Increasing the electrolyte capacity of alkaline Zn-air fuel cells by scavenging zincate with Ca(OH)2

The use of calcium hydroxide for scavenging zincate species is demonstrated to be a highly effective approach for increasing the electrolyte capacity and improving the performance of the zinc-air fuel cell system. A fundamental approach is established in this study to quantify the formation of calcium zincate as the product of scavenging and the amount of water compensation necessary for optimal performance. The good agreement between predicted and experimental results proves the validity of the proposed theoretical approach. By applying the results of theoretical predictions, both the electrolyte capacity and the cell longevity have been increased by more than 40%. It is also found that, using Ca(OH)2 to scavenge zincate species in concentrated KOH solutions, affects mostly the removal of zincate, rather than ZnO, from the electrolyte, whereas the presence of excess, free, mobile H2O plays a key role in dissolving ZnO and forming zincate. The results obtained in this study demonstrate that the proposed approach can widely and effectively be applied to all zinc-air cell systems during their discharge cycle.

A novel method for rapid and selective extraction of male DNA from rape kits using alkaline lysis and pressure cycling technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. ^ Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. ^ Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.^

Estudo in silico das interações da protease NS3-NS2B de DENV-2 com o inibidor peptídico Bz-nKRR-H com finalidades terapêuticas

Dengue virus is an important patogen that causes Dengue desease in all world, and belongs to Flavivirus gender. The virus consists of enveloped RNA with a single strand positive sense, 11Kb genome. The RNA is translated into a polyprotein precursor, wich is cleaved into 3 structural proteins (C, prM e E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5). The NS3 is a multifunctional protein, that besides to promote the polyprotein precursor cleavage, also have NTPase, helicase and RTPase activity. The NS3 needs a hydrophilic segment of 40 residues from the transmembrane NS2B protein (who acts like cofator) to realize this functions. Actually, there's no vacines available on the market, and the treatment are just symptomatic. The tetrapeptide inhibitor Bz-Nle-Lys-Arg-Arg-H (Ki de 5,8-7,0 M) was showed as a potent inhibitor μ for NS3prot in Dengue virus. That is a inteligent alternative to treat the dengue desease. The present work aimed analyse the interactions of the ligand bounded to the activity site to provid a clear and depth vision of that interaction. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant = 20 ε and 80 was used. The results demonstrate that the interaction energy of the system reached convergence at 13.5 A. Within a radius of 13,5A the most important residues were identified. Met49, Met84 and Asp81 perform interactions of hydrogen with the ligant. The Asp79 and Asp75 residues present high energy of attraction. Arg54, Arg85 and Lys 131 perform hydrogen interactions with the ligand, however, appear in BIRD graph having high repulsion energy with the inhibitor. The data also emphasizes the importance of residue Tyr161 and the involvement of the catalytic triad composed by Asp75, His51 and Ser135

Electrochemical pore formation on InP in alkaline solutions

The surface properties of InP electrodes were examined following anodization in (NH4)2S and KOH electrolytes. In both solutions, the observation of current peaks in the cyclic voltammetric curves was attributed to selective etching of the substrate and a film formation process. AFM images of samples anodized in the sulfide solution, revealed surface pitting and TEM micrographs revealed the porous nature of the film formed on top of the pitted substrate. After anodization in the KOH electrolyte, TEM images revealed that a porous layer extending 500 nm into the substrate had been formed. Analysis of the composition of the anodic products indicates the presence of In2S3 in films grown in (NH4)2S and an In2O3 phase within the porous network formed in KOH.

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus

The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

BACKGROUND: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.

METHODS: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.

RESULTS: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.

CONCLUSIONS: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

BACKGROUND: Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles.

METHODS: SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA.

RESULTS: SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum.

CONCLUSIONS: The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible association with disease pathology, while the strong reactivity of sera from experimentally infected rats against rSjB6 suggests that native SjB6 is released into host tissue and induces an immune response. This study presents a comprehensive demonstration of sequence and structural-based analysis of a secretory serpin from a trematode and suggests SjB6 may be associated with important functional roles in S. japonicum, particularly in parasite modulation of the host microenvironment.