Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands.

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91-95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11-13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.

Translating antibody-binding peptides into peptoid ligands with improved affinity and stability.

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48-57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4-7.8 10-7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.

Graduates from Accredited Paramedic Programs Have Higher Pass Rates on a National Certification Examination.

While institutional and program accreditation has long been required for physician and nursing education, paramedic program accreditation was largely voluntary in the US until recently. Our objective was to assess whether program accreditation is associated with higher pass rates and cognitive ability performance. This was a cross-sectional evaluation of the National Registry of Emergency Medical Technicians' (NREMT) Paramedic Certification cognitive examination results for graduates of US paramedic pro¬grams. Individual first-attempt and cumulative third-attempt pass rates along with first-attempt cognitive mean ability estimates (MAE) for each content area were analyzed. In 2012, 8,404 paramedic program graduates attempted the cognitive examination. The first-attempt pass rate for accredited program graduates was higher than that of their non-accredited counterparts (75.6% vs. 67.3, p<0.001). The cumulative pass rate after three attempts also was higher for accredited program graduates than for non-accredited graduates (88.9% vs 81.9%, p<0.001). Paramedic students from accredited programs demonstrated higher cognitive ability in all clinical content areas (p<0.001). Accredited program graduates had greater success on the National Paramedic Certification examination with 51% greater odds of first-time success (OR 1.51, 95%CI 1.31-1.73, p<0.001). Collectively, these results support the need for universal paramedic program accreditation.

Aging, resting metabolic rate, and oxidative damage: results from the Louisiana Healthy Aging Study.

BACKGROUND: The aging process occurs at variable rates both among and within species and may be related to the variability in oxygen consumption and free radical production impacting oxidative stress. The current study was designed to test whether nonagenarians have a relatively low metabolic rate and whether it is associated with low levels of oxidative stress relative to age. METHODS: Resting metabolic rate (RMR) and markers of oxidative stress to lipids, proteins, and DNA were measured in three groups of individuals aged 20-34 (n=47), 60-74 (n=49), and>or=90 years (n=74). RESULTS: RMR, adjusted for fat-free mass, fat mass, and sex, was lower in both older groups when compared to the young group (p