Surgical specialty and outcomes for carotid endarterectomy: evidence from the National Surgical Quality Improvement Program

Background

Carotid endarterectomy (CEA) has been performed since the 1950s and remains one of the most common surgical procedures in the United States. The procedure is performed by cardiothoracic, general, neurologic, and vascular surgeons. This study uses data from the National Surgical Quality Improvement Program (NSQIP) to examine the outcomes after CEA when performed by general or vascular surgeons.

Materials and methods

Data included 34,493 CEAs from years 2005 to 2010 recorded in the NSQIP database. Primary outcomes measured were length of stay, 30-d mortality, surgical site infection, cerebrovascular accident, myocardial infarction, and blood transfusion requirement. Secondary outcomes measured were the remaining intraoperative outcomes from the NSQIP database.

Results

After controlling for patient and surgical characteristics, patients treated by general surgeons did not have a significantly different LOS or 30-d mortality than those treated by vascular surgeons. Patients of general surgeons had nearly twice the risk of acquiring a surgical site infection (odds ratio [OR] = 1.94; P = 0.012), >1.5 times the risk of cerebrovascular accident (OR = 1.56; P = 0.008), and >1.8 times the risk of blood transfusion (OR = 1.85; P = 0.017) than those of vascular surgeons. Patients of general surgeons had less than half the risk of having a myocardial infarction (OR = 0.34; P = 0.031) than those of vascular surgeons.

Conclusions

Surgical specialty is associated with a wide range of postoperative outcomes after CEA. Additional research is needed to explore practice and cultural differences across surgical specialty that may lead to outcome differences.     

Patients with paroxysmal nocturnal hemoglobinuria demonstrate a prothrombotic clotting phenotype which is improved by complement inhibition with eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder, characterized by complement-mediated intravascular hemolysis and thrombosis. The increased incidence of PNH-driven thrombosis is still poorly understood, but unlike other thrombotic disorders, is thought to largely occur through complement-mediated mechanisms. Treatment with a C5 inhibitor, eculizumab, has been shown to significantly reduce the number of thromboembolic events in these patients. Based on previously described links between changes in fibrin clot structure and thrombosis in other disorders, our aim was to investigate clot structure as a possible mechanism of thrombosis in patients with PNH and the anti-thrombotic effects of eculizumab treatment on clot structure. Clot structure, fibrinogen levels and thrombin generation were examined in plasma samples from 82 patients from the National PNH Service in Leeds, UK. Untreated PNH patients were found to have increased levels of fibrinogen and thrombin generation, with subsequent prothrombotic changes in clot structure. No link was found between increasing disease severity and fibrinogen levels, thrombin generation, clot formation or structure. However, eculizumab treated patients showed decreased fibrinogen levels, thrombin generation and clot density, with increasing time spent on treatment augmenting these antithrombotic effects. These data suggest that PNH patients have a prothrombotic clot phenotype due to increased fibrinogen levels and thrombin generation, and that the antithrombotic effects of eculizumab are, in-part, due to reductions in fibrinogen and thrombin generation with downstream effects on clot structure.     

Deuterium equilibrium isotope effects in a supramolecular receptor for the hydrochalcogenide and halide anions

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in an arylethynyl bisurea supramolecular anion receptor and use the Perrin method of competitive titrations to study the deuterium equilibrium isotope effects (DEIE) of anion binding for HS⁻, Cl⁻, and Br⁻. This work highlights the utility and also challenges in using this method to determine EIE with highly reactive and/or weakly binding anions.

We highlight a convenient synthesis to selectively deuterate an aryl C–H hydrogen bond donor in a supramolecular anion receptor and use competitive titrations to study the deuterium equilibrium isotope effects (DEIE) in binding HS⁻, Cl⁻, and Br⁻.

Molecular recognition and host–guest binding in both biological and synthetic systems are often driven by a mixture of competitive and additive primarily non-covalent interactions. Understanding the role of each of these forces in a host–guest system can reveal insights into the driving forces behind binding and help inform on the molecular design of future hosts.^1–3 Equilibrium isotope effects (EIE), also referred to as binding isotope effects (BIE) in structural molecular biology,⁴ measure the effect of isotopic substitution on supramolecular interactions through changes in the vibrational energy of the substituted bond. These studies can be used to elucidate the complex non-covalent forces involved in host conformational changes and host–guest binding.^5–8Examples from structural molecular biology have demonstrated that EIEs can reveal mechanistic information in enzyme–ligand binding events.^4,9 Isotopic substitution in synthetic supramolecular systems has been used both for labelling purposes and for studying individual non-covalent interactions. For example, Bergman, Raymond, and coworkers used deuterium equilibrium isotope effects (DEIE) to study benzylphosphonium cation guest binding in a self-assembled supramolecular complex in aqueous solution.¹⁰ From these DEIE studies, the authors found that attractive cation⋯π interactions in the interior of the host were important for promoting guest binding, and that C–H⋯π and π⋯π interactions were relatively small contributors. In another example, Shimizu and coworkers studied the DEIE on the strength of C–H⋯π interactions in their molecular balances.¹¹ Both computational and experimental results showed that the strength of C–H⋯π and C–D⋯π interactions were about equal, settling the debate on which interaction is stronger and easing concerns about using deuteration for spectroscopic and labelling applications.Previously, we used DEIE to study Cl⁻ binding with the arylethynyl bisurea anion receptor 1^H/D (Fig. 1) in DMSO-d₆.¹² We found an experimental DEIE of 1.019 ± 0.010, which matched the computationally-predicted DEIE of 1.020. Further computational analysis determined that the DEIE was due to a distorted N–H⋯Cl⁻ hydrogen bond geometry, which resulted in changes in the C–H/D bond vibrational energy in the host–guest complex. In addition, Paneth and coworkers performed a computational study with 1^H and other hydrogen bonding supramolecular Cl⁻ receptors to determine the EIE of ^35/37Cl⁻ binding in these hosts.¹³ Because isotope effects, both equilibrium and kinetic, originate solely from changes in the vibrational energy of the isotopically labelled bond, the EIE arising from this study came from changes in the vibrational energies of the bonds in the supramolecular hosts when participating in hydrogen bonding with Cl⁻ isotopes. Indeed, a linear relationship was observed between the hydrogen bond donor (D) D–H bond lengths in the host–guest complex and the computed ^35/37Cl EIE.Open in a separate windowFig. 1Arylethynyl bisurea receptors 1^H and 1^D used in our previous DEIE study of Cl⁻ binding. Related receptors 2^H and 2^D are used in this study to avoid reaction of the nitro group with HS⁻.Previous EIE studies with receptor 1^H/D have focused on Cl⁻ binding; however, to the best of our knowledge, no work has yet investigated the EIE of hydrosulfide (HS⁻) binding in this or other systems. HS⁻ is a highly reactive anion that plays crucial roles in biology. At physiological pH, HS⁻ is favored in solution by a 3 : 1 ratio over its conjugate acid, hydrogen sulfide (H₂S). H₂S has been identified as the third physiological gasotransmitter alongside CO and NO and plays essential roles in physiological systems.¹⁵ Despite its high nucleophilicity and redox activity, HS⁻ has been observed to be bound through non-covalent interactions in the protein crystal structure of a bacterial ion channel¹⁶ and in the turn-over state of vanadium-containing nitrogenase.¹⁷ The supramolecular chemistry of HS⁻ is under-studied in synthetic supramolecular receptors, likely due to the inherent high reactivity of HS⁻. Indeed, we are aware of only three families of receptors that have been shown to reversibly bind HS⁻.^18–21Recently, we used a series of arylethynyl bisurea anion receptors to investigate and demonstrate a linear free energy relationship between the polarity of a non-traditional C–H hydrogen bond donor and the solution binding energy of HS⁻, HSe⁻, Cl⁻ and Br⁻.¹⁴ A major and unexpected finding of this study was that HS⁻ demonstrated a significant increase in sensitivity towards the polarity of the C–H hydrogen donor over HSe⁻, Cl⁻ and Br⁻. Although increasing the polarity of the C–H hydrogen bond donor did not lead to changes in selectivity between Cl⁻, Br⁻, and HSe⁻, we observed a 9-fold increase in selectivity for HS⁻ over Cl⁻, suggesting a fresh approach to selective HS⁻ recognition using non-covalent interactions. In this current study, we label the C–H hydrogen bond donor in an arylethynyl bisurea receptor with a deuterium atom (2^H/D, Fig. 1) to further investigate this apparent preference of polar C–H H-bond donors for HS⁻ over Cl⁻ and Br⁻ through DEIE.Receptor 2^H is a previously reported anion receptor for HS⁻, Cl⁻, and Br⁻ and was prepared by established methods.¹⁴ Deuterium labelling of the isotopologue 2^D was achieved by selective monodeuteration of intermediates through methods similar to those reported in the literature (Scheme 1).²² The diazonium salt 3 was synthesized in a 71% yield from 2,6-diiodo-4-trifluoromethylaniline.²³ Dediazonation in DMF-d₇ is catalyzed by FeSO₄ and allows for selective synthesis of monodeuterated intermediate 4. The deuteration step proceeds through a radical pathway that uses DMF-d₇ as the deuterium source. This deuteration reaction provides efficient deuterium incorporation even with up to 50% by volume H₂O in the reaction solution due to the differential bond strengths in DMF and H₂O.²² Sonogashira cross-coupling reaction of 4 and 4-t-butyl-2-ethynylaniline²⁴ afforded 5 in 45% yield. Subsequent addition with 4-methoxyphenyl isocyanate gave 2^D in 34% yield. Compound 2^D and intermediates were characterized through ¹H, ²H, ¹³C{¹H}, and ¹⁹F NMR spectroscopy and high-resolution mass spectrometry (see ESI^†).Open in a separate windowScheme 1Synthetic route for the selective deuteration of anion receptor 2^D.Previous work on the DEIE of Cl⁻ binding with 1^H/D in DMSO revealed an experimental isotope effect of 1.019 ± 0.010. Therefore, we expected similar small DEIEs for HS⁻, Cl⁻, and Br⁻ binding with 2^H/D. Typical methods to determine binding constants (K_a) in supramolecular systems use non-linear regression fitting of titration data. Results from this method can be affected by small errors in the known initial host and guest concentration, quality of the titration isotherm, and subsequent data fitting, which when taken together often results in 2–15% errors in K_a. To increase the precision in K^H_a/K^D_a data for this study, we used the Perrin method of competitive titrations,²⁵ which has been shown previously to reduce errors in EIE values significantly with errors as small as 0.0004.²⁶ In this method, a linearized plot of the chemical shifts of 2^H (δ_H) and 2^D (δ_D) in fast exchange with an anionic guest is fit by linear regression to eqn (1):(δ⁰_H − δ_H)(δ_D − δ^f_D) = DEIE(δ⁰_D − δ_D)(δ_H − δ^f_H)1The slope of the linear regression is equal to the DEIE of the system. Because the linear regression only relies on chemical shift values and is independent of host and guest concentration, the precision of the method is limited to the precision of the NMR instrument and quality of data fitting.In addition, ¹³C NMR spectroscopy is sensitive to isotopic labelling and can show changes in chemical shifts between isotopomers. We were able to differentiate between the ¹³C NMR signals for C^ab, C¹ and C² for free and bound 2^H and 2^D (Fig. 2a) in 10% DMSO-d₆/CD₃CN, which were similar to those reported for 1^H/D in DMSO-d₆.¹² Competitive ¹³C NMR spectroscopy titrations were performed in anaerobic and anhydrous 10% DMSO-d₆/CD₃CN at 25 °C with mixtures of 2^H and 2^D in combined concentrations between 5.71 and 13.46 mM. Aliquots of the tetrabutylammonium (TBA) salts of HS⁻, Cl⁻, and Br⁻ were added until the system had reached saturation (Titration method A in ESI^†). In an effort to decrease reactivity of HS⁻ with 2^H/D and DMSO over long periods of time and decrease oxygen and water contaminations, some titrations with HS⁻ were performed by splitting the host solution of 2^H/D between four J-young NMR tubes. For each point in the competitive titration, TBASH was added to a new solution of 2^H/D inside an N₂-filled glovebox shortly before obtaining a ¹³C NMR spectra (Titration method B in ESI^†). The C^ab, C¹ and C^{2 13}C NMR signals were tracked for 2^H and 2^D in each titration for each anion. A representative competitive titration and linearized plots for Cl⁻ binding is shown in Fig. 2.Open in a separate windowFig. 2(a) Representation of the host–guest equilibrium between 2^H/D and Cl⁻. (b) Differences in the chemical shifts between the 2^H and 2^D isotopologues are observed in the ¹³C NMR signals for the C^ab, C¹, and C² carbons. ¹³C NMR signals for the C^ab, C¹, and C² carbons in 2^H and 2^D are tracked throughout a titration. (c–e) Linearized plots from fitting the chemical shifts of the C^ab, C¹, and C² throughout a titration to eqn (1).The DEIE data calculated from tracking the chemical shifts of the C^ab, C¹ and C^{2 13}C NMR signals from Cl⁻ and Br⁻ binding are summarized in eqn (1) through linear regression is included in parentheses

Pevonedistat and azacitidine upregulate NOXA (PMAIP1) to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.     

Regulatory volume increase in rat pancreatic β-cells

 This study investigated regulatory volume increase (RVI) in rat pancreatic β-cells. Volume changes in isolated β-cells were measured by a video-imaging method. Cell shrinkage was induced by exposure to solutions made hypertonic by the addition of 100 mM mannitol. In HEPES-buffered solutions, β-cells exhibited an RVI which was almost completely abolished by 10 μM bumetanide. These data indicate that Na⁺-2Cl^–-K⁺ cotransporters make a major contribution to RVI in β-cells. In HCO₃ ^–-buffered solutions, however, an RVI was observed in the presence of 10 μM bumetanide. This bumetanide-insensitive component of RVI was inhibited by 100 μM amiloride or 100 μM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS). These data suggest that, in addition to the Na⁺-2Cl^–-K⁺ cotransporter, functionally coupled Na⁺-H⁺ exchangers and Cl^–-HCO₃ ^– exchangers may also contribute to RVI in pancreatic β-cells. Received: 3 July 1997 / Received after revision and accepted: 1 September 1997