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The deployment of proteomic analysis in clinical studies represents a significant opportunity to detect and validate biomarkers in translational medicine, improve disease understanding, and provide baseline information on population health. However, comprehensive proteome studies usually employ nanoscale chromatography and often require several hours of analysis/sample. Here, we describe a high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) methodology using 1 mm scale chromatography requiring only 15 min/sample, coupled to ion mobility-enabled mass spectrometry. The short run time effected a 6-fold increase in productivity compared with nanoscale LC/MS. The method demonstrated excellent reproducibility with retention time coefficient of variations of less than 0.05% and peak area reproducibility ranging from 5 to 15%. The 1 mm system produced similar chromatographic peak capacity values to the nanoscale miniaturized system, detecting 90% of the Escherichia coli proteins identified by the 75 μm LC/MS system (albeit based on only 75% of the peptides found by the latter). Application to the analysis of serum samples from a human prostate cancer study group resulted in the identification of a total of 533 proteins revealing the differential expression of proteins linked to patients receiving hormone-radiotherapy or undergoing surgery.

The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with90% overlap of the detected proteins.

Introduction: The advent of biologic drugs like infliximab, Etanercept, rituximab and tocilizumab has greatly improved the treatment of rheumatoid arthritis, however, increased risk of infection and high cost still remain unmet needs. A new generation of targeted therapeutics is being developed to target payload drug specifically to arthritic tissue; to concentrate the drug in the disease area and limit the off target systemic exposure. This might also reduce total effective dose.Areas covered: This article summarizes the properties and progress of targeted therapies that have been published on PubMed, and addresses their clinical potential.Expert commentary: Incredible progress with targeted therapies has already been made in the short time since the principle was first proven in animal models in 2007 when targeting payload drug to overexpressed oncofetal domain of fibronectin in inflamed arthritic joints.

Context This paper explores how structured feedback and other features of workplace-based assessment (WBA) impact on medical students' learning in the context of an evaluation of a workplace-based performance assessment: the teamwork mini-clinical evaluation exercise (T-MEX). The T-MEX enables observation-based measurement of and feedback on the behaviours required to collaborate effectively as a junior doctor within the health care team. The instrument is based on the mini-clinical evaluation exercise (mini-CEX) format and focuses on clinical encounters such as consultations with medical and allied health professionals, discharge plan preparation, handovers and team meetings. Methods The assessment was implemented during a 6-week period in 2010 with 25 medical students during their final clinical rotation. Content analysis was conducted on the written feedback provided by 23 assessors and the written reflections and action plans proposed by the 25 student participants (in 88 T-MEX forms). Semi-structured interviews with seven assessors and three focus groups with 14 student participants were conducted and the educational impact was explored through thematic analysis. Results The study enabled the identification of features of WBA that promote the development of collaborative competencies. The focus of the assessment on clinical encounters and behaviours important for collaboration provided opportunities for students to engage with the health care team and highlighted the role of teamwork in these encounters. The focus on specific behaviours and a stage-appropriate response scale helped students identify learning goals and facilitated the provision of focused feedback. Incorporating these features within an established format helped students and supervisors to engage with the instrument. Extending the format to include structured reflection enabled students to self-evaluate and develop plans for improvement. Conclusions The findings illuminate the mechanisms by which WBA facilitates learning. The educational features highlighted include effectively structured feedback processes, support for situated learning, a positive backwash (facilitation of learning through preparation for the assessment), and facilitation of informed self-assessment.

Catheter-associated urinary tract infections (CAUTI) have been associated with increases in morbidity and mortality as well as increased costs of hospitalization. At our institution, we implemented a protocol for indwelling catheter use, maintenance, and removal based on Center for Medicare and Medicaid Services (CMS) guidelines, in efforts to reduce CAUTI rates.A hospital committee of quality stewards focused on several measures which included staff education, modification of existing systems to ensure compliance, and auditing of patient care areas for catheter utilization before implementation of the protocol. Pre- and postintervention postoperative cohorts were then identified through American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) for prevalence of CAUTI. Data were collected through chart review and postdischarge patient interviews.A total of 3873 patients were identified between September 2007 and December 2010. Thirty-six patients (2.6%) were diagnosed with a CAUTI in the preintervention group (N = 1404) compared to 38 (1.5%) patients who were diagnosed with a CAUTI in the postintervention group (N = 2469). There was a 1.1% decrease in CAUTI rate after protocol implementation (P.028). This reduction in rates resulted in annual estimated savings of $81,840 to $320,540 annually.A simple, multifaceted approach consisting of staff education and changing existing processes to reflect best care practices has the potential to significantly reduce the incidence of postoperative CAUTI.

Portfolios need to be evaluated to determine whether they encourage students to develop in capabilities such as reflective practice and ethical judgment. The aims of this study were (i) to determine whether preparing a portfolio helps promote students' development in a range of capabilities including understanding ethical and legal principles, reflective practice and effective communication, and (ii) to determine to what extent the format of the portfolio affected the outcome by comparing the experiences of students at two different medical schools. A questionnaire was designed to evaluate undergraduate medical students' experiences of completing a portfolio at two medical schools. A total of 526 (45% response rate) students answered the on-line questionnaire. Students from both medical schools gave the highest ranking for the portfolio as a trigger for reflective practice. 63% of students agreed their portfolio helped them develop reflective practice skills (p 0.001), whereas only 22% disagreed. 48% of students agreed portfolios helped them understand ethical and legal principles whereas 29% disagreed (p 0.001). In contrast, only 34% of students thought the portfolio helped them to develop effective communication. Students perceive portfolio preparation as an effective learning tool for the development of capabilities such as understanding ethical and legal principles and reflective practice, whereas other capabilities such as effective communication require complementary techniques and other modes of assessment.

Objective To isolate recombinant antibodies with specificity for human arthritic synovium and to develop targeting reagents with joint-specific delivery capacity for therapeutic and/or diagnostic applications. Methods In vivo single-chain Fv (scFv) antibody phage display screening using a human synovial xenograft model was used to isolate antibodies specific to the microvasculature of human arthritic synovium. Single-chain Fv antibody tissue-specific reactivity was assessed by immunostaining of synovial tissues from normal controls and from patients with rheumatoid arthritis and osteoarthritis, normal human tissue arrays, and tissues from other patients with inflammatory diseases displaying neovasculogenesis. In vivo scFv antibody tissue-specific targeting capacity was examined in the human synovial xenograft model using both 125I-labeled and biotinylated antibody. Results We isolated a novel recombinant human antibody, scFv A7, with specificity for the microvasculature of human arthritic synovium. We showed that in vivo, this antibody could efficiently target human synovial microvasculature in SCID mice transplanted with human arthritic synovial xenografts. Our results demonstrated that scFv A7 antibody had no reactivity with the microvasculature or with other cellular components found in a comprehensive range of normal human tissues including normal human synovium. Further, we showed that the reactivity of the scFv A7 antibody was not a common feature of neovasculogenesis associated with chronic inflammatory conditions. Conclusion Here we report for the first time the identification of an scFv antibody, A7, that specifically recognizes an epitope expressed in the microvasculature of human arthritic synovium and that has the potential to be developed as a joint-specific pharmaceutical.

Mounting evidence points to the soluble oligomers of amyloid β (Aβ) peptide as important neurotoxic species in Alzheimer's disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aβ peptide self-assembly is still under debate. Here, Aβ1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aβ1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers.

The computational simulation of complete proteomic data sets and their utility to validate detection and interpretation algorithms, to aid in the design of experiments and to assess protein and peptide false discovery rates is presented. The simulation software has been developed for emulating data originating from data-dependent and data-independent LC-MS workflows. Data from all types of commonly used hybrid mass spectrometers can be simulated. The algorithms are based on empirically derived physicochemical liquid and gas phase models for proteins and peptides. Sample composition in terms of complexity and dynamic range, as well as chromatographic, experimental and MS conditions, can be controlled and adjusted independently. The effect of on-column amounts, gradient length, mass resolution and ion mobility on search specificity will be demonstrated using tryptic peptides from human and yeast cellular lysates simulated over five orders of magnitude in dynamic range. Initial justification of the simulated data sets is achieved by comparing and contrasting the in silico simulated data to experimentally derived results from a 48 protein mixture, spanning a similar magnitude of five orders of magnitude. Additionally, experimental data from replicate and dilutions series experiments will be utilized to determine error rates at the peptide and protein level with respect to mass, area, retention and drift time. The data presented reveal a high degree of similarity at the ion detection, peptide and protein level when analyzed under similar conditions.

A recent ECVAM workshop considered how to reduce falsely predictive positive results when undertaking. in vitro genotoxicity testing, and thus to avoid unnecessary follow-up with tests involving animals. As it was anticipated that modified versions of existing assays as well as new assays might contribute to a solution, an expert panel was asked to identify a list of chemicals that could be used in the evaluation of such assays. Three categories of test chemicals were chosen comprising a total of 62 compounds. This paper provides test results for these chemicals using the GreenScreen HC assay. All tests were carried out in triplicate, by multiple operators, with and without S9, using invariant protocols. Group 1 chemicals should be detected as positive in. in vitro mammalian cell genotoxicity tests: 18/20 (90%) were reproducibly positive in GreenScreen HC. Group 2 chemicals should give negative results in. in vitro genotoxicity tests: 22/23 (96%) were reproducibly negative in GreenScreen HC. Overall concordance for Groups 1 and 2 is 93%. Group 3 chemicals should give negative results in. in vitro mammalian cell genotoxicity tests, but have been reported to induce chromosomal aberrations or. Tk mutations in mouse lymphoma cells, often at high concentrations or at high levels of cytotoxicity: 13/17 (76%) were reproducibly negative in GreenScreen HC. Of the four positive compounds in Group 3. p-nitrophenol was only positive at the top dose (10. mM), 2,4-DCP is an. in vivo genotoxin, and two chemicals are antioxidant compounds that may be acting as pro-oxidants in the hyperoxic conditions of cell culture. Overall, these predictive figures are similar to those from other studies with the GreenScreen HC assay and confirm its high specificity, which in turn minimizes the generation of falsely predictive positive results. © 2009 Elsevier B.V.

Complex carbohydrates are macromolecules biosynthesized in nontemplate-type processes, bearing specific glycoepitopes involved in crucial recognition processes such as cell differentiation and cell-cell interactions. Chemical structure of single components in complex mixtures can be analyzed by mass spectrometry for determination of the size and sequence of monosaccharides involved, branching patterns, and substitution by fucose and sialic acids. For de novo identification of glycoforms in human urinome containing N- and O-free and amino acid-linked oligosaccharides, a novel method of ion mobility tandem mass spectrometry followed by computer-assisted assignment is described. Distinct patterns of ions nested specifically by their m/z values and their drift time are observed by IMS-MS. An additional peak capacity for identification of time-separated m/z values in the IMS TOF MS mode for differentiation of singly, doubly, and triply charged molecular ion species by ion mobility separation contributes to significant reduction of carbohydrate complexity in a given mass window. Profiling of glycoforms from human urinome represents a highly efficient approach for biomarker discovery and differential glycotarget identification, demonstrating potential for diagnosis of human diseases, as for congenital disorders of glycosylation.

Students in the six-year undergraduate medicine program at UNSW must submit a portfolio which demonstrates inter alia their development in teamwork skills. Much of the feedback they need to develop these skills, as well as the evidence they require to document their achievements, can only come from their peers. The eMed Teamwork system, developed for this purpose, is a computer-based system which gathers feedback from peers in project groups. The feedback submitted to the system is available to the recipient for formative purposes, and becomes part of both the author's and the recipient's portfolios for later summative assessment. This dual use ensures that the feedback is thoughtful and constructive and the system operates without significant moderation by teachers.

In 2001 the University of New South Wales Faculty of Medicine embarked on designing a curriculum-management system to support the development and delivery of its new, fully integrated, outcome-based, six-year undergraduate medicine program. The Web-enabled curriculum-management system it developed is known as eMed, and it comprises a suite of integrated tools used for managing graduate outcomes, content, activities, and assessment in the new program. The six main tools are a curriculum map, timetable, student portfolio, peer feedback tool, assessment tracking, and results tools. The eMed functions were determined by organizational and curricular needs, and a business management perspective guided its development. The eMed project was developed by a multidisciplinary team, and its successful development was achieved mostly by methodically identifying the scope of each tool and the business processes it supports. Evaluation results indicated a high level of user acceptance and approval. The eMed system is a simple yet effective educational technology system that allows users to evaluate and improve the curriculum in real time. As a second-generation curriculum-management system, eMed is much more than an educational administration system; it is a knowledge network system used by staff and students to transform data and information into knowledge and action. The integration of learning and assessment activities data in the one system gives a depth of curriculum information that is unusual and that allows for data-based decision making. Technologically, eMed helps to keep the medicine program up to date. Organizationally, it strengthens the school’s data-driven decision-making process and knowledge network culture.

The differentiation of a stem cell is dependent on the environmental cues that it receives and can be modulated by the expression of different master regulators or by secreted factors or inducers. The use of genetically modified stem cells to express the required factors can direct differentiation along the requisite pathway. This approach to the engineering of stem cells is important, as control of the pluripotentiality of stem cells is necessary in order to avoid unwanted growth, migration or differentiation to nontarget tissues. The authors provide an overview of the stem cell engineering field, highlighting challenges and solutions, and focusing on recent developments in therapeutic applications in areas such as autoimmunity, CNS lesions, bone and joint diseases, cancer and myocardial infarction.

Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3zeta chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3zeta. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.

One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A.

Despite the increasing popularity of data-independent acquisition workflows, data-dependent acquisition (DDA) is still the prevalent method of LC-MS-based proteomics. DDA is the basis of isobaric mass tagging technique, a powerful MS2 quantification strategy that allows coanalysis of up to 10 proteomics samples. A well-documented limitation of DDA, however, is precursor coselection, whereby a target peptide is coisolated with other ions for fragmentation. Here, we investigated if additional peptide purification by traveling wave ion mobility separation (TWIMS) can reduce precursor contamination using a mixture of Saccharomyces cerevisiae and HeLa proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments, we find that TWIMS provides a remarkable improvement (on average 2.85 times) in the signal-to-noise ratio for sequence ions. We also report that TWIMS reduces reporter ions contamination by around one-third (to 14-15% contamination) and even further (to 6-9%) when combined with a narrowed quadrupole isolation window. We discuss challenges associated with applying TWIMS purification to isobaric mass tagging experiments, including correlation between ion m/z and drift time, which means that coselected peptides are expected to have similar mobility. We also demonstrate that labeling results in peptides having more uniform m/z and drift time distributions than observed for unlabeled peptides. Data are available via ProteomeXchange with identifier PXD001047.

Summary: To evaluate the role of perinatal transmission in the spread of hepatitis C virus (HCV), we screened a cohort of pregnant intravenous drug using (IVDU) women for HCV antibody detection; where seropositive HCV RNA detection by polymerase chain reaction (PCR) was found we followed the infants longitudinally for HCV antibody and HCV RNA. Serum prevalence for HCV for this population was 80% with HCV RNA detected in 50%. Recruitment and follow-up over a 3-year period of a cohort of 83 seropositive women, their 91 newborns and 16 siblings of newborns, showed that there had been a 3% perinatal transmission rate with 1 sibling also infected. These positive cases were defined as transient in 1 case (HCV RNA positive by PCR at 1 month, but seronegative and HCV RNA negative at 10 months of age), 2 unevaluable (HCV RNA positive at 2 months of age, but patients lost to follow-up), and 1 chronic infection in a child at 34 months (positive HCV RNA and seropositive 34-month sibling). Maternal HCV RNA levels for those with infected infants was a mean 40-fold greater than those whose babies were uninfected, although this did not reach statistical significance. Of the remaining infants, the majority (93%) had lost passively acquired maternal antibodies by 9 months of age and all by 12 months. Of 18 women who were HCV seropositive and breast feeding (66% of whom were HCV RNA positive in their sera), none had detectable HCV RNA in breast milk. Hence we conclude that transmission of HCV from mother to infant appears to be of low frequency and positivity appears to correlate with maternal circulating viral load.

Purpose Teamwork is an important and challenging area of learning during the transition from medical graduate to intern. This preliminary investigation examined the psychometric and logistic properties of the Teamwork Mini-Clinical Evaluation Exercise (T-MEX) for the workplace-based assessment of key competencies in working with health care teams. Method The authors designed the T-MEX for direct observation and assessment of six collaborative behaviors in seven clinical situations important for teamwork, feedback, and reflection. In 2010, they tested it on University of New South Wales senior medical students during their last six-week clinical term to investigate its overall utility, including validity and reliability. Assessors rated students in different situations on the extent to which they met expectations for interns for each collaborative behavior. Both assessors and students rated the tool’s usefulness and feasibility. Results Assessment forms for 88 observed encounters were submitted by 25 students. The T-MEX was suited to a broad range of collaborative clinical practice situations, as evidenced by the encounter types and the behaviors assessed by health care team members. The internal structure of the behavior ratings indicated construct validity. A generalizability study found that eight encounters were adequate for highstakes measurement purposes. The mean times for observation and feedback and the participants’ perceptions suggested usefulness for feedback and feasibility in busy clinical settings. Conclusions Findings suggest that the T-MEX has good utility for assessing trainee competence in working with health care teams. It fills a gap within the suite of existing tools for workplace-based assessment of professional attributes.

In this study the involvement of oxidative stress in type 1 diabetes mellitus autoimmunity and the possible association with rheumatoid arthritis (RA) was investigated. We tested the hypothesis that oxidative stress induced by chronic hyperglycaemia triggers post-translational modifications and thus the formation of neo-antigens in type 1 diabetes, similar to the ones found in RA.Collagen type II (CII), a known autoantigen in RA, was treated with ribose and various reactive oxygen species (ROS). Levels of antibodies specific to native and ROS-modified CII (ROS-CII) were compared in type 1 diabetes, type 2 diabetes and healthy controls, and related to the HLA genotype.Significantly higher binding to ROS-CII vs native CII was observed in type 1 diabetic patients possessing the HLA-DRB1*04 allele irrespective of variables of glucose control (blood glucose or HbA(1c)). Type 1 diabetic patients carrying a DRB1*04 allele with the shared epitope showed the highest risk for ROS-CII autoimmunity, while the DRB1*0301 allele was protective. Conversely, native CII autoimmunity was not associated with any specific DRB1 allele. Positive and inverse seroconversion rates of response to ROS-CII were high in DRB1*04-positive type 1 diabetic patients.Hyperglycaemia and oxidative stress may trigger genetically controlled autoimmunity to ROS-CII and may explain the association between type 1 diabetes mellitus and RA.

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

To accurately determine the quantitative change of peptides and proteins in complex proteomics samples requires knowledge of how well each ion has been measured. The precision of each ions’ calculated area is predicated on how uniquely it occupies its own space in m/z and elution time. Given an initial assumption that prior to the addition of the “heavy” label, all other ion detections are unique, which is arguably untrue, an initial attempt at quantifying the pervasiveness of ion interference events in a representative binary SILAC experiment was made by comparing the centered m/z and retention time of the ion detections from the “light” variant to its “heavy” companion. Ion interference rates were determined for LC-MS data acquired at mass resolving powers of 20 and 40 K with and without ion mobility separation activated. An ion interference event was recorded, if present in the companion dataset was an ion within ± its Δ mass at half-height, ±15 s of its apex retention time and if utilized by ±1 drift bin. Data are presented illustrating a definitive decrease in the frequency of ion interference events with each additional increase in selectivity of the analytical workflow. Regardless of whether the quantitative experiment is a composite of labeled samples or label free, how well each ion is measured can be determined given knowledge of the instruments mass resolving power across the entire m/z scale and the ion detection algorithm reporting both the centered m/z and Δ mass at half-height for each detected ion. Given these measurements, an effective resolution can be calculated and compared with the expected instrument performance value providing a purity score for the calculated ions’ area based on mass resolution. Similarly, chromatographic and drift purity scores can be calculated. In these instances, the error associated to an ions’ calculated peak area is estimated by examining the variation in each measured width to that of their respective experimental median. Detail will be disclosed as to how a final ion purity score was established, providing a first measure of how accurately each ions’ area was determined as well as how precise the calculated quantitative change between labeled or unlabelled pairs were determined. Presented is how common ion interference events are in quantitative proteomics LC-MS experiments and how ion purity filters can be utilized to overcome and address them, providing ultimately more accurate and precise quantification results across a wider dynamic range.

Objective To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. Methods We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti–ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti–ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti–ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II–Fc fusion protein (mTNFRII-Fc) was also investigated. Results The anti–ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti–ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. Conclusion Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis.

This paper summarises the recent RANZCOG Indigenous Women's Health Meeting with recommendations on how the College and its membership can act now to improve the health of Aboriginal and Torres Strait Islander women and infants.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label-free nanoscale LC-MS has been applied in this study to validate a Triton X-114-based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane-bound proteins with an enrichment of over 62% for this protein subset. LC-MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane-bound proteins. This approach should be applicable to a wider scale study of disease-associated changes in the cardiac membrane subproteome.

