Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant ...“buffer” used, ammonium acetate (AmAc) with pK a values of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0–7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pH-sensitive interactions. Consequently, the discovery of volatile, ESI-compatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pK a values of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome c and myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein–ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.
Liquid–liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical ...basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length-dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.
•We surveyed birds across multiple-use landscapes of the Brazilian Amazon.•Production areas are of low importance for the conservation of Amazonian bird biodiversity.•Disturbed primary forests retain ...most bird species but lack some sensitive taxa.•Beta-diversity is modified by land-use, declining with increasing land-use intensity.
Habitat loss and degradation is the most pervasive threat to tropical biodiversity worldwide. Amazonia sits at the frontline of efforts to both improve the productivity of tropical agriculture and prevent the loss of biodiversity. To date our understanding of the biodiversity impacts of agricultural expansion in Amazonia is restricted to findings from small scale studies that typically assess the importance of a limited number of land-use types. Here we investigate local and landscape-scale responses of Amazonian avian assemblages to land-cover changes across a gradient of land-use intensity ranging from undisturbed primary forest to mechanised agriculture in 36 drainage catchments distributed across two large regions of the eastern Brazilian Amazon. We found that species richness of forest-associated birds declined progressively along this gradient, accompanied by marked shifts in assemblage composition. We found significant changes in species composition, but not richness, between primary forests that had been subject to different levels of disturbance from logging and fire. Secondary forests retained levels of species richness intermediate between primary forests and production areas, but lacked many forest-dependent species. Production areas (arable crops, cattle pastures and plantation forests) all retained far fewer species than any forest habitat, and were largely dominated by taxa commonly associated with open areas. Diversity partitioning revealed that species composition varied the most among undisturbed forest transects, and steadily decreased with increasing forest degradation and land-use intensity. Our results emphasise the importance of protecting both remaining areas of primary forest in private lands, as well as protecting the same forests from further disturbance events.
Background
Frailty is a customized marker of biological age that helps to gauge an individual’s functional physiologic reserve and ability to react to stress and is associated with increased ...postoperative morbidity and mortality. In order to mitigate frailty preoperatively, the concept of prehabilitation has entered the forefront which encompasses multidisciplinary interventions to improve health and lessen the incidence of postoperative decline. The purpose of this study is to investigate the impact of prehabilitation on postoperative outcomes in frail, surgical patients.
Methods
A comprehensive literature search was performed by two independent researchers according to PRISMA guidelines. Inclusion criteria were (1) a randomized controlled trial, case–control or observational study; (2) prehabilitation intervention; (3) frailty assessment; and (4) surgical intervention.
Results
There were five articles included in the review. Evaluation of these articles demonstrated prehabilitation may improve operative outcomes in frail surgical patients. There were no assessments as to whether prehabilitation was cost-effective although it was feasible. Prehabilitation programs should include elements of exercise, nutrition, and psychosocial counseling. Frailty should be assessed with a validated index in surgical patients who may undergo prehabilitation.
Conclusion
Prehabilitation in frail surgical patients may be the appropriate process through which providers can lessen operative risk. Currently, however, there is little evidence supporting the use of prehabilitation in this population with only five studies identified in this systematic review. More randomized controlled trials are clearly needed.
The Suzuki–Miyaura cross-coupling reaction is one of the most important reactions for pharmaceutical and fine chemical synthesis, performed using both homogeneous and heterogeneous catalysis. In this ...work, we cross-link poly(methylhydrosiloxane) (PMHS) with tri(ethylene glycol divinyl ether) to create a versatile and readily accessible gel catalyst support for Suzuki–Miyaura cross-coupling reactions in a pseudoheterogeneous manner. The Si–H units present on the PMHS backbone act dually as the cross-linking site and the reducing agent to anchor and reduce palladium(II) acetate to active palladium(0). The PMHS-supported Pd catalyst is then packed into a stainless-steel flow reactor to create a cartridgelike reactor for the continuous operation of a model Suzuki–Miyaura cross-coupling reaction. We systematically investigate the role of reaction temperature, catalyst loading, cross-linking density, and gel particle size on the transient and steady-state behavior of the cartridge flow reactor through an automated flow chemistry platform. The PMHS-supported catalytic particles demonstrate minimal deactivation and leaching over a continuous (80 h) Suzuki–Miyaura cross-coupling reaction at a 30 min nominal residence time at a relatively high reaction temperature of 95 °C. The developed modular flow chemistry strategy equipped with the cartridge flow reactor enables accelerated studies of the fundamental and applied characteristics of gel-supported catalysts while providing increased safety, higher throughput, and removal of the separation step needed for catalyst recovery compared to homogeneous cross-coupling reactions in batch reactors.
BACKGROUND:Previous research has identified a number of patient and operative factors associated with anastomotic leak after colectomy; however, a study that examines these factors on a national ...level with direct coding for anastomotic leak is lacking.
OBJECTIVE:The purpose of this work was to identify risk factors associated with anastomotic leak on a national level and quantify the additional morbidity and mortality experienced by these patients.
DESIGN:We performed a retrospective analysis of patients who underwent segmental colectomy with anastomosis from the 2012 American College of Surgeons National Surgical Quality Improvement Program colectomy procedure-targeted database. Anastomotic leak was defined as minor leak requiring percutaneous intervention or major leak requiring laparotomy. Multivariate logistic regression was used to determine predictors of anastomotic leak and its impact on postoperative outcomes.
SETTINGS:This study was conducted at a tertiary university department.
PATIENTS:This study includes 13,684 patients who underwent segmental colectomy with anastomosis at American College of Surgeons National Surgical Quality Improvement Program–affiliated hospitals in 2012.
MAIN OUTCOME MEASURES:The primary outcome studied was anastomotic leak.
RESULTS:The overall leak rate was 3.8%. Male sex, steroid use, smoking, open approach, operative time, and preoperative chemotherapy were associated with increased anastomotic leaks and diverting ileostomy with decreased incidence of leaks on multivariate analysis. Increased length of stay (13 vs 5 days; p < 0.001) and increased 30-day mortality (6.8% vs 1.6%; p < 0.001) were also seen in patients who experienced leaks. These patients also experienced increased readmission rates (43.5% vs 8.3%; p < 0.001) and were 37 times more likely to require reoperation as a complication of their primary procedure (p < 0.001).
LIMITATIONS:The main limitations of this study include its retrospective nature and the limited 30-day outcomes recorded in the American College of Surgeons National Surgical Quality Improvement Program database.
CONCLUSIONS:This study identified patient and operative risk factors for anastomotic leak on a national scale. It also demonstrates that these patients have increased morbidity and 30-day mortality rates, experience multiple readmissions to the hospital, and have a higher likelihood of requiring further operative intervention.
Activation of the AMP-Activated Kinase by Antidiabetes Drug Metformin Stimulates Nitric Oxide Synthesis In Vivo by Promoting
the Association of Heat Shock Protein 90 and Endothelial Nitric Oxide ...Synthase
Bradley J. Davis 1 ,
Zhonglin Xie 1 2 ,
Benoit Viollet 3 and
Ming-Hui Zou 1 2
1 Vascular Research Laboratory, Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, Tennessee
2 Division of Endocrinology, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma
3 Department of Genetics, Development and Molecular Pathology, Cochin Institute, University of Paris V, National Institute of
Health and Medical Research U567, National Center of Scientific Research UMR8104, Paris, France
Address correspondence and reprint requests to Ming-Hui Zou, MD, PhD, Medicine Endocrinology, BSEB 325, Department of Medicine,
University of Oklahoma Health Science Center, 941 Stanton L. Young Blvd., Oklahoma City, OK 73104. E-mail: ming-hui-zou{at}ouhsc.edu
Abstract
Metformin, one of most commonly used drugs for the treatment of type 2 diabetes, improves vascular endothelial functions and
reduces cardiovascular events in patients with type 2 diabetes, although its mechanisms remain unknown. The current study
aimed to elucidate how metformin improves endothelial functions. Exposure of cultured bovine aortic endothelial cells (BAECs)
to clinically relevant concentrations of metformin (50–500 μmol/l) dose-dependently increased serine-1179 (Ser1179) phosphorylation
(equal to human Ser1179) of endothelial nitric oxide (NO) synthase (eNOS) as well as its association with heat shock protein
(hsp)-90, resulting in increased activation of eNOS and NO bioactivity (cyclic GMP). These effects of metformin were mimicked
or completely abrogated by adenoviral overexpression of a constitutively active 5′-AMP–activated kinase (AMPK) mutant or a
kinase-inactive AMPK-α, respectively. Furthermore, administration of metformin as well as 5-aminoimidazole-4-carboxamide ribonucleoside,
an AMPK agonist, significantly increased eNOS Ser1179 phosphorylation, NO bioactivity, and coimmunoprecipitation of eNOS with
hsp90 in wild-type C57BL6 mice but not in AMPK-α1 knockout mice, suggesting that AMPK is required for metformin-enhanced eNOS
activation in vivo. Finally, incubation of BAECs with clinically relevant concentrations of metformin dramatically attenuated
high-glucose (30 mmol/l)–induced reduction in the association of hsp90 with eNOS, which resulted in increased NO bioactivity
with a reduction in overexpression of adhesion molecules and endothelial apoptosis caused by high-glucose exposure. Taken
together, our results indicate that metformin might improve vascular endothelial functions in diabetes by increasing AMPK-dependent,
hsp90-mediated eNOS activation.
AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside
AMPK, 5′-AMP–activated kinase
AMPK-CA, constitutively active AMPK kinase
BAEC, bovine aortic endothelial cell
cGMP, cyclic GMP
eNOS, endothelial nitric oxide synthase
FBS, fetal bovine serum
FITC, fluorescein isothiocyanate
GFP, green fluorescence protein
hsp, heat shock protein
ICAM, intracellular adhesion molecule
l-NAME, l-nitro-arginine methyl ester
3-NT, 3-nitrotyrosine
Ser1179, serine-1179
SOD, superoxide dismutase
Thr172, threonine-172
PDK, phosphoinositide-dependent kinase
VCAM, vascular cell adhesion molecule
Footnotes
Accepted October 31, 2005.
Received August 17, 2005.
DIABETES
Subcellular compartmentalization of biomolecules and their reactions is common in biology and provides a general strategy for improving and/or controlling kinetics in metabolic pathways that contain ...multiple sequential enzymes. Enzymes can be colocalized in multiprotein complexes, on scaffolds or inside subcellular organelles. Liquid organelles formed by intracellular phase coexistence could provide an additional means of sequential enzyme colocalization. Here we use experiment and computation to explore the kinetic consequences of sequential enzyme compartmentalization into model liquid organelles in a crowded polymer solution. Two proteins of the de novo purine biosynthesis pathway, ASL (adenylosuccinate lyase, Step 8) and ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase, Steps 9 and 10), were studied in a polyethylene glycol/dextran aqueous two-phase system. Dextran-rich phase droplets served as model liquid compartments for enzyme colocalization. In this system, which lacks any specific binding interactions between the phase-forming polymers and the enzymes, we did not observe significant rate enhancements from colocalization for the overall reaction under our experimental conditions. The experimental results were used to adapt a mathematical model to quantitatively describe the kinetics. The mathematical model was then used to explore additional, experimentally inaccessible conditions to predict when increased local concentrations of enzymes and substrates can (or cannot) be expected to yield increased rates of product formation. Our findings indicate that colocalization within these simplified model liquid organelles can lead to enhanced metabolic rates under some conditions, but that very strong partitioning into the phase that serves as the compartment is necessary. In vivo, this could be provided by specific binding affinities between components of the liquid compartment and the molecules to be localized within it.
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