Extending the substrate scope of a Baeyer–Villiger monooxygenase by multiple-site mutagenesis

Baeyer–Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer–Villiger monooxygenases with new specificities.     

The influence of phytohormones on the properties of wheat phospholipid monolayers at the water-air interface

The surface behaviour of monolayers of wheat phospholipids in the presence of phytohormones introduced into the water phase was studied using Langmuir's method. The phospholipids were extracted from the plasmalemma of non-embryogenic (NE) and embryogenic (E) calli initiated from two types of explant: immature inflorescences (inf) and embryos (emb). The surface properties were investigated in model systems of monolayers of mixed phospholipids with: 1) natural amphiphile composition (PL); 2) a determined hydrophobic part (16:0) and the natural percentage composition of the hydrophilic part (PPL); and 3) a determined hydrophilic part (PC) and the natural percentage composition of the hydrophobic part (HPL). The lower limit values of the molecular area (A(lim)) were observed for NE rather than for E monolayers in all the investigated systems (PL, PPL and HPL). The collapse pressure (pi(coll)) of the monolayer decreased in the order PPL>PL>HPL, indicating the high stability of monolayers containing saturated hydrocarbon chains. The injection of non-surface-active phytohormones into the water subphase and the subsequent formation of natural and also artificial phospholipid monolayers of E and NE causes a decrease in monolayer stability against collapse and molecular close packing. As a result of their amphipathic (hydrophilic-hydrophobic) structure, the surface properties of E phospholipids are probably optimal for these systems. The decreasing stability of the NE monolayer caused by the presence of the phytohormone seems to be advantageous in terms of membrane preparation for the differentiation process. All the investigated lipid monolayers (highly) stimulated the adsorption of indole-3-acetic acid (to the highest extent/degree) (among the examined phytohormones) from the subphase. Zearalenone had a significant influence on the surface properties of NE PPL and NE HPL monolayers. This may be connected with the ability of this phytohormone to affect the non-embryogenic structure of wheat. An anomalous temperature effect was observed in the presence of indole-3-acetic acid (IAA) in the bulk; phospholipid monolayers of embryogenic calli induced from embryos (E emb) when the temperature decreased from 25 to 15 degrees C. This phenomenon is ascribed to the dehydration of the polar groups in the monolayer     

How can malaria rapid diagnostic tests achieve their potential? A qualitative study of a trial at health facilities in Ghana

Malaria is still a major public health problem in Brazil, with approximately 306 000 registered cases in 2009, but it is estimated that in the early 1940s, around six million cases of malaria occurred each year. As a result of the fight against the disease, the number of malaria cases decreased over the years and the smallest numbers of cases to-date were recorded in the 1960s. From the mid-1960s onwards, Brazil underwent a rapid and disorganized settlement process in the Amazon and this migratory movement led to a progressive increase in the number of reported cases. Although the main mosquito vector (Anopheles darlingi) is present in about 80% of the country, currently the incidence of malaria in Brazil is almost exclusively (99,8% of the cases) restricted to the region of the Amazon Basin, where a number of combined factors favors disease transmission and impair the use of standard control procedures. Plasmodium vivax accounts for 83,7% of registered cases, while Plasmodium falciparum is responsible for 16,3% and Plasmodium malariae is seldom observed. Although vivax malaria is thought to cause little mortality, compared to falciparum malaria, it accounts for much of the morbidity and for huge burdens on the prosperity of endemic communities. However, in the last few years a pattern of unusual clinical complications with fatal cases associated with P. vivax have been reported in Brazil and this is a matter of concern for Brazilian malariologists. In addition, the emergence of P. vivax strains resistant to chloroquine in some reports needs to be further investigated. In contrast, asymptomatic infection by P. falciparum and P. vivax has been detected in epidemiological studies in the states of Rondonia and Amazonas, indicating probably a pattern of clinical immunity in both autochthonous and migrant populations. Seropidemiological studies investigating the type of immune responses elicited in naturally-exposed populations to several malaria vaccine candidates in Brazilian populations have also been providing important information on whether immune responses specific to these antigens are generated in natural infections and their immunogenic potential as vaccine candidates. The present difficulties in reducing economic and social risk factors that determine the incidence of malaria in the Amazon Region render impracticable its elimination in the region. As a result, a malaria-integrated control effort - as a joint action on the part of the government and the population - directed towards the elimination or reduction of the risks of death or illness, is the direction adopted by the Brazilian government in the fight against the disease.     

Excess mortality in persons with severe mental disorders: a multilevel intervention framework and priorities for clinical practice,policy and research agendas

Excess mortality in persons with severe mental disorders (SMD) is a major public health challenge that warrants action. The number and scope of truly tested interventions in this area remain limited, and strategies for implementation and scaling up of programmes with a strong evidence base are scarce. Furthermore, the majority of available interventions focus on a single or an otherwise limited number of risk factors. Here we present a multilevel model highlighting risk factors for excess mortality in persons with SMD at the individual, health system and socio‐environmental levels. Informed by that model, we describe a comprehensive framework that may be useful for designing, implementing and evaluating interventions and programmes to reduce excess mortality in persons with SMD. This framework includes individual‐focused, health system‐focused, and community level and policy‐focused interventions. Incorporating lessons learned from the multilevel model of risk and the comprehensive intervention framework, we identify priorities for clinical practice, policy and research agendas.     

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Species distribution and <Emphasis Type="Italic">in vitro</Emphasis> antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

Background  

In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.