STRUCTURAL BASIS OF SUBSTRATE RECOGNITION IN THIMET OLIGOPEPTIDASE AND DEVELOPMENT OF NANOPARTICLES FOR THERAPEUTIC ENZYME DELIVERY

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes is how they are able to recognize a variety of seemingly unrelated amino acid sequences as cleavage sites. We are investigating the basis for this general substrate recognition in neuropeptidases using thimet oligopeptidase (TOP) as a model. Crystal structures of TOP in complex with a variety of substrates and inhibitors shed light on the mechanisms underlying substrate recognition and pave the way for re-targeting substrate recognition in these enzymes. Nano test tube particles have been proposed as a means of delivering therapeutics such as enzymes. However, the template synthesis method for nano test tube production does not produce therapeutic quantities. In order to take full advantage of re-engineered neuropeptidases a new method for nano test tube synthesis has been developed. We show that a non-destructive template synthesis methodology can be applied to produce nano test tube particles in quantities useful for therapeutic enzyme immobilization

Structures of EccB\u3csub\u3e1\u3c/sub\u3e and EccD\u3csub\u3e1\u3c/sub\u3e from the Core Complex of the Mycobacterial ESX-1 Type VII Secretion System

Background: The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. Results: This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB1 and EccD1. The periplasmic domain of EccB1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD1has a ubiquitin-like fold and forms a dimer with a negatively charged groove. Conclusions: These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies

Recovery of excreted n-butanol from genetically engineered cyanobacteria cultures: Process modelling to quantify energy and economic costs of different separation technologies

The photoautotrophic production of excreted biofuels from genetically engineered cyanobacteria and microalgae represents a new and promising alternative to conventional algal fuel technologies. N-butanol is a particularly promising fuel product, as it can be directly used in petroleum engines, and has been successfully expressed in species of Synechococcus elongates 7942 and Synechocystis sp. PCC 6803. However, the high energy requirements of recovering butanol from dilute mixtures can easily outweigh the energy content of the fuel and must be carefully assessed and optimized. Consequently, the recovery of butanol was modelled using four of the most promising butanol separation technologies (distillation, gas stripping, pervaporation and ionic liquid extraction) to calculate the minimum butanol culture concentrations required to render the process energy-positive. With a breakeven concentration of only 3.7 g L -1 , ionic liquid extraction proved much more efficient than the distillation base-case scenario (9.3 g L -1 ), whilst neither pervaporation (10.3 g L -1 ) nor gas stripping (16.9 g L -1 ) could compete on an energy basis with distillation. Despite this, due to the high costs of the ionic liquid solvent, the lowest capital costs are obtained for distillation (pilot plant scale, butanol culture concentrations of 10 g L -1 ), whilst pervaporation carries the lowest utility costs, as a result of its low electrical energy demand. Although currently achieved maximum n-butanol culture concentrations are significantly below the calculated break-even values for all four technologies, the present work provides an important threshold for future strain development. Moreover, the recovery of side-products from purged biomass could help to reduce the costs associated with biofuel production

Processing EOS MLS Level-2 Data

A computer program performs level-2 processing of thermal-microwave-radiance data from observations of the limb of the Earth by the Earth Observing System (EOS) Microwave Limb Sounder (MLS). The purpose of the processing is to estimate the composition and temperature of the atmosphere versus altitude from .8 to .90 km. "Level-2" as used here is a specialists f term signifying both vertical profiles of geophysical parameters along the measurement track of the instrument and processing performed by this or other software to generate such profiles. Designed to be flexible, the program is controlled via a configuration file that defines all aspects of processing, including contents of state and measurement vectors, configurations of forward models, measurement and calibration data to be read, and the manner of inverting the models to obtain the desired estimates. The program can operate in a parallel form in which one instance of the program acts a master, coordinating the work of multiple slave instances on a cluster of computers, each slave operating on a portion of the data. Optionally, the configuration file can be made to instruct the software to produce files of simulated radiances based on state vectors formed from sets of geophysical data-product files taken as input

Dose-escalation study of a second-generation non-ansamycin HSP90 inhibitor, onalespib (AT13387), in combination with imatinib in patients with metastatic gastrointestinal stromal tumour

AbstractBackgroundGastrointestinal stromal tumours (GIST) treated with the tyrosine kinase inhibitor (TKI) imatinib can become resistant when additional mutations in the receptor tyrosine kinases KIT or PDGFRA block imatinib activity. Mutated KIT requires the molecular chaperone heat-shock protein 90 (HSP90) to maintain stability and activity. Onalespib (AT13387) is a potent non-ansamycin HSP90 inhibitor. We hypothesised that the combination of onalespib and imatinib may be safe and effective in managing TKI-resistant GIST.Patients and methodsIn this dose-escalation study, we evaluated the safety and efficacy of combination once-weekly intravenous onalespib for 3 weeks and daily oral imatinib in 28-d cycles. Twenty-six patients with TKI-resistant GIST were enrolled into four sequential dose cohorts of onalespib (dose range, 150–220 mg/m2) and imatinib 400 mg. The relationship between tumour mutational status (KIT/PDGFRA) and efficacy of treatment was explored.ResultsCommon onalespib-related adverse events were diarrhoea (58%), nausea (50%), injection site events (46%), vomiting (39%), fatigue (27%), and muscle spasms (23%). Overall, 81% of patients reported more than one onalespib-related gastrointestinal disorder. Nine patients (35%) had a best response of stable disease, including two patients who had KIT mutations known to be associated with resistance to imatinib and sunitinib. Disease control at 4 months was achieved in five patients (19%), and median progression-free survival was 112 d (95% confidence interval 43–165). One patient with PDGFRA-mutant GIST had a partial response for more than 376 d.ConclusionThe combination of onalespib plus imatinib was well tolerated but exhibited limited antitumour activity as dosed in this TKI-resistant GIST patient population.Trial registration ID: clinicaltrials.gov: NCT0129420

Detection of intrinsic source structure at ~3 Schwarzschild radii with Millimeter-VLBI observations of SAGITTARIUS A*

We report results from very long baseline interferometric (VLBI) observations of the supermassive black hole in the Galactic center, Sgr A*, at 1.3 mm (230 GHz). The observations were performed in 2013 March using six VLBI stations in Hawaii, California, Arizona, and Chile. Compared to earlier observations, the addition of the APEX telescope in Chile almost doubles the longest baseline length in the array, provides additional {\it uv} coverage in the N-S direction, and leads to a spatial resolution of $\sim$30 $\mu$as ($\sim$3 Schwarzschild radii) for Sgr A*. The source is detected even at the longest baselines with visibility amplitudes of $\sim$4-13% of the total flux density. We argue that such flux densities cannot result from interstellar refractive scattering alone, but indicate the presence of compact intrinsic source structure on scales of $\sim$3 Schwarzschild radii. The measured nonzero closure phases rule out point-symmetric emission. We discuss our results in the context of simple geometric models that capture the basic characteristics and brightness distributions of disk- and jet-dominated models and show that both can reproduce the observed data. Common to these models are the brightness asymmetry, the orientation, and characteristic sizes, which are comparable to the expected size of the black hole shadow. Future 1.3 mm VLBI observations with an expanded array and better sensitivity will allow a more detailed imaging of the horizon-scale structure and bear the potential for a deep insight into the physical processes at the black hole boundary.Comment: 11 pages, 5 figures, accepted to Ap

Devotions for Advent 2022 Canticles of Luke

Each week of this Advent devotional will focus on one of the four Lukan canticles, putting it in its context as well as making connections to other portions of Scripture. At the end of this Advent season, may we, like those who have gone before us, “sing to the Lord, bless his name; tell of his salvation from day to day” (Ps. 96:2). Many thanks to all the CSL and CTSFW students who contributed devotional reflections. A special note of thanks to my counterpart, Zachary Roll, who organized this effort at Concordia Seminary, St. Louis who has been a joy to work with and to get to know. A final note of thanks to Kim Hosier in the print shop and Rev. Dr. Paul Grime for their aid in completing this devotional booklet.https://scholar.csl.edu/osp/1021/thumbnail.jp