1H, 13C,and 15N backbone and side-chain chemical shift assignments of the free and bound forms of the human PTPN11 second SH2 domain

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the ¹H, ¹⁵N and ¹³C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.     

Impact of Agricultural Practices on the Zea mays L. Endophytic Community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

Secondary-structure characterization of two proficient kinase deoxyribozymes

Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.     

Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis

Organ growth results from the progression of component cells through subsequent phases of proliferation and expansion before reaching maturity. We combined kinematic analysis, flowcytometry, and microarray analysis to characterize cell cycle regulation during the growth process of leaves 1 and 2 of Arabidopsis (Arabidopsis thaliana). Kinematic analysis showed that the epidermis proliferates until day 12; thereafter, cells expand until day 19 when leaves reach maturity. Flowcytometry revealed that endoreduplication occurs from the time cell division rates decline until the end of cell expansion. Analysis of 10 time points with a 6k-cDNA microarray showed that transitions between the growth stages were closely reflected in the mRNA expression data. Subsequent genome-wide microarray analysis on the three main stages allowed us to categorize known cell cycle genes into three major classes: constitutively expressed, proliferative, and inhibitory. Comparison with published expression data obtained from root zones corresponding to similar developmental stages and from synchronized cell cultures supported this categorization and enabled us to identify a high confidence set of 131 proliferation genes. Most of those had an M phase-dependent expression pattern and, in addition to many known cell cycle-related genes, there were at least 90 that were unknown or previously not associated with proliferation.     

Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl purine nucleosides as inhibitors of hepatitis C virus RNA replication

A series of purine nucleosides containing the 2'-deoxy-2'-fluoro-2'-C-methylribofuranosyl moiety were synthesized and evaluated as potential inhibitors of the hepatitis C virus in vitro. Of the nucleosides that were synthesized, only those possessing a 2-amino group on the purine base reduced the levels of HCV RNA in a subgenomic replicon assay.     

Priming of Wheat with the Green Leaf Volatile Z-3-Hexenyl Acetate Enhances Defense against Fusarium graminearum But Boosts Deoxynivalenol Production

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.Biogenic volatile organic compounds (BVOCs) are known regulators of communication of sedentary plants with their direct environment (Dudareva et al., 2006). Besides attracting pollinators (Pichersky and Gershenzon, 2002), repelling insect herbivores (Birkett et al., 2010), and exerting direct antimicrobial properties (Friedman et al., 2002), BVOCs can act as an alarm signal to warn neighboring plants of an imminent herbivorous or pathogen attack (Heil and Ton, 2008) or serve as an intraplant signal for the induction of resistance (Karban et al., 2006). Engelberth et al. (2004) found that maize (Zea mays) seedlings emitted the green leaf volatiles (GLVs) Z-3-hexenal, Z-3-hexenol (Z-3-HOL), and Z-3-hexenyl acetate (Z-3-HAC) after they had been infested with caterpillars of Spodoptera exigua. Neighboring uninfested seedlings that had been exposed to these GLVs subsequently showed a considerable higher production of the plant defense hormone jasmonic acid (JA) after treatment with caterpillar regurgitant. This form of induced resistance is called priming. Plants in a primed state display faster and/or stronger activation of defense pathways when challenged by microbial pathogens, herbivorous insects, or abiotic stresses (Conrath, 2009). Exposure to these priming signals does not entail a direct activation of costly defense mechanisms but rather a stronger up-regulation of defense pathways when the plant is actually under attack (van Hulten et al., 2006). Besides resulting in a stronger induction of the JA pathway, priming also has been shown to enhance defense associated with the salicylic acid (SA) pathway, which plays a critical role in plant defense against biotrophic pathogens (Conrath et al., 2006; Jung et al., 2009).The lion’s share of attention on the use of GLVs in induced resistance has been directed to plant-insect interactions. However, the literature regarding priming by GLVs in plant-pathogen interactions remains scarce (Heil, 2014). Few studies have been performed investigating the effect of priming by GLVs on plant-fungus interactions (Scala et al., 2013a, and refs. therein). For example, hexanoic acid, a molecule with a similar structure to GLVs, has been shown to act as a priming agent in tomato (Solanum lycopersicum) plants against an infection by the necrotrophic fungus Botrytis cinerea, leading to a reduced accumulation of reactive oxygen species in primed plants (Vicedo et al., 2009; Kravchuk et al., 2011; Finiti et al., 2014). Since the GLVs E-2-hexenal (E-2-HAL), Z-3-HOL, E-2-hexenol, and Z-3-HAC also have been reported to be emitted by perennial ryegrass (Lolium perenne) after infection with Fusarium poae (Pańka et al., 2013) and by wheat (Triticum aestivum) seedlings after infection with Fusarium graminearum (Piesik et al., 2011), one may speculate that GLVs not only serve as a priming agent against the impending threat of herbivorous insects but rather constitute a general warning and priming mechanism against insects, bacteria, and fungi alike.Fusarium head blight (FHB) is an important disease in cereals caused by a complex of Fusarium spp., of which the hemibiotroph F. graminearum is one of the most prevalent (Parry et al., 1995; Goswami and Kistler, 2004; Audenaert et al., 2009). Besides yield losses of up to 40%, FHB also confers quality losses because of the production of mycotoxins such as deoxynivalenol (DON; Parry et al., 1995; Bottalico and Perrone, 2002; Vanheule et al., 2014).The hemiobiotrophic nature of F. graminearum entails that its lifestyle is characterized by a biotrophic phase followed by a necrotrophic phase. During the biotrophic phase, spores will germinate and hyphae will grow extracellularly and intercellularly. To counteract fungal colonization during the biotrophic phase, the host plant will accumulate hydrogen peroxide (H₂O₂) to induce programmed cell death. However, H₂O₂ acts as a signal for F. graminearum to produce DON, which in turn creates a positive feedback loop leading to increased H₂O₂ and DON production, clearing the way for F. graminearum to further colonize the host plant (Desmond et al., 2008). Plant defense against the biotrophic and necrotrophic phases generally has been linked to SA- and JA-related pathways, respectively (Glazebrook, 2005). This was also found in the study by Ding et al. (2011). They reported higher endogenous SA concentrations during the first hours of infection, followed by a rise in JA concentrations later on. However, plant defense against pathogens is regulated by a whole array of plant hormones, between which an intricate cross talk exists (Pieterse et al., 2012). One of the best-studied antagonistic signaling pathways is between SA and JA (Thaler et al., 2002; Pieterse et al., 2012). Research investigating the hormonal modulation of plant immunity has been done primarily in dicots. The negative relationship between SA and JA also seems to be conserved in rice (Oryza sativa), another monocot (De Vleesschauwer et al., 2013). Because of the presence of this possible antagonistic signaling and the hemibiotrophic lifestyle of F. graminearum, it is important to look more closely to the effect of priming on these two defense pathways in wheat.Here, we show that preexposure of wheat to the GLV Z-3-HAC primes wheat plants for an enhanced defense against a future infection with F. graminearum. Furthermore, our results indicate that pretreatment with Z-3-HAC leads to a stronger activation of JA-related defense while exerting suppressive effects on SA-responsive gene expression. Lastly, we found evidence that enhanced plant defense led to increased DON production by F. graminearum.     

Clonal Population Structure and Antimicrobial Resistance of Campylobacter jejuni in Chicken Meat from Belgium

