growth factor ? (TGF?), which promotes collagen expression and the differentiation of smooth muscle ?-actin (SMA)-positive myofibroblasts. Aortic Carboxypeptidase-like Protein (ACLP) is an ECM protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP (rACLP) induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast to myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGF? signaling. Treatment of fibroblasts with rACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGF? receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGF? receptor. These findings indicate that ACLP stimulates the fibroblast to myofibroblast transition by promoting SMA expression via TGF? signaling and promoting collagen expression through a TGF? receptor independent pathway.
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New insights on membrane mediated effects of 1?,25-dihydroxy vitamin D3 signaling in the musculoskeletal system.
Steroids
PUBLISHED: 12-01-2013
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1?,25-Dihydroxy vitamin D3 [1?,25(OH)2D3] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. Two receptors have been implicated to play roles in 1?,25(OH)2D3 mediated rapid signaling, the classical nuclear vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3). Long term efforts to investigate the roles of these two receptors demonstrated thatPdia3 is located in caveolae, where it interacts with phospholipase A2 (PLA2) activating protein (PLAA) and caveolin-1 (Cav-1) to initiate rapid signaling via Ca(++)/calmodulin-dependent protein kinase II (CaMKII), PLA2, phospholipase C (PLC), protein kinase C (PKC), and ultimately the ERK1/2 family of mitogen activated protein kinases (MAPK). VDR is present on the plasma membrane, and it is required for 1?,25(OH)2D3 induced rapid activation of Src. PDIA3+/- mice demonstrate an impaired musculoskeletal phenotype. Moreover, our studies examining mineralization of pre-osteoblasts in 3D culture have shown the physiological importance of Pdia3 and VDR interaction: knockdown of Pdia3 or VDR is characterized by impaired mineralization of the constructs.
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Quality Assessment of Mental Health Care by People with Severe Mental Disorders: A Participatory Research Project.
Community Ment Health J
PUBLISHED: 11-30-2013
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This study assessed the perceived quality of care by consumers with severe mental disorders. A questionnaire investigating service quality was developed by a consumer focus group and filled by 204 consumers. In five areas the negative evaluations exceeded or closely approximated the positive ones: choice of professionals, waiting times, information about illness and medications. All five do not refer to the outcomes of care, but to the concept of responsiveness. The results confirmed that people with severe mental disorders can give value judgments on various aspects of care. However, even in a service strongly oriented towards community care, the consumers needs in sensitive areas concerning choices, respect and autonomy are not met. The application of the concept of responsiveness to quality improvement may help services to meet consumers expectations.
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Role of ER?36 in membrane-associated signaling by estrogen.
Steroids
PUBLISHED: 11-16-2013
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Traditionally, steroid hormones such as the vitamin D3 metabolites, testosterone and dihydrotesterone, and 17?-estradiol act through cytosolic and nuclear receptors that directly interact with DNA to alter gene transcription and regulate cellular development. However, recent studies focused on rapid and membrane effects of steroid hormones have given invaluable insight into their non-classical mechanisms of action. In some cases, the traditional receptors were implicated as acting also in the plasma membrane as membrane-associated receptors. However, recent data has demonstrated the presence of an alternative splicing variant to traditional estrogen receptor ? known as ER?36, which is present in the plasma membranes of several different cell types including several cancer cell types and even in some normal cells including cartilage and bone cells. The physiological effects that result from the membrane activation of ER?36 may vary from one cell type to another, but the mechanism of action appears to use similar pathway such as the activation of various protein kinases and phospholipases leading to the activation of signaling cascades that result in rapid, non-genomic responses. These rapid responses can affect cell proliferation and apoptotic signaling, indirectly activate downstream genomic signaling through phosphorylation cascades of transcription factors, and crosstalk with classical pathways via interaction with classical receptors. This review describe the data from the last several years discusses the non-classical, rapid, and membrane-associated cellular responses to steroid hormones, particularly 17?-estradiol, through the classical receptors ER? and ER? and various non-classical receptors, especially estrogen receptor-?36 (ER?36).
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Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation.
J Biomech
PUBLISHED: 11-01-2013
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Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Youngs modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.
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Risk of colorectal cancer in Crohns disease patients with colonic involvement and stenosing disease in a population-based cohort from Hungary.
J Gastrointestin Liver Dis
PUBLISHED: 10-01-2013
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Since data is limited regarding the risk of colorectal cancer (CRC) in Crohns disease (CD) patients who present with stenosing disease in the colon, this study was undertaken to assess CRC risk in such patients, using a population-based, Veszprem province database, which includes incidental patients diagnosed between January 1, 1977 and December 31, 2011.
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Evaluation of the carcinogenicity of inorganic arsenic.
Crit. Rev. Toxicol.
PUBLISHED: 09-18-2013
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Inorganic arsenic (iAs) at high exposures is a human carcinogen, affecting mainly the urinary bladder, lung and skin. We present an assessment of the mode of action (MOA) of iAss carcinogenicity based on the United States Environmental Protection Agency/International Programme on Chemical Safety (USEPA/IPCS) framework, focusing primarily on bladder cancer. Evidence is presented for a MOA involving formation of reactive trivalent metabolites interacting with critical cellular sulfhydryl groups, leading to cytotoxicity and regenerative cell proliferation. Metabolism, kinetics, cell transport, and reaction with specific proteins play a critical role in producing the effects at the cellular level, regardless of cell type, whether urothelium, lung epithelium or epidermis. The cytotoxicity induced by iAs results in non-cancer toxicities, and the regenerative cell proliferation enhances development of epithelial cancers. In other tissues, such as vascular endothelium, different toxicities develop, not cancer. Evidence supporting this MOA comes from in vitro investigations on animal and human cells, from animal models, and from epidemiological studies. This MOA implies a non-linear, threshold dose-response relationship for both non-cancer and cancer end points. The no effect levels in animal models (approximately 1?ppm of water or diet) and in vitro (>0.1?µM trivalent arsenicals) are strikingly consistent. Cancer effects of iAs in humans generally are not observed below exposures of 100-150?ppb in drinking water: below these exposures, human urine concentrations of trivalent metabolites are generally below 0.1?µM, a concentration not associated with bladder cell cytotoxicity in in vitro or animal models. Environmental exposures to iAs in most of the United States do not approach this threshold.
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Evaluating the additivity of perfluoroalkyl acids in binary combinations on peroxisome proliferator-activated receptor-? activation.
Toxicology
PUBLISHED: 08-07-2013
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Perfluoroalkyl acids (PFAAs) are found globally in the environment, detected in humans and wildlife, and are typically present as mixtures of PFAA congeners. Mechanistic studies have found that responses to PFAAs are mediated in part by PPAR?. Our previous studies showed that individual PFAAs activate PPAR? transfected into COS-1 cells. The goal of the current study was to determine if binary combinations of perfluorooctanoic acid (PFOA) and another PFAA act in an additive fashion to activate PPAR? in the mouse one-hybrid in vitro model. COS-1 cells were transiently transfected with mouse PPAR? luciferase reporter construct and exposed to either vehicle control (0.1% DMSO or water), PPAR? agonist (WY14643, 10?M), PFOA at 1-128?M, perfluorononanoic acid (PFNA) at 1-128?M, perfluorohexanoic acid (PFHxA) at 8-1024?M, perfluorooctane sulfonate (PFOS) at 4-384?M or perfluorohexane sulfonate (PFHxS) at 8-2048?M to generate sigmoidal concentration-response curves. In addition, cells were exposed to binary combinations of PFOA+either PFNA, PFHxA, PFOS or PFHxS in an 8×8 factorial design. The concentration-response data for individual chemicals were fit to sigmoidal curves and analyzed with nonlinear regression to generate EC50s and Hillslopes, which were used in response-addition and concentration-addition models to calculate predicted responses for mixtures in the same plate. All PFOA+PFAA combinations produced concentration-response curves that were closely aligned with the predicted curves for both response addition and concentration addition at low concentrations. However, at higher concentrations of all chemicals, the observed response curves deviated from the predicted models of additivity. We conclude that binary combinations of PFAAs behave additively at the lower concentration ranges in activating PPAR? in this in vitro system.
