Epigenetic biosensors for bacteriophage detection and phage receptor discrimination

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvAB^ON subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvAB^ON subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed biosensors able to detect LPS, as well as protein-binding coliphages. Moreover, the combination of these tools allowed to identify the bacterial receptor triggering phage infection. The epigenetic opvAB::gfp biosensor thus comes in different flavours to detect a wide range of bacteriophages and identify the type of receptor they recognize.     

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

ABSTRACT

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.     

Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera

The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) plays a key role in regulating and modulating various physiological and behavioral processes in both protostomes and deuterostomes. The specific functions of serotonin are mediated by its binding to and subsequent activation of membrane receptors. The vast majority of these receptors belong to the superfamily of G-protein-coupled receptors. We report here the in vivo expression pattern of a recently characterized 5-HT₁ receptor of the honeybee Apis mellifera (Am5-HT_1A) in the mushroom bodies. In addition, we summarize current knowledge on the distribution of serotonin and serotonin receptor subtypes in the brain and specifically in the mushroom bodies of the fruit fly Drosophila melanogaster and the honeybee. Functional studies in these two species have shown that serotonergic signaling participates in various behaviors including aggression, sleep, circadian rhythms, responses to visual stimuli, and associative learning. The molecular, pharmacological, and functional properties of identified 5-HT receptor subtypes from A. mellifera and D. melanogaster will also be summarized in this review.     

Ratiometric fluorescent scaffold giving discrete response towards iodide ion: a combined experimental and DFT study

A novel ratiometric fluorescent receptor (Z)‐2‐(4‐[diethylamino]‐2‐hydroxybenzylideneamino) pyridine‐3‐carbaldehyde (3) bearing one phenolic OH and one aldehyde group as recognition sites was synthesized and characterized. The anion recognition behaviour of receptor 3 was evaluated by various spectroscopic (UV‐visible, fluorescence and ¹H nuclear magnetic resonance) methods and was validated by computational studies. The receptor showed fast response time, excellent selectivity and reproducibility towards iodide ion detection among the other surveyed anions, with a binding constant of 6.12 × 10⁴ M^?1 and a detection limit of 0.24 μM, thus confirming its potential applicability as a fluorescent sensor for iodide Copyright © 2014 John Wiley & Sons, Ltd.     

In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens

Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.     

Endothelial progenitor cells: Characterization, <Emphasis Type="Italic">in vitro</Emphasis> expansion,and prospects for autologous cell therapy

Injection of hematopoietic stem cells or endothelial progenitor cells (EPCs) expanded ex vivo has been shown to augment neovascularization in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. The limited quantity of EPCs in the circulation has been the main obstacle to clinical trials. Several authors have therefore attempted to expand these cells ex vivo in order to obtain a homogeneous cell therapy product. One possible means of expanding EPCs ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation promotes cell proliferation and C-X-C chemokine receptor type 4 (CXCR4) dependent migration and differentiation, with an overall angiogenic effect. This review summarizes the results and rationale of clinical trials of angiogenic therapy, the nature of EPCs, the different methods of ex vivo expansion, and current methods of quantification.     

A Method for Structure–Activity Analysis of Quorum-Sensing Signaling Peptides from Naturally Transformable Streptococci

Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure–function relationships. However, CSP variants have also been identified in many species, and each specifically activates its cognate receptor. How such minor changes dramatically affect the specificity of these peptides remains unclear. Structure–activity analysis of these peptides may provide clues for understanding the specificity of signaling peptide–receptor interactions. Here, we use the Streptococcus mutans CSP as an example to describe methods of analyzing its structure–activity relationship. The methods described here may provide a platform for studying quorum-sensing signaling peptides of other naturally transformable streptococci.     

嗜角蛋白真菌的界定、研究方法及其应用价值

嗜角蛋白真菌(keratinophilic fungi)是指能降解角蛋白并能利用其作为唯一营养源的真菌类群,大部分隶属于爪甲团囊菌目(Onygenales)、散囊菌目(Eurotiales)和肉座菌目(Hypocreales).由于该类群分布广、种类多、营养方式多样,其研究方法和技术手段也在不断进步,加深了其研究的深度...     

