Safety of bailout stenting after paclitaxel-coated balloon angioplasty.

BACKGROUND: Bailout stenting after suboptimal paclitaxel-coated balloon (PCB) angioplasty is required in up to 28% of cases. We sought to compare the safety of bailout stenting with drug-eluting stents (DES) compared with the more established combination of PCB with bare metal stents (BMS). METHODS: We retrospectively evaluated all patients who had stents implanted owing to suboptimal PCB angioplasty results between January 2010 and April 2015. Endpoints analyzed were major adverse cardiac events (MACE) - defined as cardiovascular death, nonfatal myocardial infarction (MI), and target lesion revascularization (TLR) - as well as major and minor bleeding. RESULTS: Baseline clinical characteristics were comparable with a high proportion of diabetics in both groups (50.0% vs. 45.8%, p = 0.74). BMS and DES sizes were similar (mean diameter 2.72 ± 0.50 mm vs. 2.89 ± 0.56 mm, p = 0.20, length 25.22 ± 13.47 mm vs. 28.08 ± 9.08 mm, p = 0.47). Outcomes were comparable at the end of 1 year (MACE 12.2% vs. 9.5%, p = 1.00, TLR 6.1% vs. 4.8%, p = 1.00, MI 0% vs. 4.8%, p = 0.30). There was no case of stent thrombosis or major bleeding, and the rates of minor bleeding were similar (4.2% vs. 4.8%, p = 1.00). CONCLUSION: Our initial experience using DES instead of BMS as a bailout after suboptimal PCB results shows that the procedure is safe and effective at 1 year.

Thermodynamic analysis of ANS binding to partially unfolded α-lactalbumin: correlation of endothermic to exothermic changeover with formation of authentic molten globules.

A fluorescent reporter, 8-anilino-1-naphthalene sulfonic acid (ANS), can serve as a reference molecule for conformational transition of a protein because its aromatic carbons have strong affinity with hydrophobic cores of partially unfolded molten globules. Using a typical calcium-binding protein, bovine α-lactalbumin (BLA), as a model protein, we compared the ANS binding thermodynamics to the decalcified (10 mM EDTA treated) apo-BLA at two representative temperatures: 20 and 40 °C. This is because the authentic molten globule is known to form more heavily at an elevated temperature such as 40 °C. Isothermal titration calorimetry experiments revealed that the BLA-ANS interactions at both temperatures were entropy-driven, and the dissociation constants were similar on the order of 10(-4) M, but there was a dramatic changeover in the binding thermodynamics from endothermic at 20 °C to exothermic at 40 °C. We believe that the higher subpopulation of authentic molten globules at 40 °C than 20 °C would be responsible for the results, which also indicate that weak binding is sufficient to alter the ANS binding mechanisms. We expect that the thermodynamic properties obtained from this study would serve as a useful reference for investigating the binding of other hydrophobic ligands such as oleic acid to apo-BLA, because oleic acid is known to have tumor-selective cytotoxicity when complexed with partially unfolded α-lactalbumin. Copyright © 2016 John Wiley & Sons, Ltd.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

Gastric digestion of α-lactalbumin in adult human subjects using capsule endoscopy and nasogastric tube sampling.

In the present study, structural changes in the milk protein α-lactalbumin (α-LA) and its proteolysis were investigated for the potential formation of protein-fatty acid complexes during in vivo gastric digestion. Capsule endoscopy allowed visualisation of the digestion of the test drinks, with nasogastric tubes allowing sampling of the gastric contents. A total of ten healthy volunteers had nasogastric tubes inserted into the stomach and ingested test drinks containing 50 g/l of sucrose and 25 g/l of α-LA with and without 4 g/l of oleic acid (OA). The samples of gastric contents were collected for analysis at 3 min intervals. The results revealed a rapid decrease in the pH of the stomach of the subjects. The fasting pH of 2·31 (SD 1·19) increased to a pH maxima of pH 6·54 (SD 0·29) after ingestion, with a subsequent decrease to pH 2·22 (SD 1·91) after 21 min (n 8). Fluorescence spectroscopy and Fourier transform IR spectroscopy revealed partial protein unfolding, coinciding with the decrease in pH below the isoelectric point of α-LA. The activity of pepsin in the fasting state was found to be 39 (SD 12) units/ml of gastric juice. Rapid digestion of the protein occurred: after 15 min, no native protein was detected using SDS-PAGE; HPLC revealed the presence of small amounts of native protein after 24 min of gastric digestion. Mirocam® capsule endoscopy imaging and video clips (see the online supplementary material) revealed that gastric peristalsis resulted in a heterogeneous mixture during gastric digestion. Unfolding of α-LA was observed during gastric transit; however, there was no evidence of a cytotoxic complex being formed between α-LA and OA.

Application of novel AI-based algorithms to biobank data: uncovering of new features and linear relationships.

We independently analyzed two large public domain datasets that contain 1H-NMR spectral data from lung cancer and sex studies. The biobanks were sourced from the Karlsruhe Metabolomics and Nutrition (KarMeN) study and Bayesian Automated Metabolite Analyzer for NMR data (BATMAN) study. Our approach of applying novel artificial intelligence (AI)-based algorithms to NMR is an attempt to globalize metabolomics and demonstrate its clinical applications. The intention of this study was to analyze the resulting spectra in the biobanks via AI application to demonstrate its clinical applications. This technique enables metabolite mapping in areas of localized enrichment as a measure of true activity while also allowing for the accurate categorization of phenotypes.

In Silico Analysis of Therapeutic Antibody Aggregation and the Influence of Glycosylation.

The aggregation of therapeutic antibodies is a major issue for the pharmaceutical industry leading to loss of drug quality, increased dosage, and unwanted immune responses such as the production of anti-drug antibodies (ADA). As aggregation can occur at various stages of development and storage, much work has been performed to reduce or eliminate it. In this report we analyzed four antibodies available in the PDB (1IGT, 1IGY, 1HZH, and 5DK3) using the online software UCSF Chimera to study the structural features of the proteins and the associated N-linked glycans in the CH2 domains of the Fc region. To study antibody aggregation in silico we used the online software TANGO and AGGRESCAN to identify aggregation prone regions (APR) in the antibodies and the influence of the Fc glycans on hydrophobic and aromatic residues present in the APRs. In the 3D structures of 1IGT and 1IGY the glycan chains are in close enough proximity to influence and protect these hydrophobic regions. However, in the 3D structures of 1HZH and 5DK3 the glycans do not appear to influence the likely APRs of the antibodies. Therefore, in these structures we modified the Fc glycan regions by adjusting the glycosylated asparagine side chains and glycosidic bonds. We successfully adjusted the glycan chains of 1HZH and 5DK3 and reduced the distance between them and the APRs to show potential influence on aggregation. However, similar to 5DK3, the influence of glycosylation on the APRs of the antibody was limited due to the size of the glycans present in the 3D structure. This report is based on in silico studies to show how antibody glycans can influence aggregation.

The role of exercise right heart catheterization to guide pulmonary hypertension therapy in older adults.

The spectrum of patients referred for suspected pulmonary arterial hypertension (PAH) includes a population with clinical features suggestive of pulmonary hypertension due to left heart disease (PH-LHD). Even after right heart catheterization (RHC) performed at rest, it can be a challenge to identify patients who will clearly benefit from PAH drug therapy. Therefore, the objective of this study was to evaluate the role of exercise RHC to influence decisions regarding prescription of PAH drug therapy in this population. A retrospective cohort study was conducted of older adults with risk factors for PH-LHD and suspected PH referred for exercise RHC. One year follow-up was conducted to record clinical outcomes, all changes in PAH drug therapy, and changes in patient-reported quality of life. The final cohort included 61 patients, mean age of 69 ± 10; 44% and 34% had a history of coronary artery disease and atrial fibrillation respectively. Exercise changed the proportional breakdown of hemodynamic diagnoses from 36% No PH, 44% PAH, and 20% PH-LHD at rest to 15% No PH, 36% PAH, and 49% PH-LHD. Although a significant proportion of patients were reclassified as PH-LHD, there was an overall increase in the proportion of patients receiving PAH drug therapy, particularly for those with PAH confirmed by exercise RHC. A total of 11 PAH drug prescriptions were employed before exercise RHC increasing to 24 after (p = 0.002). Patients receiving PAH therapy demonstrated significant improvement in self-reported quality of life. Exercise RHC appeared to influence selection of PAH drug therapy.

A scientific journey from discovery to validation of efficacy in cancer patients: HAMLET and alpha1-oleate.

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented.

A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds.

CD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.

Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

Salient Features of Monomeric Alpha-Synuclein Revealed by NMR Spectroscopy.

Elucidating the structural details of proteins is highly valuable and important for the proper understanding of protein function. In the case of intrinsically disordered proteins (IDPs), however, obtaining the structural details is quite challenging, as the traditional structural biology tools have only limited use. Nuclear magnetic resonance (NMR) is a unique experimental tool that provides ensemble conformations of IDPs at atomic resolution, and when studying IDPs, a slightly different experimental strategy needs to be employed than the one used for globular proteins. We address this point by reviewing many NMR investigations carried out on the α-synuclein protein, the aggregation of which is strongly correlated with Parkinson's disease.

Size Determination of Protein Oligomers/Aggregates Using Diffusion NMR Spectroscopy.