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17Beta-Estradiol Promotes Aggressive Laryngeal Cancer Through Membrane-Associated Estrogen Receptor-Alpha 36.
Horm Cancer
PUBLISHED: 07-25-2013
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17?-estradiol (E2) plays a key role in tumorigenesis by enhancing cell survivability and metastasis through its cytoplasmic receptors. Recently, a variant of estrogen receptor alpha, ER?36 has been implicated as a substantial mediator of E2s proliferative and antiapoptotic effects through rapid membrane-associated signaling, and cancers previously regarded as hormone-independent due to the absence of traditional receptors, may in fact be susceptible to E2. Despite rising from a secondary sex organ and having a clear gender disposition, laryngeal cancer is not uniformly accepted as hormone dependent, even in the face of compelling evidence of E2 responsiveness. The aim of this study was to further elucidate the role of E2 in the tumorigenesis of laryngeal cancer, both in vitro and in vivo. ER?36 presence was evaluated in membranes of the laryngeal carcinoma cell line, Hep2, as well as in laryngeal tumor samples. In vitro ER?36 was found to mediate rapid activation of protein kinase C and phospholipase D by E2, leading to increased proliferation and protection against chemotherapy-induced apoptosis. Furthermore, in response to E2 activation of ER?36, an upregulation of angiogenic and metastatic factors was observed. Clinical analysis of laryngeal tumors revealed a similar association between the amount of ER?36 and VEGF and indicated a role in lymph node metastasis. These findings present compelling evidence of ER?36-dependent E2 signaling in laryngeal cancer. Thus, targeting ER?36 may reduce the deleterious effects of E2 in laryngeal cancer, ultimately suggesting the importance of antiestrogen therapy or the production of novel drugs that specifically target ER?36.
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Electrical polarization of titanium surfaces for the enhancement of osteoblast differentiation.
Bioelectromagnetics
PUBLISHED: 07-24-2013
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Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner.
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Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1?,25(OH)(2)D(3).
Cell. Signal.
PUBLISHED: 07-02-2013
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1?,25-Dihydroxyvitamin D3 (1?,25(OH)2D3) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1?,25(OH)2D3. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1?,25(OH)2D3 changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1?,25(OH)2D3 regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1?,25(OH)2D3 and subsequently regulating osteoblast biology.
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Osteoblast response to nanocrystalline calcium hydroxyapatite depends on carbonate content.
J Biomed Mater Res A
PUBLISHED: 06-20-2013
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Normal bone mineral is a carbonated-apatite, but there are limited data on the effect of mineral containing carbonate on cell response. We characterized surface chemical compositions of three experimental carbonated hydroxyapatite (CO3 (2-) HA) substrates and investigated their effect on osteoblast differentiation. Carbonate was incorporated into the hydroxyapatite powders while phosphate and hydroxyl groups were shown to be reduced by analyzing the chemical composition of the substrate surfaces. CO3 (2) (-) HA powders with increasing carbonate concentrations designated as C1 (3.88%), C2 (4.85%), and C3 (5.82%) were molded, pressed, and fired into 14 mm discs. We observed that calcium phosphate ratios increased monotonically with increasing carbonate content, whereas differentiation of MG63 cells decreased. CO3 (2) (-) HA surfaces also affected factor production. Addition of carbonate caused a 70% reduction in osteoprotegerin (OPG) compared to cultures on pure HA, but the effect of carbonate was not dose-dependent. Low carbonate content reduced VEGF-A by 80%, but higher levels of carbonate reversed this effect in a concentration dependent manner, with the C3 VEFG-A levels approximately twice that of C1 levels. These observations collectively indicate that bone cells are sensitive to carbonate content in bone mineral and the effects of carbonate substitution vary with the outcome being measured. Overall, this study provides a preliminary understanding of how carbonate substitution within hydroxyapatite modulates cellular behavior. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
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CX3CR1-dependent renal macrophage survival promotes Candida control and host survival.
