Transcriptome Analysis of the Brucella abortus BvrR/BvrS Two-Component Regulatory System

International audienceBackgroundThe two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions.Methodology/Principal FindingsA total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered.Conclusions/SignificanceAll these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche

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

Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol–Gel Coating

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol–gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems

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

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

Bioactive potential of silica coatings and its effect on the adhesion of proteins to titanium implants

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC–MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work

Síntesis de nano-semiconductores de SnO₂ por combustión de geles con adición de iones Al³⁺, Ga³⁺ e In³⁺

Se sintetizaron por sol-gel y combustión de geles nanosemiconductores de SnO₂ con adición de 10% de Al³⁺5% de Ga³⁺ y 2% de In³⁺. Los cuales mostraron sensibilidad en la detección de CO, que es uno de los gases tóxicos mayormente generados en el ambiente, producido por la combustión de otros gases, gasolina, queroseno, carbón, petróleo, tabaco, madera, estufas, calentadores, calefactores, motores encendidos, entre otros. El CO es un gas incoloro e inodoro, por lo que es necesario contar con nuevos materiales para sensores que muestren la concentración de este gas. Por DRX, las fases cristalinas correspondieron con la Casiterita. Las variaciones de peso determinadas por ATD/TG, mostraron cambios en los estados de oxidación de los cationes añadidos. La detección de CO en la superficie del nanosemiconductor, fue determinada por la disminución o caída de la resistencia eléctrica.We synthesized by sol-gel and gels combustion nanosemiconductors of SnO₂ with addition of 10% A1³⁺ 5% Ga³⁺% y 2% In³⁺. This showed sensitivity in the detection of CO, which one of the most generated toxic gases in the environment, produced by the combustion of other gases, gasoline, kerosene, coal, oil, tobacco, wood, stoves, heaters, motors lit, among others. CO is colorless and odorless gas, so it is necessary to have new materials for sensors that show the concentration ofthis gas, which is high risk in health. By XRD, the crystalline phases corresponded with the Casiterite. The weight variations determined by ATD/TG showed changes in the oxidation states of the added cations. The detection of CO on the nanosemiconductor surface was determined by the decrease or fall of the electrical resistance

C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

AbstractInsufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process

Silica-gelatin hybrid sol-gel coatings: A proteomic study with biocompatibility implications

Osseointegration, including the foreign body reaction to biomaterials, is an immune‐modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation‐driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic Collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol–gel formulations and their combination with gelatin were applied as coatings on sandblasted, acid‐etched titanium substrates, and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin‐containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings