The Pan London Emergency Cardiac Surgery service: Coordinating a response to the COVID-19 pandemic.

Over the last 4 months, the novel coronavirus, SARS-CoV-2, has caused a significant economic, political, and public health impact on a global scale. The natural history of the disease and surge in the need for invasive ventilation has required the provision of intensive care beds in London to be reallocated. NHS England have proposed the formation of a Pan-London Emergency Cardiac surgery (PLECS) service to provide urgent and emergency cardiac surgery for the whole of London. In this initial report, we outline our experience of setting up and delivering a pan-regional service for the delivery of urgent and emergency cardiac surgery with a focus on maintaining a COVID-free in-hospital environment. In doing so, we hope that other regions can use this as a starting point in developing their own region-specific pathways if the spread of coronavirus necessitates similar measures be put in place across the United Kingdom.

Insights into the Structure of Sulfolobus Nucleoid Using Engineered Sac7d Dimers with a Defined Orientation.

The structure of Archaeal chromatin or nucleoid is believed to have characteristics similar to that found in both eukaryotes and bacteria. Recent comparative studies have suggested that DNA compaction in Archaea requires a bridging protein (e.g., Alba) along with either a wrapping protein (e.g., a histone) or a bending protein such as Sac7d. While X-ray crystal structures demonstrate that Sac7d binds as a monomer to create a significant kink in duplex DNA, the structure of a multiprotein-DNA complex has not been established. Using cross-linked dimers of Sac7d with a defined orientation, we present evidence that indicates that Sac7d is able to largely coat duplex DNA in vivo by binding in alternating head-to-head and tail-to-tail orientations. Although each Sac7d monomer promotes a significant kink of nearly 70°, coated DNA is expected to be largely extended because of compensation of repetitive kinks with helical symmetry.

Elucidating the Mechanism of Self-Healing in Hydrogel-Lead Halide Perovskite Composites for Use in Photovoltaic Devices.

Since the emergence of organometal halide perovskite (OMP) solar cells, there has been growing interest in the benefits of incorporating polymer additives into the perovskite precursor, in terms of both photovoltaic device performance and perovskite stability. In addition, there is interest in the self-healing properties of polymer-incorporated OMPs, but the mechanisms behind these enhanced characteristics are still not fully understood. Here, we study the role of poly(2-hydroxyethyl methacrylate) (pHEMA) in improving the stability of methylammonium lead iodide (MAPI, CH3NH3PbI3) and determine a mechanism for the self-healing of the perovskite-polymer composite following exposure to atmospheres of differing relative humidity, using photoelectron spectroscopy. Varying concentrations of pHEMA (0-10 wt %) are incorporated into a PbI2 precursor solution during the conventional two-step fabrication method for producing MAPI. It is shown that the introduction of pHEMA results in high-quality MAPI films with increased grain size and reduced PbI2 concentration compared with pure MAPI films. Devices based on pHEMA-MAPI composites exhibit an improved photoelectric conversion efficiency of 17.8%, compared with 16.5% for a pure MAPI device. pHEMA-incorporated devices are found to retain 95.4% of the best efficiency after ageing for 1500 h in 35% RH, compared with 68.5% achieved from the pure MAPI device. The thermal and moisture tolerance of the resulting films is investigated using X-ray diffraction, in situ X-ray photoelectron spectroscopy (XPS), and hard XPS (HAXPES). It is found that exposing the pHEMA films to cycles of 70 and 20% relative humidity leads to a reversible degradation, via a self-healing process. Angle-resolved HAXPES depth-profiling using a non-destructive Ga Kα source shows that pHEMA is predominantly present at the surface with an effective thickness of ca. 3 nm. It is shown using XPS that this effective thickness reduces with increasing temperature. It is found that N is trapped in this surface layer of pHEMA, suggesting that N-containing moieties, produced during reaction with water at high humidity, are trapped in the pHEMA film and can be reincorporated into the perovskite when the humidity is reduced. XPS results also show that the inclusion of pHEMA enhances the thermal stability of MAPI under both UHV and 9 mbar water vapor pressure.

The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity.

E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.

Obesity and diabetes are major risk factors for epicardial adipose tissue inflammation.