J. Clin. Invest.
PUBLISHED: 05-29-2013
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Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.
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Chaperone properties of pdia3 participate in rapid membrane actions of 1?,25-dihydroxyvitamin d3.
Mol. Endocrinol.
PUBLISHED: 05-09-2013
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Protein disulfide isomerase family A, member 3 (Pdia3) mediates many of the plasma membrane (PM)-associated rapid responses to 1?,25-dihydroxyvitamin D3 (1?,25[OH]2D3). It is not well understood how Pdia3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1?,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for Pdia3s ER chaperone function, and examined their role in responses to 1?,25(OH)2D3. Pdia3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for Pdia3. Finally, we determined whether palmitoylation and/or myristoylation were required for Pdia3-mediated responses to 1?,25(OH)2D3. Overexpressing the Pdia3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1?,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overexpressing wild-type Pdia3 (Pdia3Ovr cells). Cells overexpressing Pdia3 with K214A and C406S mutations had PKC activity comparable to untreated controls, indicating that the native response to 1?,25(OH)2D3 also was blocked. Overexpressing Pdia3[-KDEL] increased PM localization and augmented baseline PKC, but the stimulatory effect of 1?,25(OH)2D3 was comparable to that seen in wild-type cultures. In contrast, 1?,25(OH)2D3 increased prostaglandin E2 in Pdia3[±KDEL] cells. Although neither palmitoylation nor myristoylation was required for PM association of Pdia3, myristoylation was needed for PKC activation. These data indicate that both the chaperone functional domains and the subcellular location of Pdia3 control rapid membrane responses to 1?,25(OH)2D3.
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Social support provided by and strain experienced by African-American cancer caregivers.
Support Care Cancer
PUBLISHED: 05-07-2013
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Cancer is the second most common condition among people over 50, behind only dementia, associated with caregiving. As treatments improve, the number of cancer caregivers will increase. However, there is limited research about African-American cancer caregivers (AACCs).
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Controlled release of rat adipose-derived stem cells from alginate microbeads.
Biomaterials
PUBLISHED: 05-06-2013
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Cell-based therapies have potential for tissue regeneration but poor delivery methods lead to low viability or dispersal of cells from target sites, limiting clinical utility. Here, we developed a degradable and injectable hydrogel to deliver stem cells for bone regeneration. Alginate microbeads <200 ?m are injectable, persist at implantation sites and contain viable cells, but do not readily degrade in-vivo. We hypothesized that controlled release of rat adipose-derived stem cells (ASCs) from alginate microbeads can be achieved by incorporating alginate-lyase in the hydrogel. Microbeads were formed using high electrostatic potential. Controlled degradation was achieved through direct combination of alginate-lyase and alginate at 4 °C. Results showed that microbead degradation and cell release depended on the alginate-lyase to alginate ratio. Viability of released cells ranged from 87% on day 2 to 71% on day 12. Monolayer cultures of released ASCs grown in osteogenic medium produced higher levels of osteocalcin and similar levels of other soluble factors as ASCs that were neither previously encapsulated nor exposed to alginate-lyase. Bmp2, Fgf2, and Vegfa mRNA in released cells were also increased. Thus, this delivery system allows for controlled release of viable cells and can modulate their downstream osteogenic factor production.
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Development and evaluation of a calibrator material for nucleic acid-based assays for diagnosing aspergillosis.
J. Clin. Microbiol.
PUBLISHED: 04-24-2013
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Twelve laboratories evaluated candidate material for an Aspergillus DNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 10(10) units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid.
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Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1?,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.
J Tissue Eng Regen Med
PUBLISHED: 04-09-2013
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1?,25-Dihydroxyvitamin D3 [1?,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1?,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1?,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1?,25(OH)2D3, or BMP2, or with 1?,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(?-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1?,25(OH)2D3?+?BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1?,25(OH)2D3?+?BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (?CT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1?,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1?,25(OH)2D3?+?BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1?,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright © 2013 John Wiley & Sons, Ltd.
