Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

The acute lethality of total-body irradiation (TBI) involves damage to multiple organs, including bone marrow and intestine. Ionizing radiation mitigators that are effective when delivered 24 h or later after TBI include the anti-apoptotic drug, JP4-039 and the anti-necroptotic drug, necrostatin-1. In contrast to effective delivery of JP4-039 at 24 h after TBI, necrostatin-1 is most effective when delivery is delayed until 48 h, a time that correlates with the elevation of necroptosis-inducing inflammatory cytokines and necroptosis-induced serine phosphorylation of receptor-interacting serine/threonine-protein kinase-3 (RIP3) in tissues. The goal of this work was to determine whether administration of JP4-039 influenced the optimal delivery time for necrostatin-1. We measured daily levels of 33 proteins in plasma compared to intestine and bone marrow of C57BL/6NTac female mice over a 7-day time period after 9.25 Gy TBI (LD). Protein responses to TBI in plasma were different from those measured in intestine or bone marrow. In mice that were given JP4-039 at 24 h after TBI, we delayed necrostatin-1 delivery for 72 h after TBI based on measured delay in RIP-3 kinase elevation in marrow and intestine. Sequential delivery of these two radiation mitigator drugs significantly increased survival compared to single drug administration.

Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2 mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg (B6) and the most prevalent human genotype Fanca (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-β-resistant SMAD3 (129/Sv) mice and double-knockout SMAD3 Fancd2 (129/Sv) mice treated daily with TGF-β receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-β mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg and Fanca mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.

IMPORTANCEIntense interest exists in novel ω-3 formulations with high bioavailability to reduce blood triglyceride (TG) levels. OBJECTIVE To determine the phase 3 efficacy and safety of a naturally derived krill oil with eicosapentaenoic acid and docosahexaenoic acid as both phospholipid esters (PLs) and free fatty acids (FFAs) (ω-3-PL/FFA [CaPre]), measured by fasting TG levels and other lipid parameters in severe hypertriglyceridemia. DESIGN, SETTING, AND PARTICIPANTS This study pooled the results of 2 identical randomized, double-blind, placebo-controlled trials.

Introduction The surging popularity of all-terrain vehicles (ATV) in the United States has caused an “epidemic of injuries and mortality.” The U.S. Consumer Product Safety Commission reported 99,600 injuries and 426 fatalities from ATV accidents in 2013. The aim of this study was to examine the relationship between helmet use and positive toxicology screenings on outcomes in ATV accident victims. Methods This is a retrospective study of patients admitted to a Level 1 Trauma Center in southwestern West Virginia following an ATV accident between 2005 and 2013. Data were obtained from the institution’s Trauma Registry. Results A total of 1,857 patients were admitted during the study period with 39 (1.9%) reported deaths. Positive serum alcohol and/or urine drug screens were obtained in 66.4% of the patients tested (n = 1,293). Those with positive screenings were 9.5% less likely to utilize a helmet (13.2% vs. 22.7%, p < 0.001); and the lack of helmet use was associated with an increase in traumatic brain injury (57.1% vs. 41.7%, p < 0.001). Positivity for substances or the lack of helmet use was significantly associated with higher morbidity. Lack of helmet use resulted in a 3.94-fold increase in the risk of discharge in a vegetative state or death. Conclusions Drugs and alcohol use may predispose riders to be less likely to wear helmets and significantly increase the risk of a poor clinical outcome following an ATV accident. Rigorous efforts should be made to enhance safety measures through educational endeavors and amendment of current regulations to promote safe and responsible use of ATVs. Practical applications Modification of regulatory requirements should be considered in order to mandate the wearing of helmets during ATV operation. In addition, expansion of safety programs should be considered in an effort to improve availability, affordability and awareness of safe ATV practices.