Diffusion-ordered spectroscopy (DOSY) is a widely used NMR technique for the identification of different chemical moieties/compounds contained in mixtures and has been successfully employed for the separation of small molecules based on hydrodynamic radii. Herein we show that DOSY can also be applied for the size determination of larger biomolecules such as proteins and protein oligomers/aggregates. Proof-of-principle is first shown with a cross-linked oligomeric protein mixture where the hydrodynamic volumes of each component are estimated and subsequently verified with size-exclusion HPLC and SDS polyacrylamide gel electrophoresis. We then determine the sizes of protein oligomers contained in a protein solution subjected under amyloid fibrillogenesis conditions. These studies aim to provide insight into the kinetics behind protein aggregation involved in amyloidosis as well as to determine the hydrodynamic radii of proteins within the mixture.

Synechial angle closure pattern in Chinese chronic primary angle-closure glaucoma patients.

PURPOSE: The aim of this retrospective clinical study is to investigate the pattern of synechial angle closure in a group of Hong Kong Chinese with chronic primary angle-closure glaucoma. METHODS: The anterior angle of the upper, lower, medial, and lateral quadrants of 84 eyes from Hong Kong Chinese patients with chronic primary angle-closure glaucoma were assessed by the same examiner using indentation gonioscopy. RESULTS: The incidences of synechial angle closure at the superior, nasal, inferior, and temporal quadrants were 98% (83/84), 77% (65/84), 31% (26/84), and 94% (79/84), respectively. The Fisher exact test showed that the incidence of synechial angle closure at the superior quadrant was significantly higher than the nasal (P = 0.005) and inferior (P < 0.001) quadrants, but not the temporal quadrant (P = 0.07). CONCLUSION: Synechial angle closure is circumferential with asymmetry in these Chinese patients with chronic primary angle-closure glaucoma. The superior and temporal quadrants of the anterior angle may be the earliest sites of the synechial angle closure. The anterior angle may close synechially at the superior and temporal quadrants with gradual extension on the nasal quadrant, until the angle may close at the inferior quadrant.

Chloride and sodium transport across bovine ciliary body/epithelium (CBE).

PURPOSE: To study the chloride and sodium ion transports across the bovine ciliary body/epithelium (CBE) by a modified Ussing-Zerahn type chamber. METHODS: Isolated bovine CBE preparations were mounted in a modified Ussing-type chamber and the transepithelial electrical parameters were monitored. The inward (stroma to aqueous) and outward (aqueous to stroma) fluxes of 36[Cl] chloride and 22[Na] sodium ions across the CBE were measured under short-circuited conditions. The effect of 0.1 mM of furosemide and bumetanide on the chloride transport were studied. RESULTS: The potential difference (PD), the resistance and the short-circuit current (SCC) across the isolated bovine ciliary body were found to be -0.20+/-0.01 mV (aqueous negative), 75+/-1 omegacm2 and -2.70+/-0.17 microAcm(-2) (mean+/-SEM, n=50) respectively. A statistically significant net inward chloride ion flux of 1.12+/-0.41 microEq h(-1)cm(-2) (p < 0.01) was found (n=15). The net chloride transport was abolished when 0.1 mM furosemide (82% inhibition) and 0.1 mM bumetanide (100% inhibition) were applied bilateral. No significant net sodium ion flux was detected. CONCLUSIONS: Electrolyte and fluid transport across the bovine CBE may be via a bumetanide and furosemide-sensitive chloride transport mechanism. The Na-K-2Cl cotransporter plays a significant role in the trans-CBE chloride transport. The net chloride flux/current was about 12 times higher than the measured SCC, suggesting that the chloride ion transport may be coupled to other ion species.

Temperature and urea induced denaturation of the TRP-cage mini protein TC5b: A simulation study consistent with experimental observations.

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20-residue Trp-cage mini-protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force-field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR-NOE atom-atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40-90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force-field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side-chain side-chain contact (Trp6-Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.

Space analysis: a comparison between sonic digitization (DigiGraph Workstation) and the digital caliper.

The introduction of the DigiGraph Workstation permits the use of sonic digitization to measure lateral cephalometric values, mesiodistal tooth size and arch perimeter discrepancy as a one-stop diagnostic record taking set-up. This study compared the reproducibility of mesiodistal total tooth widths and arch perimeter values, on plaster casts, given by the DigiGraph Workstation and by digital calipers. Forty-seven sets of plaster casts of Southern Chinese children (mean age 12.5 years) comprised the sample. Arch perimeter was measured using calipers in six segments from the distal of the first permanent molar to its antimere in each arch. The total mesiodistal widths of all teeth, excluding second and third molars, were also measured. The difference between the available arch perimeter and the total tooth widths was taken as the arch perimeter discrepancy. Sonic digitization of the study casts was completed according to instructions of the DigiGraph software. Paired t-tests and F-tests were used to compare the two methods. Compared with manual measurement, there was an over-estimation of the total tooth widths by 1 mm in the mandible and 0.5 mm in the maxilla, and an arch perimeter discrepancy of 1.6 mm in the mandible and 0.4 mm in the maxilla when using the sonic method. The sonic digitization was not as reproducible as the digital caliper and its clinical usefulness in evaluating the space problem of an individual malocclusion should be interpreted with caution.

Retinal nerve fiber layer measurement by nerve fiber analyzer in normal subjects and patients with glaucoma.

OBJECTIVE: To identify the pattern of retinal nerve fiber layer (RNFL) loss in glaucoma using the Nerve Fiber Analyzer (NFA). DESIGN: Case-control study. PARTICIPANTS: A total of 80 normal and 75 age- and race-matched chronic open-angle glaucoma subjects were recruited. INTERVENTION: The RNFL thickness was assessed with a scanning laser polarimeter (Nerve Fiber Analyzer GDX, Laser Diagnostic Technologies Inc., San Diego, CA). Analysis of variance was used to compare the corresponding RNFL measurement indices of the different groups. MAIN OUTCOME MEASURES: Superior/nasal (S/N) and inferior/nasal (I/N) ratios of NFA are sensitive parameters to differentiate between glaucoma and nonglaucoma subjects. RESULTS: Peripapillary RNFL measurements at the superior and inferior regions were significantly lower in the glaucoma group (P<0.001) but were similar in temporal and nasal regions (P>0.05). Using nasal value as reference, S/N and I/N ratios were significantly lower in the glaucoma groups (P<0.001) and also decreased with increasing severity of glaucoma. CONCLUSIONS: In glaucoma, RNFL was more susceptible to loss in the superior and inferior regions than in the nasal and temporal regions. The S/N and I/N ratio parameters provided by the NFA appeared to give further discriminating ability in early glaucoma.

Nerve fiber analyzer and short-wavelength automated perimetry in glaucoma suspects: a pilot study.

PURPOSE: To test the relationship between the results of short-wavelength automatic perimetry (SWAP) and retinal nerve fiber layer (RNFL) measurements with scanning laser polarimetry (Nerve Fiber Analyzer, NFA) in age-matched normal subjects, glaucoma suspects, and early glaucoma patients. DESIGN: Case-control study. PARTICIPANTS AND METHODS: Thirty-eight normal subjects, 32 glaucoma suspects, and 14 early glaucoma patients were recruited. All subjects underwent RNFL assessment by NFA, achromatic visual field testing (24-2 threshold), and repeated SWAP (24-2 threshold blue-on-yellow). MAIN OUTCOME MEASURES: Mean deviation (MD) of visual field testing and RNFL values were obtained. RESULTS: Glaucoma suspects were divided into two groups according to their SWAP results: high risk (with SWAP abnormalities) and low risk (with normal SWAP result). No statistically significant difference in SWAP MD and RNFL values were observed between normal and low-risk groups (P > 0.05), but these values were found to be significantly lower in high-risk and early glaucoma groups (P < 0.01). CONCLUSIONS: This study suggests that RNFL examination by NFA may be a useful test for the early detection of glaucomatous damage of glaucoma suspects. It appears to provide agreement with SWAP abnormalities and is more sensitive than conventional standard automated perimetry.

Nerve fibre layer measurement of the Hong Kong Chinese population by scanning laser polarimetry.

PURPOSE: To obtain normal retinal nerve fibre layer (RNFL) measurements by scanning laser polarimetry in a local Hong Kong Chinese population and to identify the correlation of the measurements with age. METHODS: One hundred and fifty-nine normal Hong Kong Chinese volunteers of different ages were recruited for this study. RNFL values were measured using a Nerve Fibre Analyzer GDx (Laser Diagnostic Technologies, CA). RESULTS: Mean peripapillary RNFL measurements at the superior, inferior, temporal and nasal regions were 95.0 +/- 15.6, 97.0 +/- 16.3, 47.0 +/- 9.0 and 43.9 +/- 15.1 microns respectively (mean +/- SD). There was a highly significant negative correlation in average RNFL values with increasing age (approximately 1.9 microns per decade, p = 0.001). A significant negative correlation with increasing age was also identified in the RNFL values in all four regions and they decreased by similar amounts (p < 0.05). No correlation with age was observed for relative ratio parameters such as superior/nasal ratio, inferior/nasal ratio and max. modulation (p > 0.05). CONCLUSION: RNFL values of the Hong Kong Chinese population appeared to decrease over time cross-sectionally. Since they decreased by similar amounts at four regions, use of relative ratio parameters for analysis has an advantage over absolute values, as they were not affected by age.