Index-Based Pricing Models in Strategic Freight Procurement

This extended abstract addresses index-based pricing models between shippers and carriers in strategic freight procurement. The increasingly unpredictable transportation cost developments, caused by a variety of different international crises, led to the need for shippers and carriers to find an equitable form of collaboration. Index-based pricing models are one way to regulate the price adjustment between shipper and carrier during the contract period due to cost developments. However, one obstacle to agreeing on such models is to find the correct index for the respective cost component that is adequate for both shipper and carrier and reflects the actual cost development of the carrier. In semi-structured expert interviews, we investigated which strategies are used to reflect volatile cost developments, how index-based pricing models work, and where problems are seen in this context. Furthermore, a broader study can lead to confirmation or further classification of the results. The abstract describes index-based pricing models with focus on road freight, but the mechanism will work for other transportation modes as well

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases

AbstractBackground: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases.Results: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 Å resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open α/β-protein class and can be described as a four-layered α/α/β/α type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven α/β repeats resulting in an all parallel central β sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed β sheet. Inboth subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices.Conclusions: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 Å with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor

Native mass spectrometry can effectively predict PROTAC efficacy

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC

City of Rosemount: Stormwater Management Plan

Report and presentation completed by students enrolled in PA 5242: Environmental Planning, Policy, and Decision Making, taught by Carissa Schively Slotterback in spring 2015.This project was completed as part of a year-long partnership between the City of Rosemount and the University of Minnesota’s Resilient Communities Project (http://www.rcp.umn.edu). Storm water management is becoming a hot issue in urban planning and design fields. With climate change creating more intense and more frequent storm events, and as urban development continues, increased runoff jeopardizes the water quality of Minnesota’s lakes and rivers and raises the risk of severe localized flooding. The goal of this project was to identify opportunities or design strategies for storm water infiltration in the City of Rosemount that balance aesthetics and functionality, reduce long-term maintenance costs, manage winter snow melt, and provide opportunities for other passive uses. In collaboration with city project lead Andy Brotzler, Public Works Director for the City of Rosemount, a team of students in PA 5242: Environmental Planning, Policy, and Decision Making researched both traditional and new best management practices and provided recommendations for the City of Rosemount regarding which practices it should implement, in addition to strategies for community involvement. A final report and presentation from the project are available.This project was supported by the Resilient Communities Project (RCP), a program at the University of Minnesota that convenes the wide-ranging expertise of U of M faculty and students to address strategic local projects that advance community resilience and sustainability. RCP is a program of the Center for Urban and Regional Affairs (CURA). More information at http://www.rcp.umn.ed

Structural basis for the disaggregase activity and regulation of Hsp104

The Hsp104 disaggregase is a two-ring ATPase machine that rescues various forms of non-native proteins including the highly resistant amyloid fibers. The structural-mechanistic underpinnings of how the recovery of toxic protein aggregates is promoted and how this potent unfolding activity is prevented from doing collateral damage to cellular proteins are not well understood. Here, we present structural and biochemical data revealing the organization of Hsp104 from Chaetomium thermophilum at 3.7 angstrom resolution. We show that the coiled-coil domains encircling the disaggregase constitute a 'restraint mask' that sterically controls the mobility and thus the unfolding activity of the ATPase modules. In addition, we identify a mechanical linkage that coordinates the activity of the two ATPase rings and accounts for the high unfolding potential of Hsp104. Based on these findings, we propose a general model for how Hsp104 and related chaperones operate and are kept under control until recruited to appropriate substrates

Activation by sub-stoichiometric inhibition

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities