Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing the catalytic efficiency of de novo enzymes by several orders of magnitude without relying on directed evolution and high-throughput screening. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (kcat/KM 125 M-1 s-1) and KE70 (kcat/KM 57 M-1 s-1), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display kcat/KM values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models, with catalytic contacts present as designed and transition-state root-mean-square deviations of ≤0.65 Å. Our work shows how ensemble-based design can generate efficient artificial enzymes by exploiting the true conformational ensemble to design improved active sites.

Design of efficient artificial enzymes using crystallographically-enhanced conformational sampling.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing catalytic efficiency of de novo enzymes to a level comparable to their natural counterparts without relying on directed evolution. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (kcat/KM 125 M-1 s-1) and KE70 (kcat/KM 57 M-1 s-1), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display kcat/KM values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models. Our work shows how computational design can generate efficient artificial enzymes by exploiting the true conformational ensemble to more effectively stabilize the transition state.

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

[Risk factors and clinical evolution of carbapenemase-producing Klebsiella pneumoniae infections in a university hospital in Spain. Case-control study]. / Factores de riesgo y evolución clínica de las infecciones causadas por Klebsiella pneumoniae productora de carbapenemasas en un hospital universitario de España. Estudio de casos y controles.

OBJECTIVE: Carbapenemase-producing Enterobacterias is a global health hazard due to their ease of transmission, difficulty of treatment, and their personal and economic impact. We analyze the factors associated with an increased risk of infection by Klebsiella pneumoniae carbapenemase-producing bacteria (KPC) and factors related to poor prognosis. METHODS: We designed a case-control study. KPC isolates were taken during an outbreak in a hospital in Madrid. A logistic regression was performed with the main variables. RESULTS: Sixteen cases of clinically documented infections were isolated. Overall mortality rates in the cases group was 25%. The most frequent location was blood (37.5%) followed by urine (25%). All but one were OXA-48. Regarding factors related to an increased risk of developing infection, only previous exposure to antibiotics presented statistical significance difference OR 13 (2.40-70.46). With respect to the overall mortality, the presence of pneumonia OR 25 (1.93-323.55) or the use of invasive mechanical ventilation was associated with greater risk 15 OR 33 (1.92-122.8) For attributable mortality only invasive ventilation had a significant association OR 18 (1.48-218.95). CONCLUSIONS: Exposure to previous antibiotics is an independent risk factor for developing KPC infection, adjusted for all other clinical and demographic variables. Risk factors such as the presence of pneumonia or the use of invasive mechanical ventilation were associated with a worse prognosis in terms of overall and attributable mortality.

Karyological characterization of the endemic Iberian rock lizard, Iberolacerta monticola (Squamata, Lacertidae): insights into sex chromosome evolution.

Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species-specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.

Discrimination and mental health in Ecuadorian immigrants in Spain.

BACKGROUND: The aim of this study was to examine the effects of ethnic discrimination on the mental health of Ecuadorian immigrants in Spain and to assess the roles of material and social resources. METHODS: Data were taken from the "Neighbourhood characteristics, immigration and mental health" survey conducted in 2006 in Spain. Psychological distress measured as "Possible Psychiatric Case" (PPC) was measured by the GHQ-28. A logistic regression was fitted to assess the association between PPC and discrimination. Interactions of discrimination with social and material resources were tested using product terms. RESULTS: Some 28% of the participants met our definition of PPC. About 20% of those who reported no discrimination were PPCs, rising to 30% of those who sometimes felt discriminated against and 41% of those who continually perceived discrimination. The OR for continuous discrimination was 12 (95% CI 3.5 to 40.3) among those with high financial strain, and 10 (2.4 to 41.7) when there was lack of economic support. Emotional support had an independent effect on PPC (OR 1.8, 95% CI 1.0 to 3.6, for those who reported having no friends). Social integration through a community group or association was positively related to the probability of being a PPC (OR 1.7, 95% CI 1.0 to 2.9). CONCLUSION: Ethnic discrimination is associated with psychological distress in these Ecuadorian immigrants in Spain. Discrimination effects may be exacerbated among those facing economic stress and those without economic support. These particularly vulnerable immigrants should be the subject of social and health interventions.

[Primary cardiac tumors: a review of 29 cases]. / Tumores cardíacos primários: revisión de 29 casos.

A series of 29 cardiac tumors were operated in Asturias Central Hospital between 1974 and 1994. There were 23 benign and 6 malignant tumors. Among the benign neoplasms, 22 were myxomas and 1 lipoma; there were 6 remalignant tumors, one pulmonary artery sarcoma, two rhabdomyosarcoma, one mesothelioma and two lymphomas. We review the clinical findings of these patients. Long-term results were excellent in patients with benign lesions, and only one recurrence was found. In patients with malignant tumors, surgical procedures were only palliative and aimed at prolonging life; hence, prognosis remained unchanged.

[A suicide attempt with oral verapamil: a case report]. / Intento de autólisis con verapamilo oral: presentación de un caso.

We report a case of a 29-years-old patient who tried to commit suicide by taking a high dose of verapamil (22 pills containing 240 mg, each, equivalent to 5.28 g). Ten hours after the ingestion he presented a complete atrioventricular heart block which was resolved with medical treatment presenting a normal ECG 36 hours later. To our knowledge this is the only published case of intoxication caused by such a high dose of verapamil.