Search

Abstract In proteomics, spectral archives organize the enormous amounts of publicly available peptide tandem mass spectra by similarity, offering opportunities for error correction and novel discoveries. Here we adapt an indexing algorithm developed by Facebook for organizing online multimedia resources to tandem mass spectra and achieve practically instantaneous retrieval and clustering of approximate nearest neighbors in a large spectral archive. An interactive web-based graphical user interface enables the user to view a query spectrum in its clustered neighborhood, which facilitates contextual validation of peptide identifications and exploration of the dark proteome.

Abstract Background The high diversity and complexity of the microbial community make it a formidable challenge to identify and quantify the large number of proteins expressed in the community. Conventional metaproteomics approaches largely rely on accurate identification of the MS/MS spectra to their corresponding short peptides in the digested samples, followed by protein inference and subsequent taxonomic and functional analysis of the detected proteins. These approaches are dependent on the availability of protein sequence databases derived either from sample-specific metagenomic data or from public repositories. Due to the incompleteness and imperfections of these protein sequence databases, and the preponderance of homologous proteins expressed by different bacterial species in the community, this computational process of peptide identification and protein inference is challenging and error-prone, which hinders the comparison of metaproteomes across multiple samples. Results We developed metaSpectraST, an unsupervised and database-independent metaproteomics workflow, which quantitatively profiles and compares metaproteomics samples by clustering experimentally observed MS/MS spectra based on their spectral similarity. We applied metaSpectraST to fecal samples collected from littermates of two different mother mice right after weaning. Quantitative proteome profiles of the microbial communities of different mice were obtained without any peptide-spectrum identification and used to evaluate the overall similarity between samples and highlight any differentiating markers. Compared to the conventional database-dependent metaproteomics analysis, metaSpectraST is more successful in classifying the samples and detecting the subtle microbiome changes of mouse gut microbiomes post-weaning. metaSpectraST could also be used as a tool to select the suitable biological replicates from samples with wide inter-individual variation. Conclusions metaSpectraST enables rapid profiling of metaproteomic samples quantitatively, without the need for constructing the protein sequence database or identification of the MS/MS spectra. It maximally preserves information contained in the experimental MS/MS spectra by clustering all of them first and thus is able to better profile the complex microbial communities and highlight their functional changes, as compared with conventional approaches. tag the videobyte in this section as ESM4 Video Abstract

Biofilm is made up of microbes and their extracellular matrix, making microorganisms highly tolerant, resistant, and resilient to a wide range of antimicrobials. Biofilm treatment with conventional antimicrobial agents can accelerate the evolution and spread of resistance due to the reduced efficacy and increased gene transfer and differentiation within biofilms. Therefore, effective biofilm-targeting compounds are currently highly sought after. In the present study, we identified elasnin as a potent biofilm-targeting compound against methicillin-resistant Staphylococcus aureus (MRSA). Elasnin effectively inhibited biofilm formation and especially eradicated the pre-formed biofilms of MRSA with low cytotoxicity and low risk of resistance development and retains its activity in a chronic wound biofilms model. A comprehensive mechanistic study using multi-omics and confocal and scanning electron microscopy revealed that elasnin induced the biofilm matrix destruction in a time-dependent manner and interfered with the cell division during the exponential phase, primarily by repressing the expression of virulence factors. Cells released from the elasnin-treated biofilms exhibited a defective appearance and became more sensitive to beta-lactam antibiotic penicillin G. Through gene overexpression and deletion assay, we discovered the key role of sarZ during elasnin-induced biofilm eradication. Overall, the present study identified elasnin as a potent biofilm eradicator against MRSA that harbors potential to be developed for biofilm removal and chronic wound treatment, and provided new insights into the molecular targets for biofilm eradication in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly dangerous pathogen, and daptomycin has been increasingly used to treat its infections in clinics. Recently, several groups have shown that tolerance and resistance of microbes can evolve rapidly under cyclic antibiotic exposure. We have previously shown that the same tolerance and resistance development occurs in MRSA treated with daptomycin in an adaptive laboratory evolution (ALE) experiment. In the present study, we performed proteomic analysis to compare six daptomycin-tolerant and resistant MRSA strains that were evolved from the same ancestral strain. The strain with a higher tolerance level than the others had the most different proteome and response to antibiotic treatment, resembling those observed in persister cells, which are small subpopulations of bacteria that survive lethal antibiotics treatment. By comparing the proteome changes across strains with similar phenotypes, we identified the key proteins that play important roles in daptomycin tolerance and resistance in MRSA. We selected two candidates to be confirmed by gene overexpression analysis. Overexpression of EcsA1 and FabG, which were up-regulated in all of the tolerant evolved strains, led to increased daptomycin tolerance in wild-type MRSA. The proteomics data also suggested that cell wall modulations were implicated in both resistance and tolerance, but in different ways. While the resistant strains had peptidoglycan changes and a more positive surface charge to directly repel daptomycin, the tolerant strains possessed different cell wall changes that do not involve the peptidoglycan nor alterations of the surface charge. Overall, our study showed the differential proteome profiles among multiple tolerant and resistant strains, pinpointed the key proteins for the two phenotypes and revealed the differences in cell wall modulations between the daptomycin-tolerant/resistant strains.