Campylobacter jejuni is one of the most important causes of human diarrhea worldwide. In the present work, multilocus sequence typing was used to study the genotypic diversity of 145 C. jejuni isolates from 135 chicken meat preparations sampled across Belgium. Isolates were further typed by pulsed-field gel electrophoresis, and their susceptibilities to six antimicrobials were determined. Fifty-seven sequence types (STs) were identified; 26.8% of the total typed isolates were ST-50, ST-45, or ST-257, belonging to clonal complex CC-21, CC-45, or CC-257, respectively. One clonal group comprised 22% (32/145) of all isolates, originating from five different companies and isolated over seven sampling months. Additionally, 53.1% of C. jejuni isolates were resistant to ciprofloxacin, and 48.2% were resistant to tetracycline; 28.9% (42/145) of all isolates were resistant to both ciprofloxacin and tetracycline. The correlation between certain C. jejuni clonal groups and resistance to ciprofloxacin and tetracycline was notable. C. jejuni isolates assigned to CC-21 (n = 35) were frequently resistant to ciprofloxacin (65.7%) and tetracycline (40%); however, 90% (18/20) of the isolates assigned to CC-45 were pansusceptible. The present study demonstrates that certain C. jejuni genotypes recur frequently in the chicken meat supply. The results of molecular typing, combined with data on sample sources, indicate a possible dissemination of C. jejuni clones with high resistance to ciprofloxacin and/or tetracycline. Whether certain clonal groups are common in the environment and repeatedly infect Belgian broiler flocks or whether they have the potential to persist on farms or in slaughterhouses needs further investigation.Campylobacter jejuni is among the most common bacterial causes of human gastroenteritis worldwide (4, 23). Infected humans exhibit a range of clinical symptoms from mild, watery diarrhea to severe inflammatory diarrhea (14). In addition, C. jejuni has been identified as an important infectious trigger for Guillain-Barré syndrome, the most common cause of acute flaccid paralysis in polio-free regions (16). Another issue of concern regarding Campylobacter is the increase in antimicrobial resistance appearing in various regions around the world (1). Infection with an antimicrobial-resistant Campylobacter strain may lead to a suboptimal outcome of antimicrobial treatment or even to treatment failure (11).Consumption of contaminated water and raw milk has been implicated in campylobacteriosis outbreaks (23). However, the majority of human cases are sporadic, and consumption or mishandling of contaminated raw or undercooked poultry meat is believed to be an important source of infection. Risk assessment studies, outbreak investigations, and case-control reports all incriminate chicken meat as a major source, perhaps the major source, of food-borne transmission (14, 17, 32, 48). In Belgium in 1999, a controlled withdrawal of poultry products from sale due to alleged dioxin contamination resulted in a 40% reduction in the frequency of human campylobacteriosis (44). Thereafter and since the year 2000, the Campylobacter contamination of Belgian poultry carcasses and meat has been monitored by the Federal Agency for the Safety of the Food Chain, and the rate of positive samples is regarded as high. In 2006, 55.5% of cecal samples (n = 6,443) from Belgian broilers at slaughter tested positive for Campylobacter (3). In 2007, an industry-focused survey reported that 48% of Belgian chicken meat preparations (n = 656) were contaminated with Campylobacter (19).Molecular typing is an important tool in elucidating the diversity and transmission routes of Campylobacter isolates contaminating the food chain. In the United States, molecular analysis of Campylobacter spp. from poultry production and processing environments showed that many of the clones found within a flock are present in the final products, although the diversity of Campylobacter isolates in the final product was lower than that observed in the flock (22). Furthermore, numerous molecular epidemiological studies indicate that the genotypes of C. jejuni isolated from human cases overlap those of poultry origin (17, 47). Various molecular typing methods for the study of the population structure of Campylobacter are currently available (46). Among these, the multilocus sequence typing (MLST) approach is an emerging tool for research on the population structure and molecular epidemiology of Campylobacter. The technique is highly reproducible, portable, and easy to interpret, and results can be shared through a publicly accessible online database (31, 34). As such, MLST is becoming an important tool for studying the molecular epidemiology of Campylobacter in a global context. The accumulation of sequence typing data generated from different countries and settings could allow the creation of more-sophisticated models of the epidemiology and evolution of bacterial pathogens and the development of improved approaches for combating their spread (41).In Belgium, there is a paucity of information regarding the population structure of Campylobacter in the chicken meat supply. No population-based surveys have been conducted to investigate the molecular epidemiology of C. jejuni in chicken meat at points close to human consumption. In this study, MLST and pulsed-field gel electrophoresis (PFGE) were used to characterize the diversity of, and clonal relationships among, 145 C. jejuni isolates from Belgian chicken meat preparations. In addition, we characterized the antimicrobial resistance in this collection and correlated it with C. jejuni genotypes.     

Pyrrolo[1,2-a][1,4]benzodiazepine: a novel class of non-azole anti-dermatophyte anti-fungal agents

Broad screening revealed compound 1a to be a novel anti-fungal agent with high specificity towards dermatophytes. The anti-fungal structure-activity relationship of this novel class of 5,6-dihydro-4H-pyrrolo[1,2-a][1,4]benzodiazepines is described together with its mode of action that appeared to be the inhibition of squalene epoxidase. Preliminary in vivo results of the most active compounds are also reported.