Cost containment: an experience with surgeon education and universal preference cards at two institutions.

BACKGROUND: As the cost of health care increases in the US, focus has been placed upon efficiency, cost reduction, and containment of spending. Operating room costs play a significant role in this spending. We investigated whether surgeon education and universal preference cards can have an impact on reducing the disposable supply costs for common laparoscopic general surgery procedures. METHODS: General surgeons at two institutions participated in an educational session about the costs of the operative supplies used to perform laparoscopic appendectomies and cholecystectomies. All the surgeons at one institution agreed upon a universal preference card, with other supplies opened only by request. At the other, no universal preference cards were created, and surgeons were free to modify their own existing preference cards. Case cost data for these procedures were collected for each institution pre- (July 2014-December 2014) and post-intervention (February 2015-November 2017). RESULTS: At the institution with an education only program, there was no statistically significant change in supply costs after the intervention. At the institution that intervened with the combined education and universal preference card program, there was a statistically significant supply cost decrease for these common laparoscopic procedures combined. This significant cost decrease persisted for each appendectomies and cholecystectomies when analyzed independently as well (p = 0.001 and p < 0.001 respectively). CONCLUSIONS: In this study, surgeon education alone was not effective in reducing operating room disposable supply costs. Surgeon education, combined with the implementation of universal preference cards, significantly maintains reductions in operating room supply costs. As health care costs continue to increase in the US and internationally, universal preference cards can be an effective tool to contain cost for common laparoscopic general surgery procedures.

Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.

The role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics.

HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR (15)N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.

AMP-activated protein kinase ß-subunit requires internal motion for optimal carbohydrate binding.

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the ß-subunit, for which there are two isoforms (ß(1) and ß(2)). Muscle-specific ß(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous ß(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of ß(2)-CBM is concurrent with an increase in flexibility in ß(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to ß(1)-CBM, unbound ß(2)-CBM showed microsecond-to-millisecond motion at the base of a ß-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this ß-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from ß(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the ß(2)-CBM mutant. Insertion of a threonine into the background of ß(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.

Automated NMR relaxation dispersion data analysis using NESSY.

BACKGROUND: Proteins are dynamic molecules with motions ranging from picoseconds to longer than seconds. Many protein functions, however, appear to occur on the micro to millisecond timescale and therefore there has been intense research of the importance of these motions in catalysis and molecular interactions. Nuclear Magnetic Resonance (NMR) relaxation dispersion experiments are used to measure motion of discrete nuclei within the micro to millisecond timescale. Information about conformational/chemical exchange, populations of exchanging states and chemical shift differences are extracted from these experiments. To ensure these parameters are correctly extracted, accurate and careful analysis of these experiments is necessary. RESULTS: The software introduced in this article is designed for the automatic analysis of relaxation dispersion data and the extraction of the parameters mentioned above. It is written in Python for multi platform use and highest performance. Experimental data can be fitted to different models using the Levenberg-Marquardt minimization algorithm and different statistical tests can be used to select the best model. To demonstrate the functionality of this program, synthetic data as well as NMR data were analyzed. Analysis of these data including the generation of plots and color coded structures can be performed with minimal user intervention and using standard procedures that are included in the program. CONCLUSIONS: NESSY is easy to use open source software to analyze NMR relaxation data. The robustness and standard procedures are demonstrated in this article.

A dynamic pharmacophore drives the interaction between Psalmotoxin-1 and the putative drug target acid-sensing ion channel 1a.

Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a-ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a ß-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.

relaxGUI: a new software for fast and simple NMR relaxation data analysis and calculation of ps-ns and µs motion of proteins.

Investigation of protein dynamics on the ps-ns and µs-ms timeframes provides detailed insight into the mechanisms of enzymes and the binding properties of proteins. Nuclear magnetic resonance (NMR) is an excellent tool for studying protein dynamics at atomic resolution. Analysis of relaxation data using model-free analysis can be a tedious and time consuming process, which requires good knowledge of scripting procedures. The software relaxGUI was developed for fast and simple model-free analysis and is fully integrated into the software package relax. It is written in Python and uses wxPython to build the graphical user interface (GUI) for maximum performance and multi-platform use. This software allows the analysis of NMR relaxation data with ease and the generation of publication quality graphs as well as color coded images of molecular structures. The interface is designed for simple data analysis and management. The software was tested and validated against the command line version of relax.

Macromolecular NMR spectroscopy for the non-spectroscopist: beyond macromolecular solution structure determination.

A strength of NMR spectroscopy is its ability to monitor, on an atomic level, molecular changes and interactions. In this review, which is intended for non-spectroscopist, we describe major uses of NMR in protein science beyond solution structure determination. After first touching on how NMR can be used to quickly determine whether a mutation induces structural perturbations in a protein, we describe the unparalleled ability of NMR to monitor binding interactions over a wide range of affinities, molecular masses and solution conditions. We discuss the use of NMR to measure the dynamics of proteins at the atomic level and over a wide range of timescales. Finally, we outline new and expanding areas such as macromolecular structure determination in multicomponent systems, as well as in the solid state and in vivo.

