A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1

Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection

Update on treatment of follicular non-Hodgkin’s lymphoma: focus on potential of bortezomib

Danielle M Brander, Anne W BeavenDuke University Medical Center, Durham, NC, USAAbstract: Follicular lymphoma is predominantly managed as a chronic disease, with intermittent chemo/immunotherapy reserved for symptomatic progression. It is considered incurable with conventional treatments, and current therapeutic options are associated with significant toxicities that are especially limiting in older patients. Bortezomib (PS-341; Velcade®), a first-in-class drug targeting the proteolytic core subunit of the 26S proteasome, has emerged as a therapeutic alternative in follicular lymphoma, with promising preclinical data and efficacy in patients with other hematological malignancies. Several clinical trials were conducted with bortezomib for the treatment of non-Hodgkin’s lymphoma. As a single agent, overall responses in follicular lymphoma varied greatly (16%–41%), with weekly bortezomib showing less neurotoxicity than twice-weekly regimens, but with concern about decreased responses. Combination with rituximab was projected to improve the efficacy of bortezomib, but this resulted in increased toxicities and questionable added benefit. Although the largest Phase III study in follicular lymphoma of bortezomib plus rituximab versus rituximab alone demonstrated a significant progression-free survival difference, the absolute difference was small (12.8 months versus 11 months). Combining bortezomib with established regimens, such as rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP), or rituximab-bendamustine also did not show definite benefit, and many of these studies did not meet their primary endpoint when bortezomib failed to improve responses or survival to the degree anticipated. In a disease where the goal of treatment is palliative and affected patients often have other medical and treatment-related comorbidities, decisions regarding therapies which carry risks of additional toxicities must be considered carefully. Conclusive evidence of the ability of bortezomib to improve patient outcomes meaningfully and to justify the added toxicity is lacking, but limitations in cross-trial comparisons are recognized. Large randomized trials and investigations of combinations with promising novel targeted agents will aid in determining the role of bortezomib, if any, in the future treatment of follicular lymphoma.Keywords: bortezomib, follicular lymphoma, proteasome inhibito

Knee replacement.

Knee-replacement surgery is frequently done and highly successful. It relieves pain and improves knee function in people with advanced arthritis of the joint. The most common indication for the procedure is osteoarthritis. We review the epidemiology of and risk factors for knee replacement. Because replacement is increasingly considered for patients younger than 55 years, improved decision making about whether a patient should undergo the procedure is needed. We discuss assessment of surgery outcomes based on data for revision surgery from national joint-replacement registries and on patient-reported outcome measures. Widespread surveillance of existing implants is urgently needed alongside the carefully monitored introduction of new implant designs. Developments for the future are improved delivery of care and training for surgeons and clinical teams. In an increasingly ageing society, the demand for knee-replacement surgery will probably rise further, and we predict future trends. We also emphasise the need for new strategies to treat early-stage osteoarthritis, which will ultimately reduce the demand for joint-replacement surgery

Effects of two pre-workout supplements on concentric and eccentric force production during lower body resistance exercise in males and females: a counterbalanced, double-blind, placebo-controlled trial

Abstract Background Pre-workout supplements purportedly enhance feelings of energy, reduce fatigue and improve exercise performance. The purpose of this study was to examine the performance effects of caffeinated and non-caffeinated multi-ingredient pre-workout supplements. Methods In a counterbalanced, double-blind, placebo-controlled design, eccentric and concentric force production during lower body resistance exercise on a mechanized squat device were assessed after supplement ingestion. Repetitions-in-reserve/RPE and subjective feelings of energy, focus and fatigue were also examined. Twenty-one resistance-trained adults (12 F, 9 M) completed three conditions in random order: caffeinated supplement, non-caffeinated supplement and placebo. Subjects were not informed of the presence of a placebo condition. Thirty minutes after supplement ingestion, a 3-repetition maximum test and 5 sets of 6 repetitions were completed using the squat device. Each repetition involved 4-s eccentric and concentric phases, and the force signal throughout each repetition was sampled from a load cell contained within the squat device. The scaled and filtered force signals were analyzed using customized software. Repeated measures analysis of variance and appropriate follow-up analyses were utilized to compare dependent variables, and relevant effect sizes (d) were calculated. Results Supplement or placebo ingestion led to similar subjective responses (p > 0.05). Energy (+8 to 44%; d = 0.3 to 0.8) and focus (+8 to 25%; d = 0.3 to 0.5) were acutely increased by supplement or placebo ingestion and decreased as the exercise session progressed. Fatigue was acutely decreased by supplement or placebo ingestion (−7 to 38%; d = −0.1 to −0.6) and increased as the exercise session progressed. Eccentric and concentric forces were unimproved by supplementation during the exercise sets for both sexes. In the non-caffeinated supplement condition only, maximal eccentric force production was lower during sets 3 to 5, as compared to set 1 (p < 0.05). Effect size data indicated that both the caffeinated and non-caffeinated supplements may contribute to small increases in concentric force production in males (+5 to 20%, d = 0.2 to 0.4 relative to placebo), but not females. Conclusions As compared to placebo, caffeinated and non-caffeinated multi-ingredient pre-workout supplements failed to improve concentric and eccentric force production. In males, effect size data indicate a possible small benefit of supplementation on concentric force production, although this was not statistically significant. When resistance-trained subjects were unaware of the presence of a placebo, resistance exercise performance was similar regardless of whether a placebo or multi-ingredient supplement was ingested