Interactions Between Rhinanthus minor and Its Hosts: A Review of Water, Mineral Nutrient and Hormone Flows and Exchanges in the Hemiparasitic Association

At the ecological level, the effects of the facultative root hemiparasite Rhinanthus minor on the structure and functioning of its host communities are relative well described; yet until recently, the mechanistic basis for parasitic plant-driven community change and the physiological basis for the host-parasite interaction were poorly understood. Empirical incremental flow models, based on the increase in water, mineral nutrients, carbon assimilates or phytohormones between two defined time points, have been successfully employed to investigate the physiology of resource acquisition by- and distribution within host-parasitic plant associations. In this study we review the application of these empirical flow models to Rhinanthus-host associations showing the extent of and physiological basis of resource abstraction from the host and how this is profoundly influenced by soil nutrient status. We show that Rhinanthus primarily abstracts water and mineral nutrients via the apoplastic pathway through direct lumen-lumen connections with little resource acquisition via symplastic pathways. Nutrient status of the soil is shown to significantly influence the resource acquisition. We also investigate the hormonal regulation of resource acquisition by Rhinanthus showing pivotal roles for the key for the phytohormones abscisic acid (ABA) and cytokinins.     

Strategies for the plant-based expression of dengue subunit vaccines

Despite significant efforts in many countries, there is still no commercially viable dengue vaccine. Currently, attention is focused on the development of either live attenuated vaccines or live attenuated chimaeric vaccines using a variety of backbones. Alternate vaccine approaches, such as whole inactivated virus and subunit vaccines are in the early stages of development, and are each associated with different problems. Subunit vaccines offer the advantage of providing a uniform antigen of well-defined nature, without the added risk of introducing any genetic material into the person being inoculated. Preliminary trials of subunit vaccines (using dengue E protein) in rhesus monkeys have shown promising results. However, the primary disadvantages of dengue subunit vaccines are the low levels of expression of dengue proteins in mammalian or insect cells, as well as the added unknown risks of antigens produced from mammalian cells containing other potential sources of contamination. In the past two decades, plants have emerged as an alternative platform for expression of biopharmaceutical products, including antigens of bacterial, fungal or viral origin. In the present minireview, we highlight the current plant expression technologies used for expression of biopharmaceutical products, with an emphasis on plants as a production system for dengue subunit vaccines.     

Characterization of a Leishmania stage‐specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence

Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein‐encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co‐immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene‐deleted parasites (Ldp27^?/?) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27^?/? parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.     

Longitudinal Genome-Wide Association of Cardiovascular Disease Risk Factors in the Bogalusa Heart Study

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recent genome-wide association (GWA) studies have pinpointed many loci associated with CVD risk factors in adults. It is unclear, however, if these loci predict trait levels at all ages, if they are associated with how a trait develops over time, or if they could be used to screen individuals who are pre-symptomatic to provide the opportunity for preventive measures before disease onset. We completed a genome-wide association study on participants in the longitudinal Bogalusa Heart Study (BHS) and have characterized the association between genetic factors and the development of CVD risk factors from childhood to adulthood. We report 7 genome-wide significant associations involving CVD risk factors, two of which have been previously reported. Top regions were tested for replication in the Young Finns Study (YF) and two associations strongly replicated: rs247616 in CETP with HDL levels (combined P = 9.7×10⁻²⁴), and rs445925 at APOE with LDL levels (combined P = 8.7×10⁻¹⁹). We show that SNPs previously identified in adult cross-sectional studies tend to show age-independent effects in the BHS with effect sizes consistent with previous reports. Previously identified variants were associated with adult trait levels above and beyond those seen in childhood; however, variants with time-dependent effects were also promising predictors. This is the first GWA study to evaluate the role of common genetic variants in the development of CVD risk factors in children as they advance through adulthood and highlights the utility of using longitudinal studies to identify genetic predictors of adult traits in children.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

The effect of mGlu8 deficiency in animal models of psychiatric diseases

The metabotropic glutamate receptor subtype 8 (mGlu₈) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu₈ deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu₈-deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu₈-deficient mice. Furthermore, novel object recognition, chlordiazepoxide-facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu₈ deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine-induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post-traumatic stress disorder. We conclude that mGlu₈ might be a potential target for disorders with pathophysiological changes in brain areas where mGlu₈ modulates glutamate and gamma-amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.     

Inhibition of the Activity of Both Domains of Lysostaphin through Peptidoglycan Modification by the Lysostaphin Immunity Protein

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.Lysostaphin is a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus (18) that lyses susceptible staphylococci by hydrolyzing the polyglycine cross bridges in their cell wall peptidoglycans (3). The lysostaphin gene sequence was independently determined in 1987 by two groups (8, 13). BLAST analysis (1) of mature lysostaphin revealed two domains: an N-terminal catalytic domain (CAT), which is a member of the M23 family of zinc metalloendopeptidases, and a C-terminal cell wall targeting domain (CWT), which is a member of the SH3b domain family (Fig. ​(Fig.11 A).Open in a separate windowFIG. 1.(A) Schematic diagram of mature lysostaphin, the recombinant catalytic domain (rCAT) (lysostaphin residues 1 to 148), and the recombinant cell wall targeting domain (rCWT) (lysostaphin residues 149 to 246). The numbers represent the beginning and end of the domains, and the solid boxes indicate the N-terminal His₆ tag of the recombinant proteins. (B) SDS-PAGE analysis of rCAT and rCWT purified by a nickel affinity column. Mobilities of molecular mass standards are given on the left side of the gel.The lysostaphin endopeptidase resistance gene (epr or lif) encodes a FemABX-like immunity protein that is located adjacent to the lysostaphin gene on the plasmid pACK1 in S. simulans bv. staphylolyticus (4, 7, 20). Members of the FemABX family of proteins are nonribosomal peptidyl transferases that are involved in the addition of cross bridge amino acids during peptidoglycan subunit synthesis in the cytoplasm (15). In S. simulans bv. staphylolyticus, the lysostaphin immunity protein inserts serines in place of some glycines during peptidoglycan synthesis, which provides resistance to lysostaphin (4, 20).Originally it was suggested that the incorporation of serines in these peptidoglycan cross bridges gave increased resistance to lysostaphin because of the inability of the enzyme to hydrolyze glycyl-serine or seryl-glycine bonds (4, 14, 16). Others later reported that the CWT specifically binds to the polyglycine cross bridges in staphylococci (6) and the binding of CWT to producer-strain cells was less than that to susceptible cells (2). However, the ability of the enzyme or its targeting domain to bind to purified peptidoglycans from staphylococci containing the lysostaphin resistance gene has not been determined. Therefore, we determined if the modification to staphylococcal peptidoglycan cross bridges made by the lysostaphin immunity protein affected the activity of the binding domain, the catalytic domain, or both.