The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Protective effect and economic impact of insecticide application methods on barley

The objective of this work was to evaluate the protective effect of different forms of insecticide application on the transmission of yellow dwarf disease in barley cultivars, as well as to determine the production costs and the net profit of these managements. The experiments were carried out during 2011 and 2012 growing seasons, using the following managements at main plots: T1, seed treatment with insecticide (ST) + insecticide on shoots at 15-day interval; T2, just ST; T3, insecticide applied on shoots, when aphid control level (CL) was reached; T4, without insecticide; and T5, ST + insecticide on shoots when CL was reached. Different barley cultivars - BRS Cauê, BRS Brau and MN 6021 - were arranged in the subplots. Insecticides lambda cyhalothrin (pyrethroid) and thiamethoxam (neonicotinoid) were used. There were differences on yellow dwarf disease index in both seasons for the different treatments, while damage to grain yield was influenced by year and aphid population. Production costs and net profit were different among treatments. Seed treatment with insecticide is sufficient to reduce the transmission of yellow dwarf disease in years with low aphid population pressure, while in years with larger populations, the application of insecticide on shoots is also required.

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin.

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.

egr-4, a target of EGFR signaling, is required for the formation of the brian primordial and head regeneration in planarians

During the regeneration of freshwater planarians, polarity and patterning programs play essential roles in determining whether a head or a tail regenerates at anterior or posterior-facing wounds. This decision is made very soon after amputation. The pivotal role of the Wnt/β-catenin and Hh signaling pathways in re-establishing anterior-posterior (AP) polarity has been well documented. However, the mechanisms that control the growth and differentiation of the blastema in accordance with its AP identity are less well understood. Previous studies have described a role of Smed-egfr-3, a planarian epidermal growth factor receptor, in blastema growth and differentiation. Here, we identify Smed-egr-4, a zinc-finger transcription factor belonging to the early growth response gene family, as a putative downstream target of Smed-egfr-3. Smed-egr-4 is mainly expressed in the central nervous system and its silencing inhibits anterior regeneration without affecting the regeneration of posterior regions. Single and combinatorial RNA interference to target different elements of the Wnt/β-catenin pathway, together with expression analysis of brain- and anterior-specific markers, revealed that Smed-egr-4: (1) is expressed in two phases - an early Smed-egfr-3-independent phase and a late Smed-egfr-3-dependent phase; (2) is necessary for the differentiation of the brain primordia in the early stages of regeneration; and (3) that it appears to antagonize the activity of the Wnt/β-catenin pathway to allow head regeneration. These results suggest that a conserved EGFR/egr pathway plays an important role in cell differentiation during planarian regeneration and indicate an association between early brain differentiation and the proper progression of head regeneration.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

Quantile Estimation in Non-Stationary Streaming Data

We provide an incremental quantile estimator for Non-stationary Streaming Data. We propose a method for simultaneous estimation of multiple quantiles corresponding to the given probability levels from streaming data. Due to the limitations of the memory, it is not feasible to compute the quantiles by storing the data. So estimating the quantiles as the data pass by is the only possibility. This can be effective in network measurement. To provide the minimum of the mean-squared error of the estimation, we use parabolic approximation and for comparison we simulate the results for different number of runs and using both linear and parabolic approximations.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

New evidence of neuroprotection by lactate after transient focal cerebral ischaemia: extended benefit after intracerebroventricular injection and efficacy of intravenous administration.

BACKGROUND: Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously. METHODS: Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na L-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na L-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h. RESULTS: Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm(3), n = 7 vs. 12.1 ± 3.8 mm(3), n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm(3), n = 12 vs. 29.6 ± 25.4 mm(3), n = 12, p < 0.05). CONCLUSIONS: The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human bodyassociated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

Techniques de gastrostomie chez l'enfant et expérience lausannoise avec le bouton de gastrostomie à ballonnet (Mic-Key)

Résumé : Ce travail comprend deux parties : La première partie a pour but de présenter une revue des techniques de gastrostomie chez l'enfant. La gastrostomie est, par définition, un tractus fistuleux entre l'estomac et la paroi abdominale. Le but de la gastrostomie est de permettre la décompression gastrique, la nutrition entérale et l'apport médicamenteux. Les indications et contre-indications à la confection et utilisation de la gastrostomie sont détaillées dans ce travail. Historiquement, les premières gastrostomies étaient d'origine accidentelle ou infectieuse (fistule gastro-cutanée), incompatibles avec la vie. Sedillot, en 1845 décrivit la première gastrostomie chirurgicale sans cathéter, qui avait comme désavantage la présence de fuites. Depuis, les techniques se sont multipliées en évoluant vers la continence et l'utilisation de cathéters. En 1979 Gauderer décrivit pour la première fois une technique percutanée, réalisée sur un enfant âgé de 5 mois. Cette technique est appelée « Percutaneous Endoscopic Gastrostomy » (PEG). Elle a ensuite été élargie à la population adulte. Actuellement, il existe une grande multiplicité de techniques par abord « laparotomique », laparoscopique ou percutanée (endoscopique ou radiologique). Ces techniques peuvent être combinées. Toutes ces techniques nécessitent la présence intermittente ou continue d'un dispositif, qui permet le maintient de la gastrostomie ouverte et évite les fuites gastriques. Ces dispositifs sont multiples; initialement il s'agissait de cathéters rigides (bois, métal, caoutchouc). Ensuite ils ont été fabriqués en silicone, ce qui les rend plus souples et mieux tolérés par le patient. Pour éviter leur dislocation, ils possèdent un système d'amarrage intra-gastrique tel que : un champignon (Bard®), un ballonnet (Foley®, Mic-Key®), ou une forme spiralée du cathéter (« pig-tail ») et possèdent un système d'amarrage extra-gastrique (« cross-bar »). En 1982, Gauderer créa le premier dispositif à fleur de peau : le bouton de gastrostomie (BG). Actuellement, il en existe deux types : à champignon (Bard®) et à ballonnet (Mic-Key®). Il existe plusieurs types de complications liées à la technique opératoire, à la prise en charge et au matériel utilisé. Une comparaison des différentes techniques, matériaux utilisés et coûts engendrés est détaillée dans ce travail. La deuxième partie de ce travail est dédiée aux BG et plus spécifiquement au BG à ballonnet (Mic-Key®). Nous présentons les différents boutons et les techniques spécifiques. Le BG est inséré soit dans une gastrostomie préformée, soit directement lors de la confection d'une gastrostomie par laparotomie, laparoscopie ou de façon percutanée. Les complications liées au BG sont rapportées. D'autres utilisations digestives ou urologiques sont décrites. Nous présentons ensuite notre expérience avec 513 BG à ballonnet (Mic-Key®) dans une revue de 73 enfants. La pose du BG est effectuée dans une gastrostomie préformée sans recours à une anesthésie générale. La technique choisie pour la confection de la gastrostomie dépend de la pathologie de base, de l'état général du patient, de la nécessité d'une opération concomitante et du risque anesthésique. Nous apportons des précisions sur le BG telles que la dimension en fonction de l'âge, la durée de vie, et les causes qui ont amené au changement du BG. Nos résultats sont comparés à ceux de la littérature. Sur la base de notre expérience et après avoir passé en revue la littérature spécialisée, nous proposons des recommandations sur le choix de la technique et le choix du matériel. Ce travail se termine avec une réflexion sur le devenir de la gastrostomie. Si le futur consiste à améliorer et innover les techniques et les matériaux, des protocoles destinés à la standardisation des techniques, à la sélection des patients et à l'enseignement des soins devraient s'en suivre. La prise en charge de l'enfant ne se limite pas à la sélection appropriée de la technique et des matériaux, mais il s'agit avant tout d'une approche multidisciplinaire. La collaboration entre le personnel soignant, la famille et l'enfant est essentielle pour que la prise en charge soit optimale et sans risques.

Targeting Enterococcus faecalis Biofilms with Phage Therapy.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

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.

The CXCR4/CXCR7/CXCL12 Axis Is Involved in a Secondary but Complex Control of Neuroblastoma Metastatic Cell Homing.

Neuroblastoma (NB) is one of the most deadly solid tumors of the young child, for which new efficient and targeted therapies are strongly needed. The CXCR4/CXCR7/CXCL12 chemokine axis has been involved in the progression and organ-specific dissemination of various cancers. In NB, CXCR4 expression was shown to be associated to highly aggressive undifferentiated tumors, while CXCR7 expression was detected in more differentiated and mature neuroblastic tumors. As investigated in vivo, using an orthotopic model of tumor cell implantation of chemokine receptor-overexpressing NB cells (IGR-NB8), the CXCR4/CXCR7/CXCL12 axis was shown to regulate NB primary and secondary growth, although without any apparent influence on organ selective metastasis. In the present study, we addressed the selective role of CXCR4 and CXCR7 receptors in the homing phase of metastatic dissemination using an intravenous model of tumor cell implantation. Tail vein injection into NOD-scid-gamma mice of transduced IGR-NB8 cells overexpressing CXCR4, CXCR7, or both receptors revealed that all transduced cell variants preferentially invaded the adrenal gland and typical NB metastatic target organs, such as the liver and the bone marrow. However, CXCR4 expression favored NB cell dissemination to the liver and the lungs, while CXCR7 was able to strongly promote NB cell homing to the adrenal gland and the liver. Finally, coexpression of CXCR4 and CXCR7 receptors significantly and selectively increased NB dissemination toward the bone marrow. In conclusion, CXCR4 and CXCR7 receptors may be involved in a complex and organ-dependent control of NB growth and selective homing, making these receptors and their inhibitors potential new therapeutic targets.

Paperin hydrofobiliimauksen purkautumiseen vaikuttavat mekanismit

Työssä tutkittiin liimauksen reversiota ja häviämistä erilaisilla hydrofobiliimoilla alkaalisessa paperinvalmistuksessa käytettävien täyteaineiden kanssa. Liimauksen reversiota ja häviämistä arvioitiin arkkimuotilla valmistetuista koearkeista mittaamalla hydrofobiliimautuneisuutta kuvaavia ominaisuuksia kuten raakareunan absorptio ja kontaktikulma. Liimauksen reversiota kiihdytettiin lämpökäsittelyllä ja UV-säteilytyksellä. Liimauksen häviämistä tutkittiin muuttamalla viiraveden alkaalisuutta ja käyttämällä kalsiumkarbonaattia täyteaineena. Verrattaessa käytettyjä hydrofobiliimoja ASA1+AKD -liimayhdistelmällä ja AKD -liimauksella saavutettiin paperille paras liimautuneisuus. ASA2 -liimauksella saavutettiin paperille parempi liimautuneisuus kuin ASA1 -liimalla. Kalsiumkarbonaatilla täytetyillä arkeilla mitattiin korkeampi liimautuneisuus kuin kaoliinilla täytetyillä arkeilla. Kokeellisessa osassa tutkittiin myös 4 tuntia kestävän 105°C lämpökäsittelyn ja UV-säteilytyksen sekä viiraveden alkaalisuuden vaikutusta hydrofobiliimauksen reversioon ja häviämiseen. Liimauksen reversiota aikaansai UV-säteily ja lämpökäsittely. UV-säteily ja lämpökäsittely näyttävät aikaansaavan kovalenttisen esterisidoksen katkeamisen tai hydrolysoitumisen liimamolekyylin ja selluloosan karboksyyliryhmän välillä. Liimauksen reversiota havaittiin jokaisella hydrofobiliimalla. Liimauksen häviämistä aikaansai korkea viiraveden alkaalisuus (520 ppm CaCO3). Liimauksen häviämisen aiheuttamaa alhaisempaa liimautuneisuutta havaittiin myös viiraveden alkaalisuuden ollessa normaalia tasoa (250 ppm CaCO3), kun täyteaineena käytettiin saostettua kalsiumkarbonaattia.

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.