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Biphasic fusion of the murine posterior frontal suture.
Plast. Reconstr. Surg.
PUBLISHED: 04-02-2013
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Craniosynostosis is the premature fusion of cranial sutures early in development. Mice are commonly used to study the mechanisms driving both normal and pathologic cranial suture development. Despite their frequency of use as a model, the time course of bone formation and mineralization during fusion of mouse posterior frontal suture is not well defined.
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Tailoring adipose stem cell trophic factor production with differentiation medium components to regenerate chondral defects.
Tissue Eng Part A
PUBLISHED: 03-28-2013
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Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50??g/mL ascorbic acid-2-phosphate (AA2P), 100?nM dexamethasone (Dex), 10?ng/mL transforming growth factor (TGF)-?1, and 100?ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-?1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24?h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-?2, TGF-?3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-?2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-?2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-?1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-?2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASCs production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.
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Rough titanium alloys regulate osteoblast production of angiogenic factors.
Spine J
PUBLISHED: 03-20-2013
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Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin ?2?1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK.
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Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations.
Clin. Infect. Dis.
PUBLISHED: 03-13-2013
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Fluconazole (FLC) resistance is common in C. glabrata and echinocandins are often used as first-line therapy. Resistance to echinocandin therapy has been associated with FKS1 and FKS2 gene alterations.
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Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes.
Toxicology
PUBLISHED: 02-13-2013
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While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPAR?). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPAR?. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPAR? agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPAR? in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPAR?. Rankings were conducted on the limited dataset. In mouse hepatocytes, the pattern was similar to that previously observed in the COS-1 reporter cell assay. With the exception of PFHxA, longer chain PFAA carboxylates were the most active. The pattern was similar in human hepatocytes, although PFDA and PFOS showed higher activity than previously observed while PFOA showed somewhat less activity. These data reflect inherent challenges in using primary hepatocytes to predict toxicological response.
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Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group.
Vaccine
PUBLISHED: 02-08-2013
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In 2003 the World Health Organization (WHO) convened a working group and published a set of standard methods for studies measuring nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus). The working group recently reconvened under the auspices of the WHO and updated the consensus standard methods. These methods describe the collection, transport and storage of nasopharyngeal samples, as well as provide recommendations for the identification and serotyping of pneumococci using culture and non-culture based approaches. We outline the consensus position of the working group, the evidence supporting this position, areas worthy of future research, and the epidemiological role of carriage studies. Adherence to these methods will reduce variability in the conduct of pneumococcal carriage studies undertaken in the context of pneumococcal vaccine trials, implementation studies, and epidemiology studies more generally so variability in methodology does not confound the interpretation of study findings.
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Testing for departures from additivity in mixtures of perfluoroalkyl acids (PFAAs).
Toxicology
PUBLISHED: 02-07-2013
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This study is a follow-up to a paper by Carr et al. that determined a design structure to optimally test for departures from additivity in a fixed ratio mixture of four perfluoroalkyl acids (PFAAs) using an in vitro transiently-transfected COS-1 PPAR? reporter model with a mixing ratio that is based on average serum levels in NHANES subjects. Availability of information regarding potential for additivity of PFAAs in mixtures is critically important for risk assessors who are concerned with the ability of the compounds to affect human health and impact ecological systems. It is clear that exposures are not to single compounds, but to mixtures of the PFAAs. This paper presents the results from the data collected using the design from Carr et al. along with subsequent analyses that were performed to classify the relationships among mixtures of PFAAs. A non-linear logistic additivity model was employed to predict relative luciferase units (RLU), an indicator of PPAR? activation. The results indicated a less than additive relationship among the four PFAAs. To determine if the possible "antagonism" is from the competition among or between carboxylates and sulfonates, four different binary mixtures were also studied. There was a less than additive relationship in all four binary mixtures. These findings are generally similar to two other reports of interfering interactions between PFAAs in mixtures. The most conservative interpretation for our data would be an assumption of additivity (and lack of a greater than additive interaction), with a potential for antagonistic interactions.
