A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.     

Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated Dungeness crab Metacarcinus magister

In the present study of the marine Dungeness crabs Metacarcinus magister, the long term effects of high environmental ammonia (HEA) on hemolymph ammonia and urea concentrations, branchial ammonia excretion rates and mRNA expression levels of the crustacean Rh-like ammonia transporter (RhMM), H(+)-ATPase (subunit B), Na(+)/K(+)-ATPase (α-subunit) and Na(+)/H(+)-exchanger (NHE) were investigated. Under control conditions, the crabs' hemolymph exhibited a total ammonia concentration of 179.3±14.5μmol L(-1), while urea accounted for 467.2±33.5μmol L(-1), respectively. Both anterior and posterior gills were capable of excreting ammonia against a 16-fold inwardly directed gradient. Under control conditions, mRNA expression levels of RhMM were high in the gills in contrast to very low expression levels in all other tissues investigated, including the antennal gland, hepatopancreas, and skeletal muscle. After exposure to 1mmol L(-1) NH(4)Cl, hemolymph ammonia increased within the first 12h to ca. 500μmol L(-1) and crabs were able the keep this hemolymph ammonia level for at least 4 days. During this initial period, branchial RhMM and H(+)-ATPase (subunit B) mRNA expression levels roughly doubled. After 14 days of HEA exposure, hemolymph ammonia raised up to environmental levels, whereas urea levels increased by ca. 30%. At the same time, whole animal ammonia and urea excretion vanished. Additionally, branchial RhMM, H(+)-ATPase, Na(+)/K(+)-ATPase and NHE mRNA levels decreased significantly after long term HEA exposure, whereas expression levels of RhMM in the internal tissues increased substantially. Interestingly, crabs acclimated to HEA showed no mortality even after 4 weeks of HEA exposure. This suggests that M. magister possesses a highly adaptive mechanism to cope with elevated ammonia concentrations in its body fluids, including an up-regulation of an Rh-like ammonia transporter in the internal tissues and excretion or storage of waste nitrogen in a so far unknown form.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

Evaluation of absolute peptide quantitation strategies using selected reaction monitoring

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.     

Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector

Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between patients in our study (245 m).     

Evolution and diversity of the Golgi

The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.