Learning from Protein Engineering by Deconvolution of Multi-Mutational Variants.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. Following the establishment of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was introduced. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. During the past decade, this phenomenon was shown to be general. In some studies, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations were performed in order to shed light on the origin of non-additivity at all stages of an evolutionary upward climb. Data of complete deconvolution can be used to construct unique multi-dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional fitness landscapes. Along a related line, biochemists have long tested the result of introducing two point mutations in an enzyme for mechanistic reasons, followed by a comparison of the respective double mutant in so-called double mutant cycles, which originally showed only additive effects, but more recently also uncovered cooperative and antagonistic non-additive effects. We conclude with suggestions for future work, and call for a unified overall picture of non-additivity and epistasis.

Polymeric Nanotubes as Drug Delivery Vectors─Comparison of Covalently and Supramolecularly Assembled Constructs.

Rod-shaped nanoparticles have been identified as promising drug delivery candidates. In this report, the in vitro cell uptake and in vivo pharmacokinetic/bio-distribution behavior of molecular bottle-brush (BB) and cyclic peptide self-assembled nanotubes were studied in the size range of 36-41 nm in length. It was found that BB possessed the longest plasma circulation time (t1\2 > 35 h), with the cyclic peptide system displaying an intermediate half-life (14.6 h), although still substantially elevated over a non-assembling linear control (2.7 h). The covalently bound BB underwent substantial distribution into the liver, whereas the cyclic peptide nanotube was able to mostly circumvent organ accumulation, highlighting the advantage of the inherent degradability of the cyclic peptide systems through their reversible aggregation of hydrogen bonding core units.

Cyclic Peptide-Polymer Nanotubes as Efficient and Highly Potent Drug Delivery Systems for Organometallic Anticancer Complexes.

Functional drug carrier systems have potential for increasing solubility and potency of drugs while reducing side effects. Complex polymeric materials, particularly anisotropic structures, are especially attractive due to their long circulation times. Here, we have conjugated cyclic peptides to the biocompatible polymer poly(2-hydroxypropyl methacrylamide) (pHPMA). The resulting conjugates were functionalized with organoiridium anticancer complexes. Small angle neutron scattering and static light scattering confirmed their self-assembly and elongated cylindrical shape. Drug-loaded nanotubes exhibited more potent antiproliferative activity toward human cancer cells than either free drug or the drug-loaded polymers, while the nanotubes themselves were nontoxic. Cellular accumulation studies revealed that the increased potency of the conjugate appears to be related to a more efficient mode of action rather than a higher cellular accumulation of iridium.

Systematic study of the structural parameters affecting the self-assembly of cyclic peptide-poly(ethylene glycol) conjugates.

Self-assembling cyclic peptides (CP) consisting of amino acids with alternating d- and l-chirality form nanotubes by hydrogen bonding, hydrophobic interactions, and π-π stacking in solution. These highly dynamic materials are emerging as promising supramolecular systems for a wide range of biomedical applications. Herein, we discuss how varying the polymer conformation (linear vs. brush), as well as the number of polymer arms per peptide unimer affects the self-assembly of PEGylated cyclic peptides in different solvents, using small angle neutron scattering. Using the derived information, strong correlations were drawn between the size of the aggregates, solvent polarity, and its ability to compete for hydrogen bonding interactions between the peptide unimers. Using these data, it could be possible to engineer cyclic peptide nanotubes of a controlled length.

Secondary Self-Assembly of Supramolecular Nanotubes into Tubisomes and Their Activity on Cells.

The properties and structures of viruses are directly related to the three-dimensional structure of their capsid proteins, which arises from a combination of hydrophobic and supramolecular interactions, such as hydrogen bonds. The design of synthetic materials demonstrating similar synergistic interactions still remains a challenge. Herein, we report the synthesis of a polymer/cyclic peptide conjugate that combines the capability to form supramolecular nanotubes via hydrogen bonds with the properties of an amphiphilic block copolymer. The analysis of aqueous solutions by scattering and imaging techniques revealed a barrel-shaped alignment of single peptide nanotubes into a large tubisome (length: 260 nm (from SANS)) with a hydrophobic core (diameter: 16 nm) and a hydrophilic shell. These systems, which have a structure that is similar to those of viruses, were tested in vitro to elucidate their activity on cells. Remarkably, the rigid tubisomes are able to perforate the lysosomal membrane in cells and release a small molecule into the cytosol.

Triblock Copolymer Nanovesicles for pH-Responsive Targeted Delivery and Controlled Release of siRNA to Cancer Cells.

New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and polyglycerolmethacrylate (poly-GMA), and a central weakly basic block, polyimidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatized and underivatized copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above ∼2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor overexpressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher uptake of ds-DNA loaded folate-functional polymersomes in KB cells compared to nontargeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31 and 23% knockdown in target protein expression, respectively.

Extreme synergistic mutational effects in the directed evolution of a baeyer-villiger monooxygenase as catalyst for asymmetric sulfoxidation.

Structure-based directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to phenyl acetone monooxygenase (PAMO), a thermally robust Baeyer-Villiger monooxygenase, in the quest to access a mutant which displays reversed enantioselectivity in the asymmetric sulfoxidation of prochiral thioethers. Whereas WT PAMO leads to 90% ee in the sulfoxidation of p-methylbenzyl methyl thioether with preference for the (S)-sulfoxide, the evolved mutant I67Q/P440F/A442N/L443I is 95% (R)-selective in the reaction of this and other thioethers. Partial deconvolution of the (R)-selective mutant with generation of the respective four single mutants shows that all of them are (S)-selective, which points to pronounced synergism (cooperative nonadditivity) when they interact in concert. Complete deconvolution with formation of all combinatorial forms of the respective double and triple mutants allows the designed construction of a fitness landscape featuring all 24 upward pathways leading from WT to the (R)-selective quadruple mutant. In all 24 trajectories strong cooperative mutational effects were found as well, which indicates that such mutational changes in enzymes constitute nonlinear systems. A theoretical analysis based on induced fit docking explains many of the observed effects on a molecular level.

Diagnostic yield of transbronchial cryobiopsy in interstitial lung disease: a randomized trial.

BACKGROUND AND OBJECTIVE: Transbronchial lung biopsy (TBLB) is required for evaluation in selected patients with interstitial lung disease (ILD). The diagnostic yield of histopathologic assessment is variable and is influenced by factors such as the size of samples and the presence of crush artefacts left by conventional biopsy forceps. We compared the diagnostic yield and safety of TBLB with cryoprobe sampling versus conventional forceps sampling. METHODS: This randomized clinical trial analysed data for 77 patients undergoing TBLB for evaluation of ILD; patients were assigned to either a conventional-forceps group or a cryoprobe group. Two pathologists assessed the tissue samples and agreed on histopathologic diagnoses. We also compared the duration of procedures, complications and sample-quality variables. RESULTS: The most frequent diagnosis observed in the cryoprobe group was non-specific interstitial pneumonia. Histopathologic diagnoses were identified in more cases in the cryoprobe group (74.4%) than in the conventional-forceps group (34.1%) (P < 0.001), and the diagnostic yield was higher in the cryoprobe group (51.3% vs 29.1% in the conventional forceps group; P = 0.038). A larger mean area of tissue was harvested by cryoprobe (14.7 ± 11 mm(2) ) than by conventional forceps (3.3 ± 4.1 mm(2)) (P < 0.001). More grade 2 bleeding (not statistically significant) occurred in the cryoprobe group (56.4%) than in the conventional-forceps group (34.2%). No differences in other complications were observed. CONCLUSIONS: TBLB by cryoprobe is safe and potentially useful in the diagnosis of ILD. Larger multisite randomized trials are required to confirm the potential benefits of this procedure. Clinical trial registration at ClinicalTrials.gov: NCT01064609.

Many pathways in laboratory evolution can lead to improved enzymes: how to escape from local minima.

