Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH 2 . A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. mechanism of action | respiratory chain | hypoosmotic stress response | metallocenes

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

Objectives To evaluate the uptake and outcome of expectant management of spontaneous first trimester miscarriage in an early pregnancy assessment unit. Participants 1096 consecutive patients with a diagnosis of spontaneous first trimester miscarriage. Methods Each miscarriage was classified as complete, incomplete, missed, or anembryonic on the basis of ultrasonography. Women who needed treatment were given the choice of expectant management or surgical evacuation of retained products of conception under general anaesthesia. Women undergoing expectant management were checked a few days after transvaginal bleeding had stopped, or they were monitored at weekly intervals for four weeks. Main outcome measures A complete miscarriage (absence of transvaginal bleeding and endometrial thickness < 15 mm), the number of women completing their miscarriage within each week of management, and complications (excessive pain or transvaginal bleeding necessitating hospital admission or clinical evidence of infection). Results Two patients with molar pregnancies were excluded, and 37% of the remainder (408/1094) were classified as having had a complete miscarriage. 70% (478/686) of women with retained products of conception chose expectant management; of these, 27 (6%) were lost to follow up. A successful outcome without surgical intervention was seen in 81% of cases (367/451). The rate of spontaneous completion was 91% (201/221) for those cases classified as incomplete miscarriage, 76% (105/138) for missed miscarriage, and 66% (61/92) for anembryonic pregnancy. 70% of women completed their miscarriage within 14 days of classification (84% for incomplete miscarriage and 52% for missed miscarriage and anembryonic pregnancy). Conclusions Most women with retained products of conception chose expectant management. Ultrasonography can be used to advise patients on the likelihood that their miscarriage will complete spontaneously within a given time.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.