Vibration response imaging: protocol for a systematic review.

The concept of lung sounds conveying information regarding lung physiology has been used extensively in clinical practice, particularly with physical auscultation using a stethoscope. Advances in computer technology have facilitated the construction of dynamic visual images derived from recorded lung sounds. Arguably, the most significant progress in this field was the development of the commercially available vibration response imaging (VRI) (Deep Breeze Ltd, Or-Akiva, Israel). This device provides a non-invasive, dynamic image of both lungs constructed from sounds detected from the lungs using surface sensors. In the literature, VRI has been utilized in a multitude of clinical and research settings. This systematic review aims to address three study questions relating to whether VRI can be used as an evaluative device, whether the images generated can be characterized, and which tools and measures have been used to assess these images. This systematic review will involve implementing search strategies in five online journal databases in order to extract articles relating to the application of VRI. Appropriate articles will be identified against a set of pre-determined eligibility criteria and assessed for methodological quality using a standardized scale. Included articles will have data extracted by the reviewers using a standardized evidence table. A narrative synthesis based on a standardized framework will be conducted, clustering evidence into three main groups; one for each of the study questions. A meta-analysis will be conducted if two or more research articles meet pre-determined criteria that allow quantitative synthesis to take place. This systematic review aims to provide a complete overview of the scope of VRI in the clinical and research settings, as well as to discuss methods to interpret the data obtained from VRI. The systematic review intends to help clinicians to make informed decisions on the clinical applicability of the device, to allow researchers to identify further potential avenues of investigation, and to provide methods for the evaluation and interpretation of dynamic and static images. The publication and registration of this review with PROSPERO provides transparency and accountability, and facilitates the appraisal of the proposed systematic review against the original design. PROSPERO registration number: CRD42013003751

P40 and P90 from Mpn142 are targets of multiple processing events on the surface of mycoplasma pneumoniae

© 2015 by the authors. Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9-84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30

Terminomics methodologies and the completeness of reductive dimethylation: A meta-analysis of publicly available datasets

© 2019 by the authors. Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data

Correlation of the International Prostate Symptom Score bother question with the Benign Prostatic Hyperplasia Impact Index in a clinical practice setting

To evaluate the association between the International Prostate Symptom Score (IPSS) bother question (BQ) and a validated disease-specific quality-of-life questionnaire, the Benign Prostatic Hyperplasia (BPH) Impact Index (BPH-II), using the BPH Registry and Patient Survey database. PATIENTS AND METHODS The BPH Registry and Patient Survey is a multicentre, longitudinal, observational database of management practices and patient outcomes in a population of patients with BPH in the USA, managed with watchful waiting or pharmacotherapy. Men enrolled in the BPH Registry who completed the IPSS BQ and the four-item BPH-II at enrolment were identified. The association between the IPSS BQ score and the BPH Impact Index was assessed using Spearman rank correlation. RESULTS At baseline (enrolment visit), 6439 men (mean age 66 years) completed the IPSS BQ and the BPH-II. The mean (sd) score of the IPSS BQ was 2.5 (1.4) and of the BPH-II was 2.8 (2.8). Based on responses to the BPH-II, at least half the men reported that their urinary symptoms were associated with physical discomfort, worry about their health, and bothersomeness. The IPSS BQ score was significantly correlated ( P  < 0.001) with the BPH-II ( r  = 0.68) and each of its four questions (physical discomfort, r  = 0.52; worry about health, r  = 0.53; bothersomeness of trouble with urination, r  = 0.67; and time kept from usual activities, r  = 0.44). CONCLUSIONS The IPSS BQ score has a strong and positive correlation with the BPH-II among men enrolled in the BPH Registry. The IPSS BQ is a convenient tool for assessing disease-specific quality of life when determining treatment strategies and evaluating treatment outcomes in men with BPH.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72931/1/j.1464-410X.2008.07574.x.pd

A comprehensive guide for performing sample preparation and top-down protein analysis

© 2017 by the authors. Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform0s abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism0s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer

N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen

© 2017 The Author(s). Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

© 2016 The Authors. Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endopro-teolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions.

Background: Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV. Results: Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIVrtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4β7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques. Conclusion: Taken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus