No abstract

MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments.Francisella tularensis is the causative agent of tularemia, a zoonotic disease that is typically transmitted by inhalation, by the bite of an infected arthropod, or by the ingestion of contaminated water. F. tularensis subsp. tularensis, subsp. holarctica, and subsp. mediasiatica are pathogens of humans and various other mammals (12). F. tularensis subsp. novicida (F. novicida) is infectious for immunocompromised humans only (22). All F. tularensis subspecies and F. novicida cause severe disease in mice, though their lethal doses vary in accordance to their propensity to cause human disease (12). Human pathogenic F. tularensis are highly infectious: fewer than 10 bacteria inoculated by the respiratory route can cause the disease in humans and result in a high incidence of mortality if untreated (12). The mechanisms that cause high F. tularensis infectivity and virulence are unknown. One possible mechanism is the remarkable ability of F. tularensis to evade innate immune responses early in infection. In mice infected with F. tularensis, proinflammatory cytokines and chemokines are usually measured only after a significant systemic organ burden and inflammatory granulocyte infiltration occurs (4, 9, 11).

We investigated whether cognitive decline could be explained by resting-state electroencephalography (EEG) biomarkers measured in prefrontal regions that reflect the slowing of intrinsic EEG oscillations. In an aged population dwelling in a rural community (total = 496, males = 165, females = 331), we estimated the global cognitive decline using the Mini-Mental State Examination (MMSE) and measured resting-state EEG parameters at the prefrontal regions of Fp1 and Fp2 in an eyes-closed state. Using a tertile split method, the subjects were classified as T3 (MMSE 28–30, N = 162), T2 (MMSE 25–27, N = 179), or T1 (MMSE ≤ 24, N = 155). The EEG slowing biomarkers of the median frequency, peak frequency and alpha-to-theta ratio decreased as the MMSE scores decreased from T2 to T1 for both sexes (−5.19 ≤ t-value ≤ −3.41 for males and −7.24 ≤ t-value ≤ −4.43 for females) after adjusting for age and education level. Using a double cross-validation procedure, we developed a prediction model for the MMSE scores using the EEG slowing biomarkers and demographic covariates of sex, age and education level. The maximum intraclass correlation coefficient between the MMSE scores and model-predicted values was 0.757 with RMSE = 2.685. The resting-state EEG biomarkers showed significant changes in people with early cognitive decline and correlated well with the MMSE scores. Resting-state EEG slowing measured in the prefrontal regions may be useful for the screening and follow-up of global cognitive decline in elderly individuals.

Early transplant dysfunction and failure because of immunological and nonimmunological factors still presents a significant clinical problem for transplant recipients. A critical unmet need is the noninvasive detection and prediction of immune injury such that acute injury can be reversed by proactive immunosuppression titration. In this study, we used iTRAQ -based proteomic discovery and targeted ELISA validation to discover and validate candidate urine protein biomarkers from 262 renal allograft recipients with biopsy-confirmed allograft injury. Urine samples were randomly split into a training set of 108 patients and an independent validation set of 154 patients, which comprised the clinical biopsy-confirmed phenotypes of acute rejection (AR) (n ‫؍‬ 74), stable graft (STA) (n ‫؍‬ 74), chronic allograft injury (CAI) (n ‫؍‬ 58), BK virus nephritis (BKVN) (n ‫؍‬ 38), nephrotic syndrome (NS) (n ‫؍‬ 8), and healthy, normal control (HC) (n ‫؍‬ 10). A total of 389 proteins were measured that displayed differential abundances across urine specimens of the injury types (p < 0.05) with a significant finding that SUMO2 (small ubiquitin-related modifier 2) was identified as a "hub" protein for graft injury irrespective of causation. Sixtynine urine proteins had differences in abundance (p < 0.01) in AR compared with stable graft, of which 12 proteins were up-regulated in AR with a mean fold increase of 2.8. Nine urine proteins were highly specific for AR because of their significant differences (p < 0.01; fold increase >1.5) from all other transplant categories (HLA class II protein HLA-DRB1, KRT14, HIST1H4B, FGG, ACTB, FGB, FGA, KRT7, DPP4). Increased levels of three of these proteins, fibrinogen beta (FGB; p ‫؍‬ 0.04), fibrinogen gamma (FGG; p ‫؍‬ 0.03), and HLA DRB1 (p ‫؍‬ 0.003) were validated by ELISA in AR using an independent sample set. The fibrinogen proteins further segregated AR from BK virus nephritis (FGB p ‫؍‬ 0.03, FGG p ‫؍‬ 0.02), a finding that supports the utility of monitoring these urinary proteins for the specific and sensitive noninvasive diagnosis of acute renal allograft rejection. Molecular & Cellular Proteomics

We obtained insight into normal lung function by proteome analysis of bronchoalveolar lavage fluid (BALF) from six normal human subjects using a "Lyse-N-Go' shotgun proteomic protocol. Intra-sample variation was calculated using three different label-free methods, (i) protein sequence coverage; (ii) peptide spectral counts and (iii) peptide single-ion current areas (PICA), which generates protein expression data by summation of the area under the curve for a given peptide single-ion current trace and then adding values for all peptides from that same parent protein.PICA gave the least intra-subject variability and was used to calculate differences in protein expression between the six subjects. We observed an average threefold inter-sample variability, which affects analysis of changes in protein expression that occur in different diseases. We detected 167 unique proteins with >100 proteins detected in each of the six individual BAL samples, 42 of which were common to all six subjects. Gene ontology analysis demonstrated enrichment of several biological processes in the lung, reflecting its expected role in gas exchange and host defense as an immune organ. The same biological processes were enriched compared to either plasma or total genome proteome, suggesting an active enrichment of plasma proteins in the lung rather than passive capillary leak.