A pair of receptor‐like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol‐induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col‐0 and Cvi‐0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol‐induced stress condition led to identification of EGM1 and EGM2, a pair of tandem‐duplicated genes encoding receptor‐like kinases that are potentially involved in signaling of mannitol‐associated stress responses. Using various genetic approaches, we identified two non‐synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor‐like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.     

Olfactory receptors in aquatic and terrestrial vertebrates

In species representing different levels of vertebrate evolution, olfactory receptor genes have been identified by molecular cloning techniques. Comparing the deduced amino-acid sequences revealed that the olfactory receptor gene family of Rana esculenta resembles that of Xenopus laevis, indicating that amphibians in general may comprise two classes of olfactory receptors. Whereas teleost fish, including the goldfish Carassius auratus, possess only class I receptors, the `living fossil' Latimeria chalumnae is endowed with both receptor classes; interestingly, most of the class II genes turned out to be pseudogenes. Exploring receptor genes in aquatic mammals led to the discovery of a large array of only class II receptor genes in the dolphin Stenella Coeruleoalba; however, all of these genes were found to be non-functional pseudogenes. These results support the notion that class I receptors may be specialized for detecting water-soluble odorants and class II receptors for recognizing volatile odorants. Comparing the structural features of both receptor classes from various species revealed that they differ mainly in their extracellular loop 3, which may contribute to ligand specificity. Comparing the number and diversity of olfactory receptor genes in different species provides insight into the origin and the evolution of this unique gene family. Accepted: 29 July 1998     

A study of comparative modelling,simulation and molecular dynamics of CXCR3 receptor with lipid bilayer

The G-coupled receptors seen on the cell surface are composites with a lipid bilayer. The chemokines are kind of G-coupled receptor which majorly involved in the activation and downstream signalling of the cell. In general, many G-coupled receptors lack their 3D structures which become a hurdle in the drug designing process. In this study, comparative modelling of the CXCR3 receptor was carried out, structure evaluation was done using various tools and softwares. Additionally, molecular dynamics and docking were performed to prove the structural quality and architecture. Interestingly, the studies like toggle switch mechanism, lipid dynamics, virtual screening were carried out to find the potent antagonist for the CXCR3 receptor. During virtual screening 14,303 similar molecules were retrieved among them only four compounds have an ability to interact with a crucial amino acid residue of an antagonist. Hence, these screened compounds can serve as a drug candidate for a CXCR3 receptor, but further in vitro and in vivo studies are ought to do to prove its same efficacy.     

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane‐bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor‐interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi‐fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1‐1 and etr1‐2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.     

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including ΔGzPPG1;ΔGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

Comparative Localization of Receptors for Plasmin and for Urokinase on MCF7 Cells

The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment. On unfixed cells, these methods gave the same heterogeneous patterns of surface staining, consisting of contours and grains, localized mainly at the upper nonadherent face of the tumor cells by confocal microscopy. Only a part of the cells was stained. When both receptors were characterized together, their presence was found on the same cells and they gave almost superimposable patterns in many cases, as shown by confocal microscopy. In contrast, when MCF 7 cells were fixed or permeabilized before staining, quite different patterns were observed: almost all the cells were labeled. The staining was mainly cytoplasmic and localized preferentially in the center and close to the upper face of the cells. Similar results were found with antiserum against urokinase receptor and monoclonal antivinculin antibody. It is likely that the receptor for urokinase and the receptor for plasmin have similar localizations on MCF 7 cells, thus resulting in a functional cooperation.     

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide‐binding leucine‐rich repeat (NLR) protein with a Toll‐like interleukin‐1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely‐related Nicotiana sylvestris and the distantly‐related beet plant (Beta vulgaris). Roq1 was found to co‐immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.