A synthetic approach to 'click' neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry.

The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards 'click' neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide-alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our 'click' neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

Harnessing ester bond chemistry for protein ligation.

The hydrolysis potential of ester bonds in covalently cross-linking proteins is captured in our novel protein ligation technology. This new type of "molecular superglue" based on the spontaneously-formed Thr-Gln ester bonds found in cell-surface adhesins, affords a unique mechanism to both rationally assemble and disassemble complex protein nanomaterials.

Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R.

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Site-specific cross-linking of collagen peptides by lysyl advanced glycation endproducts.

Cross-linking of proteins by advanced glycation endproducts (AGEs) causes a host of pathological conditions but their exact roles are unknown. Cross-linking lysyl AGEs were synthesized and incorporated into two types of collagen peptides. The utility of these cross-linked peptides for biochemical investigations was demonstrated by proteolysis studies and circular dichroism.

Total Chemical Synthesis of an Orf Virus Protein, ORFV002, an Inhibitor of the Master Gene Regulator NF-κB.

ORFV002 is a novel orf viral protein (117 Aa) that inhibits nuclear events through the regulation of the transcriptional activity of NF-κB, a master regulator of human gene expression (Diel et al., J Virol 2011, 85, 264-275). It is identified as the first nuclear inhibitor of NF-κB produced by orf virus (ORFV) and no homologues in other genera of the Chordopoxvirinae subfamily have been reported to date (Diel et al., J Virol 2011, 85, 264-275). Our molecular structure predictions suggest that ORFV002 may mimic part of IκB, an inhibitor and natural human partner of NF-κB. Recent advances in total chemical synthesis of proteins have provided solutions in overcoming challenges of current recombinant methods of protein isolation for structure elucidation. Aided by Boc solid phase peptide synthesis and native chemical ligation, ORFV002 was successfully synthesized in multimilligram amounts in good yield and high purity.

The significance of definitions of success in program evaluation.

In program evaluation, determining whether a program is reaching its goals is key to evaluating program success. Having an understanding of the stated goals of each organization within a system rather than simply knowing the formal system goals can aid in program development and inform policy making. Evaluability assessments were used initially as a means of learning how Philadelphia delinquency prevention programs define success. Subsequent surveys of programs were conducted to ascertain whether any changes in definitions of success had occurred. Examination of programs' definitions of success over time has facilitated program development and policy making.

Intramuscular cimetidine and ranitidine as prophylaxis against gastric aspiration syndrome--a randomized double-blind study.

Preoperative cimetidine 300 mg or ranitidine in 50 and 100 mg doses were administered intramuscularly to 120 patients in a randomized double-blind study. The volume and pH of gastric aspirate samples obtained after tracheal intubation and before extubation were measured. The pH of gastric aspirate was higher following ranitidine 100 mg than ranitidine 50 mg or cimetidine 300 mg at both intubation and extubation (p = 0.006). In addition, fewer patients tended to be "at risk" of pulmonary aspiration syndrome (pH less than or equal to 2.5) after ranitidine 100 mg than ranitidine 50 mg or cimetidine 300 mg. Preoperative intramuscular ranitidine 100 mg was found to be suitable for use in protection against gastric aspiration syndrome.

A study of young-age and mid-life homicidal women admitted to a psychiatric hospital for pre-trial evaluation.

The authors compared a group of young female homicidal offenders with a group of middle-aged homicidal women as to demographic data, psychopathology, physical disorders and type of victims chosen. Young women tend to have low socioeconomic status, have antisocial personality disorder, and/or schizophrenia as psychiatric diagnoses and most likely kill their children, while mid-life women tend to have slightly higher socioeconomic status, suffer from affective disorder and alcoholism and have more frequent physical disorders and most likely murder their spouses. A significant finding noted among mid-life women is the high frequency of physical abuse by husbands who later become their homicide victims. Treatment implications of these findings are noted.

Differences between midlife female offenders and those younger than 40.

The authors studied 66 women referred for forensic evaluation and classified them into two age groups: 48 women 17-39 years old composed a young adult group and 18 women 40-54 years old composed a midlife group. The midlife group included a significantly larger number of first-time offenders with a higher frequency of medical as well as psychiatric disorders. None of the women in the midlife group was diagnosed as having antisocial personality disorder, but this was the most common diagnosis among the younger women. The authors note the implications of these findings for treatment of older female offenders, especially those in midlife.