Cold adaptation in the marine bacterium,Sphingopyxis alaskensis,assessed using quantitative proteomics

The cold marine environment constitutes a large proportion of the Earth's biosphere. Sphingopyxis alaskensis was isolated as a numerically abundant bacterium from several cold marine locations, and has been extensively studied as a model marine bacterium. Recently, a metabolic labelling platform was developed to comprehensively identify and quantify proteins from S. alaskensis. The approach incorporated data normalization and statistical validation for the purpose of generating highly confident quantitative proteomics data. Using this approach, we determined quantitative differences between cells grown at 10°C (low temperature) and 30°C (high temperature). Cold adaptation was linked to specific aspects of gene expression: a dedicated protein‐folding system using GroESL, DnaK, DnaJ, GrpE, SecB, ClpB and PPIase; polyhydroxyalkanoate‐associated storage materials; a link between enzymes in fatty acid metabolism and energy generation; de novo synthesis of polyunsaturated fatty acids in the membrane and cell wall; inorganic phosphate ion transport by a phosphate import PstB homologue; TonB‐dependent receptor and bacterioferritin in iron homeostasis; histidine, tryptophan and proline amino acid metabolism; and a large number of proteins without annotated functions. This study provides a new level of understanding on how important marine bacteria can adapt to compete effectively in cold marine environments. This study is also a benchmark for comparative proteomic analyses with other important marine bacteria and other cold‐adapted organisms.     

Phylogenetic representativeness: a new method for evaluating taxon sampling in evolutionary studies

Background  

Taxon sampling is a major concern in phylogenetic studies. Incomplete, biased, or improper taxon sampling can lead to misleading results in reconstructing evolutionary relationships. Several theoretical methods are available to optimize taxon choice in phylogenetic analyses. However, most involve some knowledge about the genetic relationships of the group of interest (i.e., the ingroup), or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications.     

Dense and sparse aggregations in complex motion: Video coupled with simulation modeling

Investigations into the complex behaviors of aggregations of highly mobile animals have not used the link between image processing technology and simulation modeling fruitfully to address many fundamental ecological issues. Examples include population censusing, which remains difficult despite the demonstrated ecological importance of assessing abundance and density of organisms. We introduce a theoretical framework that connects thermal infrared video imaging with an individual-based simulation model—an approach that appears to be applicable to a diverse set of aggregated, highly mobile, nocturnal animals. To demonstrate the framework two applications are presented. The first is a dense aggregation of Brazilian free-tailed bats (Tadarida brasiliensis) that exhibits an emergence pattern that has complex dynamics and the second is a sparse local aggregation of agricultural pest moths whose dynamics are insipid. The first application uses individual-based modeling to mimic the behavior in the video of bats during a nightly emergence from a cave and to provide reliable estimates of the numbers, and associated error bounds. The second application uses video recordings of sparse aggregations to provide consistent estimates of the numbers of flying noctuid moths recorded over a corn and cotton-dominated agroecosystem in south-central Texas. This does not use a mathematical model because error estimates tend to be small.     

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein ��2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.     

Annexin A1: a central player in the anti-inflammatory and neuroprotective role of microglia

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer's disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an "eat me" signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.     

A study of the effects of ‘Candidatus Phytoplasma mali’ on the psyllid Cacopsylla melanoneura (Hemiptera: Psyllidae)

Cacopsylla melanoneura is a univoltine psyllid vector of ‘Candidatus Phytoplasma mali’, the etiological agent of apple proliferation (AP), a severe disease in European apple orchards. The influence of ‘Ca. P. mali’ on the fitness of C. melanoneura was studied. In the spring of 2007, male-female pairs of field-collected adults were exposed to ‘Ca. P. mali’-infected or healthy ‘Golden Delicious’ apple shoots. Exposure to these diseased shoots did not affect the life span of the adult psyllids. However, significantly fewer eggs were laid on the diseased shoots. Furthermore, fewer of the eggs that were laid on the infected plants hatched. Data suggest a detrimental effect of AP phytoplasma on the fitness of C. melanoneura.     

Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses

Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.     

Porcine circovirus type 2 (PCV2) antigen localisation and post-weaning multisystemic wasting syndrome (PMWS) in free-ranging wild boar (<Emphasis Type="BoldItalic">Sus scrofa</Emphasis> ssp <Emphasis Type="BoldItalic">scrofa</Emphasis>) in Italy

Wild boar (Sus scrofa ssp scrofa) is a host of porcine circovirus 2 (PCV-2), and infection in this species has been reported in many countries. This study sampled tissues from 348 wild boars in Italy in the provinces of Bologna, Padua and Pisa. No clinical signs or macroscopic lesions were recorded in the sampled animals. Immunolocalisation for PCV-2 was performed on tissues, and viral antigen was disclosed in 38 animals, 11 immunohistochemistry (IHC) positive out of 148 (7.43%) sampled in northern Italy and 27 immunofluorescence (IF) positive out of 200 (13.5%) sampled in central Italy, respectively. Histologically, 19 cases displayed post-weaning multisystemic wasting syndrome (PMWS) histological lesions, but PCV2 was only proved in nine of them (three from Bologna and Padua by IHC and six from Pisa by IF). Animals aged 7 to 8 months were the most affected by PCV2 infection, suggesting an important role of this age class in the epidemiology of the virosis in the tested populations. Moreover, the study confirmed that wild boar can be a host of PCV-2, and showed that the virus and PMWS is widespread and endemic in wild boar in Italy as in other countries.