Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.Em espectrometria de massas (MS) no modo de ionização eletrospray (ESI), as amostras podem ser analisadas com um método prévio de separação (cromatografia líquida ou eletroforese capilar) ou por infusão direta da amostra. A injeção direta apresenta um grande benefício que é a redução do volume de amostra consumido e a possibilidade de amostragem contínua por um período estendido. Este maior tempo de amostragem possibilita a análise completa através de fragmentações sucessivas por espectrometria de massas, crítico quando se busca elucidação estrutural de novos compostos, ou quando se dispõe de um analisador de massas do tipo captura de íons. Neste trabalho, descrevemos uma metodologia de deposição de filme estável na extremidade cônica dos dispositivos de boro-silicato, visando o desenvolvimento de nanoinjetores estáticos. A formação de uma camada dupla de titânio e ouro proporcionou uma excelente fixação do filme, resultando em um nanoinjetor para amostragem por ionização/formação do aerossol no sistema de espectrometria de massas. O objetivo deste filme é manter o contato elétrico no sistema. Os nanoinjetores apresentaram repetibilidade e estabilidade elevadas por mais de 100 min de amostragem contínua com apenas 10 µL de amostra.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Departamento de Ciências Exatas e da TerraUniversidade de São Paulo Instituto de Ciências BiomédicasUniversidade de São Paulo Instituto de Química de São CarlosUNIFESP, Depto. de Ciências Exatas e da TerraSciEL

Imunidade inata no intestino de carrapatos: hemocidinas e outros agentes antimicrobianos

Ticks are vectors of various pathogens, resulting in severe human and animal health problems. The midgut of these arthropods constitutes the primary pathogen-vector interface, and microbial control in this organ may be mediated by the activity of antimicrobial peptides derived from the digestion of heme-containing proteins (hemocidins), lysozymes and defensins, as well as protease inhibitors and oxidative stress. Thus, vector competence in ticks is directly related with their immune system.Os carrapatos são vetores de diversos organismos patogênicos, resultando em graves problemas para a saúde humana e animal. O intestino destes artrópodes constitui a interface primária patógeno-vetor, e o controle microbiano neste órgão pode ser mediado pela atividade de peptídeos antimicrobianos derivados da digestão de hemeproteínas (hemocidinas), lisozimas e defensinas, além de inibidores de proteases e estresse oxidativo. Sendo assim, a competência vetorial dos carrapatos está diretamente relacionada ao seu sistema imune

Gomesin, a peptide produced by the spider Acanthoscurria gomesiana, is a potent anticryptococcal agent that acts in synergism with fluconazole

Gomesin is an 18-residue cysteine-rich antimicrobial peptide produced by hemocytes of the spider Acanthoscurria gomesiana. in the present study, the antifungal properties of gomesin against Cryptococcus neoformans, the etiologic agent of cryptococcosis, were evaluated. Gomesin bound to the cell surface of cryptococci, which resulted in cell death associated with membrane permeabilization. Antifungal concentrations of gomesin were not toxic for human brain cells. Supplementation of cryptococcal cultures with the peptide (1 mu M) caused a decrease in capsule expression and rendered fungal cells more susceptible to killing by human brain phagocytes. the possible use of gomesin in combination with fluconazole, a standard antifungal drug, was also evaluated. in association with fluconazole, gomesin concentrations with low antimicrobial activity (0.1-1 mu M) inhibited fungal growth and enhanced the antimicrobial activity of brain phagocytes. These results reveal the potential of gomesin to promote inhibition of cryptococcal growth directly or by enhancing the effectiveness of host defenses.Univ Fed Rio de Janeiro, Inst Microbiol Prof Paulo Goes, Dept Microbiol Geral, Lab Estudos Integrados Bioquim Microbiana, BR-21941590 Rio de Janeiro, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-05508 São Paulo, BrazilUniv Texas, Dept Biol Sci, El Paso, TX 79968 USAUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilWeb of Scienc

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH–I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

<p>Abstract</p> <p>Background</p> <p>Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen <it>Anaplasma marginale </it>(Rickettsiales: Anaplasmataceae), is vectored by <it>Rhipicephalus </it>(<it>Boophilus</it>)<it>microplus </it>in many tropical and subtropical regions of the world. <it>A. marginale </it>undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in <it>Dermacentor variabilis </it>and cultured <it>Ixodes scapularis </it>tick cells in response to infection with <it>A. marginale</it>. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in <it>R. microplus </it>male salivary glands in response to <it>A. marginale </it>infection. Additionally, a <it>R. microplus</it>-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to <it>A. marginale </it>infection.</p> <p>Results</p> <p>Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male <it>R. microplus </it>salivary glands infected with <it>A. marginale</it>. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with <it>A. marginale</it>. The impact of selected tick genes on <it>A. marginale </it>infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced <it>A. marginale </it>infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher <it>A. marginale </it>infection levels in tick cells.</p> <p>Conclusions</p> <p>Characterization of differential gene expression in salivary glands of <it>R. microplus </it>in response to <it>A. marginale </it>infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in <it>A. marginale </it>infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.</p

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis

<p>Abstract</p> <p>Background</p> <p>Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider <it>Acanthoscurria gomesiana</it>. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. <it>Candida albicans</it> is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution.</p> <p>Results</p> <p>Treatment with gomesin effectively reduced <it>Candida albicans </it>in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-α, IFN-γ and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and <it>C. albicans</it>-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mic</p> <p>Conclusions</p> <p>Gomesin proved to be effective against experimental <it>Candida albicans</it> infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin's mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.</p

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. the results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). in contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide. (c) 2006 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Chem, Dept Biochem, BR-05508 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Parasitol, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample