Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

Ectodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase Cδ (PKCδ). Downregulation of PKCδ inhibits neuregulin release and reduces ex vivo neurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate.     

Intraspecific and intergenerational differences in plant–soil feedbacks

Interactions between plant and soil communities are known to play an integral role in shaping ecosystems. Plants influence the composition of soil communities and soil communities in turn influence plant performance. Such a plant–soil feedback may incur selection pressure on plants and the associating soil community. However, the evolutionary consequences of these above–belowground feedback interactions remain largely speculative. Here we assess whether plant–soil feedback effects differ between intraspecific plant populations and between generations within the same plant population. We used two populations of Trifolium pratense and assessed their performance when grown in association with their home versus away soil biota. Both populations were colonized by distinct microbial communities and performed better with their own home soil communities than with the soil community from the other intraspecific population, demonstrating intraspecific positive feedback effects of home soil. In one of the two populations, we found that plant performance and the root associated microbiota community differed between parental and progeny plants when inoculated with their own home soil. Differences in root associated community characteristics could explain more than 80% of the variation in performance among the progeny and parental plants. Our results highlight that intraspecific differences in both plant and associated soil communities shape plant–soil feedback effects, and consequently indicate that plant–soil feedback can influence the direction of selection between intraspecific plant populations.     

Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance

Introductionbla_OXA-48, bla_NDM-1 and bla_CTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described bla_OXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the bla_SHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania).MethodsSequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank.ResultsComparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the bla_OXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible.ConclusionThis work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.     

V-REVCOMP: automated high-throughput detection of reverse complementary 16S rRNA gene sequences in large environmental and taxonomic datasets

Reverse complementary DNA sequences - sequences that are inadvertently given backwards with all purines and pyrimidines transposed - can affect sequence analysis detrimentally unless taken into account. We present an open-source, high-throughput software tool -v-revcomp (http://www.cmde.science.ubc.ca/mohn/software.html) - to detect and reorient reverse complementary entries of the small-subunit rRNA (16S) gene from sequencing datasets, particularly from environmental sources. The software supports sequence lengths ranging from full length down to the short reads that are characteristic of next-generation sequencing technologies. We evaluated the reliability of v-revcomp by screening all 406 781 16S sequences deposited in release 102 of the curated SILVA database and demonstrated that the tool has a detection accuracy of virtually 100%. We subsequently used v-revcomp to analyse 1 171 646 16S sequences deposited in the International Nucleotide Sequence Databases and found that about 1% of these user-submitted sequences were reverse complementary. In addition, a nontrivial proportion of the entries were otherwise anomalous, including reverse complementary chimeras, sequences associated with wrong taxa, nonribosomal genes, sequences of poor quality or otherwise erroneous sequences without a reasonable match to any other entry in the database. Thus, v-revcomp is highly efficient in detecting and reorienting reverse complementary 16S sequences of almost any length and can be used to detect various sequence anomalies.     

Cytomegalovirus viremia as a risk factor for mortality prior to antiretroviral therapy among HIV-infected gold miners in South Africa

Background

Cytomegalovirus (CMV) viremia has been shown to be an independent risk factor for increased mortality among HIV-infected individuals in the developing world. While CMV infection is nearly ubiquitous in resource-poor settings, few data are available on the role of subclinical CMV reactivation on HIV.

Methods

Using a cohort of mineworkers with stored plasma samples, we investigated the association between CMV DNA concentration and mortality prior to antiretroviral therapy availability.

Results

Among 1341 individuals (median CD4 count 345 cells/µl, 70% WHO stage 1 or 2, median follow-up 0.9 years), 70 (5.2%) had CMV viremia at baseline; 71 deaths occurred. In univariable analysis CMV viremia at baseline was associated with a three-fold increase in mortality (hazard ratio [HR] 3.37; 95% confidence intervals [CI] 1.60, 7.10). After adjustment for CD4 count, WHO stage and HIV viral load (N = 429 with complete data), the association was attenuated (HR 2.27; 95%CI 0.88, 5.83). Mortality increased with higher CMV viremia (≥1,000 copies/ml vs. no viremia, adjusted HR 3.65, 95%CI: 1.29, 10.41). Results were similar using time-updated CMV viremia.

Conclusions

High copy number, subclinical CMV viremia was an independent risk factor for mortality among male HIV-infected adults in South Africa with relatively early HIV disease. Studies to determine whether anti-CMV therapy to mitigate high copy number viremia would increase lifespan are warranted.     

Antimicrobial Properties of a Novel Silver-Silica Nanocomposite Material

Nanotechnology enables development and production of novel silver-based composite materials. We used in vitro tests to demonstrate the antimicrobial activity of a silver-silica nanocomposite compared to the activities of conventional materials, such as silver nitrate and silver zeolite. A silver-silica-containing polystyrene material was manufactured and shown to possess strong antimicrobial properties.Many applications, including medicine and food production and storage, would benefit greatly from incorporation of safe and inexpensive long-lasting biocides into polymers, paints, or textiles (1, 5). The antimicrobial effect of silver additives is broadly used in various injection-molded plastic products, in textiles (13), and in coating-based applications, including air ducts, countertops, and food preparation areas (12). Some important advantages of silver-based antimicrobials are their excellent thermal stability and their health and environmental safety (19). However, like the use of all biocide products, the use of silver is strictly controlled by various national laws and control agencies. In the United States, the Environmental Protection Agency has regulated the use of silver as a biocide since 1954 (2) under the Federal Insecticide Fungicide and Rodenticide Act. In the European Union, a European biocide product directive (EU/BPD/98) imposes regulatory requirements on the use and claims associated with all biocide products (3).In the past few years, there has been a tremendous push for development of inorganic nanoparticles with structures that exhibit novel physical, chemical, and biological properties (34). In particular, the potential benefits of nano-silver materials have been recognized by many industries due to the strong antimicrobial activity of silver against a broad spectrum of bacteria, viruses, and fungi and the low frequency of development of resistance (10, 30).Generally, silver-based antimicrobial additives consist of silver ions integrated into inert matrices consisting of ceramic, glass, or zeolite. Other silver additives based on silver salts or metallic silver may be readily incorporated into thermoplastic polymers, such as polyethylene, polypropylene, polystyrene, or nylon (5). The bactericidal efficacy of silver-containing polymers is based on the release of silver ions (Ag⁺) through interaction with a liquid watery phase (19). Although the antimicrobial effects of silver ions and salts have been intensively studied, the mechanism of the inhibitory action of silver on microbes is still not fully understood. It has been proposed that silver ions interact with disulfide or sulfhydryl groups of enzymes, causing structural changes that lead to disruption of metabolic processes followed by cell death (8, 11). The inhibitory action of silver nanoparticles is also based on the release of Ag⁺ (20, 24). Exposure of microorganisms to silver nanoparticles was shown to result in strong antimicrobial activity (6, 9, 26, 33). In addition to the increased surface area and associated increased potential for the release of Ag⁺, when dispersed in liquid suspensions, silver nanoparticles may accumulate in the bacterial cytoplasmic membrane, causing a significant increase in permeability and cell death (33), and penetrate bacterial cells (28). Recently, it has been suggested that the antimicrobial mechanism of silver nanoparticles may also be related to membrane damage due to free radicals that are derived from the surface of the nanoparticles (16). This bactericidal activity also appears to be dependent on the size and shape of the silver nanoparticles (25).In this study, we evaluated the properties of a novel silver-silica nanocomposite material (HeiQ AGS-20; HeiQ Materials, Bad Zurzach, Switzerland) used as an antimicrobial additive and compared its efficacy to the efficacies of the conventional silver additives silver nitrate (AgNO₃; 63.5% Ag) and silver zeolite (38% Ag bound to type A zeolite; Sigma-Aldrich, Buchs, Switzerland). The novel silver-silica nanocomposite material was produced using an industrial flame spray pyrolysis process. This process involves combustion of a flammable solvent containing homogeneously dissolved compounds as the source of components for the synthesis of the material (14, 21, 22). A representative transmission electron micrograph of the silver-silica material is shown in Fig. ​Fig.1.1. The nanocomposite consists of silver nanoparticles embedded in a matrix of amorphous silicon dioxide (SiO₂). The SiO₂ fine structure consists of aggregate matrix particles with an average diameter of approximately 1 μm (Fig. ​(Fig.1A).1A). Silver metal particles are located on the surface of the silica and are also embedded within the matrix (Fig. ​(Fig.1B).1B). High-magnification scanning transmission electron microscopy imaging of a localized region of a nanoparticle indicated that each silica particle contains many small silver metal particles with a typical diameter between 1 and 10 nm (Fig. ​(Fig.1C).1C). The specific surface area of the nanocomposite powder, as measured by nitrogen adsorption (7), is typically about 250 m²/g, a value which is consistent with the open structure of the silica aggregate shown in Fig. ​Fig.1.1. It can be concluded that, upon contact with moisture, the pure silver particles act as a source that releases silver ions, which represent the active antimicrobial principle (27). Some key advantages of the novel nanocomposite are the dispersion of the discrete silver particles throughout the silica (which prevents agglomeration of the silver particles), the small diameter of the silver particles (which results in a large surface area and release of a large amount of Ag⁺, which results in high antimicrobial efficiency), and the small size of the silver-silica composite (ca. 1 μm) (which allows the material to be uniformly dispersed and readily incorporated into a variety of substrates, including synthetic fibers, plastics, and other thin or delicate materials). The silica structure acts as a convenient carrier for incorporating the fine silver particles into plastics, textiles, and coatings. A further advantage is that the immobilization of silver nanoparticles within the silica structure limits the potential for release and disposal of the nanoparticles themselves. This property may be highly desirable because of the possible abilities of nanoparticles to cross biological membranes and other barriers (31).Open in a separate windowFIG. 1.(A) Transmission electron micrograph showing an amorphous silicon dioxide aggregate particle (gray structure) together with numerous supported silver metal particles (dark spots). (B) Scanning transmission electron micrograph of the structure shown in panel A, providing better contrast between the silica structure (gray) and the silver metal particles (bright spots). (C) Higher magnification of the region in panel B enclosed in a box. The silver metal particles are typically between 1 and 10 nm in diameter. Transmission electron microscope and scanning transmission electron microscope images were obtained using an FEI Tecnai F30 FEG microscope operated at 300 kV.The microorganisms and growth conditions used for antimicrobial testing are shown in Table ​Table1.1. The MICs for all combinations of silver materials and microorganisms were determined by preparing twofold serial dilutions of the additives in an appropriate growth medium (Table ​(Table1).1). The tubes were then inoculated with 10⁷ CFU/ml from overnight cultures of the bacteria or 10⁶ CFU/ml for Candida albicans and incubated on a shaker (180 rpm) for 24 h. The MIC was defined as the lowest concentration of the silver additive at which no visual turbidity of the growth medium developed. The minimal bactericidal concentration (MBC) was determined by surface plating 0.2-ml aliquots from the nonturbid tubes, followed by incubation at 37°C for 24 h. The MBC was defined as the lowest concentration of silver additive resulting in less than 200 colonies per plate (corresponding to a killing rate of more than 4 logs). Aspergillus niger spores were harvested by floating the spores in densely grown lawns on malt extract agar plates in an extraction buffer (0.1% [vol/vol] Tween 20, 145 mM sodium chloride, 20 mM sodium phosphate; pH 7.4) and removing them. The MIC was determined by spreading approximately 200 spores on malt extract agar plates containing serial dilutions of the silver additives (Table ​(Table1)1) and was defined as the lowest concentration that prevented visible growth after 72 h of incubation at 30°C.

TABLE 1.

Comparison of the antimicrobial activities of silver nanocomposite powder, silver nitrate, and silver zeolite

Airway closure on imaging relates to airway hyperresponsiveness and peripheral airway disease in asthma

The regional pattern and extent of airway closure measured by three-dimensional ventilation imaging may relate to airway hyperresponsiveness (AHR) and peripheral airways disease in asthmatic subjects. We hypothesized that asthmatic airways are predisposed to closure during bronchoconstriction in the presence of ventilation heterogeneity and AHR. Fourteen asthmatic subjects (6 women) underwent combined ventilation single photon emission computed tomography/computed tomography scans before and after methacholine challenge. Regional airway closure was determined by complete loss of ventilation following methacholine challenge. Peripheral airway disease was measured by multiple-breath nitrogen washout from which S(cond) (index of peripheral conductive airway abnormality) was derived. Relationships between airway closure and lung function were examined by multiple-linear regression. Forced expiratory volume in 1 s was 87.5 ± 15.8% predicted, and seven subjects had AHR. Methacholine challenge decreased forced expiratory volume in 1 s by 23 ± 5% and increased nonventilated volume from 16 ± 4 to 29 ± 13% of computed tomography lung volume. The increase in airway closure measured by nonventilated volume correlated independently with both S(cond) (partial R(2) = 0.22) and with AHR (partial R(2) = 0.38). The extent of airway closure induced by methacholine inhalation in asthmatic subjects is greater with increasing peripheral airways disease, as measured by ventilation heterogeneity, and with worse AHR.     

Completeness of Follow-Up Determines Validity of Study Findings: Results of a Prospective Repeated Measures Cohort Study

Background

Current reporting guidelines do not call for standardised declaration of follow-up completeness, although study validity depends on the representativeness of measured outcomes. The Follow-Up Index (FUI) describes follow-up completeness at a given study end date as ratio between the investigated and the potential follow-up period. The association between FUI and the accuracy of survival-estimates was investigated.

Methods

FUI and Kaplan-Meier estimates were calculated twice for 1207 consecutive patients undergoing aortic repair during an 11-year period: in a scenario A the population’s clinical routine follow-up data (available from a prospective registry) was analysed conventionally. For the control scenario B, an independent survey was completed at the predefined study end. To determine the relation between FUI and the accuracy of study findings, discrepancies between scenarios regarding FUI, follow-up duration and cumulative survival-estimates were evaluated using multivariate analyses.

Results

Scenario A noted 89 deaths (7.4%) during a mean considered follow-up of 30±28months. Scenario B, although analysing the same study period, detected 304 deaths (25.2%, P<0.001) as it scrutinized the complete follow-up period (49±32months). FUI (0.57±0.35 versus 1.00±0, P<0.001) and cumulative survival estimates (78.7% versus 50.7%, P<0.001) differed significantly between scenarios, suggesting that incomplete follow-up information led to underestimation of mortality. Degree of follow-up completeness (i.e. FUI-quartiles and FUI-intervals) correlated directly with accuracy of study findings: underestimation of long-term mortality increased almost linearly by 30% with every 0.1 drop in FUI (adjusted HR 1.30; 95%-CI 1.24;1.36, P<0.001).

Conclusion

Follow-up completeness is a pre-requisite for reliable outcome assessment and should be declared systematically. FUI represents a simple measure suited as reporting standard. Evidence lacking such information must be challenged as potentially flawed by selection bias.     

Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae

Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.     

Betamethasone activation of CTP: Cholinephosphate cytidylyltransferase is mediated by fatty acids

The purpose of the present study was to determine the mechanisms by which glucocorticoids increase the activity of CTP: cholinephosphate cytidylyltransferase, a key enzyme required for the synthesis of surfactant phosphatidylcholine. Lung cytidylyltransferase exists as an inactive, light form low in lipids (L-form) and an active, heavy form high in lipid content (H-form). In vivo, fatty acids stimulate and aggregate the inactive L-form to the active H-form. In vivo, betamethasone increases the amount of H-form while decreasing the amount of L-form in fetal lung. There is also a coordinate increase in total free fatty acids in the H-form. In the present study, we used gas chromatography–mass spectrometry to measure the fatty acid species associated with the H-forms in fetal rat lung after the mothers were treated with betamethasone (1 mg/kg). In vivo, betamethasone increased the total amount of free fatty acids associated with the H-form by 62%. Further, the hormone selectively increased the mass of myristic and oleic acids in H-form by 52 and 82%, respectively. However, betamethasone produced the greatest increase in the amount of H-form linoleic acid, which increased fourfold relative to control. In vitro, each of the fatty acids increased L-form activity in a dose-dependent manner; however, linoleic acid was the most potent. Linoleic and oleic acids also effectively increased L-form aggregations. These observations suggest that in vivo glucocorticoids elevate the level of specific fatty acids which convert cytidylyltransferase to the active form. © 1995 Wiley-Liss, Inc.