Directed evolution is a method to tune the properties of enzymes for use in organic chemistry and biotechnology, to study enzyme mechanisms, and to shed light on darwinian evolution in nature. In order to enhance its efficacy, iterative saturation mutagenesis (ISM) was implemented. This involves: 1) randomized mutation of appropriate sites of one or more residues; 2) screening of the initial mutant libraries for properties such as enzymatic rate, stereoselectivity, or thermal robustness; 3) use of the best hit in a given library as a template for saturation mutagenesis at the other sites; and 4) continuation of the process until the desired degree of enzyme improvement has been reached. Despite the success of a number of ISM-based studies, the question of the optimal choice of the many different possible pathways remains unanswered. Here we considered a complete 4-site ISM scheme. All 24 pathways were systematically explored, with the epoxide hydrolase from Aspergillus niger as the catalyst in the stereoselective hydrolytic kinetic resolution of a chiral epoxide. All 24 pathways were found to provide improved mutants with notably enhanced stereoselectivity. When a library failed to contain any hits, non-improved or even inferior mutants were used as templates in the continuation of the evolutionary pathway, thereby escaping from the local minimum. These observations have ramifications for directed evolution in general and for evolutionary biological studies in which protein engineering techniques are applied.

Enhancing the efficiency of directed evolution in focused enzyme libraries by the adaptive substituent reordering algorithm.

Directed evolution is a broadly successful strategy for protein engineering in the quest to enhance the stereoselectivity, activity, and thermostability of enzymes. To increase the efficiency of directed evolution based on iterative saturation mutagenesis, the adaptive substituent reordering algorithm (ASRA) is introduced here as an alternative to traditional quantitative structure-activity relationship (QSAR) methods for identifying potential protein mutants with desired properties from minimal sampling of focused libraries. The operation of ASRA depends on identifying the underlying regularity of the protein property landscape, allowing it to make predictions without explicit knowledge of the structure-property relationships. In a proof-of-principle study, ASRA identified all or most of the best enantioselective mutants among the synthesized epoxide hydrolase from Aspergillus niger, in the absence of peptide seeds with high E-values. ASRA even revealed a laboratory error from irregularities of the reordered E-value landscape alone.

Predicting nocturnal hypoventilation in hypercapnic chronic obstructive pulmonary disease patients undergoing long-term oxygen therapy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients are very sensitive to changes in pulmonary mechanics and central ventilation control during sleep and may develop significant gas exchange alterations with increased hypoxemia and hypercapnia. Oxygen therapy improves nocturnal desaturation but can worsen hypoventilation. OBJECTIVES: To analyze the prevalence of nocturnal hypoventilation (NHV) in hypercapnic COPD patients and to determine predictive factors for this phenomenon. METHODS: This was a prospective multicenter study which enrolled 80 clinically stable COPD patients with hypercapnic respiratory failure who fulfilled the conventional criteria for long-term oxygen therapy (LTOT). All patients had undergone pulmonary function testing, blood gas analysis, and respiratory polygraphy. Arterial blood gas samples were obtained while patients were awake and during sleep. NHV was considered when an increase in PaCO2 >10 mm Hg was observed in any nocturnal arterial blood gas sample as compared to the awake levels. RESULTS: Seventeen patients (21%) developed NHV. NHV was associated with the values of BMI, hemoglobin, hematocrits, DLCO, and PaO2 reached after oxygen administration. In the logistic regression analysis BMI (OR 1.26, 95% CI 1.068-1.481; p = 0.006) and the diurnal increase of PaO2 after O2 (OR 0.89, 95% CI 0.807-0.972; p = 0.010) were the variables that best discriminated with a sensitivity of 82% and a specificity of 78%. CONCLUSIONS: NHV is a relatively common finding in stable hypercapnic COPD patients undergoing LTOT and it is related to a higher BMI and lower PaO2 after oxygen administration.

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and ß-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.

Prediction of postoperative pulmonary complications in a population-based surgical cohort.

