AutoPrime: selecting primers for expressed sequences

A selection of evaluations from Faculty of 1000 covering microarrays to study antibiotic resistance, identifying SUMO substrate proteins, parallel SNP genotyping, intron origin and evolution and variation in CpG-island methylation in humans.     

Priming of inducible defenses protects Norway spruce against tree-killing bark beetles

Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short-lived model plants such as Arabidopsis, but little is known about this phenomenon in long-lived plants like spruce. We compared the effects of pretreatment with sublethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48-year-old Norway spruce (Picea abies) trees to mass attack by a tree-killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees but largely avoided fungus-inoculated trees and MeJA-treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91-fold higher terpene concentrations compared with untreated trees, whereas application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus-inoculated trees is a result of direct induction of defenses, whereas resistance in MeJA-treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.     

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.     

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.     

Sediment microbial communities in Great Boiling Spring are controlled by temperature and distinct from water communities

Great Boiling Spring is a large, circumneutral, geothermal spring in the US Great Basin. Twelve samples were collected from water and four different sediment sites on four different dates. Microbial community composition and diversity were assessed by PCR amplification of a portion of the small subunit rRNA gene using a universal primer set followed by pyrosequencing of the V8 region. Analysis of 164 178 quality-filtered pyrotags clearly distinguished sediment and water microbial communities. Water communities were extremely uneven and dominated by the bacterium Thermocrinis. Sediment microbial communities grouped according to temperature and sampling location, with a strong, negative, linear relationship between temperature and richness at all taxonomic levels. Two sediment locations, Site A (87–80 °C) and Site B (79 °C), were predominantly composed of single phylotypes of the bacterial lineage GAL35 ($\widehat{p}$=36.1%), Aeropyrum ($\widehat{p}$=16.6%), the archaeal lineage pSL4 ($\widehat{p}$=15.9%), the archaeal lineage NAG1 ($\widehat{p}$=10.6%) and Thermocrinis ($\widehat{p}$=7.6%). The ammonia-oxidizing archaeon ‘Candidatus Nitrosocaldus'' was relatively abundant in all sediment samples <82 °C ($\widehat{p}$=9.51%), delineating the upper temperature limit for chemolithotrophic ammonia oxidation in this spring. This study underscores the distinctness of water and sediment communities in GBS and the importance of temperature in driving microbial diversity, composition and, ultimately, the functioning of biogeochemical cycles.     

Hyaluronan and N-ERC/Mesothelin as Key Biomarkers in a Specific Two-Step Model to Predict Pleural Malignant Mesothelioma

Purpose

Diagnosis of malignant mesothelioma is challenging. The first available diagnostic material is often an effusion and biochemical analysis of soluble markers may provide additional diagnostic information. This study aimed to establish a predictive model using biomarkers from pleural effusions, to allow early and accurate diagnosis.

Patients and Methods

Effusions were collected prospectively from 190 consecutive patients at a regional referral centre. Hyaluronan, N-ERC/mesothelin, C-ERC/mesothelin, osteopontin, syndecan-1, syndecan-2, and thioredoxin were measured using ELISA and HPLC. A predictive model was generated and validated using a second prospective set of 375 effusions collected consecutively at a different referral centre.

Results

Biochemical markers significantly associated with mesothelioma were hyaluronan (odds ratio, 95% CI: 8.82, 4.82–20.39), N-ERC/mesothelin (4.81, 3.19–7.93), CERC/mesothelin (3.58, 2.43–5.59) and syndecan-1 (1.34, 1.03–1.77). A two-step model using hyaluronan and N-ERC/mesothelin, and combining a threshold decision rule with logistic regression, yielded good discrimination with an area under the ROC curve of 0.99 (95% CI: 0.97–1.00) in the model generation dataset and 0.83 (0.74–0.91) in the validation dataset, respectively.

Conclusions

A two-step model using hyaluronan and N-ERC/mesothelin predicts mesothelioma with high specificity. This method can be performed on the first available effusion and could be a useful adjunct to the morphological diagnosis of mesothelioma.     

Effect of PEEP and Tidal Volume on Ventilation Distribution and End-Expiratory Lung Volume: A Prospective Experimental Animal and Pilot Clinical Study

Introduction

Lung-protective ventilation aims at using low tidal volumes (V_T) at optimum positive end-expiratory pressures (PEEP). Optimum PEEP should recruit atelectatic lung regions and avoid tidal recruitment and end-inspiratory overinflation. We examined the effect of V_T and PEEP on ventilation distribution, regional respiratory system compliance (C_RS), and end-expiratory lung volume (EELV) in an animal model of acute lung injury (ALI) and patients with ARDS by using electrical impedance tomography (EIT) with the aim to assess tidal recruitment and overinflation.

Methods

EIT examinations were performed in 10 anaesthetized pigs with normal lungs ventilated at 5 and 10 ml/kg body weight V_T and 5 cmH₂O PEEP. After ALI induction, 10 ml/kg V_T and 10 cmH₂O PEEP were applied. Afterwards, PEEP was set according to the pressure-volume curve. Animals were randomized to either low or high V_T ventilation changed after 30 minutes in a crossover design. Ventilation distribution, regional C_RS and changes in EELV were analyzed. The same measures were determined in five ARDS patients examined during low and high V_T ventilation (6 and 10 (8) ml/kg) at three PEEP levels.

Results

In healthy animals, high compared to low V_T increased C_RS and ventilation in dependent lung regions implying tidal recruitment. ALI reduced C_RS and EELV in all regions without changing ventilation distribution. Pressure-volume curve-derived PEEP of 21±4 cmH₂O (mean±SD) resulted in comparable increase in C_RS in dependent and decrease in non-dependent regions at both V_T. This implied that tidal recruitment was avoided but end-inspiratory overinflation was present irrespective of V_T. In patients, regional C_RS differences between low and high V_T revealed high degree of tidal recruitment and low overinflation at 3±1 cmH₂O PEEP. Tidal recruitment decreased at 10±1 cmH₂O and was further reduced at 15±2 cmH₂O PEEP.

Conclusions

Tidal recruitment and end-inspiratory overinflation can be assessed by EIT-based analysis of regional C_RS.     

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.