The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate ...the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-κB and stimulates tumour necrosis factor-α production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.
Background Asthma is a heterogeneous disease characterized by abnormal airway pathophysiology and susceptibility to different stimuli, as exemplified by a subset of patients with exercise-induced ...bronchoconstriction. Induced sputum provides a noninvasive method to sample airway biofluids that are enriched in proteins. Objective We hypothesized that novel mechanisms in the pathogenesis of asthma might be revealed by studying the patterns of protein expression in induced sputum. Methods We used shotgun proteomics to analyze induced sputum from 5 healthy subjects and 10 asthmatic patients, including 5 with exercise-induced bronchoconstriction. Differential protein expression among asthmatic patients, asthma subphenotypes, and control subjects was determined by using spectral counting and computational methods. Results Using Gene Ontology analysis, we defined the functional landscape of the induced sputum proteome and applied network analysis to construct a protein interaction map for this airway compartment. Shotgun proteomics analysis identified a number of proteins the differential enrichment or depletion of which robustly distinguished asthmatic patients from healthy control subjects and captured the effects of exercise on induced sputum proteome. Functional and network analysis identified key processes, including proteolytic activity, that are known contributors to airway remodeling. Importantly, this approach highlighted previously unrecognized roles for differentially expressed proteins in pathways implicated in asthma, such as modulation of phospholipase A2 by secretoglobin, a putative role for S100A8/9 in human asthma, and selective upregulation of complement component 3a in response to exercise in asthmatic patients. Conclusion Computationally intensive analysis of induced sputum proteome is a powerful approach to understanding the pathophysiology of asthma and a promising methodology to investigating other diseases of the airways.
We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded ...affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.
Background:
Although posterior glenohumeral instability is becoming an increasingly recognized cause of shoulder pain, the role of posterior glenoid bone loss on outcomes remains incompletely ...understood.
Purposes:
To prospectively determine the amount of bone loss associated with posterior instability events and to determine predisposing factors based on preinstability imaging.
Study Design:
Cross-sectional study; Level of evidence, 3.
Methods:
A total of 1428 shoulders were evaluated prospectively for ≥4 years. At baseline, a subjective history of shoulder instability was ascertained for each patient, and bilateral noncontrast magnetic resonance imaging (MRI) scans of the shoulders were obtained regardless of any reported history of shoulder instability. The cohort was prospectively followed during the study period, and those who were diagnosed with posterior glenohumeral instability were identified. Postinjury MRI scans were obtained and compared with the screening MRI scans. Glenoid version, perfect-circle-based bone loss was measured for each patient's pre- and postinjury MRI scans using previously described methods.
Results:
Of the 1428 shoulders that were prospectively followed, 10 shoulders sustained a first-time posterior instability event and 3 shoulders sustained a recurrent posterior instability event. At baseline, 11 of 13 shoulders had some amount of glenoid dysplasia and/or bone loss. The change in glenoid bone loss was 5.4% along the axis of greatest loss (95% CI, 3.8%-7.0%; P = .009), 4.4% at the glenoid equator (95% CI, 2.7%-6.2%; P = .016), and 4.2% of total glenoid area (95% CI, 2.9%-5.3%; P = .002). Recurrent glenoid instability was associated with a greater amount of absolute bone loss along the axis of greatest loss compared with first-time instability (recurrent: 16.8% ± 1.1%; 95% CI, 14.6%-18.9%; first-time: 10.0% ± 1.5%; 95% CI, 7.0%-13.0%; P = .005). Baseline glenoid retroversion ≥10° was associated with a significantly greater percentage of bone loss along the axis of greatest loss (≥10° of retroversion: 13.5% ± 2.0%; 95% CI, 9.6%-17.4%; <10° of retroversion: 8.5% ± 0.8%; 95% CI, 7.0%-10.0%; P = .045).
Conclusions:
Posterior glenohumeral instability events were associated with glenoid bone loss of 5%. The amount of glenoid bone loss after a recurrent posterior glenohumeral instability event was greater than that after first-time instability. Glenoid retroversion ≥10° was associated with a greater amount of posterior glenoid bone loss after a posterior instability event.