BACKGROUND: Current knowledge of the risk for postoperative pulmonary complications (PPCs) rests on studies that narrowly selected patients and procedures. Hypothesizing that PPC occurrence could be predicted from a reduced set of perioperative variables, we aimed to develop a predictive index for a broad surgical population. METHODS: Patients undergoing surgical procedures given general, neuraxial, or regional anesthesia in 59 hospitals were randomly selected for this prospective, multicenter study. The main outcome was the development of at least one of the following: respiratory infection, respiratory failure, bronchospasm, atelectasis, pleural effusion, pneumothorax, or aspiration pneumonitis. The cohort was randomly divided into a development subsample to construct a logistic regression model and a validation subsample. A PPC predictive index was constructed. RESULTS: Of 2,464 patients studied, 252 events were observed in 123 (5%). Thirty-day mortality was higher in patients with a PPC (19.5%; 95% [CI], 12.5-26.5%) than in those without a PPC (0.5%; 95% CI, 0.2-0.8%). Regression modeling identified seven independent risk factors: low preoperative arterial oxygen saturation, acute respiratory infection during the previous month, age, preoperative anemia, upper abdominal or intrathoracic surgery, surgical duration of at least 2 h, and emergency surgery. The area under the receiver operating characteristic curve was 90% (95% CI, 85-94%) for the development subsample and 88% (95% CI, 84-93%) for the validation subsample. CONCLUSION: The risk index based on seven objective, easily assessed factors has excellent discriminative ability. The index can be used to assess individual risk of PPC and focus further research on measures to improve patient care.

Directed evolution of an enantioselective epoxide hydrolase: uncovering the source of enantioselectivity at each evolutionary stage.

Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.

Assessment of a primary and tertiary care integrated management model for chronic obstructive pulmonary disease.

BACKGROUND: The diagnosis and treatment of patients with chronic obstructive pulmonary disease (COPD) in Spain continues to present challenges, and problems are exacerbated when there is a lack of coordinated follow-up between levels of care. This paper sets out the protocol for assessing the impact of an integrated management model for the care of patients with COPD. The new model will be evaluated in terms of 1) improvement in the rational utilization of health-care services and 2) benefits reflected in improved health status and quality of life for patients. METHODS/DESIGN: A quasi-experimental study of the effectiveness of a COPD management model called COPD PROCESS. The patients in the study cohorts will be residents of neighborhoods served by two referral hospitals in Barcelona, Spain. One area comprises the intervention group (n = 32,248 patients) and the other the control group (n = 32,114 patients). The study will include pre- and post-intervention assessment 18 months after the program goes into effect. Analyses will be on two datasets: clinical and administrative data available for all patients, and clinical assessment information for a cohort of 440 patients sampled randomly from the intervention and control areas. The main endpoints will be the hospitalization rates in the two health-care areas and quality-of-life measures in the two cohorts. DISCUSSION: The COPD PROCESS model foresees the integrated multidisciplinary management of interventions at different levels of the health-care system through coordinated routine clinical practice. It will put into practice diagnostic and treatment procedures that are based on current evidence, multidisciplinary consensus, and efficient use of available resources. Care pathways in this model are defined in terms of patient characteristics, level of disease severity and the presence or absence of exacerbation. The protocol covers the full range of care from primary prevention to treatment of complex cases.

The ADMIT series--issues in inhalation therapy. 2. Improving technique and clinical effectiveness.

Aerosol inhalation is considered the optimal route for administering the majority of drugs for the treatment of obstructive airways diseases. A number of Pressurised Metered-Dose and Dry Powder Inhalers are available for this purpose. However, inhalation of therapeutic aerosols is not without difficulty; it requires precise instructions on the inhalation manoeuvre, which is different from spontaneous normal breathing. Also, the characteristics of the inhaler device have to be suitable for the user. Available data indicate a frequent lack of knowledge demonstrated by health professionals and patients on the inhalation manoeuvre and handling of inhalers, resulting in a reduction of therapeutic benefit. This paper reviews the literature concerning the fundamental aspects of inhaler devices, inhalation manoeuvre and device selection, in an attempt to increase the knowledge of, and to optimise the clinical use of, therapeutic inhalers.

Constructing and analyzing the fitness landscape of an experimental evolutionary process.

Iterative saturation mutagenesis (ISM) is a promising approach to more efficient directed evolution, especially for enhancing the enantioselectivity and/or thermostability of enzymes. This was demonstrated previously for an epoxide hydrolase (EH), after five sets of mutations led to a stepwise increase in enantioselectivity. This study utilizes these results to illuminate the nature of ISM, and identify the reasons for its operational efficacy. By applying a deconvolution strategy to the five sets of mutations and measuring the enantioselectivity factors (E) of the EH variants, DeltaDeltaG( not equal) values become accessible. With these values, the construction of the complete fitness-pathway landscape is possible. The free energy profiles of the 5!=120 evolutionary pathways leading from the wild-type to the best mutant show that 55 trajectories are energetically favored, one of which is the originally observed route. This particular pathway was analyzed in terms of epistatic effects operating between the sets of mutations at all evolutionary stages. The degree of synergism increases as the stepwise evolutionary process proceeds. When encountering a local minimum in a disfavored pathway, that is, in the case of a dead end, choosing another set of mutations at a previous stage puts the evolutionary process back on an energetically favored trajectory. The type of analysis presented here might be useful when evaluating other mutagenesis methods and strategies in directed evolution.

Effect of incorrect use of dry powder inhalers on management of patients with asthma and COPD.

BACKGROUND: Incorrect usage of inhaler devices might have a major influence on the clinical effectiveness of the delivered drug. This issue is poorly addressed in management guidelines. METHODS: This article presents the results of a systematic literature review of studies evaluating incorrect use of established dry powder inhalers (DPIs) by patients with asthma or chronic obstructive pulmonary disease (COPD). RESULTS: Overall, we found that between 4% and 94% of patients, depending on the type of inhaler and method of assessment, do not use their inhalers correctly. The most common errors made included failure to exhale before actuation, failure to breath-hold after inhalation, incorrect positioning of the inhaler, incorrect rotation sequence, and failure to execute a forceful and deep inhalation. Inefficient DPI technique may lead to insufficient drug delivery and hence to insufficient lung deposition. As many as 25% of patients have never received verbal inhaler technique instruction, and for those that do, the quality and duration of instruction is not adequate and not reinforced by follow-up checks. CONCLUSIONS: This review demonstrates that incorrect DPI technique with established DPIs is common among patients with asthma and COPD, and suggests that poor inhalation technique has detrimental consequences for clinical efficacy. Regular assessment and reinforcement of correct inhalation technique are considered by health professionals and caregivers to be an essential component of successful asthma management. Improvement of asthma and COPD management could be achieved by new DPIs that are easy to use correctly and are forgiving of poor inhalation technique, thus ensuring more successful drug delivery.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

[Management according to the Global Initiative for Asthma guidelines of patients with near-fatal asthma reduces morbidity and mortality]. / El tratamiento según la guía de la Global Initiative for Asthma (GINA) reduce la morbimortalidad de los pacientes con asma de riesgo vital.

OBJECTIVE: To assess the effect of adequate outpatient care as defined by guidelines of the Global Initiative for Asthma (GINA) on the long-term outcome of near-fatal asthma. PATIENTS AND METHODS: Fifty-three patients who had experienced a near-fatal attack of asthma were treated according to the GINA guidelines and followed for a mean of 49 months (intervention group). Clinical and spirometric measurements corresponding to the periods before the attack (obtained retrospectively) and after the attack (obtained prospectively) were compared to measurements from 40 near-fatal asthma patients who had not been managed according to the GINA guidelines and who were followed for a mean of 51 months (historic control group). RESULTS: There were no deaths in the intervention group and 6 deaths (15%) in the control group (P=.005). The mean (SD) number of new near-fatal asthma attacks was significantly lower in the intervention group (0.17 [0.61]) than in the control group (1.6 [1]) (P< .001). Emergency visits following a near-fatal asthma attack decreased from 0.9 (1.8) to 0.3 (0.6) in the intervention group and hospital admissions decreased from 3.4 (5.1) to 0.5 (1.4) (P< .001). Eosinophil count decreased from 390 (411) x l0(9) cells/L to 159 (121) x l0(9) cells/L (P=.01) and forced expiratory volume in 1 second increased from 68% (23%) of predicted to 76% (20%) (P=.006). CONCLUSIONS: Management according to the GINA guidelines of patients who had experienced a near-fatal asthma attack was associated with a decrease in asthma morbidity and mortality.