Luciana Maria de Hollanda

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G → C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.

Boron nitride nanotubes chemically functionalized with glycol chitosan for gene transfection in eukaryotic cell lines.

Nanostructured materials have been widely studied concerning their potential biomedical applications, primarily to selectively carry specific drugs or molecules within a tissue or organ. In this context, boron nitride nanotubes (BNNTs) have generated considerable interest in the scientific community because of their unique properties, presenting good chemical inertness and high thermal stability. Among the many applications proposed for BNNTs in the biomedical field in recent years, the most important include their use as biosensors, nanovectors for the delivery of proteins, drugs, and genes. In the present study, BNNTs were synthesized, purified, and functionalized with glycol chitosan through a chemical process, yielding the BNNT-GC. The size of BNNT-GC was reduced using an ultrasound probe. Two samples with different sizes were selected for in vitro assays. The nanostructures were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA), and dynamic light scattering (DLS). The in vitro assays MTT and neutral red (NR) were performed with NIH-3T3 and A549 cell lines and demonstrated that this material is not cytotoxic. Furthermore, the BNNT-GC was applied in gene transfection of plasmid pIRES containing a gene region that express a green fluorescent protein (GFP) in NIH-3T3 and A549 cell lines. The gene transfection was characterized by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Our results suggest that BNNT-GC has moderate stability and presents great potential as a gene carrier agent in nonviral-based therapy, with low cytotoxicity and good transfection efficiency.

Carbon nanoparticles for gene transfection in eukaryotic cell lines

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Pathogenic and opportunistic respiratory bacteria-induced apoptosis

Several pathogenic or opportunistic bacteria have the ability to either induce or inhibit host cell apoptosis. The capacity to modulate cell pathways that result in the induction or delay of host cell apoptosis is considered to be an important bacterial virulence mechanism. These processes could be mediated by different host cell signaling pathways that are subverted by the bacteria. Pathogens are able to activate apoptotic proteins, such as caspases, or inactivate anti-apoptotic proteins, such as NFkB and the MAPKKs, or even up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. The bacteria-induced apoptotic or anti-apoptotic processes are often related with the fact that the bacteria acquire the ability to reach the host tissues. However, apoptosis is also considered to be a host defense mechanism against infectious agents. Thus, the apoptosis phenomenon plays a central role in host-pathogen interactions.

Effect of mesoporous silica under Neisseria meningitidis transformation process: environmental effects under meningococci transformation

BackgroundThis study aimed the use of mesoporous silica under the naturally transformable Neisseria meningitidis, an important pathogen implicated in the genetic horizontal transfer of DNA causing a escape of the principal vaccination measures worldwide by the capsular switching process. This study verified the effects of mesoporous silica under N. meningitidis transformation specifically under the capsular replacement.Methodswe used three different mesoporous silica particles to verify their action in N. meningitis transformation frequency.Resultswe verified the increase in the capsular gene replacement of this bacterium with the three mesoporous silica nanoparticles.Conclusionthe mesouporous silica particles were capable of increasing the capsule replacement frequency in N. meningitidis.

Haemophilus influenzae porine ompP2 gene transfer mediated by graphene oxide nanoparticles with effects on transformation process and virulence bacterial capacity

BackgroundH. influenzae is a natural competent bacterium that can uptake DNA from the environment and recombine into bacterial genome. The outbreaks of Brazilian purpuric fever, heavily polluted areas of a different H. influenzae biogroup - aegyptius - as well as gene transference between Neisseria meningitis make the transformation process an important evolutionary factor. This work studied the horizontal transference of the omp P2 gene from a multiresistant strain of H. influenzae 07 (NTHi), under the influence of graphene oxide nanoparticles in order to mimic an atmosphere rich in suspended particles and this way verify if the CFU transformants number was increased.Material and methodsIn this article the gene ompP2 was transformed into different strains of H. influenzae mediated or not by graphene oxide nanoparticles in suspension, followed by the adhesion tests in Hec-1B (human endometrium adenocarcinoma) and A549 (pulmonary epithelial carcinoma) cells lines. The transformation frequency and the adhesion capacity were determined in all the mutants to which the ompP2 gene was transferred and compared to their wild type strains.ResultsThe nanoparticles increased the transformation ratio of one particular strain isolated from a pneumonia case. The adhesion patterns to A549 and Hec1b cell lines of these mutated bacteria has their capacity increased when compared to the wild type.ConclusionsGraphene oxide nanoparticles aid the transformation process, helping to increase the number of CFUs, and the mutants generated with the omp P2 gene from a H. influenzae resistant strain not only present a chloramphenicol resistance but also have an increased adherence patterns in A549 and Hec1B cell lines.

Effects of hyperbaric oxygen on Pseudomonas aeruginosa susceptibility to imipenem and macrophages

BACKGROUND The seriousness to treat burn wounds infected with Pseudomonas aeruginosa led us to examine whether the effect of the carbapenem antibiotic imipenem is enhanced by hyperbaric oxygen (HBO). MATERIALS & METHODS The effects of HBO (100% O2, 3 ATA, 5 h) in combination with imipenen on bacterial counts of six isolates of P. aeruginosa and bacterial ultrastructure were investigated. Infected macrophages were exposed to HBO (100% O2, 3 ATA, 90 min) and the production of reactive oxygen species monitored. RESULTS HBO enhanced the effects of imipenen. HBO increased superoxide anion production by macrophages and likely kills bacteria by oxidative mechanisms. CONCLUSION HBO in combination with imipenem can be used to kill P. aeruginosa in vitro and such treatment may be beneficial for the patients with injuries containing the P. aeruginosa.

Effects of multi-walled carbon nanotubes (MWCNT) under Neisseria meningitidis transformation process.

BackgroundThis study aimed at verifying the action of multi-walled carbon nanotubes (MWCNT) under the naturally transformable Neisseria meningitidis against two different DNA obtained from isogenic mutants of this microorganism, an important pathogen implicated in the genetic horizontal transfer of DNA, causing the escape of the principal vaccination measured worldwide by the capsular switching process.Materials and methodsThe bacterium receptor strain C2135 was cultivated and had its mutant DNA donor M2 and M6, which received a receptor strain and MWCNT at three different concentrations. The inhibition effect of DNAse on the DNA in contact with nanoparticles was evaluated.ResultsThe results indicated an in increase in the transformation capacity of N. meninigtidis in different concentrations of MWCNT when compared with negative control without nanotubes. A final analysis of the interaction between DNA and MWCNT was carried out using Raman Spectroscopy.ConclusionThese increases in the transformation capacity mediated by MWCNT, in meningococci, indicate the interaction of these particles with the virulence acquisition of these bacteria, as well as with the increase in the vaccination escape process.

Chemical Modifications of PhTX-I Myotoxin from Porthidium hyoprora Snake Venom: Effects on Structural, Enzymatic, and Pharmacological Properties

We recently described the isolation of a basic PLA2 (PhTX-I) from Porthidium hyoprora snake venom. This toxin exhibits high catalytic activity, induces in vivo myotoxicity, moderates footpad edema, and causes in vitro neuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.