ABSTRACT Adaptive laboratory evolution (ALE) is a useful tool to study the evolution of antibiotic tolerance in bacterial populations under diverse environmental conditions. The role of population bottlenecks in the evolution of tolerance has been investigated in Escherichia coli, but not in a more clinically relevant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used ALE to evolve MRSA under repetitive daptomycin treatment and incorporated population bottlenecks following antibiotic exposure. We observed that the populations finally attained a tolerance mutation in the yycH gene after 2 weeks of evolution with population bottlenecks, and additional mutations in yycI and several other genes further increased the tolerance level. The tolerant populations also became resistant to another glycopeptide antibiotic, vancomycin. Through proteomics, we showed that yycH and yycI mutations led to the loss of function of the proteins and downregulated the WalKR two-component system and the downstream players, including the autolysin Atl and amidase Sle1, which are important for cell wall metabolism. Overall, our study offers new insights into the evolution of daptomycin tolerance under population bottlenecking conditions, which are commonly faced by pathogens during infection; the study also identified new mutations conferring daptomycin tolerance and revealed the proteome alterations in the evolved tolerant populations. IMPORTANCE Although population bottlenecks are known to influence the evolutionary dynamics of microbial populations, how such bottlenecks affect the evolution of tolerance to antibiotics in a clinically relevant methicillin-resistant S. aureus (MRSA) pathogen are still unclear. Here, we performed in vitro evolution of MRSA under cyclic daptomycin treatment and applied population bottlenecks following the treatment. We showed that under these experimental conditions, MRSA populations finally attained mutations in yycH, yycI, and several other genes that led to daptomycin tolerance. The discovered yycH and yycI mutations caused early termination of the genes and loss of function of the proteins, and they subsequently downregulated the expression of proteins controlled by the WalKR two-component system, such as Atl and Sle1. In addition, we compared our proteomics data with multiple studies on distinct daptomycin-tolerant MRSA mutants to identify proteins with a consistent expression pattern that could serve as biological markers for daptomycin tolerance in MRSA.

The genus Streptomyces is known to harbor numerous biosynthetic gene clusters (BGCs) of potential utility in synthetic biology applications. However, it is often difficult to link uncharacterized BGCs with the secondary metabolites they produce. Proteomining refers to the strategy of identifying active BGCs by correlating changes in protein expression with the production of secondary metabolites of interest. In this study, we devised a shotgun proteomics-based workflow to identify active BGCs during fermentation when a variety of compounds are being produced. Mycelia harvested during the non-producing growth phase served as the background. Proteins that were differentially expressed were clustered based on the proximity of the genes in the genome to highlight active BGCs systematically from label-free quantitative proteomics data. Our software tool is easy-to-use and requires only 1 point of comparison where natural product biosynthesis was significantly different. We tested our proteomining clustering method on three Streptomyces species producing different compounds. In Streptomyces coelicolor A3(2), we detected the BGCs of calcium-dependent antibiotic, actinorhodin, undecylprodigiosin, and coelimycin P1. In Streptomyces chrestomyceticus BCC24770, 7 BGCs were identified. Among them, we independently re-discovered the type II PKS for albofungin production previously identified by genome mining and tedious heterologous expression experiments. In Streptomyces tenebrarius, 5 BGCs were detected, including the known apramycin and tobramycin BGC as well as a newly discovered caerulomycin A BGC in this species. The production of caerulomycin A was confirmed by LC-MS and the inactivation of the caerulomycin A BGC surprisingly had a significant impact on the secondary metabolite regulation of S. tenebrarius. In conclusion, we developed an unbiased, high throughput proteomics-based method to complement genome mining methods for the identification of biosynthetic pathways in Streptomyces sp.

Radiation dermatitis is a common adverse effect of radiotherapy (RT) in breast cancer patients. Although radiation dermatitis is reported by either the clinician or the patient, previous studies have shown disagreement between clinician-reported outcomes (CROs) and patient-reported outcomes (PROs). This review evaluated the extent of discordance between CROs and PROs for radiation dermatitis. Studies reporting both clinician and patient-reported outcomes for external beam RT were eligible. Nine studies met the inclusion criteria for the systematic review, while 8 of these studies were eligible for inclusion in a meta-analysis of acute and late skin toxicities. We found an overall agreement between CROs and PROs of acute skin colour change, fibrosis and/or retraction, and moist desquamation (p > 0.005). Reporting of late breast pain, breast edema, skin colour change, telangiectasia, fibrosis and/or retraction and induration/fibrosis alone (p > 0.005) were also in agreement between clinicians and patients. Our meta-analysis revealed a greater reporting of acute breast pain by patients (RR = 0.89, 95% CI 0.87–0.92, p

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

ABSTRACT Elasnin is a new antibiofilm compound that was recently reported to have excellent activity against methicillin-resistant Staphylococcus aureus (MRSA) biofilms. In this study, we established that elasnin also has antibacterial activity against growing S. aureus planktonic cells. To explore elasnin’s potential as an antibiotic, we applied adaptive laboratory evolution (ALE) and produced evolved strains with elevated elasnin tolerance. Interestingly, they were more sensitive toward daptomycin and lysostaphin. Whole-genome sequencing revealed that all of the evolved strains possessed a single point mutation in a putative phosphate transport regulator. Subsequently, they exhibited increased intracellular phosphate (Pi) and polyphosphate levels. Inhibition of the phosphate transport regulator gene changed the phenotype of the wild type to one resembling those observed in the evolved strains. Proteomics and transcriptomics analyses showed that elasnin treatment resulted in the downregulation of many proteins related to cell division and cell wall synthesis, which is important for the survival of growing exponential-phase cells. Other downregulated processes and factors were fatty acid metabolism, glycolysis, the two-component system, RNA degradation, and ribosomal proteins. Most importantly, transport proteins and proteins involved in oxidative phosphorylation and the phosphotransferase system were more upregulated in the evolved strain than in the ancestral strain, indicating that they are important for elasnin tolerance. Overall, this study showed that elasnin has antibacterial activity against growing S. aureus cells and revealed the altered processes due to elasnin treatment and those associated with its tolerance. IMPORTANCE Besides the excellent antibiofilm properties of elasnin, we discovered that it can also kill growing methicillin-resistant Staphylococcus aureus (MRSA) planktonic cells. We subjected MRSA cells to an in vitro evolution experiment, and the resulting evolved strains exhibited increased elasnin tolerance, reduced growth rate, loss of pigmentation, and an increased proportion of small-colony formation, and they became more sensitive toward daptomycin and lysostaphin. Through multiomics analysis, we uncovered the affected processes in growing S. aureus planktonic cells following elasnin treatment, including the downregulation of cell wall synthesis, cell division, and some genes/proteins for the two-component system. These findings suggest that elasnin suppressed processes important for the cells’ survival and adaptation to environmental stresses, making it an ideal drug adjuvant candidate. Overall, our study provides new insights into the mechanism of elasnin in S. aureus planktonic cells and pointed out the potential application of elasnin in clinics.

ABSTRACT Elasnin is a recently reported antibiofilm agent that is effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Remarkably, we observed that elasnin has a superior activity in eradicating daptomycin-resistant MRSA strain biofilm, with a lower minimum biofilm eradication concentration (MBEC) value of 0.625 μg/mL, compared to 2.5 μg/mL for the wild type. Confocal microscopy further confirmed the higher biofilm eradication on the daptomycin-resistant strain, displaying ∼53% decrease in cell density upon elasnin treatment, while the wild-type strain was only decreased by ∼15%. Quantitative proteomics revealed that the daptomycin-resistant strain has a lower expression of the membrane, cell wall, and extracellular proteins, and also proteins involved in the arginine biosynthesis, pathogenesis, and cell adhesion compared to the wild type, which may result in weaker biofilm development. This study highlights the potential clinical application of elasnin through its superior biofilm eradication activity against a daptomycin-resistant MRSA strain, and revealed the associated processes governing this superior activity through proteomics analysis. IMPORTANCE Due to the increased use of daptomycin for the treatment of MRSA infections, the emergence of daptomycin-resistant strains has become prevalent in recent years. In this study, we discovered that elasnin, a newly reported antibiofilm compound, has a superior activity in eradicating daptomycin-resistant MRSA strain biofilms compared to the wild type. Follow-up analysis revealed the reason behind this superior activity, which is the lower expression of key proteins that play a role in pathogenesis and cell adhesion in the daptomycin-resistant strain, leading to weaker biofilm development. This showcases the potential use of elasnin in clinical settings where daptomycin-resistant strains and biofilm formation are prevalent. Altogether, our study provides new insights into the mechanism of elasnin in MRSA biofilm cells and identified its superior biofilm eradicating activity in the daptomycin-resistant strain.

Although the phenotype of increased tolerance and/or resistance was commonly observed in evolved populations from typical adaptive laboratory evolution (ALE) experiments, a wide variety of mutations that underlie those phenotypes have been discovered. Therefore, performing ALE experiments in multiple populations in parallel would serve the purpose of mining for different tolerant/resistant mutants and would be useful to explore the diverse population dynamics of evolution.

Introduction Measurement-based care (MBC) represents the approach of regularly using symptom rating scales to guide patient care decisions in mental healthcare. MBC is an effective, feasible and acceptable approach to enhance clinical outcomes in various disciplines, including medicine, psychology, social work and psychotherapy. Yet, it is infrequently used by clinicians, potentially due to limited education for care providers. The objective of this scoping review is to survey the characteristics of MBC educational programmes for undergraduate, graduate and postgraduate clinical trainees in mental healthcare.Methods and analysis Using database-tailored search strategies, we plan on searching Medline, PsycINFO, Embase, CINAHL and Cochrane Central for relevant studies. Thereafter, we will analyse the selected studies to extract information on the delivery of educational programmes, the clinical and educational outcomes of these programmes, and the potential enablers and barriers to MBC education. In this paper, we articulate the protocol for this scoping review.Ethics and dissemination This scoping review does not require research ethics approval. The findings from this scoping review will be incorporated into the creation of a novel MBC curriculum and handbook. Results will be disseminated at appropriate national or international conferences, as well as in a peer-reviewed journal publication.

Summary: Recent discoveries indicate that tolerance and resistance could rapidly evolve in bacterial populations under intermittent antibiotic treatment. In the present study, we applied antibiotic combinations in laboratory experiments to generate novel methicillin-resistant Staphylococcus aureus strains with distinct phenotypes (tolerance, resistance, and suppressed tolerance), and compared their proteome profiles to uncover the adaptation mechanisms. While the tolerant strains have very different proteomes than the susceptible ancestral strain, the resistant strain largely resembles the ancestral in terms of their proteomes. Our proteomics data and other assays support the connection between the detected mutations to the observed phenotypes, confirming the general understanding of tolerance and resistance mechanisms. While resistance directly counteracts the action mechanism of the antibiotic, tolerance involves complex substantial changes in the cells’ biological process to achieve survival advantages. Overall, this study provides insights into the existence of diverse evolutionary pathways for tolerance and resistance development under different treatment scenarios.

Background: During recovery from stroke, the contralesional motor cortex (M1) may undergo maladaptive changes that contribute to impaired interhemispheric inhibition (IHI). Transcranial direct current stimulation (tDCS) with the cathode over contralesional M1 may inhibit this maladaptive plasticity, normalize IHI, and enhance motor recovery.Objective: The objective of this systematic review and meta-analysis was to evaluate available evidence to determine whether cathodal tDCS on contralesional M1 enhances motor re-learning or recovery post-stroke more than sham tDCS.Methods: We searched OVID Medline, Embase, and the Cochrane Central Register of Controlled Trials for participants with stroke (>1 week post-onset) with motor impairment and who received cathodal or sham tDCS to contralesional M1 for one or more sessions. The outcomes included a change in any clinically validated assessment of physical function, activity, or participation, or a change in a movement performance variable (e.g., time, accuracy). A meta-analysis was performed by pooling five randomized controlled trials (RCTs) and comparing the change in Fugl–Meyer upper extremity scores between cathodal and sham tDCS groups.Results: Eleven studies met the inclusion criteria. Qualitatively, four out of five cross-over design studies and three out of six RCTs reported a significant effect of cathodal vs. sham tDCS. In the quantitative synthesis, cathodal tDCS (n = 65) did not significantly reduce motor impairment compared to sham tDCS (n = 67; standardized mean difference = 0.33, z = 1.79, p = 0.07) with a little observed heterogeneity (I2 = 5%).Conclusions: The effects of cathodal tDCS to contralesional M1 on motor recovery are small and consistent. There may be sub-populations that may respond to this approach; however, further research with larger cohorts is required.

Recent laboratory evolution studies have shown that upon repetitive antibiotic treatments, bacterial populations will adapt and eventually became tolerant and resistant to the drug. Drug tolerance rapidly evolves upon frequent, intermittent antibiotic treatments, and such emerging drug tolerance seems to be specific to the treatment conditions, complicating clinical practice. Moreover, it has been shown that tolerance often promotes the development of resistance, which further reinforces the need of clinical diagnostics for antibiotic tolerance to reduce the occurrence of acquired resistance. Here, we discuss the laboratory evolution studies that were performed to track the development of tolerance in bacterial populations, and highlight the urgency of developing a comprehensive knowledge base of various tolerance phenotypes and their detection in clinics. Finally, we propose future directions for basic research in this growing field.

IntroductionColchicine may inhibit inflammasome signaling and reduce proinflammatory cytokines, a purported mechanism of COVID-19 pneumonia. The aim of this systematic review and meta-analysis is to report on the state of the current literature on the use of colchicine in COVID-19 and to investigate the reported clinical outcomes in COVID-19 patients by colchicine usage.MethodsThe literature was searched from January 2019 through January 28, 2021. References were screened to identify studies that reported the effect of colchicine usage on COVID-19 outcomes including mortality, intensive care unit (ICU) admissions, or mechanical ventilation. Studies were meta-analyzed for mortality by the subgroup of trial design (RCT vs observational) and ICU status. Studies reporting an risk ratio (RR), odds ratio (OR) and hazard ratio (HR) were analyzed separately.ResultsEight studies, reporting on 16,248 patients, were included in this review. The Recovery trial reported equivalent mortality between colchicine and non-colchicine users. Across the other studies, patients who received colchicine had a lower risk of mortality-HR of 0.25 (95% CI: 0.09, 0.66) and OR of 0.22 (95% CI: 0.09, 0.57). There was no statistical difference in risk of ICU admissions between patients with COVID-19 who received colchicine and those who did not-OR of 0.26 (95% CI: 0.06, 1.09).ConclusionColchicine may reduce the risk of mortality in individuals with COVID-19. Further prospective investigation may further determine the efficacy of colchicine as treatment in COVID-19 patients in various care settings of the disease, including post-hospitalization and long-term care.

ABSTRACT Through adaptive laboratory evolution (ALE) experiments, it was recently found that when a bacterial population was repetitively treated with antibiotics, they will adapt to the treatment conditions and become tolerant to the drug. In this study, we utilized an ampicillin-tolerant Escherichia coli population isolated from an ALE experiment to study the mechanisms of persistence during ampicillin treatment and resuscitation. Interestingly, the persisters of this population exhibit filamentous morphology upon ampicillin treatment, and the filaments are getting longer over time. Proteomics analysis showed that proteins involved in carbohydrate metabolism are upregulated during antibiotic treatment, in addition to those involved in the oxidative stress response. Bacterial SOS response, which is associated with filamentation, was found to be induced on account of the increasing expression of RecA. Measurement of endogenous reactive oxygen species (ROS) revealed that the population have ∼100-fold less ROS generation under ampicillin treatment than the wild type, leading to a lower mutagenesis rate. Single-cell observations through time-lapse microscopy show that resuscitation of the filaments is stochastic. During resuscitation, proteins involved in the tricarboxylic acid (TCA) cycle, glyoxylate cycle and glycolytic processes, and ATP generation are downregulated, while ribosomal proteins and porins are upregulated in the filaments. One particular protein, ElaB, was upregulated by over 7-fold in the filaments after 3 h of resuspension in fresh medium, but its expression went down after the filaments divided. Knockout of elaB increased persistence on wild-type E. coli, and upon resumption of growth, mutants lacking elaB have a higher fraction of small colony variants (SCVs) than the wild type. IMPORTANCE Persisters are a subpopulation of cells with enhanced survival toward antibiotic treatment and have the ability to resume normal growth when the antibiotic stress is lifted. Although proteomics is the most suitable tool to study them from a system-level perspective, the number of persisters that present naturally is too few for proteomics analysis, and thus the complex mechanisms through which they are able to survive antibiotic stresses and resuscitate in fresh medium remain poorly understood. To overcome that challenge, we studied an evolved Escherichia coli population with elevated persister fraction under ampicillin treatment and obtained its proteome profiles during antibiotic treatment and resuscitation. We discovered that during treatment with ampicillin, this tolerant population employs an active oxidative stress response and exhibits lower ROS levels than the wild type. Moreover, an inner membrane protein which has implications in various stress responses, ElaB, was found to be highly upregulated in the persisters during resuscitation, and its knockout caused increased formation of small colony variants after ampicillin treatment, suggesting that ElaB is important for persisters to resume normal growth.

