Integrating Internal Fragments in the Interpretation of Top-Down Sequencing Data of Larger Oligonucleotides.

In the context of direct top-down analysis or concerted bottom-up characterization of nucleic acid samples, the waning yield of terminal fragments as a function of precursor ion size poses a significant challenge to the gas-phase sequencing of progressively larger oligonucleotides. In this report, we examined the behavior of oligoribonucleotide samples ranging from 20 to 364 nt upon collision-induced dissociation (CID). The experimental data showed a progressive shift from terminal to internal fragments as a function of size. The systematic evaluation of experimental factors, such as collision energy, precursor charge, sample temperature, and the presence of chaotropic agents, showed that this trend could be modestly alleviated but not suppressed. This inexorable effect, which has been reported also for other activation techniques, prompted a re-examination of the features that have traditionally discouraged the utilization of internal fragments as a source of sequence information in data interpretation procedures. Our simulations highlighted the ability of internal fragments to produce self-consistent ladders with either end corresponding to each nucleotide in the sequence, which enables both proper alignment and correct recognition of intervening nucleotides. In turn, contiguous ladders display extensive overlaps with one another and with the ladders formed by terminal fragments, which unambiguously constrain their mutual placement within the analyte sequence. The experimental data borne out the predictions by showing ladders with extensive overlaps, which translated into uninterrupted "walks" covering the entire sequence with no gaps from end to end. More significantly, the results showed that combining the information afforded by internal and terminal ladders resulted in much a greater sequence coverage and nucleotide coverage depth than those achievable when either type of information was considered separately. The examination of a series of 58-mer oligonucleotides with high sequence homology showed that the assignment ambiguities engendered by internal fragments did not significantly exceed those afforded by the terminal ones. Therefore, the balance between potential benefits and perils of including the former makes a compelling argument for the development of integrated data interpretation strategies, which are better equipped for dealing with the changing fragmentation patterns obtained from progressively larger oligonucleotides.

Examining human factors and ergonomics aspects in a manufacturing organisation's metrics system: measuring up to stakeholder needs.

This research examines the status of human factors and ergonomics (HF/E) metrics in the case context of product realisation in an electronics manufacturing company. Interactions with 100+ stakeholders over a five year period were thematically analysed for metrics-related views and content. A disconnect between engineering metrics and HF/E metrics was evident. Engineers and HF/E specialists expressed different understandings of the gap between the disciplines and how to generate HF/E metrics that would fit the organisation. Other emerging themes provided insight for metrics development including improving indicator relatability, considerations for communication of information, and barriers to implementation of metrics. The results led to seven recommendations to help guide practitioners in developing and refining HF/E metrics as part of an organisation's metrics system. This macroergonomic case study provides key points for consideration when developing HF/E focussed metrics to support organisations being more proactive with HF/E in work system design. Practitioner summary: Metrics' presence, stakeholder views on metrics, and metrics-related content in a case organisation were thematically analysed with a macroergonomics focus. Human factors and ergonomics metrics (HF/E) were disconnected from engineering metrics thus limiting the design team's ability to handle human factors in design. Factors influencing HF/E metrics creation and integration were identified, resulting in seven recommendations for developing HF/E metrics.

Developing a simulation tool to quantify biomechanical load and quality of care in nursing.

Nursing is a high musculoskeletal disorder (MSD) risk job with high workload demands. This study combines Digital Human Modelling (DHM) and Discrete Event Simulation (DES) to address the need for tools to better manage MSD risk. This novel approach quantifies physical-workload, work-performance, and quality-of-care, in response to varying geographical patient-bed assignments, patient-acuity levels, and nurse-patient ratios. Lumbar loads for 86 care-delivery tasks in an acute care hospital unit were used as inputs in a DES model of the care-delivery process, creating a shift-long time trace of the biomechanical load. Peak L4/L5 compression and moment were 3574 N and 111.58 Nm, respectively. This study reports trade-offs in all three experiments: (i) increasing geographical patient-bed assignment distance decreased L4/L5 compression (8.8%); (ii) increased patient-acuity decreased L4/L5 moment (4%); (iii) Increased nurse-patient ratio decreased L4/L5 compression (10%) and moment (17%). However, in all experiments, Quality of care indicators deteriorated (20, 19, and 29%, respectively).Practitioner Summary: This research has the potential to support decision-makers by developing a simulation tool that quantifies the impact of varying operational and design-policies in terms of biomechanical-load and quality of care. The demonstrator-model reports: as geographical patient-bed distance, patient-acuity levels, and nurse-patient ratios increase, biomechanical-load reduces, and quality of care deteriorates.

Modelling the impacts of COVID-19 on nurse workload and quality of care using process simulation.

