Bioinformatic Discovery of a Cambialistic Monooxygenase.

Dinuclear monooxygenases mediate challenging C-H bond oxidation reactions throughout nature. Many of these enzymes are presumed to exclusively utilize diiron cofactors. Herein we report the bioinformatic discovery of an orphan dinuclear monooxygenase that preferentially utilizes a heterobimetallic manganese-iron (Mn/Fe) cofactor to mediate an O2-dependent C-H bond hydroxylation reaction. Unlike the structurally similar Mn/Fe-dependent monooxygenase AibH2, the diiron form of this enzyme (SfbO) exhibits a nascent enzymatic activity. This behavior raises the possibility that many other dinuclear monooxygenases may be endowed with the capacity to harness cofactors with a variable metal content.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor.

Manganese cofactors activate strong chemical bonds in many essential enzymes. Yet very few manganese-dependent enzymes are known to functionalize ubiquitous carbon-hydrogen (C-H) bonds, and those that catalyze this important reaction display limited intrinsic reactivity. Herein, we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis requires manganese to functionalize a C-H bond possessing a bond dissociation enthalpy (BDE) exceeding 100 kcal/mol. Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site that utilizes a manganese ion at the locus for O 2 activation and substrate coordination. Accordingly, this enzyme represents the first documented Mn-dependent monooxygenase in biology. Related proteins are widespread in microorganisms suggesting that many uncharacterized monooxygenases may utilize manganese-containing cofactors to accomplish diverse biological tasks.

Postharvest Application of Acibenzolar-S-Methyl Activates Salicylic Acid Pathway Genes in Kiwifruit Vines.

The plant defence inducer Actigard® (acibenzolar-S-methyl [ASM]) is applied before flowering and after fruit harvest to control bacterial canker in kiwifruit caused by Pseudomonas syringae pv. actinidiae. Pre-flowering application of ASM is known to upregulate defence gene expression; however, the effect of postharvest ASM on defence gene expression in the vine is unknown. In this study, the expression of eight "defence marker" genes was measured in the leaves of Actinidia chinensis var. chinensis, "Zesy002," and Actinidia chinensis var. deliciosa, "Hayward," vines after postharvest treatment with ASM and/or copper. There were two orchards per cultivar with harvest dates approximately three weeks apart for investigating potential changes in responsiveness to ASM during the harvest period. In all trials, postharvest ASM induced the expression of salicylic-acid-pathway defence genes PR1, PR2, PR5, BAD, DMR6, NIMIN2, and WRKY70. Gene upregulation was the greatest at 1 day and 7 days after treatment and declined to the control level after 3 weeks. In "Zesy002", the ASM-induced response was greater at the early harvest site than at the late harvest site. This decline was concomitant with leaf yellowing and a reduction in RNA yield. Effects of postharvest ASM on gene expression did not persist into the following spring, nor were vines conditioned to respond more strongly to pre-flowering ASM application.

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta.

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C-H Bonds of Aromatic 2,5-Diketopiperazines.

Cytochrome P450 enzymes generally functionalize inert C-H bonds, and thus, they are important biocatalysts for chemical synthesis. However, enzymes that catalyze both aliphatic and aromatic hydroxylation in the same biotransformation process have rarely been reported. A recent biochemical study demonstrated the P450 TxtC for the biosynthesis of herbicidal thaxtomins as the first example of this unique type of enzyme. Herein, the detailed characterization of substrate requirements and biocatalytic applications of TxtC are reported. The results reveal the importance of N-methylation of the thaxtomin diketopiperazine (DKP) core on enzyme reactions and demonstrate the tolerance of the enzyme to modifications on the indole and phenyl moieties of its substrates. Furthermore, hydroxylated, methylated, aromatic DKPs are synthesized through a biocatalytic route comprising TxtC and the promiscuous N-methyltransferase Amir_4628; thus providing a basis for the broad application of this unique P450.

High-Yield Production of Herbicidal Thaxtomins and Thaxtomin Analogs in a Nonpathogenic Streptomyces Strain.

Thaxtomins are virulence factors of most plant-pathogenic Streptomyces strains. Due to their potent herbicidal activity, attractive environmental compatibility, and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the U.S. Environmental Protection Agency. However, the low yield of thaxtomins in native Streptomyces producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 after chromosomal integration. The production of thaxtomins and nitrotryptophan analogs was observed using liquid chromatography-mass spectrometry (LC-MS) analysis. When the engineered S. albus J1074 was cultured in the minimal medium Thx defined medium supplemented with 1% cellobiose (TDMc), the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than that in S. scabiei 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/liter. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in S. albus J1074 was capitalized on to produce one unnatural fluorinated analog, 5-fluoro-thaxtomin A (5-F-thaxtomin A), whose structure was elucidated by a combination of MS and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to produce thaxtomins and analogs thereof with high yield, fostering their agricultural applications.IMPORTANCE Thaxtomins are agriculturally valuable herbicidal natural products, but the productivity of native producers is limiting. Heterologous expression of the thaxtomin gene cluster in S. albus J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineered S. albus J1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products.

Environmental health needs and launching of an environmental health protection unit in Pakistan.

Pakistan is seriously confronted by many complex and difficult environmental challenges related to air, water, soil, forests and food including issues such as climate change. The close link between environment and health is neither well understood nor appreciated. The annual cost of environmental degradation in Pakistan has been estimated to be around US $4.0 billion orat least 6% of the country's GDP. Up to 35% of the burden of disease is attributable to environmental hazards and risk factors and most of this burden is preventable. A systematic process for identifying environmental health needs and issues as well as the efforts made by the government of Pakistan and the World Health Organization in establishing and launching an environmental health protection unit are described. Also presented are the mission, functions, structure (operational and logistical) and technical requirements as well as sustainability aspects of the environmental health protection unit.

Safe water supply in emergencies and the need for an exit strategy to sustain health gains: lessons learned from the 2005 earthquake in Pakistan.

The bacteriological quality of drinking-water supply of five major urban centres affected by the October 2005 earthquake in Pakistan were assessed in three phases: onset of emergency, during emergency response and post-emergency. A total of 1850 samples were randomly collected from the study area during each phase, and faecal coliforms were detected in 100%, 28% and 91% in Battagram, 81%, 22% and 77% in Mansehra, 100%, 27% and 92% in Rawalakot, 100%, 23% and 65% in Bagh and in 30%, 14% and 5% in Muzaffarabad respectively. Faecal contamination was high during the onset ofemergency and post-emergency phases in four out ofthe five surveyed towns. The organization of a timely emergency response intervention depends on the level of preparedness of local water-supply service providers as well as on their institutional capacities. Bacteriological water-quality improvements in emergencies may not be sustained unless complemented by a proper exit strategy.

POSSUM and P-POSSUM for risk-adjusted audit of patients undergoing emergency laparotomy.

BACKGROUND: The Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity (POSSUM) is a scoring system that is used widely to predict 30-day mortality and morbidity rates. The Portsmouth predictor modification (P-POSSUM) was developed to overcome the overprediction of mortality by POSSUM, especially in low-risk patients. In this prospective study, the validity of POSSUM and P-POSSUM was tested in patients undergoing emergency laparotomy in a referral hospital of a developing country. METHODS: Some 120 patients who underwent emergency laparotomy in a single unit were studied. Predicted morbidity and mortality rates were calculated by POSSUM and P-POSSUM equations using both linear regression and the exponential methods of analysis. These were compared with actual outcomes. RESULTS: When the linear method of analysis was used POSSUM overpredicted morbidity, and there was a significant difference between the observed and predicted values (observed to expected (O : E) ratio 0.68). The prediction was more accurate when the exponential method was used (O : E ratio 0.91). POSSUM also significantly overpredicted mortality when analysed by the linear method (O : E ratio 0.39), but the prediction improved when exponential analysis was used (O : E ratio 0.62). Applying linear and exponential analyses for P-POSSUM, the O : E ratios for mortality were 0.66 and 0.88 respectively. CONCLUSION: If analysed correctly POSSUM is a good predictor of morbidity and mortality in patients undergoing emergency laparotomy. P-POSSUM predicts mortality equally well. Both equations may be used for risk-adjusted surgical audit of patients undergoing emergency laparotomy.

Modelo biopsicosocial para el afronte de pacientes farmacodependientes / Biopsychosocial model for therapeutic approach of drug dependent patients

El presente artículo describe el modelo biopsicosocial para pacientes fármacodependientes basado en intervenciones biológicas, psicológicas y sociales. Se describen las características y procedimientos específicos del modelo con la participación de la familia, terceros, el afronte interdisciplinario, la promoción de la participación no profesional, de organizaciones extrainstitucionales y el seguimiento a largo plazo.

The authors describe the Biopsychosocial Model por treatment of drug dependent patients based on biological, psychological and social interventions. The characteristics and specific procedures which includes family and third persons participation, interprofessional approach, promotion of non-professional participations and extrainstitutional organizations as well as the long term follow up are described.