Gcg-XTEN: An Improved Glucagon Capable of Preventing Hypoglycemia without Increasing Baseline Blood Glucose

While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels.Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN.The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon

Barriers encountered during enrollment in an internet-mediated randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Online technology is a promising resource for conducting clinical research. While the internet may improve a study's reach, as well as the efficiency of data collection, it may also introduce a number of challenges for participants and investigators. The objective of this research was to determine the challenges that potential participants faced during the enrollment phase of a randomized controlled intervention trial of Stepping Up to Health, an internet-mediated walking program that utilized a multi-step online enrollment process.</p> <p>Methods</p> <p>We conducted a quantitative content analysis of 623 help tickets logged in a participant management database during the enrollment phase of a clinical trial investigating the effect of an automated internet-mediated walking intervention. Qualitative coding was performed by two trained coders, and 10% of the sample was coded by both coders to determine inter-coder reliability. Quantitative analyses included standard descriptive statistics on ticket characteristics and theme frequency, and a Poisson regression analysis identified characteristics of potential participants who reported more frequent problems during enrollment.</p> <p>Results</p> <p>In total, 880 potential participants visited the study website and 80% completed the enrollment screening. Of the potential participants who visited the study website, 38% had help tickets logged in the participant management database. The total number of help tickets associated with individual potential participants ranged from 0 to 7 (M = .71). Overall, 46% of help tickets were initiated by email and 54% were initiated by phone. The most common help ticket theme was issues related to the study process (48%). The next most prominent theme was discussion related to obtaining medical clearance (34%), followed by issues related to pedometers and uploading (31%). Older individuals, women, and those with lower self-rated internet ability were more likely to report problems during the enrollment process.</p> <p>Conclusion</p> <p>Prospective participants in an online clinical trial encountered a number of barriers to enrollment that led them to request help from study staff. Questions about the complex enrollment process itself were common. In a complex multi-step enrollment process, providing personalized feedback to potential participants indicating their status within the enrollment process may be beneficial.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NCT00729040</p

Isolation and characterization of pBM400 : a 53 kb plasmid from Bacillus megaterium QM B1551

Includes bibliographical references (pages [77]-84)Bacillus megaterium QM B1551 has seven indigenous plasmids ranging from 5.5 kb to 165 kb. Our laboratory is studying the role of these plasmids in the cell. We have cloned, sequenced, and characterized five replicons. The fourth largest plasmid, pBM400, has been partially characterized with respect to replication and 18.6 kb sequenced. The sequenced region, designated rrn-clone II, harbors a ribosomal RNA operon (6.3 kb) and a replication clone (12.3 kb). An Xbal fragment of mi-clone II is unique to pBM400. To further characterize pBM400, the plasmid must be isolated into a plasmidless strain. Since the wild-type pBM400 does not contain a phenotypic marker, a kanamycin gene (from pDG792) was introduced into the plasmid by first inserting the Xbal fragment and a temperature-sensitive origin of replication (pE194ts) into the vector pJM103 (CmR). A kanamycin gene was then inserted into the Xbal fragment to generate pSMl, which was transformed into the wild-type strain (seven plasmids) selecting for kanamycin resistance. A transformant was grown at 46°C with kanamycin selection to select for homologous recombination into pBM400. A colony displaying KmR Cms suggested that a double crossover had occurred, thus integrating only the kanamycin gene into pBM400. The integration of the kanamycin gene was further verified by polymerase chain reaction (PCR) and Southern hybridization. The pBM400::kan was then transformed into PY361, a plasmidless strain of B. megaterium. The resultant transformant, PV627 (KmR), was shown to contain only pBM400::kan by agarose gel electrophoresis, PCR, and Southern hybridization analyses. To further analyze the isolated plasmid, restriction analysis of pBM400 was performed with various enzymes. A total size of approximately 53 kb without the kanamycin gene was found. Additionally, partial libraries of EcoRI and Pstl fragments of pBM400 in pJM103 were constructed. Subsequently, all ends of these fragments were sequenced and a restriction map was constructed. Sequence analysis revealed similarities to genes for copper/cadmium resistance, styrene degradation, transposase, integration/recombination, and also to unknown genes from the B. subtilis genome. PV627 was also tested for the presence of a second homologous replicon, as well as for megacin (bacteriocin) production and resistance, neither of which were found on pBM400.M.S. (Master of Science

Sequencing and Characterization of pBM400 from Bacillus megaterium QM B1551

Bacillus megaterium QM B1551 plasmid pBM400, one of seven indigenous plasmids, has been labeled with a selectable marker, isolated, completely sequenced, and partially characterized. A sequence of 53,903 bp was generated, revealing a total of 50 predicted open reading frames (ORFs); 33 were carried on one strand and 17 were carried on the other. These ORFs comprised 57% of the pBM400 sequence. Besides the replicon region and a complete rRNA operon that have previously been described, several interesting genes were found, including genes for predicted proteins for cell division (FtsZ and FtsK), DNA-RNA interaction (FtsK, Int/Rec, and reverse transcriptase), germination (CwlJ), styrene degradation (StyA), and heavy metal resistance (Cu-Cd export and ATPase). Three of the ORF products had high similarities to proteins from the Bacillus anthracis virulence plasmid pXO1. An insertion element with similarity to the IS256 family and several hypothetical proteins similar to those from the chromosomes of other Bacillus and Lactococcus species were present. This study provides a basis for isolation and sequencing of other high-molecular-weight plasmids from QM B1551 and for understanding the role of megaplasmids in gram-positive bacteria. The genes carried by pBM400 suggest a possible role of this plasmid in the survival of B. megaterium in hostile environments with heavy metals or styrene and also suggest that there has been an exchange of genes within the gram-positive bacteria, including pathogens

High-Throughput Screening of Small Molecule Libraries using SAMDI Mass Spectrometry

High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of “label-free” assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100 000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications