110 research outputs found
A Three-Protein Panel to Support the Diagnosis of Sepsis in Children
Sepsis is a syndrome without a standard validated diagnostic test. Early recognition is crucial. Serum proteome analysis in children with sepsis may identify new biomarkers. This study aimed to find suitable blood biomarkers for an early diagnosis of sepsis. An analytical observational case-control study was carried out in a single center. Children admitted to a Pediatric Intensive Care Unit with clinical diagnosed sepsis were eligible for study. A proteomic analysis conducted by mass spectrometry was performed. Forty patients with sepsis and 24 healthy donors were recruited. Proteomics results revealed 44 proteins differentially expressed between patients and healthy controls. Six proteins were selected to be validated: lactoferrin, serum amyloid-A1 (SAA-1), complement factor B, leucine-rich alpha-2 glycoprotein (LRG1), soluble interleukin-2 alpha chain receptor (sCD25) and soluble haptoglobin-hemoglobin receptor. Our results showed that sCD25, SAA-1, and LRG1 had high levels of specificity and sensitivity, as well as an excellent area under the ROC curve (>0.9). Our study provides a serum proteomic analysis that identifies new diagnostic biomarkers in sepsis. SAA-1, sCD25 and LRG1 were able to separate septic from healthy donor, so they could be used together with other clinical and analytical features to improve sepsis diagnosis in children.This work was funded by Research Projects from University of Basque Country (US10/02) and from the Basque Government (SAIO10-PE10BF02, SAIO12-PE12BF002, 2012111052, 2019111056). CICbioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County, the ProteoRed-ISCIII (Grant PRB3 IPT17/0019), CIBERehd Network and Severo Ochoa Grant (SEV-2016-0644)
Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo
The interactions of implanted biomaterials with the host organism determine the success or failure of an implantation. Normally, their biocompatibility is assessed using in vitro tests. Unfortunately, in vitro and in vivo results are not always concordant; new, effective methods of biomaterial characterisation are urgently needed to predict the in vivo outcome. As the first layer of proteins adsorbed onto the biomaterial surfaces might condition the host response, mass spectrometry analysis was performed to characterise these proteins. Four distinct hybrid sol-gel biomaterials were tested. The in vitro results were similar for all the materials examined here. However, in vivo, the materials behaved differently. Six of the 171 adsorbed proteins were significantly more abundant on the materials with weak biocompatibility; these proteins are associated with the complement pathway. Thus, protein analysis might be a suitable tool to predict the in vivo outcomes of implantations using newly formulated biomaterials
Development and characterisation of strontium-doped sol-gel coatings to optimise the initial bone regeneration processes
Strontium plays an important role in bone regeneration; it promotes the differentiation and maturation of osteoblasts and inhibits the activity of osteoclasts. Our principal objective in this study was to formulate new organic-inorganic hybrid sol-gel coatings applied to titanium discs. These coatings were functionalised with different amounts of SrCl2 and examined using in vitro tests and proteomics. The chemical and morphological characteristics of obtained coatings were scrutinised. The in vitro evaluation using the MC3T3-E1 osteoblasts and RAW264.7 macrophages showed the osteogenic and anti-inflammatory effects of strontium doping. The proteomic assay identified 111 different proteins adhering to the coatings. Six of these proteins reduced their adhesion affinity as a result of Sr-doping, whereas 40 showed increased affinity. Moreover, the proteomic analysis revealed osteogenic and anti-inflammatory properties of these biomaterials. The analysis also showed increased adhesion of proteins related to the coagulation system. We can conclude that proteomic methods are invaluable in developing new biomaterials and represent an important tool for predicting the biocompatibility of dental implants
Structural basis for assembly of vertical single β-barrel viruses
The vertical double beta-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 angstrom resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single beta-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double beta-barrel MCPs.Peer reviewe
Identification of a panel of serum protein markers in early stage of sepsis and its validation in a cohort of patients
Background: Sepsis is a life-threatening illness with a challenging diagnosis. Current serum biomarkers are not sensitive enough for diagnosis. With the aim of finding proteins associated with sepsis, serum protein profile was compared between patients and healthy donors and serum classical inflammatory proteins were analyzed in both groups. Methods: Serum protein profiles were characterized by two-dimensional electrophoresis (2DE). Identification of the proteins was carried out by mass spectrophotometry and their validation was performed by Enzyme-Linked-lmmunoSorbent Assay (ELISA) in a cohort of 85 patients and 67 healthy donors. Seven classical inflammatory proteins were analyzed in the same cohort by ELISA: interleukin-2 receptor alpha-chain (sCD25), scavenger receptor cysteine
Osteointegración de implantes de titanio con superficies activas. Un estudio proteómico
Titanium is a biomaterial largely used on dental implant
manufacturing. However, as a consequence of its
intrinsically low bioactivity, the development of distinct
superficial treatments in order to enhance its osseointegration
properties is being studied. In this sense,
the use of titanium implants with a higher level of
roughness has been broadened, recurring to the application
of sand-blasted acid-etched
surface treatments. In this
article, a study of two distinct
titanium surface treatments
has been carried out, regarding
the physico-chemical
properties (roughness, hydrophilicity
and chemical composition)
of each, as well as the
pattern of adhered proteins onto
each surface (proteomic study).
Hence, mass spectrometry
analysis allowed the detection
of 2 18 d istinct a dsorbed p roteins,
being 37 of those related
to bone regenerative processes
and dental implant integration.
Moreover, using differential
quantification between associated proteins, comparing
surfaces, it was observable a greater affinity of
APOE, ANT3 and PROC proteins to the treated surface,
directly linked to the bone regenerative process.
On the other hand, the treated surface displays lower
affinity of CO3 protein. The variations between the
adsorbed protein profiles could be an explanation for
distinct in vivo outcomes.El titanio es un biomaterial ampliamente empleado en
la fabricación de implantes dentales, sin embargo, como
consecuencia de su baja bioactividad se han desarrollado
distintos tratamientos superficiales buscando
una mejora en su capacidad de osteintegración. De esta
forma, se ha extendido el uso de implantes de titanio
con un mayor grado de rugosidad gracias a la aplicación
de un tratamiento de granallado, al que le sigue
un tratamiento de ataque ácido. En este artículo se ha
llevado a cabo un estudio de discos de titanio con dos
tipos de superficie: sin tratamiento alguno y con tratamiento
de granallado más ataque ácido. El estudio
reveló diferencias físico-químicas (rugosidad, hidrofilia
y composición química) tras la aplicación del tratamiento
superficial, pero también en cuanto al perfil
de proteínas adheridas a cada superficie (estudio
proteómico). Así, la espectrometría de masas permitió
la caracterización de las proteínas adsorbidas en ambos
tipos de superficies. El análisis permitió la identificación
de 218 proteínas distintas, pudiendo relacionar
37 de ellas con el proceso de regeneración ósea y en
consecuencia con la osteointegración de un implante
dental. Además, tras la cuantificación diferencial entre
proteínas asociadas, antes y después de aplicar el tratamiento
superficial mencionado, se observó que tras
su aplicación se producía un aumento en la afinidad
de las proteínas APOE, ANT3 y PROC, directamente
relacionadas con el proceso de regeneración ósea. Por
el contrario, la proteína CO3 se adhería a esta superficie
en menor proporción. Estas variaciones de los perfiles
de proteínas podrían explicar la diferencia encontrada
en la respuesta de las distintas superficies al ser
caracterizadas en cuanto a su comportamiento in vivo
Differential profile of protein expression on human keratocytes treated with autologous serum and plasma rich in growth factors (PRGF)
Purpose
The main objective of this study is to compare the protein expression of human keratocytes treated with Plasma rich in growth factors (PRGF) or autologous serum (AS) and previously induced to myofibroblast by TGF-beta 1 treatment.
Methods
Blood from healthy donor was collected and processed to obtain AS and PRGF eye drops. Blood derivates were aliquoted and stored at-80 degrees C until use. Keratocyte cells were pretreated for 60 minutes with 2.5 ng/ml TGF-beta 1. After that, cells were treated with PRGF, AS or with TGF-beta 1 (control). To characterize the proteins deregulated after PRGF and AS treatment, a proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS was carried out.
Results
Results show a catalogue of key proteins in close contact with a myofibroblastic differentiated phenotype in AS treated-cells, whereas PRGF-treated cells show attenuation on this phenotype. The number of proteins downregulated after PRGF treatment or upregulated in AS-treated cells suggest a close relationship between AS-treated cells and cytoskeletal functions. On the other hand, proteins upregulated after PRGF-treatment or downregulated in AS-treated cells reveal a greater association with processes such as protein synthesis, proliferation and cellular motility.
