Kaliandra de Almeida Gonçalves

Size-selective silver nanoparticles: future of biomedical devices with enhanced bactericidal properties

Silver nanoparticles (AgNPs) are attracting attention due to their bactericidal activity and consequent possible biomedical applications. The key to their broad-acting and potent biocidal property seems to be based on the size-related mechanism by which AgNPs act on different bacteria strains. Here, we report the synthesis and successful size-selective fractionation of AgNPs obtained through chemical reduction of silver nitrate in ethylene glycol using polyvinylpyrrolidone as a protective agent. A combination of characterization techniques (UV-vis spectroscopy, transmission electron microscopy and small-angle X-ray scattering) is employed to differentiate the two size-fractionated samples. From the analyses, it is evidenced that AgNPs are mainly spherical and have their radius centered at ∼8.5 and ∼11.0 nm. The nanoparticles bactericidal efficacy is investigated using the disk diffusion test against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus lysodeikticus. Although both fractionated samples present bactericidal activity against all four tested bacteria (one Gram negative and three Gram positives), those presenting smaller size own enhanced antibacterial properties.

Tailored Silica-Antibiotic Nanoparticles: Overcoming Bacterial Resistance with Low Cytotoxicity

New and more aggressive antibiotic resistant bacteria arise at an alarming rate and represent an ever-growing challenge to global health care systems. Consequently, the development of new antimicrobial agents is required to overcome the inefficiency of conventional antibiotics and bypass treatment limitations related to these pathologies. In this study, we present a synthesis protocol, which was able to entrap tetracycline antibiotic into silica nanospheres. Bactericidal efficacy of these structures was tested against bacteria that were susceptible and resistant to antibiotics. For nonresistant bacteria, our composite had bactericidal efficiency comparable to that of free-tetracycline. On the other hand, the synthesized composites were able to avoid bacterial growth of resistant bacteria while free-tetracycline has shown no significant bactericidal effect. Finally, we have investigated the cytotoxicity of these nanoparticles against mammalian cells to check any possible poisoning effect. It was found that these nanospheres are not apoptosis-inducers and only a reduction on the cell replication rate was seen when compared to the control without nanoparticles.

Functionalized Silica Nanoparticles As an Alternative Platform for Targeted Drug-Delivery of Water Insoluble Drugs

The selective action of drugs in tumor cells is a major problem in cancer therapy. Most chemotherapy drugs act nonspecifically and damage both cancer and healthy cells causing various side effects. In this study, the preparation of a selective drug delivery system, which is able to act as a carrier for hydrophobic and anticancer drugs is reported. Amino-functionalized silica nanoparticles loaded with curcumin were successfully synthesized via sol-gel approach and duly characterized. Thereafter, the targeting ligand, folate, was covalently attached to amino groups of nanoparticle surface through amide bond formation. The cytotoxic effect of nanoparticles on prostate cancer cells line was evaluated and compared to normal cells line (prostate epithelial cell). Cytotoxicity experiments demonstrated that folate-functionalized nanoparticles were significantly cytotoxic to tumor cells, whereas normal cells were much less affected by the presence of these structures.

Solution structure of the human signaling protein RACK1

BackgroundThe adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development.ResultsIn this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion.ConclusionOur SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Mechanism of interaction between colloids and bacteria as evidenced by tailored silica–lysozyme composites

The ever increasing antibiotic resistance levels in pathogenic and non-pathogenic bacteria have boosted the search for effective controlling methods of bacterial infections. In this context, we prepared colloidal silica–lysozyme composites to evaluate the interaction between these structures and bacteria. Lysozyme was chosen since it is a protein that holds bactericidal properties and that, in the reaction process, acts as a catalyst which favors silica hydrolysis and condensation. The high entrapment yield of lysozyme in the silica cage (approximately 95%) does not change the secondary structure of the protein resulting in a material with superior bactericidal properties. The antimicrobial differences when silica–lysozyme composites are tested against Escherichia coli and Staphylococcus aureus are used to propose an interaction mechanism between colloids and bacteria. Bactericidal properties of the composites are attributed to the ultra-structural organization of the composite which, due to the positive surface charge, is attracted by the negative bacterial cell wall while lysozyme is delivered.

Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery.

Ki‐1/57 physically interacts with RPL38 by two hybrid (View interaction)

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Tailored Silica Nanoparticles Surface to Increase Drug Load and Enhance Bactericidal Response

Nanoparticles’ surface properties can be used as triggers to regulate or even enhance biological response and generate tailored structures to substitute conventional antibiotics. Here, silica nanoparticles surface was duly tuned in order to increase the water-insoluble drug load (curcumin) and improve the antibacterial activity. Our main motivation was based on the electrostatic attraction between the positively charged amino groups and the negatively charged curcumin and/or bacteria membrane. In addition, the variation of amino grafting amount on silica nanoparticles indicated that the grafting increase was directly related to the extent of drug entrapped into the nanoparticles as well as to the bactericidal activity. The combination of amino-functionalized silica nanoparticles associated with the presence of curcumin allowed to produce a dual bactericidal system that shows promising perspective for its use in biomedical applications.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkins lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.