AMPK beta subunits display isoform specific affinities for carbohydrates.

AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

Sirolimus and everolimus reduce albumin endocytosis in proximal tubule cells via an angiotensin II-dependent pathway.

BACKGROUND: The proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) often induce proteinuria due to glomerular but also tubular dysfunction in transplant patients. The beneficial effect of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II (Ang II) type 1 receptor blockers (ARB) has been reported. AIM: This study aimed to investigate: (i) the role of an Ang II-dependent mechanism and Ang II type 1 receptor (AT(1)R) in the regulation of receptor-mediated albumin endocytosis on proximal tubular epithelial cells (PTEC) following PSI treatment; (ii) the specific roles of the albumin receptors cubilin and megalin in albumin binding and uptake in PTEC. METHODS: A human renal PTEC line (HK-2) was used in the study. The binding of Alexa 488 conjugated albumin was measured by flow cytometry. Albumin uptake and the expression of cubilin and megalin were determined by cellular ELISA. RESULTS: The administration of PSIs resulted in decreased albumin binding and uptake and downregulation of cubilin and megalin expression in PTEC. These effects were significantly reversed by the administration of an ACE-I (ramipril) or an ARB (losartan). Combined use of ramipril and losartan demonstrated additive effects on cubilin expression, but not on megalin expression or albumin binding and uptake. CONCLUSIONS: Our findings suggest that decreased albumin endocytosis in PTECs following PSI treatment involve an Ang II-dependent pathway via AT(1)R. The albumin receptor megalin may play a more crucial role than cubulin, since its expression correlated directly with albumin uptake.

Recognition and detoxification of the insecticide DDT by Drosophila melanogaster glutathione S-transferase D1.

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 A resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional (1)H,(15)N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.

Anti-HLA I antibodies induce VEGF production by endothelial cells, which increases proliferation and paracellular permeability.

Anti-human leukocyte antigen class I (HLA I) antibodies were shown to activate several protein kinases in endothelial cells (ECs), which induces proliferation and cell survival. An important phenomenon in antibody-mediated rejection is the occurrence of interstitial edema. We investigated the effect of anti-HLA I antibodies on endothelial proliferation and permeability, as one possible underlying mechanism of edema formation. HLA I antibodies increased the permeability of cultured ECs isolated from umbilical veins. Anti-HLA I antibodies induced the production of vascular endothelial growth factor (VEGF) by ECs, which activated VEGF receptor 2 (VEGFR2) in an autocrine manner. Activated VEGFR2 led to a c-Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and its degradation. Aberrant VE-cadherin expression resulted in impaired adherens junctions, which might lead to increased endothelial permeability. This effect was only observed after cross-linking of HLA I molecules by intact antibodies. Furthermore, our results suggest that increased endothelial proliferation following anti-HLA I treatment occurs via autocrine VEGFR2 activation. Our data indicate the ability of anti-HLA I to induce VEGF production in ECs. Transactivation of VEGFR2 leads to increased EC proliferation and paracellular permeability. The autocrine effect of VEGF on endothelial permeability might be an explanation for the formation of interstitial edema after transplantation.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway.

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

Proliferation signal inhibitor-induced decrease of vascular endothelial cadherin expression and increase of endothelial permeability in vitro are prevented by an anti-oxidant.

BACKGROUND: Rapamycines, sirolimus (SRL) and everolimus (ERL), are proliferation signal inhibitors (PSIs). PSI therapy often leads to edema. We hypothesized that increased oxidative stress in response to PSIs may modulate the expression of vascular endothelial (VE)-cadherin on endothelial cells (ECs) and, subsequently, vascular permeability, which in turn may be involved in the development of edema. METHODS: Experiments were performed on human umbilical vein ECs (HUVECs). Oxidative stress was measured by dichlorofluorescein-diacetate. Expression of VE-cadherin was evaluated by immunofluorescent staining and western blot analysis. Endothelial "permeability" was assessed using a transwell model. RESULTS: SRL and ERL, at concentrations of 1, 10 and 100 nmol/liter, enhanced oxidative stress (SRL: 24 +/- 12%, 29 +/- 9%, 41 +/- 13% [p < 0.05, in all three cases]; ERL: 13 +/- 10%, 27 +/- 2%, 40 +/- 12% [p < 0.05, in the latter two cases], respectively) on HUVECs, which was inhibited by the anti-oxidant, N-acetyl-cysteine (NAC) and, to a lesser extent, by the specific inhibitor of nitric oxide synthase, N-Omega-nitro-L-arginine methylester. By the use of NAC, VE-cadherin expression remained comparable with control, according to both immunocytochemistry and western blot analysis. Permeability was significantly increased by SRL and ERL at 100 nmol/liter (29.5 +/- 6.4% and 33.8 +/- 4.2%, respectively); however, co-treatment with NAC abrogated the increased permeability. CONCLUSIONS: EC homeostasis, as indicated by VE-cadherin expression, may be damaged by SRL and ERL, but resolved by the anti-oxidant NAC.