BACKGROUNDEpicardial adipose tissue (EAT) directly overlies the myocardium, with changes in its morphology and volume associated with myriad cardiovascular and metabolic diseases. However, EAT's immune structure and cellular characterization remain incompletely described. We aimed to define the immune phenotype of EAT in humans and compare such profiles across lean, obese, and diabetic patients.METHODSWe recruited 152 patients undergoing open-chest coronary artery bypass grafting (CABG), valve repair/replacement (VR) surgery, or combined CABG/VR. Patients' clinical and biochemical data and EAT, subcutaneous adipose tissue (SAT), and preoperative blood samples were collected. Immune cell profiling was evaluated by flow cytometry and complemented by gene expression studies of immune mediators. Bulk RNA-Seq was performed in EAT across metabolic profiles to assess whole-transcriptome changes observed in lean, obese, and diabetic groups.RESULTSFlow cytometry analysis demonstrated EAT was highly enriched in adaptive immune (T and B) cells. Although overweight/obese and diabetic patients had similar EAT cellular profiles to lean control patients, the EAT exhibited significantly (P ≤ 0.01) raised expression of immune mediators, including IL-1, IL-6, TNF-α, and IFN-γ. These changes were not observed in SAT or blood. Neither underlying coronary artery disease nor the presence of hypertension significantly altered the immune profiles observed. Bulk RNA-Seq demonstrated significant alterations in metabolic and inflammatory pathways in the EAT of overweight/obese patients compared with lean controls.CONCLUSIONAdaptive immune cells are the predominant immune cell constituent in human EAT and SAT. The presence of underlying cardiometabolic conditions, specifically obesity and diabetes, rather than cardiac disease phenotype appears to alter the inflammatory profile of EAT. Obese states markedly alter EAT metabolic and inflammatory signaling genes, underlining the impact of obesity on the EAT transcriptome profile.FUNDINGBarts Charity MGU0413, Abbott, Medical Research Council MR/T008059/1, and British Heart Foundation FS/13/49/30421 and PG/16/79/32419.

Cardiothoracic surgery in the midst of a pandemic: Operative outcomes and maintaining a coronavirus disease 2019 (COVID-19)-free environment.

OBJECTIVE: In the United Kingdom, the coronavirus disease 2019 (COVID-19) pandemic has led to the cessation of elective surgery. However, there remains a need to provide urgent and emergency cardiac and thoracic surgery as well as to continue time-critical thoracic cancer surgery. This study describes our early experience of implementing a protocol to safely deliver major cardiac and thoracic surgery in the midst of the pandemic. METHODS: Data on all patients undergoing cardiothoracic surgery at a single tertiary referral center in London were prospectively collated during the first 7 weeks of lockdown in the United Kingdom. A comprehensive protocol was implemented to maintain a COVID-19-free environment including the preoperative screening of all patients, the use of full personal protective equipment in areas with aerosol-generating procedures, and separate treatment pathways for patients with and without the virus. RESULTS: A total of 156 patients underwent major cardiac and thoracic surgery over the study period. Operative mortality was 9% in the cardiac patients and 1.4% in thoracic patients. The preoperative COVID-19 protocol implemented resulted in 18 patients testing positive for COVID-19 infection and 13 patients having their surgery delayed. No patients who were negative for COVID-19 infection on preoperative screening tested positive postoperatively. However, 1 thoracic patient tested positive on intraoperative bronchoalveolar lavage. CONCLUSIONS: Our early experience demonstrates that it is possible to perform major cardiac and thoracic surgery with low operative mortality and zero development of postoperative COVID-19 infection.

Structure, stability, and flexibility of ribosomal protein L14e from Sulfolobus solfataricus.

Ribosomal protein L14e is a component of the large ribosomal subunit in both archaea and eukaryotes. We report here a high-resolution NMR solution structure of recombinant L14e and show that the N-terminal 57 residues adopt a classic SH3 fold. The protein contains a tight turn between strands 1 and 2 instead of the typical SH3 RT-loop, indicating that it is unlikely to interact with neighboring ribosomal proteins using the common SH3 site for proline-rich sequences. The remainder of the protein (39 residues) forms a largely extended chain with a short helix which packs onto the surface of the SH3 domain via hydrophobic interactions. It has the potential of adopting an alternative structure to expose a hydrophobic surface for protein-protein interactions in the ribosome without disruption of the SH3 fold. (15)N relaxation data demonstrate that the majority of the C-terminal chain is well-defined on the SH3 surface. The globular protein unfolds reversibly with a T(m) of 102.8 degrees C at pH 7, making it one of the most stable SH3 domain proteins described to date. The structure of L14e is expected to serve as a model for other members of the L14e family, along with members of the COG2163 group, including L6e and L27e. Interestingly, the N-terminal sequence of L14e shows the greatest similarity of any Sulfolobus protein to the reported N-terminal sequence of Sac8b, a DNA-binding protein reported by Grote et al. ((1986) Biochim. Biophys. Acta 873, 405-413). The likelihood that L14e and Sac8b are the same protein is discussed.

Defining the stability of multimeric proteins.

The practical application of scanning calorimetry and spectroscopic methods to measure the stability of multimeric proteins is described. Oligomeric proteins are stabilized by both the intrinsic folding energy of the subunits as well as interactions between the subunits. Oligomerization results in a concentration dependence for multimer stability, which increases logarithmically with increasing concentration. Since the increase in stability does not plateau at high protein concentrations, the effect of concentration must be described quantitatively. Straightforward mathematical methods are provided for deriving the appropriate models for multimer unfolding, and methods are presented for analyzing equilibrium unfolding data and stability using the models.

Ligand-binding interactions and stability.

The reversible interaction or binding of ligands to biological macromolecules is fundamental to nearly every aspect of biochemistry and cell biology. Binding events typically do not occur in isolation in biochemistry, and are almost always coupled or linked to other reactions such as protonation changes, other ligand-binding interactions, structural transitions, and folding. It is rarely sufficient to simply state that something binds. An understanding of binding requires a measure of affinity, stoichiometry, and the contributions of linked reactions. Emphasis is placed here on defining binding and the influence of linkage on binding and stability using both spectroscopic and calorimetric data.

Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d.

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.

A new photocrosslinkable polycaprolactone-based ink for three-dimensional inkjet printing.

A new type of photocrosslinkable polycaprolactone (PCL) based ink that is suitable for three-dimensional (3D) inkjet printing has been developed. Photocrosslinkable Polycaprolactone dimethylacrylate (PCLDMA) was synthesized and mixed with poly(ethylene glycol) diacrylate (PEGDA) to prepare an ink with a suitable viscosity for inkjet printing. The ink performance under different printing environments, initiator concentrations, and post processes was studied. This showed that a nitrogen atmosphere during printing was beneficial for curing and material property optimization, as well as improving the quality of structures produced. A simple structure, built in the z-direction, demonstrated the potential for this material for the production of 3D printed objects. Cell tests were carried out to investigate the biocompatibility of the developed ink. © 2016 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1645-1657, 2017.

Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d.

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.

The hyperthermophile protein Sso10a is a dimer of winged helix DNA-binding domains linked by an antiparallel coiled coil rod.

Sso10a is a member of a group of DNA-binding proteins thought to be important in chromatin structure and regulation in the hyperthermophilic archaeon Sulfolobus solfataricus. We have determined the structure of Sso10a to 1.47A resolution directly with unlabelled native crystals by a novel approach using sulfur single-wavelength anomalous scattering (SAS) from a chromium X-ray source. The 95 amino acid residue protein contains a winged helix DNA-binding domain with an extended C-terminal alpha-helix that leads to dimerization by forming a two-stranded, antiparallel coiled-coil rod. The winged helix domains are at opposite ends of the extended coiled coil with two putative DNA-recognition helices separated by 55A and rotated by 83 degrees. Formation of stable dimers in solution is demonstrated by both analytical ultracentrifugation and differential scanning calorimetry. With a T0 of 109 degrees C, Sso10a is one of the most stable two-stranded coiled coils known. The coiled coil contains a rare aspartate residue (D69) in the normally hydrophobic d position of the heptad repeat, with two aspartate-lysine (d-g') interhelical ion pairs in the symmetrical dimer. Mutation of D69 to alanine resulted in an increase in thermal stability, indicating that destabilization resulting from the partially buried aspartate residue cannot be offset by ion pair formation. Possible DNA-binding interactions are discussed on the basis of comparisons to other winged helix proteins. The structure of Sso10a provides insight into the structures of the conserved domain represented by COG3432, a group of more than 20 hypothetical transcriptional regulators coded in the genomic sequences of both crenarchaeota and euryarchaeota.

[Clinical outcome of patients after transmyocardial laser revascularization alone and combined with coronary artery bypass grafting]. / Kliniczna ocena efektu przezsercowej rewaskularyzacji laserowej polaczonej lub niepolaczonej z pomostowaniem aortalno-wiencowym.

The aim of the study was to assess the effect of transmyocardial laser revascularization (TMLR) alone and in combination with coronary artery bypass grafting (CABG) on the angina score (CCS--Canadian Cardiovascular Society class), exercise tolerance and left ventricular function 6 months after the procedures. Sixty two patients were subjected to revascularization, 38 to sole TMLR procedure and 24 to combination CABG and TMLR (CABG/TMLR group). The angina score and exercise stress test together with radionuclide ventriculography were performed before and 6 months after the operation. The angina class and exercise tolerance were similar in both groups preoperatively. After the operation the improvement was seen in both groups with no statistical difference. The left ventricular ejection fraction were 61 +/- 8% and 54 +/- 8% (p < 0.05) before operation and after 6 months respectively. Transmyocardial laser revascularisation alone and in combination with coronary artery bypass grafting may relieve the angina and improve the exercise tolerance. However the left ventricular ejection fraction may drop significantly.

Atrial myxoma masquerading as Takayasu's arteritis.

We describe the case of a 48-year-old woman whose atrial myxoma was mistaken for vasculitis. The case report highlights the reasons why these two disorders may become confused, the dangers of initiating the wrong treatment and a simple means of avoiding misdiagnosis.

Effect of mutation of the Sac7d intercalating residues on the temperature dependence of DNA distortion and binding thermodynamics.

Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by approximately 66 degrees at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 degrees C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (DeltaC(p)) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive DeltaC(p) coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 degrees C is due in part to the distortion of DNA.

Role of a surface tryptophan in defining the structure, stability, and DNA binding of the hyperthermophile protein Sac7d.

Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein-DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (DeltaG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 degrees C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.

Solution structure, stability, and flexibility of Sso10a: a hyperthermophile coiled-coil DNA-binding protein.

Sso10a is one of a number of DNA-binding proteins from the hyperthermophile Sulfolobus solfataricus that has been associated with DNA packaging and chromatin regulation. Sequence analysis indicates that it is a member of a conserved group of archaeal transcription regulators (COG3432). We have determined the solution structure of Sso10a and show that it is a homodimer of winged-helix DNA-binding domains. The dimer interface consists of an extended antiparallel coiled coil, with the globular DNA-binding domains positioned at opposite ends of a solvent-exposed coiled-coil rod. NMR structure refinement of the elongated structure benefited not only from the inclusion of residual dipolar couplings from partially aligned samples but also the influence of anisotropic rotational diffusion on heteronuclear relaxation. An analysis of backbone mobility using (15)N relaxation rates indicated that the overall tertiary and quaternary structure is largely inflexible on the nanosecond to picosecond time scale. Amide hydrogen exchange data demonstrated that the most stable region of the protein extends from the core of the winged helices into the coiled coil. The positions of the globular heads relative to the coiled coil in solution deviate only slightly from that observed in a crystal structure. The most significant difference between the solution and crystal structures occurs in the putative DNA-binding helix-turn-helix (HTH) motif. This is the region of lowest stability in solution and a point of protein-protein contact in the crystal. Alternative conformations of the HTH motif may permit adjustment of the structure for optimal DNA binding.

Stability and flexibility in the structure of the hyperthermophile DNA-binding protein Sac7d.

Sac7d is a chromatin protein from the hyperthermophile Sulfolobus acidocaldarius that severely kinks duplex DNA with negligible change in protein structure. In previous work, the overall stability of Sac7d has been well-characterized with a global analysis of the linkage of folding, protonation, and anion binding. We extend that work here with NMR measurements of global stability as well as the distribution of stability and flexibility in the solution structure. Native state amide hydrogen exchange has been used to identify the most-protected core amide protons which exchange through global unfolding. The pH and temperature dependence of stability defined by native state exchange is in excellent agreement with the free energy surface determined by a linkage analysis of the dependence of folding on pH, salt, and temperature. These results confirm that the deltaC(P) obtained from a Kirchhoff analysis of DSC data (i.e., deltaH vs Tm) is incorrect, and an accurate description of the protein stability curve for Sac7d requires a measure of the thermodynamic contributions of protonation and anion binding. Amide hydrogen exchange, along with generalized order parameters determined by 15N relaxation data, demonstrates considerable variation in stability throughout the structure with some of the least stable regions occurring at the N- and C-termini. The most stable and inflexible region of the backbone occurs primarily in the DNA-binding beta-sheet which is responsible for bending DNA.