Proper regulation of protein levels is essential for health, and abnormal levels of proteins are hallmarks of many diseases. A number of studies have recently shown that messenger RNA levels vary ...among individuals of a species and that genetic linkage analysis can be used to identify quantitative trait loci that influence these levels. By contrast, little is known about the genetic basis of variation in protein levels in genetically diverse populations, in large part because techniques for large-scale measurements of protein abundance lag far behind those for measuring transcript abundance. Here we describe a label-free, mass spectrometry-based approach to measuring protein levels in total unfractionated cellular proteins, and we apply this approach to elucidate the genetic basis of variation in protein abundance in a cross between two diverse strains of yeast. Loci that influenced protein abundance differed from those that influenced transcript levels, emphasizing the importance of direct analysis of the proteome.
We investigated whether a single 60-min bout of whole leg, peristaltic pulse external pneumatic compression (EPC) altered select growth factor-related mRNAs and/or various phospho(p)-proteins related ...to cell growth, proliferation, inflammation and apoptosis signalling (e.g. Akt-mTOR, Jak-Stat). Ten participants (8 males, 2 females; aged 22·2 ± 0·4 years) reported to the laboratory 4 h post-prandial, and vastus lateralis muscle biopsies were obtained prior to (PRE), 1 h and 4 h post-EPC treatment. mRNA expression was analysed using real-time RT-PCR and phosphophorylated and cleaved proteins were analysed using an antibody array. No changes in selected growth factor-related mRNAs were observed following EPC. All p-proteins significantly altered by EPC decreased, except for p-rps6 (Ser235/236) which increased 31% 1 h post-EPC compared to PRE levels (P = 0·016). Notable decreases also included p-BAD (Ser112; -28%, P = 0·004) at 4 h post-EPC compared to PRE levels. In summary, an acute bout of EPC transiently upregulates p-rps6 as well as affecting other markers in the Akt-mTOR signalling cascade. Future research should characterize whether chronic EPC application promotes alterations in lower-limb musculature and/or enhances exercise-induced training adaptations.
Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative ...condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).
Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in ...the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.
Substituted triphenylamine (TPA) radical cations show great potential as oxidants and as spin-containing units in polymer magnets. Their properties can be further tuned by supramolecular assembly. ...Here, we examine how the properties of photogenerated radical cations, intrinsic to TPA macrocycles, are altered upon their self-assembly into one-dimensional columns. These macrocycles consist of two TPAs and two methylene ureas, which drive the assembly into porous organic materials. Advantageously, upon activation the crystals can undergo guest exchange in a single-crystal-to-single-crystal transformation generating a series of isoskeletal host–guest complexes whose properties can be directly compared. Photoinduced electron transfer, initiated using 365 nm light-emitting diodes, affords radicals at room temperature as observed by electron paramagnetic resonance (EPR) spectroscopy. The line shape of the EPR spectra and the quantity of radicals can be modulated by both polarity and heavy atom inclusion of the encapsulated guest. These photogenerated radicals are persistent, with half-lives between 1 and 7 d and display no degradation upon radical decay. Re-irradiation of the samples can restore the radical concentration back to a similar maximum concentration, a feature that is reproducible over several cycles. EPR simulations of a representative spectrum indicate two species, one containing two N hyperfine interactions and an additional broad signal with no resolvable hyperfine interaction. Intriguingly, TPA analogues without bromine substitution also exhibit similar quantities of photogenerated radicals, suggesting that supramolecular strategies can enable more flexibility in stable TPA radical structures. These studies will help guide the development of new photoactive materials.
Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell ...wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.
► Mass spectrometry defines several antibacterial T6SS substrates ► T6SS substrates from disparate species reveal an effector-immunity paradigm ► Informatic analysis based on shared properties defines a large T6S effector family ► Effector-immunity distribution indicates a broad role for T6SS in bacterial interactions
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