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The practice of re-melting glass was well known, certainly from the Roman period onwards. This can be seen not only in ancient literary evidence but also in the archaeological evidence, collections of broken glass have been found in, for example, Pompeii (79 AD) and the Iulia Felix shipwreck (Third century AD). Elevated levels of certain transition metals in archaeological glasses are interpreted as indications of the mixing and/or recycling of different glasses. Assumptions have been made that all glasses could be recycled, but to what extent are these valid? Why does the evidence for the recycling of glass only occur from the Roman period onwards? From the middle of the First millennium BC to the Ninth century AD, natron glass was the predominant glass type in the Mediterranean and Europe, however, plant ash glass was still in use in some areas. To test the effects on the final product of mixing different composition glass types, experimental glasses were made by mixing varying quantities of replica plant ash glass, replica natron glass, and a modern glass. At low temperatures crystalline material formed in the products containing replica plant ash glass. As the plant ash glass content increased, so too did the amount of crystalline material produced. This is due to a combination of the glass compositions and the firing temperature. It appears that natron type glass can be more easily recycled at lower temperatures, although, if a high enough temperature is used then most glass types can be recycled. Early furnace technology, i.e. the vertical heating chamber furnace, may not have been able to achieve these high temperatures, hence the widespread practice of recycling did not begin until after the invention of glassblowing which required a change in the furnace technology to the use of a horizontal heating chamber furnace. [ABSTRACT FROM AUTHOR]

In this study, the use of trace elements as a provenance indicator for Roman natron glass is evaluated. Suitable glass‐making sand raw materials are analysed for their trace elemental composition and compared to glass from known production centres. It is shown that the combined use of Nd isotopic and trace element analysis can be efficient for the provenancing of Roman glass. Trace elements associated with (de)colourants of glass are only present in small concentrations in glass‐making sands. Background levels introduced to the glass by the sand raw material are defined and a lower threshold for the concentrations influenced by recycling is determined. [ABSTRACT FROM AUTHOR]

The provenancing of Roman natron glass is one of the most challenging problems in the field of archaeometry. Although the use of Sr and Nd isotope ratios and trace element signatures as an indication of provenance has proven promising, there are still many unknowns. In this study, the influence of the different raw materials on the final Sr isotopic composition of Roman natron glass is examined. It is shown that the 87Sr/86Sr ratio in natron glass is significantly influenced by the silicate fraction of the sand used and does not always provide a clear indication of the lime source used. [ABSTRACT FROM AUTHOR]

In this study analysis of major elements and Sr--Nd isotopes is performed on 33 colourless glass fragments from two Ro- man shipwrecks discovered in the Northern Mediterranean Sea, the Iulia Felix (irst half of the third century AD) and the Ouest-Embiez (end of the second--beginning of the third century AD). Two compositional groups are deined based upon the major elements analysis, suggesting the use of diferent raw materials, and possibly the production of the glass samples in two separate factories. Sr--Nd isotopes, promising indicators for provenancing geological resources used as raw materials 87 86 in glass manufacturing, conirm the compositional groups. The Sr/ Sr signature is very close to the modern sea water signature (0·7092) for all samples, likely due to the use of shell as glass raw material. The Nd signature further subdivides the compositional groups, suggesting the use of three diferent sand raw materials for the production of glass. [ABSTRACT FROM AUTHOR]

Background: Prompt sepsis treatment is associated with improved outcomes but requires a complex series of actions by multiple clinicians. We investigated whether simply reorganizing emergency department (ED) care to expedite patients' initial evaluation was associated with shorter sepsis door-to-antibiotic times., Methods: Patients eligible for this retrospective study received IV antibiotics and demonstrated acute organ failure after presenting to one of three EDs in Utah. On May 1, 2016, the intervention ED instituted "swarming" as the default model for initial evaluation of all mid- and low-acuity patients. Swarming involved simultaneous patient evaluation by the ED physician, nurse, and technician followed by a team discussion of the initial care plan. Care was unchanged at the two control EDs. A 30-day wash-in period separated the baseline (May 16, 2015 to April 15, 2016) and post-intervention (May 16, 2016 to November 15, 2016) analysis periods. We conducted a quasi-experimental analysis comparing door-to-antibiotic time for sepsis patients at the intervention ED after versus before care reorganization, applying difference-in-differences methods to control for trends in door-to-antibiotic time unrelated to the studied intervention and multivariable regression to adjust for patient characteristics., Results: The analysis included 3,230 ED sepsis patients, including 1,406 from the intervention ED. Adjusted analyses using difference-in-differences methods to control for temporal trends unrelated to the studied intervention revealed no significant change in door-to-antibiotic time after care reorganization (-7 minutes, 95% CI -20 to 6 minutes, p = 0.29). Multivariable pre/post analyses using data only from the intervention ED overestimated the magnitude and statistical significance of outcome changes associated with ED care reorganization., Conclusions: Implementation of an ED care model involving parallel multidisciplinary assessment and early team discussion of the care plan was not associated with improvements in mid- and low-acuity sepsis patients' door-to-antibiotic time after accounting for changes in the outcome unrelated to the studied intervention., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: IDP and SMB have received research funding to their institution from Janssen Pharmaceuticals, and IDP has received research funding to his institution from Immunexpress Inc. Faron Pharmaceuticals and Sedana Medical paid SMB’s institution for his service on trial steering committees. Asahi Kasei Pharma provided research funding to the institution of IDP, SMB, JRB and DB for subject enrollment in a clinical trial. AMAG Pharmaceuticals paid JRB consultation fees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors otherwise have declared that no competing interests exist.

Asn deamidation was monitored in Ala-based octadecapeptides of varying alpha-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala --> Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of alpha-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.

Purpose: Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range., Methods: Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry., Results: The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG)., Conclusions: PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.

The effect of pH and urea on the conformation of recombinant human megakaryocyte growth and development factor (rHuMGDF) was determined by circular dichroism, intrinsic fluorescence spectroscopy, and equilibrium ultracentrifugation. The conformation of rHuMGDF was dependent on pH and urea concentration. Multiple folding forms were evidenced by multiple pH-induced transitions and urea-induced equilibrium transitions that deviated from a simple two-state process. In neutral to alkaline pH, rHuMGDF exists as a monomer, but an acid-induced conformational state self-associates to form a soluble aggregate. A folding intermediate(s) was observed with a more stable secondary structure than tertiary structure and was dependent on the pH of the urea-induced denaturation. The differences in the stabilities of the folding states were most distinct in the pH range of 4.5 to 6.5. The presence of intermediates in the folding pathway of rHuMGDF are similar to findings of previous studies of related growth factors that share a common three-dimensional structure.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.

Purpose: The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue., Methods: The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules., Results: Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation., Conclusions: We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.