A highly sensitive fluorometric assay for "enkephalinase," a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins

A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG), was synthesized as a selective substrate for the neutral metalloendopeptidase (EC 3.4.24.11) involved in enkephalin metabolism. This enzyme, designated "enkephalinase," cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG (V = 0.65 mumol/mg protein/min and Km = 45 microM) leading to a fluorescence increase related to the disappearance of intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This change was used for quantitative measurements of "enkephalinase" activity in different tissues and determination of inhibitory potency of various compounds. The substrate is not cleaved by aminopeptidase or dipeptidylaminopeptidase activities and the assay itself is rapid, convenient, and sensitive.     

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165T

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165^T. The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10⁶ M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.     

PTPN22 gene polymorphism in Egyptian alopecia areata patients and its impact on response to diphencyprone immunotherapy

PTPN22 1858C>T gene polymorphism has been associated with several autoimmune disorders including alopecia areata. The aim of the current study was to investigate the effect of the inherited genetic polymorphism 1858C>T of PTPN22 gene on the predisposition to severe forms of alopecia areata and its effect on the response to DPC treatment. To achieve our aim, PTPN22 1858C>T genotyping was performed by PCR-based restricted fragment length polymorphism (PCR-RFLP) analysis. The study included 103 Egyptian patients with extensive alopecia areata treated by DPC. Hundred healthy age and sex matched blood donors were included in the current study as a control group. Results of genotyping showed that PTPN22 CT and TT mutant genotypes were significantly higher in AA patients compared to controls and conferred increase risk of AA (OR = 2.601, 95% CI = 1.081–6.255). Statistical comparison between AA patients with wild and mutant genotypes revealed that the duration of the illness was significantly longer in those harboring the mutant genotypes. Moreover, the association of other autoimmune diseases as atopy and diabetes mellitus was higher in patients with mutant genotypes. Furthermore, PTPN22 1858C>T genetic polymorphism did not affect the patients' response to DPC immunotherapy.     

Desiccation resistance along an aridity gradient in the cactophilic fly Drosophila buzzatii: sex-specific responses to stress

Stress resistance characters are valuable tools for the study of acclimation potential, adaptive strategies and biogeographic patterns in species exposed to environmental variability. Water stress is a challenge to terrestrial arthropods because of their small size and relatively high area: volume ratio. Fruit flies have been investigated to record adaptive morphological and physiological traits, as well as to test their responses to stressful factors. In this study, we investigate the ability to cope with water stress, by examining variation in desiccation resistance in a species that lives mainly in desert lands. Specifically, we explored the genetic and ecological basis of desiccation resistance in populations of Drosophila buzzatii from Northern Argentina. We used a common garden experiment with desiccation treatments on a number of isofemale lines from four populations along an aridity gradient. Our results revealed significant among-population differentiation and substantial amounts of genetic variation for desiccation resistance. We also detected significant genotype-by-environment and genotype-by-sex interactions indicative that desiccation resistance responses of the lines assayed were environment- and sex-specific. In addition, we observed clinal variation in female desiccation resistance along gradients of altitude, temperature and humidity; that desiccation resistance is a sexually dimorphic trait, and that sexual dimorphism increased along the aridity and altitudinal gradients. Based on current evidence, we propose that the observed sex-specific responses may reflect different life history traits, and survival and reproductive strategies in different ecological scenarios.     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.     

Does the Establishment of Sustainable Use Reserves Affect Fire Management in the Humid Tropics?

Tropical forests are experiencing a growing fire problem driven by climatic change, agricultural expansion and forest degradation. Protected areas are an important feature of forest protection strategies, and sustainable use reserves (SURs) may be reducing fire prevalence since they promote sustainable livelihoods and resource management. However, the use of fire in swidden agriculture, and other forms of land management, may be undermining the effectiveness of SURs in meeting their conservation and sustainable development goals. We analyse MODIS derived hot pixels, TRMM rainfall data, Terra-Class land cover data, socio-ecological data from the Brazilian agro-census and the spatial extent of rivers and roads to evaluate whether the designation of SURs reduces fire occurrence in the Brazilian Amazon. Specifically, we ask (1) a. Is SUR location (i.e., de facto) or (1) b. designation (i.e. de jure) the driving factor affecting performance in terms of the spatial density of fires?, and (2), Does SUR creation affect fire management (i.e., the timing of fires in relation to previous rainfall)? We demonstrate that pre-protection baselines are crucial for understanding reserve performance. We show that reserve creation had no discernible impact on fire density, and that fires were less prevalent in SURs due to their characteristics of sparser human settlement and remoteness, rather than their status de jure. In addition, the timing of fires in relation to rainfall, indicative of local fire management and adherence to environmental law, did not improve following SUR creation. These results challenge the notion that SURs promote environmentally sensitive fire-management, and suggest that SURs in Amazonia will require special attention if they are to curtail future accidental wildfires, particularly as plans to expand the road infrastructure throughout the region are realised. Greater investment to support improved fire management by farmers living in reserves, in addition to other fire users, will be necessary to help ameliorate these threats.     

Segregostat: a novel concept to control phenotypic diversification dynamics on the example of Gram-negative bacteria

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advanced technical set-ups for the real-time analyses and control of population behaviour on single-cell level. In this work, set-up, design and operation of the so called segregostat are described which, in contrast to a traditional chemostat, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, i.e. phenotypic diversification dynamics of Eschericia coli and Pseudomonas putida based on outer membrane permeabilization, emphasizing the applicability and versatility of the proposed approach. Upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic features (non-permeabilized and permeabilized cells). Online analysis leads to the determination of the ratio between cells in these two states, which in turn triggers the addition of glucose pulses in order to maintain a predefined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor set-ups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single-cell approaches are required, such as synthetic co-culture processes.     

Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula

In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (q_P) affecting the maximum quantum yield of PSII (Y); the real quantum yield (?_exc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO₂ assimilation caused by PSII damage. This was clearly estimated by the Y, ?_exc and especially by the quantum yield of electron transport of PSII (Φ_PSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.     

Effects of the methanolic extract of Chrysanthemum trifurcatum (Desf.) Batt. and Trab. on rat duodenal motility

The effect of the methanolic extract of flowers of Chrysanthemum trifurcatum (Desf.) Batt. and Trab. Var. macrocephalum (viv.) Beg. on the rat duodenum smooth muscle motility was examined in vitro. The extract has shown dose-dependent stimulator effects on the amplitude of the spontaneous contractions. With 0.1 g/ml of extract, maximal stimulation was obtained. With that dose, the variation (%) was significantly 1050 +/- 13 (P<0.001) compared with control and represented 80 +/- 5.83% (P<0.001) of the maximum effect of acetylcholine. Atropine (2 microg/ml) reduced by 81 +/- 4% (P<0.05) the spasmogenic effects of C. trifurcatum and by 92 +/- 3% (P<0.05) the acetylcholine effects, while papaverine (2 microg/ml) completely inhibited the spasmogenic effects of extract. With a fixed dose of acetylcholine added (20 microg/ml), the extract increases its effect, but acetylcholine decreases its action. These results suggested that the methanolic extract of C. trifurcatum could stimulate duodenal smooth muscle contractions through muscarinic receptors. Thy explain the respective traditional use of plant in gastrointestinal problems, especially constipation.