The role of Serratia marcescens porins in antibiotic resistance

The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semi-quantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent

Assessment of insecticide resistance in eggs and neonate larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Spanish Cydia pomonella (L.) field populations have developed resistance to several insecticide groups. Diagnostic concentrations were established as the LC90 calculated on a susceptible strain (S_Spain) for five and seven insecticides and tested on eggs and neonate larvae field populations, respectively. The three most relevant enzymatic detoxification systems (mixed-function oxidases (MFO), glutathione S-tranferases (GST) and esterases (EST)) were studied for neonate larvae. In eggs, 96% of the field populations showed a significantly lower efficacy when compared with the susceptible strain (S_Spain) and the most effective insecticides were fenoxycarb and thiacloprid. In neonate larvae, a significant loss of susceptibility to the insecticides was detected. Flufenoxuron, azinphos-methyl and phosmet showed the lowest efficacy, while lambda-cyhalothrin, alpha-cypermethrin and chlorpyrifos-ethyl showed the highest. Biochemical assays showed that the most important enzymatic system involved in insecticide detoxification was MFO, with highest enzymatic activity ratios (5.1–16.6 for neonates from nine field populations). An enhanced GST and EST activities was detected in one field population, with enzymatic activity ratios of threefold and fivefold for GST and EST, respectively, when compared with the susceptible strain. The insecticide bioassays showed that the LC90 used were effective as diagnostic concentrations. Measures of MFO activity alongside bioassays with insecticide diagnostic concentrations could be used as tools for monitoring insecticide resistance in neonate larvae of C. pomonella.

Efeito da azadiractina sobre Chaetosiphon fragaefolli (Cockerell, 1901) (Hemiptera: Aphididae) na cultura do morangueiro

O pulgão-verde Chaetosiphon fragaefolli é o principal inseto-praga da cultura do morangueiro. Neste trabalho, foi avaliado o efeito da azadiractina para o controle do inseto em laboratório e casa de vegetação. Os tratamentos avaliados foram a azadiractina (Azamax®, 100; 200 e 300 ml.100L-1) comparado com o tiametoxam (Actara 250 WG®, 10 g.100L-1), lambda-cialotrina (Karate Zeon 50 CS®, 80 ml.100L-1) e uma testemunha (água). Os produtos foram pulverizados sobre plantas de morangueiro da cultivar Aromas infestadas artificialmente em casa de vegetação. A azadiractina foi equivalente a lambda-cialotrina e ao tiametoxam no controle de C. fragaefolii desde que realizada uma segunda pulverização sete dias após a primeira. A persistência biológica dos inseticidas lambda-cialotrina e tiametoxam foi superior a 28 dias, com um controle de 75% da população de pulgões, enquanto azadiractina apresentou menor persistência biológica, controlando 70% da população por sete dias.

Laajakaistainen Vivaldi-antennielementti

Diplomityössä suunniteltiin, rakennettiin ja mitattiin laajakaistainen antennielementti lineaariseen antenniryhmään. Elementti toimii mikroaaltoalueella, ja sen kaistanleveys on noin 4,8:1. Elementti koostuu kaksipuolisesta eksponentiaalisesti taperoidusta rakoantennista eli Vivaldi-antennista ja laajakaistaisesta siirtymästä liuskajohdosta kaksipuoliseen rakojohtoon. Elementin koko pienimmällä käyttötaajuudella on noin 0,31 lambda kertaa 0,34 lambda, josta antennitorven koko on vain noin 0,21 lambda kertaa 0,21 lambda. Elementti suunniteltiin HFSS-simulointiohjelman avulla ja rakennettiin kahdesta erillisestä piirilevystä puristamalla nämä yhteen alumiinisella kehyksellä. Mittauksilla varmistettiin elementin toiminta ja simulointien luotettavuus. Osoitettiin, että elementti voidaan suunnitella simulointiohjelman avulla ja rakentaa työssä käytetyllä tavalla. Osoitettiin myös, että tarvittavaa mitoitussimulointien määrää voidaan vähentää yhdistämällä erikseen mitoitetut rakoantenni ja siirtymä. Lisäksi simuloinnein osoitettiin, että elementti toimii myös ryhmässä ja että sen toimintaa voidaan parantaa kehyksen avulla.

Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Characterizing RecA-Independent Induction of Shiga toxin2-encoding Phages by EDTA Treatment

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Serious games for rehabilitation using head-mounted display and haptic devices

In the health domain, the field of rehabilitation suffers from a lack specialized staff while hospital costs only increase. Worse, almost no tools are dedicated to motivate patients or help the personnel to carry out monitoring of therapeutic exercises. This paper demonstrates the high potential that can bring the virtual reality with a platform of serious games for the rehabilitation of the legs involving a head-mounted display and haptic robot devices. We first introduce SG principles and the current context regarding rehabilitation interventions followed by the description of an original haptic device called Lambda Health System. The architecture of the model is then detailed, including communication specifications showing that lag is imperceptible for user (60Hz). Finally, four serious games for rehabilitation using haptic robots and/or HMD were tested by 33 health specialists.

Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Optimal Traffic Grooming in WDM using Lighttours

Recent developments in optical communications have allowed simpler optical devices to improve network resource utilization. As such, we propose adding a lambda-monitoring device to a wavelength-routing switch (WRS) allowing better performance when traffic is routed and groomed. This device may allow a WRS to aggregate traffic over optical routes without incurring in optical-electrical-optical conversion for the existing traffic. In other words, optical routes can be taken partially to route demands creating a sort of "lighttours". In this paper, we compare the number of OEO conversions needed to route a complete given traffic matrix using either lighttours or lightpaths

G+: Enhanced Traffic Grooming in WDM Mesh Networks using Lighttours

In this article, a new technique for grooming low-speed traffic demands into high-speed optical routes is proposed. This enhancement allows a transparent wavelength-routing switch (WRS) to aggregate traffic en route over existing optical routes without incurring expensive optical-electrical-optical (OEO) conversions. This implies that: a) an optical route may be considered as having more than one ingress node (all inline) and, b) traffic demands can partially use optical routes to reach their destination. The proposed optical routes are named "lighttours" since the traffic originating from different sources can be forwarded together in a single optical route, i.e., as taking a "tour" over different sources towards the same destination. The possibility of creating lighttours is the consequence of a novel WRS architecture proposed in this article, named "enhanced grooming" (G+). The ability to groom more traffic in the middle of a lighttour is achieved with the support of a simple optical device named lambda-monitor (previously introduced in the RingO project). In this article, we present the new WRS architecture and its advantages. To compare the advantages of lighttours with respect to classical lightpaths, an integer linear programming (ILP) model is proposed for the well-known multilayer problem: traffic grooming, routing and wavelength assignment The ILP model may be used for several objectives. However, this article focuses on two objectives: maximizing the network throughput, and minimizing the number of optical-electro-optical conversions used. Experiments show that G+ can route all the traffic using only half of the total OEO conversions needed by classical grooming. An heuristic is also proposed, aiming at achieving near optimal results in polynomial time

Desenvolvimento de método analítico por CLAE em comprimidos de Benznidazol para a Doença de Chagas

The analytical method of high performance liquid chromatography (HPLC) for the assay of benznidazole in tablets was developed and validated following the requirements of regulatory agencies. The method used as mobile phase acetonitrile:wather 1:1, a C18 column of 12.5 cm length x 4 mm id, 5 mm particles and lambda=316 nm. The statistical analysis of the results demonstrated that the method satisfies all parameters so as to be considered a safe and efficient analytical alternative of low cost for laboratory routine.

Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmaco-kinetics after a single oral dose administration

The analytical method developed to evaluate tamoxifen in dog plasma samples was precise, accurate, robust and linear in the range of 5-200 ng/mL. The limits of detection and quantification were 0.981 ng/mL and 2.97 ng/mL, respectively. Besides, the intra-day precision and accuracy variations were 8.78 and 10.16%, respectively. Tamoxifen concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (lambda=280 nm). The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies.

Desenvolvimento de método espectrofotométrico para análise de paraformaldeído em saneantes comerciais e industriais

O presente artigo descreve uma metodologia espectrofotométrica para a análise de paraformaldeído presente em amostras de saneantes comerciais e industriais, utilizando-se ácido cromotrópico (ACT), ácido clorídrico concentrado e peróxido de hidrogênio, produzindo um composto púrpura - avermelhado (lambda max = 575 nm). A lei de Beer é obedecida numa faixa de concentração de 0,8 - 4,8 mg L-1 em formaldeído, apresentando excelente coeficiente de correlação (r = 0,9999). Os valores obtidos nas análises concordaram muito favoravelmente com os obtidos pelo procedimento padrão recomendado pelo NIOSH (National Institute for Occupational Safety and Health).

Targeted Therapy Possibilities for Metastatic Melanoma

The incidence of malignant melanoma of the skin has been steadily rising worldwide during the past decades. Most early detected primary tumors can be removed surgically and the prognosis is good. However, at the same time there still is no permanent cure for metastatic melanoma and its prognosis is poor, although lately new effective drugs have emerged. In this thesis, four different approaches of experimental therapy for metastatic melanoma were studied. Endogenous cis-Urocanic acid (UCA) is found in every individual’s skin, where exposure to UV light from the sun generates it from its inactive trans conformation. Cis- UCA was found to destroy malignant melanoma cells in culture under an acidified pH and sufficient concentration through caspase-3 mediated apoptosis. Furthermore, cis-UCA is able to considerably diminish the growth rate in human melanoma tumors on living SCID mice. Using replication-competent Semliki Forest viruses, human melanoma tumors grown in SCID mice were dramatically shrunken as the fulminant production of viruses in melanoma cells leads them to apoptosis within 72 hours. Small oligopeptides attaching to melanoma cells were identified using in vivo phage display. The melanoma-specific peptides found were further tested in vitro on adenoviruses. Ultimately, the adenoviral retargeting using the peptides was tested in vivo. One peptide homed to human transferring receptor upregulated on melanoma cells. In order to kill the malignant melanoma cells with the retargeted adenoviruses, the viruses should carry genetic material producing apoptotic proteins in the cancer tissue. TIMP-3 has been identified as a good candidate for such a protein, as it inhibits malignant cell adhesion as well as promotes apoptosis through a caspase-8 pathway. It is further shown here that adenovirally delivered TIMP-3 is even more potent, as it could kill non-adherent cancer cells, lacking the fully functional death receptor signalling pathway. Adenovirally delivered TIMP-2 also showed marked antitumor effects in human malignant melanoma xenografts on SCID mice both in ex vivo and systemic delivery.