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Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate. ispartof: ANALYTICAL CHEMISTRY vol:93 issue:26 pages:9041-9048 ispartof: location:United States status: published

Exercise is an effective strategy in the prevention and treatment of metabolic diseases. Alterations in the skeletal muscle proteome, including post-translational modifications, regulate its metabolic adaptations to exercise. Here, we examined the effect of high-intensity interval training (HIIT) on the proteome and acetylome of human skeletal muscle, revealing the response of 3168 proteins and 1263 lysine acetyl-sites on 464 acetylated proteins. We identified global protein adaptations to exercise training involved in metabolism, excitation-contraction coupling, and myofibrillar calcium sensitivity. Furthermore, HIIT increased the acetylation of mitochondrial proteins, particularly those of complex V. We also highlight the regulation of exercise-responsive histone acetyl-sites. These data demonstrate the plasticity of the skeletal muscle proteome and acetylome, providing insight into the regulation of contractile, metabolic and transcriptional processes within skeletal muscle. Herein, we provide a substantial hypothesis-generating resource to stimulate further mechanistic research investigating how exercise improves metabolic health.Impact statementHostrup and colleagues identify global proteomic and acetylomic adaptations to high-intensity interval training, demonstrating adaptations to processes regulating metabolism and contraction.

The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis in vivo. To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.

Main text Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115.c, was coordinated by the Bureau International des Poids et Mesures (BIPM), the Health Sciences Authority (HSA) of Singapore and the Chinese National Institute of Metrology (NIM). Nine Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of the glycated hexapeptide of HbA1c (GE) present as the main component in the comparison sample for CCQM-K115.c. The comparison samples were prepared by HSA/BIPM from synthetic GE purchased from a commercial supplier and used as provided without further treatment or purification. GE was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of chemically synthesized peptides of known sequence, without cross-links, up to 5 kDa and modification (mono-glycation). It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used amino acid analysis (PICAA) or quantitative nuclear magnetic resonance (PICqNMR) spectroscopy with a correction for structurally-related peptide impurities approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, the BIPM, the HSA and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the GE mass fraction and the mass fraction of the peptide-related impurities as indispensable contributor regardless of the use of PICAA, PICqNMR or mass balance to determine the GE purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the GE mass fraction. In particular, it allows participants to demonstrate the efficacy of their implementation of peptide-related impurity identification and quantification. More detailed studies on the identification/quantification of peptide-related impurities revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the GE mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that that all results are directly taken for the calculation of the KCRVPepImp by use of random-effects meta-analysis (DerSimonian-Laird (DSL) variance-weighted mean). The KCRVPepImp of 45.4 mg/g is associated with a corresponding expanded uncertainty of 9.5 mg/g (k =2.26) providing a more realistic basis of evaluation for the capabilities of the participants to identify/quantify peptide-related impurities. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide-related impurity profile indicates that in most cases the major related peptide impurities have been identified and quantified. The approach selected to obtain a KCRVGE for the mass fraction of GE is based on the DerSimonian-Laird (DSL) variance-weighted mean. The DSL mean takes into account the uncertainties of the results while introducing sufficient excess variance to allow for their observed dispersion resulting in a larger expanded uncertainty U(KCRVGE). The KCRVGE for CCQM-K115.c is 628 mg/g with a corresponding expanded uncertainty of the KCRVGE of 27 mg/g (k =2.26). All GE mass fraction results are in agreement with the KCRVGE. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Main text Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115.2018, was coordinated by the Bureau International des Poids et Mesures (BIPM), the Health Sciences Authority (HSA) of Singapore and the Chinese National Institute of Metrology (NIM). Ten Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of the hexapeptide of HbA0 (VE) present as the main component in the comparison sample for CCQM-K115.2018. The comparison samples were prepared by HSA/BIPM from synthetic VE purchased from a commercial supplier and used as provided without further treatment or purification. VE was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of chemically synthesized peptides of known sequence, without cross-links, up to 5 kDa and without modification. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used amino acid analysis (PICAA) with a correction for structurally-related peptide impurities approach as the amount of material that had been provided to each participant (25 mg) was insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, the BIPM, the HSA and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the VE mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the VE purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the VE mass fraction. In particular, it allowed participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the VE mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most consistent set of results is taken for the calculation of the KCRVPepImp. The sum of the combined cis/trans VE depsipeptide impurities (only identified/quantified by NRC and confirmed by BIPM and HSA) and mass fractions of peptide related impurities that have been identified by at least two participants have been used to establish the KCRVPepImp. The KCRVPepImp of 53.0 mg/g is associated with a corresponding expanded uncertainty of 17.3 mg/g (k = 2) providing a more realistic basis of evaluation for the capabilities of the participants to identify/quantify peptide related impurities. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in all cases the major related peptide impurity, VE depsipeptide, has not been identified. The VE depsipeptide impurity was initially and uniquely identified and quantified by the NRC by the use of 1H-NMR. The related peptide impurity mass fraction results of only four participants (NRC, LGC, LNE and HSA) are in agreement with the KCRVPepImp. The NRC has identified and quantified the VE depsipeptide whereas the LGC, LNE and HSA have not identified the VE depsipeptide but accounted for that contribution. The approach selected to obtain a KCRVVE for the mass fraction of VE is based on a mass balance calculation that takes into account the most consistent set of results for the peptide related impurities KCRVPepImp, TFA mass fraction and the water mass fraction. The KCRVVE for CCQM-K115.2018 is 613 mg/g with a corresponding expanded uncertainty of the KCRVVE of 20 mg/g (k = 2). To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Disulfide bonds are critical linkages for maintaining protein structure and enzyme activity. These linkages, however, can limit peptide sequencing efforts by mass spectrometry (MS) and often require chemical reduction and alkylation. Under such conditions, information regarding cysteine connectivity is lost. Online partial disulfide reduction within the electrospray (ESI) source has recently been established as a means to identify complex cysteine linkage patterns in a liquid chromatography-MS experiment without the need for sample pre-treatment. Corona discharge (CD) is invoked as the causative factor of this in-source reduction (ISR); however, evidence remains largely circumstantial. In this study, we demonstrate that instrumental factors—nebulizing gas, ESI capillary material, organic solvent content, ESI spray needle-to-MS distance—all modulate the degree of reduction observed for the single disulfide in oxytocin, further implicating CD in ISR. Rigorous analysis of solution conditions, however, reveals that corona discharge alone can induce only minor disulfide reduction. We establish that CD-ESI of peptide solutions containing formic acid or its conjugate base results in a dramatic increase in disulfide reduction. It is also determined that ISR is exacerbated at low pH for complex peptides containing multiple disulfide bonds and possessing higher-order structure, as well as for a small protein. Overall, our results demonstrate that ESI of formate/formic acid–containing solutions under corona discharge conditions facilitates disulfide ISR, likely by a similar reduction pathway measured in γ-radiolysis studies nearly three decades ago.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Main text Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level. The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure; this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability. Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins. To reach the main text of this paper, click on Final Report.

Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Seven Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of oxytocin (OXT) present as the main component in the comparison sample for CCQM-K115.b. The comparison samples were prepared from synthetic OXT purchased from a commercial supplier and used as provided without further treatment or purification. OXT was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of chemically synthesized peptides of known sequence, with one cross-link and up to 5 kDa. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used amino acid analysis (PICAA) or quantitative nuclear magnetic resonance (PICqNMR) spectroscopy with a correction for structurally-related peptide impurities approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the OXT mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, PICqNMR or mass balance to determine the OXT purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the OXT mass fraction. In particular, it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities and the hydrolysis efficiency revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the OXT mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the Largest Consistent Subset (LCS) of results is taken for the calculation of the KCRVPepImp by use of the weighted mean. The KCRVPepImp of 31.6 mg/g is associated with a small corresponding expanded uncertainty of ±1.4 mg/g (k =2) providing a more realistic basis of evaluation for the capabilities of the participants to identify/quantify peptide related impurities. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases the major related peptide impurities have been identified and quantified. The approach selected to obtain a KCRVOXT for the mass fraction of OXT is based on random-effects meta-analysis (DerSimonian-Laird (DSL) variance-weighted mean). The DSLmean takes into account the uncertainties of the results while introducing sufficient excess variance to allow for their observed dispersion resulting in a larger expanded uncertainty U(KCRVOXT). The KCRVOXT for CCQM-K115.b is 787.2 mg/g with a corresponding expanded uncertainty of the KCRVOXT of ±12.9 mg/g. All OXT mass fraction results except the result of NMIM are in agreement with the KCRVOXT. It should be pointed out that the mass balance approaches show smaller uncertainties than PICAA or PICqNMR approaches. Mass balance approaches seem to produce slightly higher OXT mass fractions while PICAA approaches deliver slightly lower OXT mass fractions. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Motivation RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs (sRNA). Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. Results We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses. Availability and implementation The UEA sRNA Workbench is available at: http://srna-workbench.cmp.uea.ac.uk. The source code is available at: https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench Supplementary information Supplementary data are available at Bioinformatics online.

Identification and quantitation of related impurities is vital in obtaining corrected purity values for peptide certified reference materials. The sensitivity and selectivity of high-resolution mass spectrometry (MS) renders it an indispensable technique in this arena. Typical quantitation efforts involve constructing external calibration curves, although analysis of dilute peptide solutions can be complicated by analyte adsorption to vial walls, instrument tubing, etc. The standard addition method alleviates many concerns associated with this sample loss as the calibrant solutions more closely match the matrix of the samples. Yet, both strategies require acquisition of synthetic impurity peptide standards. Label-free proteomics relies on electrospray ionization (ESI)-MS signals to quantify identical peptides across multiple samples; however, peptides of differing sequence can exhibit widely disparate ESI-MS responses. This study explores the use of peak area ratios to quantitate sequence-related peptide impurities in an angiotensin II candidate certified reference material. Using synthetic standards of five abundant substances, impurity mass fractions calculated via the relative response method are in reasonable agreement with those determined from standard addition experiments, whereas external calibration measurements frequently overestimate impurity amounts. For a synthetic peptide and its related sequence impurities, the relative response method can expedite analysis and lower expenditures, and in some cases improve data quality.

Many peptides with antimicrobial activity and/or therapeutic potential contain disulfide bonds as a means to enhance stability, and their quantitation is often performed using electrospray ionization mass spectrometry (ESI-MS). Disulfides can be reduced during ESI under commonly used instrument conditions, which has the potential to hinder accurate peptide quantitation. We demonstrate that this in-source reduction (ISR) is predominantly observed for peptides infused from acidic solutions and subjected to elevated ESI voltages (3-4 kV). ISR is readily apparent in the mass spectrum of oxytocin-a small, single disulfide-containing peptide. However, subtle m/z shifts due to partial ISR of highly charged (z ≥ 3) peptides with multiple disulfide linkages may proceed unnoticed. Ion mobility (IM)-MS separates ions on the basis of charge and shape in the gas phase, and using insulin as a model system, we show that IM-MS arrival time distributions (ATDs) are particularly sensitive to partial ISR of large peptides. Isotope modeling allows for the relative quantitation of disulfide-intact and partially reduced states of the mobility-separated peptide conformers. Interestingly, hepcidin peptides ionized from acidic solutions at elevated ESI voltages undergo gas-phase compaction, ostensibly due to partial disulfide ISR. Our IM-MS results lead us to propose that residual acid is the likely cause of disparate ATDs recently measured for hepcidin from different suppliers [Anal. Bioanal. Chem. 409, 2559-2567 (2017)]. Overall, our results demonstrate the utility of IM-MS to detect partial ISR of disulfide-bonded peptides and reinforce the notion that peptide/protein measurements should be carried out using minimally activating instrument conditions. Graphical Abstract ᅟ.

RNA silencing (RNA interference, RNAi) is a complex, highly conserved mechanism mediated by short, typically 20-24 nt in length, noncoding RNAs known as small RNAs (sRNAs). They act as guides for the sequence-specific transcriptional and posttranscriptional regulation of target mRNAs and play a key role in the fine-tuning of biological processes such as growth, response to stresses, or defense mechanism.High-throughput sequencing (HTS) technologies are employed to capture the expression levels of sRNA populations. The processing of the resulting big data sets facilitated the computational analysis of the sRNA patterns of variation within biological samples such as time point experiments, tissue series or various treatments. Rapid technological advances enable larger experiments, often with biological replicates leading to a vast amount of raw data. As a result, in this fast-evolving field, the existing methods for sequence characterization and prediction of interaction (regulatory) networks periodically require adapting or in extreme cases, a complete redesign to cope with the data deluge. In addition, the presence of numerous tools focused only on particular steps of HTS analysis hinders the systematic parsing of the results and their interpretation.The UEA small RNA Workbench (v1-4), described in this chapter, provides a user-friendly, modular, interactive analysis in the form of a suite of computational tools designed to process and mine sRNA datasets for interesting characteristics that can be linked back to the observed phenotypes. First, we show how to preprocess the raw sequencing output and prepare it for downstream analysis. Then we review some quality checks that can be used as a first indication of sources of variability between samples. Next we show how the Workbench can provide a comparison of the effects of different normalization approaches on the distributions of expression, enhanced methods for the identification of differentially expressed transcripts and a summary of their corresponding patterns. Finally we describe individual analysis tools such as PAREsnip, for the analysis of PARE (degradome) data or CoLIde for the identification of sRNA loci based on their expression patterns and the visualization of the results using the software. We illustrate the features of the UEA sRNA Workbench on Arabidopsis thaliana and Homo sapiens datasets.

Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

The yin and yang of proteasomal regulation The ubiquitin-proteasome pathway regulates myriad proteins through their selective proteolysis. The small protein ubiquitin is attached, typically in many copies, to the target protein, which is then recognized and broken down by the proteasome. Shi et al. found a repeat structure in the proteasome for recognizing ubiquitin as well as ubiquitin-like (UBL) proteins. Tandem binding sites allow the proteasome to dock multiple proteins. One of the bound UBL proteins is an enzyme that cleaves ubiquitin-protein conjugates, which antagonizes degradation. Thus, the repetition of related binding sites with distinct specificity achieves a balance of positive and negative regulation of the proteasome. Science , this issue p. 10.1126/science.aad9421

The biologically active conformation of α 1 -antitrypsin (α 1 AT) and other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop (RCL) that acts as bait for the target enzyme. The protein can also adopt an inactive “latent” conformation that has the RCL inserted as a central strand in β-sheet A. This latent form is thermodynamically more stable than the active conformation. Nonetheless, folding of α 1 AT consistently yields the active state. The reasons that the metastable form is kinetically preferred remain controversial. The current work demonstrates that a carefully orchestrated folding mechanism prevents RCL insertion into sheet A. Temporal changes in solvent accessibility during folding are monitored using pulsed oxidative labeling and mass spectrometry. The data obtained in this way complement recent hydrogen/deuterium exchange results. Those hydrogen/deuterium exchange measurements revealed that securing of the RCL by hydrogen bonding of the first β‐strand in sheet C is one factor that favors formation of the active conformation. The oxidative labeling data presented here reveal that this anchoring is preceded by the formation of hydrophobic contacts in a confined region of the protein. This partial collapse sequesters the RCL insertion site early on and is therefore instrumental in steering α 1 AT towards its active conformation. RCL anchoring by hydrogen bonding starts to contribute at a later stage. Together, these two factors ensure that formation of the active conformation is kinetically favored. This work demonstrates how the use of complementary labeling techniques can provide insights into the mechanisms of protracted folding reactions.

Mass spectrometry (MS)-based protein conformational studies are a rapidly growing field. The characterization of partially disordered conformers is of particular interest because these species are not amenable to classical high-resolution techniques. Such equilibrium intermediates can often be populated by exposure to mildly acidic pH. Hydroxyl radical (·OH) introduces oxidative modifications at solvent-accessible side chains, while buried sites are protected. ·OH can be generated by laser photolysis of H(2)O(2) (fast photochemical oxidation of proteins-FPOP). The resulting labeling pattern can be analyzed by MS. The characterization of partially disordered intermediates usually involves comparative measurements under different solvent conditions. It can be challenging to separate structurally induced labeling changes from pH-mediated "secondary" effects. The issue of secondary effects in FPOP has received little prior attention. We demonstrate that with a proper choice of conditions (e.g., in the absence of pH-dependent ·OH scavengers) such undesired phenomena can be almost completely eliminated. Using apomyoglobin as a model system, we map the structure of an intermediate that is formed at pH 4. This species retains a highly protected helix G that is surrounded by partially protected helices A, B, and H. Our results demonstrate the utility of FPOP for the structural characterization of equilibrium intermediates. The near absence of an intrinsic pH dependence represents an advantage compared to hydrogen/deuterium exchange MS.

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.

Laser-induced oxidative labeling of proteins provides insights into biomolecular structures and interactions. In these experiments, the hydroxyl radical ((*)OH) formed by photolysis of H(2)O(2) generates covalent modifications that are detectable by mass spectrometry. Under conditions where individual protein molecules are irradiated only once, the short (*)OH lifetime ( approximately 1 mus) ensures that covalent modifications are formed before any oxidation-induced conformational changes take place. This feature implies that the method should be free of structural artifacts. It has been proposed that single-exposure conditions can be achieved by passing the solution through a capillary where successive laser pulses generate a string of irradiated flow segments that are well separated from one another. The current work explores the convection phenomena within the labeling capillary in more detail. The experiments are conducted at Reynolds numbers <