Relatively few studies have focused on causal factors in the development of locus of control. Here nine western European countries are studied to assess the hypothesis that the society in which one lives is important in the scores on locus of control. The data indicate greater variation resulting from country of residence on locus of control than that from the variables of life cycle, sex, and social class. Findings regarding these latter variables are congruent with previous research. In addition, being single was related to having an external locus of control.

Purpose: The purpose of our study was to determine the relationship between perceived neck disability and active cervical range of motion (ROM). Methods: Twenty-three senior center attendees aged 60 to 90 participated. Their ROM was measured using the cervical range of motion instrument (CROM). Perceived neck disability was characterized using the Neck Disability Index (NDI). Pearson correlations were used to describe the relationships between CROM measurements and the NDI scores. Results: No CROM measurement was correlated strongly or significantly with the NDI score. The correlations ranged from 0.009 for left lateral flexion to 0.411 for right rotation. Conclusions: CROM and NDI measurements were not related. Impairments in variables other than cervical ROM may need to be addressed if the reduction of neck disability is intended in elders.

A well-aligned curriculum has consistent focus on curricular goals, teaching/learning activities and assessment. Poor alignment affects the way students budget learning time and may interfere with achievement of curricular goals. We noticed students' poor attendance in clinical clerkships prior to final examinations and hypothesised that they were responding to curricular misalignment.To quantify the extent to which students and tutors perceived a mismatch between activities pertaining to their current clerkship and those they thought necessary to prepare for final examinations and internship.Medical students in final (sixth) year clerkships in medicine, surgery and critical care and clinical tutors.A self-report questionnaire on time spent in clerkship activities was administered 3 months before the final examinations.The amounts of time spent on current activities were fairly evenly spread over teaching, study and self-directed patient contacts, and observing patient care (ward rounds, operating theatre, accident and emergency, outpatient department and clinical meetings). Less time was available for recreation. Students and tutors concurred, independently, that good examination and internship preparation required a shifting of the balance. Examination preparation redistributed time from observing patient care and recreation to study and self-directed patient contacts. Internship preparation redistributed time from teaching and recreation to observing patient care.Students and tutors perceived that current activities were not well aligned with assessment requirements but were better aligned with the requirements of internship. If we want students to direct their attention towards curricular goals, we need to bring goals, teaching/learning activities and assessment into alignment.

In 2000 the Diabetes Centre and the Medical Oncology Department of St Vincent's Hospital, Sydney established a joint clinic for the teaching of final-year medical students. The clinic was established amid concerns that hospitals are increasingly focused on acute care and have few resources available for teaching about chronic conditions. The clinic aimed to improve both patient care and learning opportunities by engaging students in useful activities with chronically ill patients. The students met with their patients regularly to monitor progress, adjust medication (under supervision) and arrange support services. The students and staff from both units met once a week in a coordination meeting where cases were reviewed and learning issues discussed. Students had informal interactions with medical and allied health staff at other times. Overall the clinic provided a rich learning environment for students with a focus on the development of the integrated skills required in the care of chronically ill people, rather than on the specific medical disciplines involved.

INTRODUCTION: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis. METHODS: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis. RESULTS: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10). CONCLUSIONS: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

Section 303(d) of the Clean Water Act requires states to assess the condition of their waters and to implement plans to improve the quality of waters identified as impaired. U.S. Environmental Protection Agency guidelines require a stream segment to be listed as impaired when greater than 10% of the measurements of water quality conditions exceed numeric criteria. This can be termed a "raw score" assessment approach. Water quality measurements are samples taken from a population of water quality conditions. Concentrations of pollutants vary naturally, measurement errors may be made, and occasional violations of a standard may be tolerable. Therefore, it is reasonable to view the assessment process as a statistical decision problem. Assessment of water quality conditions must be cognizant of the possibility of type I (a false declaration of standards violation) and type II (a false declaration of no violation) errors. The raw score approach is shown to have a high type I error rate. Alternatives to the raw score approach are the Binomial test and the Bayesian Binomial approach. These methods use the same information to make decisions but allow for control of the error rates. The two statistical methods differ based on consideration of prior information about violation. Falsely concluding that a water segment is impaired results in unnecessary planning and pollution control implementation costs. On the other hand, falsely concluding that a segment is not impaired may pose a risk to human health or to the services of the aquatic environment. An approach that recognizes type I and type II error in the water quality assessment process is suggested.