Background: The influence of cigarette smoking on cancer risk has been well-studied. Similarly, exposure to ionizing radiation from radiotherapy (RT) can produce detrimental effects on an individual's health. In patients administered RT, there has been an observed relationship in other primary carcinomas. The purpose of this systematic review was to summarize the influence of cigarette smoking on outcomes post adjuvant RT in breast cancer patients. Methods: OVID Medline, Cochrane and Embase were searched and 1893 articles were identified. A total of 71 articles were included in the review. Study type, published year and sample size, age, systemic therapies, RT techniques and treatment side effects were collected if available. Results: The review found 198 different outcomes which fell into 7 categories and similar outcomes were recorded. 40% of skin reaction outcomes, 50% of cardiovascular outcomes, 71% of reconstruction outcomes, 29% of pulmonary function outcomes, 33% of mortality outcomes and 42% of secondary recurrence outcomes reported significant differences between smokers and non-smokers. None of the articles reported non-smokers to have a higher risk than smokers. Conclusion: Cigarette smoking can pose a higher risk of post-treatment complications that can influence an individual's quality of life, survival rate and/or recurrence risk. This review further assessed the impact of smoking on various patient outcomes and side-effects in the adjuvant breast RT setting. The information provided in this review suggest that smoking cessation programs would help educate patients to understand their risks of being a current or former smoker when undergoing RT.

Abstract Background The aim of this review is to describe the challenges and barriers to conducting research in long-term care facilities. Methods A literature search was conducted in Ovid MEDLINE, Embase, Cochrane Central, PsycINFO and CINAHL. Keywords used included “long term care”, “nursing home”, “research”, “trial”, “challenge” and “barrier”, etc. Resulting references were screened in order to identify relevant studies that reported on challenges derived from first-hand experience of empirical research studies. Challenges were summarized and synthesized. Results Of 1723 references, 39 articles were selected for inclusion. To facilitate understanding we proposed a classification framework of 8 main themes to categorize the research challenges presented in the 39 studies, relating to the characteristics of facility/owner/administrator, resident, staff caregiver, family caregiver, investigator, ethical or legal concerns, methodology, and budgetary considerations. Conclusions Conducting research in long-term care facilities is full of challenges which can be categorized into 8 main themes. Investigators should be aware of all these challenges and specifically address them when planning their studies. Stakeholders should be involved from an early stage and flexibility should be built into both the methodology and research budget.

Immune responses triggered by implant abutment surfaces contributed by surface-adsorbed proteins are critical in clinical implant integration. How material surface-adsorbed proteins relate to host immune responses remain unclear. This study aimed to profile and address the immunological roles of surface-adsorbed salivary proteins on conventional implant abutment materials. Standardized polished bocks (5 × 5 × 1 mm3) were prepared from titanium and feldspathic ceramic. Salivary acquired pellicle formed in vitro was examined by liquid chromatography-tandem mass spectrometry and gene ontology (GO) analysis to identify and characterize the adsorbed proteins. Out of 759 proteins identified from pooled saliva samples, 396 were found to be attached to the two materials tested—369 on titanium and 298 on ceramic, with 281 common to both. GO annotation of immune processes was undertaken to form a protein–protein interaction network, and 14 hub proteins (≥6 interaction partners) (coding genes: B2M, C3, CLU, DEFA1, HSP90AA1, HSP90AB1, LTF, PIGR, PSMA2, RAC1, RAP1A, S100A8, S100A9, and SLP1) were identified as the key proteins connecting multiple (6–9) immune processes. The results offered putative immunological prospects of implant abutment material surface-adsorbed salivary proteins, which could potentially underpin the dynamic nature of implant–mucosal/implant–microbial interactions.

Introduction: In bone metastases, the disruption of normal bone processes results in increased resorption and formation rates, which can often be quantitatively measured by biomarkers in the urine and blood. The purpose of this review is to summarize relevant studies of urinary markers used as a diagnostic and/or prognostic tool, as well as its potential and advances in directing therapy. Methods: A literature search was conducted using Ovid MEDLINE (1950 to July 2014), EMBASE (1950 to 2014 week 30) and Cochrane Central Register of Controlled Trials (3rd Quarter 2014) to identify studies that detailed the use of urinary markers in the cancer setting, specifically involving markers for bone metastases. Search terms included “urinary markers”, “cancer”, and “bone metastases”. Results: A total of 35 articles, with 24 original studies, were identified. In general, urinary markers can be used to detect early signs of bone metastases prior to skeletal imaging, but still must be used in conjunction with imaging to avoid false positive results. The use of urinary markers, such as N-telopeptide, as a prognostic tool remains controversial, but can provide information on the relative risk of skeletal related events (SREs), disease progression, as well as death. Finally, while urinary markers have shown to be potentially useful in confirming the efficacy of bone metastases treatments, exploring the appropriate dosages for treatment, and directing therapy, it is still unclear to what extent urinary markers should be reduced by. Conclusion: The potential use of urinary markers in the management of bone metastases is promising as it can allow for earlier and more convenient detection of bone metastases in comparison to other techniques. However, additional studies involving prospective clinical trials are suggested to further examine the potential of urinary markers in developing appropriate treatment strategies and endpoints, especially in developing a clearer protocol on the extent urinary markers should be reduced by to correlate with achievement of clinical benefit.

Objective: To investigate the quality of life (QOL) following palliative radiotherapy for painful bone metastases. Methods: A literature search was conducted in OvidSP Medline (1946–Jan Week 4 2014), Embase (1947–Week 5 2014), and the Cochrane Central Register of Controlled Trials (Dec 2013) databases. The search was limited to English. Subject headings and keywords included ‘palliative radiation’, ‘cancer palliative therapy’, ‘bone metastases’, ‘quality of life’, and ‘pain’. All studies (prospective or retrospective) reporting change in QOL before and after palliative radiotherapy for painful bone metastases were included. Results: Eighteen articles were selected from a total of 1730. The most commonly used tool to evaluate QOL was the Brief Pain Inventory. Seventeen studies collected data prospectively. An improvement in symptoms and functional interference scores following radiotherapy was observed in all studies. The difference in changes in QOL between responders and non responders was inconsistently reported. Conclusion: QOL improves in patients who respond to palliative radiotherapy for painful bone metastases.

Purpose: Radiation therapy is the standard treatment for symptomatic bone metastases. Several randomized control trials and meta-analyses have concluded a similar efficacy in pain relief when comparing single versus multiple fraction regimes. However, there continues to be reluctance to conform to published guidelines that recommend a single treatment for the palliation of painful bone metastases. The purpose of this literature review is to summarize international patterns of practice, and to determine if guidelines recommending single fraction treatment have been implemented in clinical care. Methods: A literature search was conducted in Ovid Medline, Embase, and Cochrane Central. Search words included, ‘bone metastases’, ‘radiation therapy’, ‘radiotherapy’, ‘patterns of practice’, and ‘dose fractionation’. Both prospective and retrospective studies that investigated the prescription of radiotherapy to bone metastases using actual patient databases were included. Articles were excluded if they investigated hypothetical scenarios. Results: Six hundred and thirteen results were generated from the literature search. Twenty-six articles met the inclusion criteria. Of these, 11 were Canadian, 8 were European, 6 were American, and 1 was Australian. The use of single fraction radiotherapy (SFRT) ranged from 3% to 75%, but was generally lower in American studies. Choice of fractionation depended on a variety of factors, including patient age, prognosis, site of irradiation, and physician experience. Conclusion: Despite the publication of robust randomized control trials, meta-analyses, and clinical practice guidelines recommending the use of a single treatment to palliate uncomplicated bone metastasis, SFRT is internationally underutilized.

Let Qλ(t,y) be the number of people present at time t with y units of remaining service time in an infinite server system with arrival rate equal to λ>0. In the presence of a non-lattice renewal arrival process and assuming that the service times have a continuous distribution, we obtain a large deviations principle for Qλ( · )/λ under the topology of uniform convergence on [0,T]×[0,∞). We illustrate our results by obtaining the most likely path, represented as a surface, to ruin in life insurance portfolios, and also we obtain the most likely surfaces to overflow in the setting of loss queues.

Angelica Radix, the roots of the genus Angelica, has been used for more than 2,000 years as a traditional medicine in Eastern Asia. The Chinese Pharmacopoeia records more than 100 herbal formulae containing Angelica roots. There are two common sources of Angelica roots, Angelica sinensis from China and A. gigas from Korea. The two species of Angelica roots differ in their chemical compositions, pharmacological properties and clinical efficacy. 1H-NMR metabolic profiling has recently emerged as a promising quality control method for food and herbal chemistry. We explored the use of 1H-NMR metabolic profiling for the quality control of Angelica Radix. Unlike previous work, we performed the metabolic profiling on hot water extracts, so as to mimic the clinically relevant preparation method. Unsupervised principle component analyses of both the full spectral profile and a selection of targeted molecules revealed a clear differentiation of three types of Angelica roots. In addition, the levels of 13 common metabolites were measured. Statistically significant differences in the levels of glucose, fructose and threonine were found between different sources of Angelica. Ferulic acid, a marker commonly used to evaluate Angelica root, was detected in our samples, but the difference in ferulic acid levels between the samples was not statistically significant. Overall, we successfully applied 1H-NMR metabolic profiling with water extraction to discriminate all three sources of Angelica roots, and obtained quantitative information of many common metabolites.

Abstract Over the past decade, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has evolved into the main proteome discovery technology. Up to several thousand proteins can now be reliably identified from a sample and the relative abundance of the identified proteins can be determined across samples. However, the remeasurement of substantially similar proteomes, for example those generated by perturbation experiments in systems biology, at high reproducibility and throughput remains challenging. Here, we apply a directed MS strategy to detect and quantify sets of pre‐determined peptides in tryptic digests of cells of the human pathogen Leptospira interrogans at 25 different states. We show that in a single LC–MS/MS experiment around 5000 peptides, covering 1680 L. interrogans proteins, can be consistently detected and their absolute expression levels estimated, revealing new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans. This is the first study that describes the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date.

Abstract The ability to analyze and understand the mechanisms by which cells process information is a key question of systems biology research. Such mechanisms critically depend on reversible phosphorylation of cellular proteins, a process that is catalyzed by protein kinases and phosphatases. Here, we present PhosphoPep, a database containing more than 10 000 unique high‐confidence phosphorylation sites mapping to nearly 3500 gene models and 4600 distinct phosphoproteins of the Drosophila melanogaster Kc167 cell line. This constitutes the most comprehensive phosphorylation map of any single source to date. To enhance the utility of PhosphoPep, we also provide an array of software tools that allow users to browse through phosphorylation sites on single proteins or pathways, to easily integrate the data with other, external data types such as protein–protein interactions and to search the database via spectral matching. Finally, all data can be readily exported, for example, for targeted proteomics approaches and the data thus generated can be again validated using PhosphoPep, supporting iterative cycles of experimentation and analysis that are typical for systems biology research.

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

BACKGROUND: For advanced pancreatic cancer, many regimens have been compared with gemcitabine (G) as the standard arm in randomized controlled trials. Few regimens have been directly compared with each other in randomized controlled trials and the relative efficacy and safety among them remains unclear. METHODS: A systematic review was performed through MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and ASCO meeting abstracts up to May 2013 to identify randomized controlled trials that included advanced pancreatic cancer comparing the following regimens: G, G+5-fluorouracil, G+ capecitabine, G+S1, G+ cisplatin, G+ oxaliplatin, G+ erlotinib, G+ nab-paclitaxel, and FOLFIRINOX. Overall survival and progression-free survival with 95% credible regions were extracted using the Parmar method. A Bayesian multiple treatment comparisons was performed to compare all regimens simultaneously. RESULTS: Twenty-two studies were identified and 16 were included in the meta-analysis. Median overall survival, progression free survival, and response rates for G arms from all trials were similar, suggesting no significant clinical heterogeneity. For overall survival, the mixed treatment comparisons found that the probability that FOLFIRINOX was the best regimen was 83%, while it was 11% for G+ nab-paclitaxel and 3% for G+ S1 and G+ erlotinib, respectively. The overall survival hazard ratio for FOLFIRINOX versus G+ nab-paclitaxel was 0.79 [0.50-1.24], with no obvious difference in toxicities. The hazard ratios from direct pairwise comparisons were consistent with the mixed treatment comparisons results. CONCLUSIONS: FOLFIRINOX appeared to be the best regimen for advanced pancreatic cancer probabilistically, with a trend towards improvement in survival when compared with other regimens by indirect comparisons.