This study investigated carbohydrate ingestion of varied doses and frequencies during a simulated cross-country skiing time trial. Ten men and three women (age: 30 ± 7 years; V ˙ O 2 m a x : 59.6 ± 5.7 mL/kg/min) completed four, 30-km classic technique roller skiing time trials on a treadmill. A 1:1 maltodextrin-fructose carbohydrate solution was provided at high (2.4 g/min; HC) and moderate (1.2 g/min; MC) ingestion rates, each at high (six feeds; HF) and low (two feeds; LF) frequencies. In the LF trials, blood glucose was elevated following carbohydrate ingestion (at 4 and 19 km) but was reduced at 14 and 29 km compared with HF strategies (P ≤ 0.05). Gastrointestinal discomfort was higher in HC-LF compared with all other trials (P ≤ 0.05). Whole-body lipid oxidation was lower and carbohydrate oxidation was higher in LF compared with HF trials (P ≤ 0.05). While performance time was not significantly different between trials (140:11 ± 15:31, 140:43 ± 17:40, 139:12 ± 15:32 and 140:33 ± 17:46 min:s in HC-HF, HC-LF, MC-HF, and MC-LF, respectively; P 0.05), it was improved with trial order (P 0.001). There was no effect of order on any other variable (P 0.05). Altering carbohydrate dose or frequency does not affect cross-country ski performance. However, low-frequency carbohydrate ingestion resulted in poorer maintenance of euglycemia, reduced lipid oxidation, and increased gastrointestinal discomfort.

Objectives Bacterial colonization of the infant gut may have important influences on the development of gastrointestinal, respiratory, and allergic disease. Early diet is a major determinant of the gut microflora. It is very difficult to carry out studies in human infants that can investigate the interaction of diet, flora, and mucosa. In this study we have developed an infant human flora-associated (IHFA) rat model to allow such investigation. Methods Germ-free infant rats were infected with fecal bacteria from exclusively breast-fed infants and were maintained on a modified infant formula for 8 weeks. The fecal and cecal contents were collected and compared with feces of breast-fed infants for bacterial populations, bacterial metabolites, and enzymes and for the ability to inhibit adhesion of pathogenic bacteria to human mucosal cells. Results The IHFA cecum and feces were dominated by lactic acid bacteria, Bifidobacterium, and lactobacilli, which were representative of the infant feces. The fecal short-chain fatty acid profile was dominated by acetic and lactic acid in a similar manner to human infant feces. Other bacterial metabolites were similar to those of the human infant. Rat intestinal samples were able to inhibit the adhesion of pathogens to mucosal cells, but to a lesser extent than the human samples. Conclusions This IHFA infant model of the intestinal flora of the breast-fed infant is considered valid for studying the effect of diet on bacterial colonization and metabolism.

The coupling of electrospray ionization (ESI) with ion mobility-mass spectrometry (IM-MS) allows structural studies on biological macromolecules in a solvent-free environment. Collision cross sections (CCSs) measured by IM-MS provide a measure of analyte size. For native proteins and their complexes, many structural features can be preserved in the gas phase, making IM-MS a powerful approach for a range of bioanalytical applications. In addition to tightly folded conformers, a large number of partially disordered proteins participate in biological processes and disease mechanisms. It remains unclear to what extent IM-MS is suitable for exploring structural properties of these semifolded species. The current work addresses this question, using myoglobin as model system. This protein follows a sequential unfolding pathway that comprises two partially disordered states, i.e., apo-myoglobin (aMb) at pH 7 and pH 4. IM-MS data acquired for these two conformers were compared to those of native holo-myoglobin (hMb) at pH 7 and extensively unfolded aMb at pH 2. When examining individual aMb charge states, the degree of gas phase unfolding is not strongly correlated with the corresponding solution behavior. A key problem is that non-native conformers generate high ESI charge states, resulting in conformational transitions caused by intramolecular electrostatic repulsion. It is possible to establish a link between solution phase and gas phase structure when normalizing CCS distributions according to their respective ESI-MS signal intensities. This approach yields CCS averages that follow the expected progression hMbpH 7aMbpH 7aMbpH 4aMbpH 2. However, this trend mainly reflects the protonation behavior of the conformers during the ESI process, rather than a genuine memory of solution structure. Overall, our data reveal that electrostatically driven expansion as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution.

Kinetic measurements can provide insights into protein folding mechanisms. However, the initial (submillisecond) stages of folding still represent a formidable analytical challenge. A number of ultrarapid triggering techniques have been available for some time, but coupling of these techniques with detection methods that are capable of providing detailed structural information has proven to be difficult. The current work addresses this issue by combining submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for our measurements. Exposure of the protein to a brief pulse of hydroxyl radical (·OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using mass spectrometry-based peptide mapping, providing spatially resolved measurements of changes in solvent accessibility. The submillisecond mixer used here improves the time resolution by a factor of 50 compared to earlier ·OH labeling experiments from our laboratory. Data obtained in this way indicate that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed A(E)G(H) scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. Major conformational changes go to completion within 0.1 s. The technique introduced here is capable of providing in-depth structural information on very short time scales that have thus far been dominated by low resolution (global) spectroscopic probes. By employing submillisecond mixing in conjunction with slower mixing techniques, it is possible to observe complete folding pathways, from fractions of a millisecond all the way to minutes.

The biologically active conformation of α₁-antitrypsin (α₁AT) and other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop (RCL) that acts as bait for the target enzyme. The protein can also adopt an inactive "latent" conformation that has the RCL inserted as a central strand in β-sheet A. This latent form is thermodynamically more stable than the active conformation. Nonetheless, folding of α₁AT consistently yields the active state. The reasons that the metastable form is kinetically preferred remain controversial. The current work demonstrates that a carefully orchestrated folding mechanism prevents RCL insertion into sheet A. Temporal changes in solvent accessibility during folding are monitored using pulsed oxidative labeling and mass spectrometry. The data obtained in this way complement recent hydrogen/deuterium exchange results. Those hydrogen/deuterium exchange measurements revealed that securing of the RCL by hydrogen bonding of the first β-strand in sheet C is one factor that favors formation of the active conformation. The oxidative labeling data presented here reveal that this anchoring is preceded by the formation of hydrophobic contacts in a confined region of the protein. This partial collapse sequesters the RCL insertion site early on and is therefore instrumental in steering α₁AT towards its active conformation. RCL anchoring by hydrogen bonding starts to contribute at a later stage. Together, these two factors ensure that formation of the active conformation is kinetically favored. This work demonstrates how the use of complementary labeling techniques can provide insights into the mechanisms of protracted folding reactions.

Summary: RNA silencing is a complex, highly conserved mechanism mediated by small RNAs (sRNAs), such as microRNAs (miRNAs), that is known to be involved in a diverse set of biological functions including development, pathogen control, genome maintenance and response to environmental change. Advances in next generation sequencing technologies are producing increasingly large numbers of sRNA reads per sample at a fraction of the cost of previous methods. However, many bioinformatics tools do not scale accordingly, are cumbersome, or require extensive support from bioinformatics experts. Therefore, researchers need user-friendly, robust tools, capable of not only processing large sRNA datasets in a reasonable time frame but also presenting the results in an intuitive fashion and visualizing sRNA genomic features. Herein, we present the UEA sRNA workbench, a suite of tools that is a successor to the web-based UEA sRNA Toolkit, but in downloadable format and with several enhanced and additional features. Availability: The program and help pages are available at http://srna-workbench.cmp.uea.ac.uk. Contact: vincent.moulton@cmp.uea.ac.uk

Considerable progress in deciphering the mechanisms of protein folding has been made. However, most work in this area has focused on single-chain systems, whereas the majority of proteins are oligomers. The spontaneous assembly of intact multi-subunit systems from disordered building blocks encompasses the formation of intramolecular as well as intermolecular contacts. Both types of interaction affect the solvent accessibility of individual protein segments. This work employs pulsed hydroxyl radical (·OH) labeling for tracking time-dependent accessibility changes during folding and assembly of the S100A11 homodimer. ·OH induces covalent modifications at exposed residues. Structural snapshots are obtained by combining ·OH labeling with rapid mixing and mass spectrometry. The free subunits are found to possess a partially non-native hydrophobic core that prevents subunit association during the initial stages of the reaction. Instead, the protein forms an early (10 ms) monomeric intermediate that exhibits reduced solvent accessibility in regions distant from helices I and IV, which constitute the dimerization interface. Subunit association is complete after 800 ms, although the protein retains significant disorder in helices II and III at this point. Subsequent consolidation of these elements leads to the native state. The experimental strategy used here could become a general tool for deciphering kinetic mechanisms of biomolecular self-assembly processes.

Elastic network models of biomolecules have proved to be relatively good at predicting global conformational changes particularly in large systems. Software that facilitates rapid and intuitive exploration of conformational change in elastic network models of large biomolecules in response to externally applied forces would therefore be of considerable use, particularly if the forces mimic those that arise in the interaction with a functional ligand. We have developed software that enables a user to apply forces to individual atoms of an elastic network model of a biomolecule through a haptic feedback device or a mouse. With a haptic feedback device the user feels the response to the applied force whilst seeing the biomolecule deform on the screen. Prior to the interactive session normal mode analysis is performed, or pre-calculated normal mode eigenvalues and eigenvectors are loaded. For large molecules this allows the memory and number of calculations to be reduced by employing the idea of the important subspace, a relatively small space of the first M lowest frequency normal mode eigenvectors within which a large proportion of the total fluctuation occurs. Using this approach it was possible to study GroEL on a standard PC as even though only 2.3% of the total number of eigenvectors could be used, they accounted for 50% of the total fluctuation. User testing has shown that the haptic version allows for much more rapid and intuitive exploration of the molecule than the mouse version.

A haptic feedback device enables a user to manipulate three dimensional structures and feel forces contained within complex data-sets such as those resulting from computational biology. However, as the data-set grows in size it becomes difficult to ensure that the user can easily interact with every part of it. One could scale the data-set down to fit into the haptic workspace, however, this could result in important features being missed. A secondary problem is enabling the user to select points efficiently within the three dimensional data set, where the perception of depth can be difficult. In this paper we present novel techniques to rapidly navigate large and complex data-sets with a haptic feedback device, whilst still permitting accurate and fast selection of points in three dimensional space. We have applied these techniques as part of software dedicated to studying the response of biomolecules to externally applied forces using elastic network models.

This review discusses various mass spectrometry (MS)-based approaches for exploring structural aspects of proteins in solution. Electrospray ionization (ESI)-MS, in particular, has found fascinating applications in this area. For example, when used in conjunction with solution-phase hydrogen/deuterium exchange (HDX), ESI-MS is a highly sensitive tool for probing conformational dynamics. The main focus of this article is a technique that is complementary to HDX, that is, the covalent labeling of proteins by hydroxyl radicals. The reactivity of individual amino acid side chains with *OH is strongly affected by their degree of solvent exposure. Thus, analysis of the oxidative labeling pattern by peptide mapping and tandem mass spectrometry provides detailed structural information. A convenient method for *OH production is the photolysis of H(2)O(2) by a pulsed UV laser, resulting in oxidative labeling on the microsecond time scale. Selected examples demonstrate the use of this technique for structural studies on membrane proteins, and the combination with rapid mixing devices for characterizing the properties of short-lived protein (un)folding intermediates.

The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (·OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as + 16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by ·OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible.

Background From the 1950s computer based renderings of molecules have been produced to aid researchers in their understanding of biomolecular structure and function. A major consideration for any molecular graphics software is the ability to visualise the three dimensional structure of the molecule. Traditionally, this was accomplished via stereoscopic pairs of images and later realised with three dimensional display technologies. Using a haptic feedback device in combination with molecular graphics has the potential to enhance three dimensional visualisation. Although haptic feedback devices have been used to feel the interaction forces during molecular docking they have not been used explicitly as an aid to visualisation. Results A haptic rendering application for biomolecular visualisation has been developed that allows the user to gain three-dimensional awareness of the shape of a biomolecule. By using a water molecule as the probe, modelled as an oxygen atom having hard-sphere interactions with the biomolecule, the process of exploration has the further benefit of being able to determine regions on the molecular surface that are accessible to the solvent. This gives insight into how awkward it is for a water molecule to gain access to or escape from channels and cavities, indicating possible entropic bottlenecks. In the case of liver alcohol dehydrogenase bound to the inhibitor SAD, it was found that there is a channel just wide enough for a single water molecule to pass through. Placing the probe coincident with crystallographic water molecules suggests that they are sometimes located within small pockets that provide a sterically stable environment irrespective of hydrogen bonding considerations. Conclusion By using the software, named HaptiMol ISAS (available from http://www.haptimol.co.uk), one can explore the accessible surface of biomolecules using a three-dimensional input device to gain insights into the shape and water accessibility of the biomolecular surface that cannot be so easily attained using conventional molecular graphics software.

The structural characterization of short-lived intermediates provides insights into the mechanisms of protein folding and unfolding. Using holo-myoglobin as a model system, this work reports the application of oxidative pulse labeling for experiments of this kind. Protein unfolding is triggered by a pH jump from 6.5 to 3.2 in 150 mM NaCl. Subsequent (.-)OH exposure at various time points using laser photolysis of H2O2 leads to covalent modifications of solvent-exposed side chains within approximately 1 mus (Hambly, D. M.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2005, 16, 2057-2063). Most of these modifications appear as 16 Da adducts in the mass spectrum of the intact protein. The overall extent of labeling increases with time, reflecting the exposure of reactive side chains that had previously been buried. Unfolding and disruption of heme-protein interactions go to completion within approximately 10 s. Spatially resolved information is obtained by monitoring the signal intensities of unmodified tryptic peptides. After 50 ms, many regions have lost most of their protection, whereas structure is retained in the B, E, F, and G helices. The BEF core remains partially folded, even after 500 ms, at which point helix G is fully unprotected. The observation of an "early" (BEFG) and a "late" (BEF) intermediate is in accord with optical stopped-flow measurements. Formation of these transient species is attributed to the persistence of heme-protein interactions during the early stages of the reaction. Overall, this work demonstrates the feasibility of laser-induced oxidative labeling as a tool for characterizing the structure of short-lived protein conformers. The combination of this approach with ultrarapid mixing or photochemical triggering should allow folding experiments in the submillisecond range.

Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here.

Small RNAs (sRNAs) are a class of short (20-25 nt) non-coding RNAs that play important regulatory roles in gene expression. An essential first step in understanding their function is to confidently identify sRNA targets. In plants, several classes of sRNAs such as microRNAs (miRNAs) and trans-acting small interfering RNAs have been shown to bind with near-perfect complementarity to their messenger RNA (mRNA) targets, generally leading to cleavage of the mRNA. Recently, a high-throughput technique known as Parallel Analysis of RNA Ends (PARE) has made it possible to sequence mRNA cleavage products on a large-scale. Computational methods now exist to use these data to find targets of conserved and newly identified miRNAs. Due to speed limitations such methods rely on the user knowing which sRNA sequences are likely to target a transcript. By limiting the search to a tiny subset of sRNAs it is likely that many other sRNA/mRNA interactions will be missed. Here, we describe a new software tool called PAREsnip that allows users to search for potential targets of all sRNAs obtained from high-throughput sequencing experiments. By searching for targets of a complete 'sRNAome' we can facilitate large-scale identification of sRNA targets, allowing us to discover regulatory interaction networks.

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A technique is presented for obtaining the elastic-plastic stress-strain distribution in axisymmetric and plane stress continuum composed of temperature dependent linear strain-hardening isotropic material. The method employs an incremental finite element computer program using the Prandtl-Reuss flow equations to derive a plastic stress-strain matrix in a manner similar to the tangent modulus method. The addition of pseudo stresses is used to develop load terms that account for changes in the material properties which occur during the increment, thus keeping the solution on the temperature dependent material stress-strain curve. The pseudo stresses also correct for any elastic over-shoot that occurs when an element changes from an elastic to a plastic state. With this technique it becomes economically feasible to perform elastic-plastic stress analyses on two-dimensional continuum subjected to transient thermal and mechanical loadings with material properties varying during multiple loading-unloading cycles.

Coal, we are all aware, is a remnant of the organic matter of plants which flourished long ago. Owing to the length of time that has elapsed since the remains were entombed, and the pressure the material has been subjected to by overlying strata of great thickness, coal now presents to the unaided eye little or no evidence of organic structure, but is a lustrous black product not at all like wood in appearance.

To address a number of difficulties with nursing documentation, a process-oriented documentation system called the Problem-Intervention-Evaluation (PIE) system was developed and implemented on a 35-bed medical unit at Craven County Hospital, New Bern, North Carolina. The PIE system simplifies the documentation process and unifies the care plan and progress notes into a complete, concise record of nursing care actually planned and provided. Informal evaluations indicate that the PIE system increases job satisfaction for nursing staff, improves the quality of documentation, and promotes continuity of care from shift to shift and from day to day. The article raises questions for further evaluation.

In the Halifax Hard Bed Coal seam it is well-known that nodules occur in abundance; these nodules are locally known as “ coal balls.” Just above the coal is a bed of shale in which nodules called “ baura pots “ are found; the latter is divided from the coal by a thin shale containing Aviculopectens in abundance; under the coal is a hard fire-clay called ganister. “Coal balls” are generally spherical, of a brown or greyish-brown colour internally, they are very hard and contain the remains of plants, in some instances being largely composed of wood fossilized by carbonate of lime; they have generally a coating of iron pyrites, which occasionally replaces the carbonate of lime and becomes the chief constituent of the ball. “ Baum pots” may be divided into two kinds—first, those consisting of successive coatings of mineral matter surrounding a central portion; second, those showing stratification like the shale in which they occur. “Baum pots” are generally oval, of a grey colour, hard and compact and often contain fossil shells. They have frequently an outer coating of iron pyrites. I am not aware that the composition of these nodules has been examined in detail, it being generally stated that they are composed of limestone coated with, iron pyrites. With a view to learn something more as to their composition I have made analyses of two “coal balls” and one “baum pot” from the Halifax district. On examination of the “coal balls” qualitatively I found the ...