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Hormonal modulation of connective tissue homeostasis and sex differences in risk for osteoarthritis of the knee.
Biol Sex Differ
PUBLISHED: 02-04-2013
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Young female athletes experience a higher incidence of ligament injuries than their male counterparts, females experience a higher incidence of joint hypermobility syndrome (a risk factor for osteoarthritis development), and post-menopausal females experience a higher prevalence of osteoarthritis than age-matched males. These observations indicate that fluctuating sex hormone levels in young females and loss of ovarian sex hormone production due to menopause likely contribute to observed sex differences in knee joint function and risk for loss of function. In studies of osteoarthritis, however, there is a general lack of appreciation for the heterogeneity of hormonal control in both women and men. Progress in this field is limited by the relatively few preclinical osteoarthritis models, and that most of the work with established models uses only male animals. To elucidate sex differences in osteoarthritis, it is important to examine sex hormone mechanisms in cells from knee tissues and the sexual dimorphism in the role of inflammation at the cell, tissue, and organ levels. There is a need to determine if the risk for loss of knee function and integrity in females is restricted to only the knee or if sex-specific changes in other tissues play a role. This paper discusses these gaps in knowledge and suggests remedies.
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Production of latex agglutination reagents for pneumococcal serotyping.
BMC Res Notes
PUBLISHED: 01-24-2013
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The current gold standard for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping.
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Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17?-estradiol involves differential regulation of plasma membrane associated estrogen receptors.
Biochim. Biophys. Acta
PUBLISHED: 01-07-2013
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Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17?-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ER? in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ER? (propylpyrazole triol, PPT) and ER? (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2s effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3F?S activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.
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Silica desiccant packets for storage and transport of Streptococcus pneumoniae and other clinically relevant species.
PLoS ONE
PUBLISHED: 01-01-2013
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Bacterial isolates are often transported between laboratories for research and diagnostic purposes. Silica desiccant packets (SDPs), which are inexpensive and do not require freezing, were evaluated for storage and recovery of bacterial isolates. Conditions such as inoculum size, swab type and temperature of storage were investigated using ten Streptococcus pneumoniae isolates. The optimized protocol was then tested using 49 additional S. pneumoniae isolates representing 40 serogroups. Overall, S. pneumoniae growth was considered satisfactory (>100 colony forming units) for 98/109 (89.9%) and 20/20 (100%) swabs after 14 days at room temperature or 28 days at 4° C, respectively. Storage in SDPs did not impact on the ability of S. pneumoniae isolates to be subsequently serotyped. When the survival of nine other clinically relevant bacterial species was tested, seven were viable after 28 days at room temperature, the exceptions being Neisseria gonorrhoeae and Haemophilus influenzae. SDPs are suitable for transport and short-term storage of bacterial species including S. pneumoniae.
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Fungal diagnostics in pneumonia.
Semin Respir Crit Care Med
PUBLISHED: 12-13-2011
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Fungal pneumonia is increasingly common, particularly in highly immunosuppressed patients, such as solid organ or hematopoietic stem cell transplant recipients, and the diagnosis is evolving. Although standard techniques such as microscopy and culture remain the mainstays of diagnosis, relatively recent advances in serological and molecular testing are important additions to the field. This article reviews the laboratory tools used to diagnose fungal respiratory disease.
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Cytokine gene polymorphisms and the outcome of invasive candidiasis: a prospective cohort study.
Clin. Infect. Dis.
PUBLISHED: 12-05-2011
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?Candida bloodstream infections cause significant morbidity and mortality among hospitalized patients. Although clinical and microbiological factors affecting prognosis have been identified, the impact of genetic variation in the innate immune responses mediated by cytokines on outcomes of infection remains to be studied.
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Myocardin-related transcription factor-A complexes activate type I collagen expression in lung fibroblasts.
J. Biol. Chem.
PUBLISHED: 11-02-2011
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Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell ?-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.