Conclusion
This proteomic analysis helps to understand the molecular events underlying AS and PRGF-driven tissue regeneration processes, providing new evidence that comes along with the modulation of TGF-beta 1 activity and the reversion of the myofibroblastic phenotype by PRGF.This study was fully supported by BTI Biotechnology Institute, a dental implant company that investigates in the fields of oral implantology and PRGF-Endoret technology. MF and FM received a salary as scientists from BTI Biotechnology Institute. EA is the Scientific Director and president of BTI Biotechnology Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Characterization of serum proteins attached to distinct sol–gel hybrid surfaces
The success of a dental implant depends on its osseointegration, an important feature of the implant biocompatibility. In this study, two distinct sol–gel hybrid coating formulations [50% methyltrimethoxysilane: 50% 3-glycidoxypropyl-trimethoxysilane (50M50G) and 70% methyltrimethoxysilane with 30% tetraethyl orthosilicate (70M30T)] were applied onto titanium implants. To evaluate their osseointegration, in vitro and in vivo assays were performed. Cell proliferation and differentiation in vitro did not show any differences between the coatings. However, four and eight weeks after in vivo implantation, the fibrous capsule area surrounding 50M50G-implant was 10 and 4 times, respectively, bigger than the area of connective tissue surrounding the 70M30T treated implant. Thus, the in vitro results gave no prediction or explanation for the 50M50G-implant failure in vivo. We hypothesized that the first protein layer adhered to the surface may have direct implication in implant osseointegration, and perhaps correlate with the in vivo outcome. Human serum was used for adsorption analysis on the biomaterials, the first layer of serum proteins adhered to the implant surface was analyzed by proteomic analysis, using mass spectrometry (LC-MS/MS). From the 171 proteins identified; 30 proteins were significantly enriched on the 50M50G implant surface. This group comprised numerous proteins of the immune complement system, including several subcomponents of the C1 complement, complement factor H, C4b-binding protein alpha chain, complement C5 and C-reactive protein. This result suggests that these proteins enriched in 50M50G surface might trigger the cascade leading to the formation of the fibrous capsule observed. The implications of these results could open up future possibilities to predict the biocompatibility problems in vivo
Insight into the antibacterial mechanism of Cu-enriched sol–gel coatings employing proteomics
Advanced antibacterial biomaterials can help reduce the severe consequences of infections. Using copper compounds is an excellent option to achieve this goal; they offer a combination of regenerative and antimicrobial functions. In this study, new CuCl2-doped sol–gel coatings were developed and physicochemically characterised. Their osteogenic and inflammatory responses were tested in vitro using human osteoblasts and THP-1 macrophages. Their antibacterial effect was evaluated using Escherichia coli and Staphylococcus aureus. The Cu influence on the adsorption of human serum proteins was analysed employing proteomics. The materials released Cu2+ and were not cytotoxic. The osteoblasts in contact with these materials showed an increased ALP, BMP2 and OCN gene expression. THP-1 showed an increase in pro-inflammatory markers related to M1 polarization. Moreover, Cu-doped coatings displayed a potent antibacterial behaviour against E. coli and S. aureus. The copper ions affected the adsorption of proteins related to immunity, coagulation, angiogenesis, fibrinolysis, and osteogenesis. Interestingly, the coatings had increased affinity to proteins with antibacterial functions and proteins linked to the complement system activation that can lead to direct bacterial killing via large pore-forming complexes. These results contribute to our understanding of the antibacterial mechanisms of Cu-biomaterials and their interaction with biological systems.This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21], Generalitat Valenciana [APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I [UJI-B2021-25] and Basque Government [MARSA21/07]. The authors would like to thank Raquel Oliver, José Ortega and Iraide Escobés for their valuable technical assistance and GMI-Ilerimplant for making the titanium discs
Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants
Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.This work was supported by Ministerio de Economía y Competitividad (MINECO) [MAT 2014-51918-C2-2-R], Universidad de Castellón [P11B2014-19], Plan de Promoción de la Investigación de la Universidad Jaume I under grant [Predoc/2014/25] and Generalitat Valenciana under grant [Grisolia/2014/016].
The authors would like to thank Antonio Coso and Jaime Franco (GMI-Ilerimplant) for their inestimable contribution to this study, and Iraida Escobes (CIC bioGUNE) for her valuable technical assistance
- …