Higher acuity levels in COVID-19 patients and increased infection prevention and control routines have increased the work demands on nurses. To understand and quantify these changes, discrete event simulation (DES) was used to quantify the effects of varying the number of COVID-19 patient assignments on nurse workload and quality of care. Model testing was based on the usual nurse-patient ratio of 1:5 while varying the number of COVID-19 positive patients from 0 to 5. The model was validated by comparing outcomes to a step counter field study test with eight nurses. The DES model showed that nurse workload increased, and the quality of care deteriorated as nurses were assigned more COVID-19 positive patients. With five COVID-19 positive patients, the most demanding condition, the simulant-nurse donned and doffed personal protective equipment (PPE) 106 times a shift, totaling 6.1 hours. Direct care time was reduced to 3.4 hours (-64% change from baseline pre-pandemic case). In addition, nurses walked 10.5km (+46% increase from base pre-pandemic conditions) per shift while 75 care tasks (+242%), on average, were in the task queue. This contributed to 143 missed care tasks (+353% increase from base pre-pandemic conditions), equivalent to 9.6 hours (+311%) of missed care time and care task waiting time increased to 1.2 hours (+70%), in comparison to baseline (pre-pandemic) conditions. This process simulation approach may be used as potential decision support tools in the design and management of hospitals in-patient care settings, including pandemic planning scenarios.

Assessing human factors and ergonomics capability in organisations - the Human Factors Integration Toolset.

This paper presents the development of a tool that allows an organisation to assess its level of human factors (HF) and ergonomics integration and maturity within the organisation. The Human Factors Integration Toolset (available at: https://www.researchgate.net/project/Human-Factors-Integration-Toolset ) has been developed and validated through a series of workshops with 45 participants from industry and academia and through industry partnered field-testing. HF maturity is assessed across five levels in 16 organisational functions based on any of 31 discrete elements contributing to HF. Summing element scores in a function determines a percent of ideal HF for the function. Industry stakeholders engaged in field-testing found the tool helped to establish the status of HF in the organisation, plan projects to further develop HF capabilities, and initiate discussions on HF for performance and well-being. Improvement suggestions included adding an IT function, refining the language for non-HF specialists, including knowledge work and creating a digital version to improve usability. Practitioner summary: A tool scoring HF capability in 16 organisation functions has been developed collaboratively. Industry stakeholders expressed a need for the tool and provided validation of tool design decisions. Field-testing improved tool usability and showed that beyond scoring HF capability, the tool created opportunities for discussions of HF-related improvement possibilities. Abbreviations: HF: human factors; HFIT: Human Factors Integration Toolset.

Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry.

Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746-750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746-750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10-20% reduction in rates.

Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction.

The heterodimer HIF-1α (hypoxia inducible factor)/HIF-ß (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics "hotspot identification" strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.

Linking human factors to corporate strategy with cognitive mapping techniques.

For human factors (HF) to avoid being considered of "side-car" status, it needs to be positioned within the organization in such a way that it affects business strategies and their implementation. Tools are needed to support this effort. This paper explores the feasibility of applying a technique from operational research called cognitive mapping to link HF to corporate strategy. Using a single case study, a cognitive map is drawn to reveal the complex relationships between human factors and achieving an organization's strategic goals. Analysis of the map for central concepts and reinforcing loops enhances understanding that can lead to discrete initiatives to facilitate integration of HF. It is recommended that this technique be used with senior managers to understand the organizations` strategic goals and enhance understanding of the potential for HF to contribute to the strategic goals.

Discovery of a novel B-Raf fusion protein related to c-Met drug resistance.

In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions: in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.

Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry.

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.

Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity.

Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids--mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1-/-) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography-mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC(50) of 247 nM. The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases.

A systematic exploration of distal arm muscle activity and perceived exertion while applying external forces and moments.

The purpose of this study was to systematically explore and describe the response of selected hand and forearm muscles during a wide range of static force and moment exertions. Twenty individuals with manual work experience performed exertions in power grip, pulp pinch and lateral pinch grips. Electromyography (EMG) from eight sites of the hand and forearm, grip force as well as ratings of perceived exertion (RPE) were monitored as each participant exerted approximately 350 short (5 s) static grip forces and external forces and moments. As expected, strong relationships were found between grip force alone without other actions and muscle activation. When the hand was used to grip and transmit forces and moments to the environment, the relationships between grip force and muscle activation were much weaker. Using grip force as a surrogate for forearm and hand tissue loading may therefore be misleading.

Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis.

The single biggest problem with solution-phase H/D exchange as a mass spectrometric probe of surface exposure in a protein (or protein complex) is back-exchange of H for D after the initial H/D exchange has been quenched. Back-exchange results in loss of pertinent data and also greatly hampers data analysis. Previously, very fast, cold (0-4 degrees C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages approximately 30%. In this report, supercritical fluid chromatography replaces HPLC as the desalting/separation technique prior to mass analysis, providing a dramatic reduction in back-exchange compared to the fast, cold HPLC methods.

Locomotion through apertures when wider space for locomotion is necessary: adaptation to artificially altered bodily states.

The objective of this study is to describe the adaptability of the central nervous system to safely cross a narrow aperture when the space required for passage is transiently extended with external objects under different locomotor constraints. In one of four locomotion forms (normal walking, walking while holding a 63-cm horizontal bar with or without rotating the shoulders to cross a door opening, and wheelchair use), nine participants were asked to pass through an aperture created by two doors (the relative aperture widths were 1.02, 1.10, and 1.20 times their maximum horizontal dimension under each form of locomotion) without a collision. The kinematic analyses showed that, when the participants rotated their shoulders while walking and holding a bar, virtually the same locomotor patterns as those during normal walking were observed: shoulder rotation was regulated well in response to the width of an aperture, and no collisions occurred. When shoulder rotations were restricted while walking and holding a bar or using a wheelchair, a large reduction in the speed of movement was observed as the participants approached the door, and, furthermore, the modulation in speed was dependent on the width of the aperture. In addition, the participants crossed at the center of aperture more accurately; nevertheless, collision sometimes occurred (more frequently, during wheelchair use). These findings reveal that movement constraints on shoulder rotation are likely to be a critical factor in determining whether quick and successful adaptation takes place.

Any way you look at it, successful obstacle negotiation needs visually guided on-line foot placement regulation during the approach phase.

In the two experiments discussed in this paper we quantified obstacle avoidance performance characteristics carried out open loop (without vision) but with different initial visual sampling conditions and compared it to the full vision condition. The initial visual sampling conditions included: static vision (SV), vision during forward walking for three steps and stopping (FW), vision during forward walking for three steps and not stopping (FW-NS), and vision during backward walking for three steps and stopping (BW). In experiment 1, we compared performance during SV, FW and BW with full vision condition, while in the second experiment we compared performance during FW and FW-NS conditions. The questions we wanted to address are: Is ecologically valid dynamic visual sampling of the environment superior to static visual sampling for open loop obstacle avoidance task? What are the reasons for failure in performing open loop obstacle avoidance task? The results showed that irrespective of the initial visual sampling condition when open loop control is initiated from a standing posture, the success rate was only approximately 50%. The main reason for the high failure rates was not inappropriate limb elevation, but incorrect foot placement before the obstacle. The second experiment showed that it is not the nature of visual sampling per se that influences success rate, but the fact that the open loop obstacle avoidance task is initiated from a standing posture. The results of these two experiments clearly demonstrate the importance of on-line visual information for adaptive human locomotion.

Protein-inhibitor complexes analyzed by alkaline capillary LC-MS.

Liquid chromatography-mass spectrometry (LC-MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein-ligand complexes. We have successfully developed LC-MS method for protein molecular weight measurement using small-bore and capillary LC-MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein-ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.

High-throughput preparative process utilizing three complementary chromatographic purification technologies.

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.

Measurement of prehensile grasp capabilities by a force and moment wrench: methodological development and assessment of manual workers.

Prehensile grasp capability is typically quantified by pinch and grasp forces. This work was undertaken to develop a methodology to assess complex, multi-axis hand exertions through the measurement of forces and moments exerted by the hand along and about three orthogonal axes originating at the grip centre; termed an external wrench. Instrumentation consisting of a modified pinch/grip dynamometer affixed to a 6 df force cube was developed to simultaneously measure three forces, three moments and the pinch/grip force about the centre of the grip. Twenty right hand dominant manual workers (10 male and 10 female), free of hand or wrist disorders, completed a variety of maximal strength tasks. The randomized block design involved three separate grips--power grip, lateral pinch and pulp pinch. Randomized within each block were three non-concurrent repetitions of isolated maximal force and moment generations along and about the three principle orthogonal axes and a maximal grip force exertion. Trials were completed while standing, with the arm abducted and elbow flexed to 90 degrees with a wrist posture near neutral. Where comparable protocols existed in the literature, forces and moments exerted were found to be of similar magnitude to those reported previously. Female and male grip strengths on a Jamar dynamometer were 302.6 N and 450.5 N, respectively. Moment exertions in a power grip (female and male) were 4.7 Nm and 8.1 Nm for pronator, 4.9 Nm and 8.0 Nm for supinator, 6.2 Nm and 10.3 Nm for radial deviator, 7.7 Nm and 13.0 Nm for ulnar deviator, 6.2 Nm and 8.2 Nm for extensor, and 7.1 Nm and 9.3 Nm for flexor moments. Correlations with and between maximal force and moment exertions were only moderate. This paper describes instrumentation that allows comprehensive characterization of prehensile force and moment capability.