Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

Cannibalism and intraguild predation in Typhlodromus exhilaratus and T. phialatus (Acari: Phytoseiidae) under laboratory conditions

Two species of Phytoseiidae are found in the same agroecosystem: Typhlodromus exhilaratus prevails in vine plots, while T. phialatus prevails in uncultivated surrounding areas. The objective of the present paper was to investigate whether the poor settlement of T. phialatus in vine plots can be explained by intraguild predation of these two species and/or cannibalism. Predatory abilities of the females on larvae and protonymphs were studied under laboratory conditions. A first experiment was conducted with only conspecific or heterospecific phytoseiid prey, in a second experiment Tetranychus urticae eggs were added to the phytoseiid prey. Oviposition, prey consumption, and escape rates of females were recorded. Oviposition and intraguild predation rates were higher for T. exhilaratus than for T. phialatus. Typhlodromus exhilaratus consumed fewer conspecifics than heterospecific phytoseiids, and oviposited when feeding on both diets. Typhlodromus phialatus consumed equal amounts of con- and heterospecifics. Although these two generalist predators belong to the type III defined by McMurtry and Croft (Annual Review of Entomology 42:291–321, 1997), our results suggest that they have different predation behaviour. However, because these results were obtained in experiments where no choice was given between the two phytoseiid species, they are difficult to link to previous studies conducted on the intraguild predation of the Phytoseiidae. The greater voracity and prolificacy of T. exhilaratus could partially explain the poor settlement of T. phialatus in vineyards and the predominance of T. exhilaratus. However, a full understanding of this phenomenon will require the study of other factors, such as susceptibility to pesticides and micro-climatic conditions, as well as the ability to cope with different food sources and host plants.     

Involvement of RD20, a member of caleosin family,in ABA-mediated regulation of germination in Arabidopsis thaliana

The RD20 gene encodes a member of the caleosin family, which is primarily known to function in the mobilization of seed storage lipids during germination. In contrast to other caleosins, RD20 expression is early-induced by water deficit conditions and we recently provided genetic evidence for its positive role in drought tolerance in Arabidopsis. RD20 is also responsive to pathogen infection and is constitutively expressed in diverse tissues and organs during development suggesting additional roles for this caleosin. This addendum describes further exploration of phenotypic alterations in T-DNA insertional rd20 mutant and knock-out complemented transgenic plants in the context of early development and susceptibility to a phytopathogenic bacteria. We show that the RD20 gene is involved in ABA-mediated inhibition of germination and does not play a significant role in plant defense against Pseudomonas syringae.Key words: ABA, Arabidopsis thaliana, biotic stress, caleosin, germination, RD20     

CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth?

Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity.     

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.     

Secretory carrier membrane protein 2 regulates exocytic insertion of NKCC2 into the cell membrane

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.     

Next Generation Sequencing Uncovers Unexpected Bacterial Pathogens in Ticks in Western Europe

Background and Aims

Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France.

Methods

RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence.

Results

We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas.

Conclusions

The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.     

Status of two cryptic species, Typhlodromus exhilaratus Ragusa and Typhlodromus phialatus Athias-Henriot (Acari: Phytoseiidae): consequences for taxonomy

Typhlodromus phialatus and T. exhilaratus are morphologically close species. Their differentiation is based on the shape of the insemination apparatus and on idiosomal setae length. However, the setae length values are often intermediate between these two species and do not allow accurate identification. Furthermore, the handful of differences in insemination apparatus shape are also questionable as a means of differentiation. Synonymy between these species has thus been questioned. Three experiments were carried out. Idiosomal seta length measurements, molecular tests and cross experiments were conducted for three populations, identified as T. exhilaratus and T. phialatus according to the shape of their insemination apparatus. The results show that the variation range of seta lengths is great and that these criteria do not allow accurate separation of these populations into two species. However, molecular tests show a species-level nucleotide differentiation between them. Cross experiments confirm this result, showing complete reproduction incompatibility between the mites bearing different insemination apparatus shapes. Therefore, T. exhilaratus and T. phialatus could be considered to be two valid species and the insemination apparatus could be considered as a pertinent diagnostic criterion at the specific level.