Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT

Quantifying the impact of runoff events on microbiological contaminant concentrations entering surface drinking source waters

Concentrations of microbiological contaminants in streams increase during rainfall-induced higher flow 'event' periods as compared to 'baseflow' conditions. If the stream feeds a drinking water reservoir, such periods of heightened pathogen loads may pose a challenge to the water treatment plant and subsequently a health concern to water consumers downstream. In order to manage this risk, it is desirable to first quantify the differences in surface water quality between baseflow and event conditions. The Event Mean Concentration (EMC) is a flow-weighted average concentration of a contaminant over the duration of a single event, proposed here as a standard parameter for quantifying the net effect of events on microbial water quality. Application of the EMC concept was assessed using flow and quality data for several events from an urbanised catchment. Expected mean EMCs were significantly larger than expected mean baseflow concentrations ( p-value≤0.012) for three microbial agents - Escherichia coli (13,000 [n = 7] v. 610 [n = 16] mpn/100 ml), Cryptosporidium (234 [n = 6] v. 51 [n = 16] oocysts/10 litres) and Campylobacter (48 [n = 5] v. 2.1 [n = 16] mpn/100 ml). These parameter estimates were complemented by estimating data variability and uncertainty in the form of second-order random variables. As such the results are in a format appropriate for potential use as components in probabilistic risk assessments evaluating the effect runoff events have on drinking water quality. © IWA Publishing 2005

Precipitation Effects on Microbial Pollution in a River : Lag Structures and Seasonal Effect Modification

Background: The river Gota Alv is a source of freshwater for 0.7 million swedes. The river is subject to contamination from sewer systems discharge and runoff from agricultural lands. Climate models projects an increase in precipitation and heavy rainfall in this region. This study aimed to determine how daily rainfall causes variation in indicators of pathogen loads, to increase knowledge of variations in river water quality and discuss implications for risk management. Methods: Data covering 7 years of daily monitoring of river water turbidity and concentrations of E. coli, Clostridium and coliforms were obtained, and their short-term variations in relation with precipitation were analyzed with time series regression and non-linear distributed lag models. We studied how precipitation effects varied with season and compared different weather stations for predictive ability. Results: Generally, the lowest raw water quality occurs 2 days after rainfall, with poor raw water quality continuing for several more days. A rainfall event of >15 mm/24-h (local 95 percentile) was associated with a three-fold higher concentration of E. coli and 30% higher turbidity levels (lag 2). Rainfall was associated with exponential increases in concentrations of indicator bacteria while the effect on turbidity attenuated with very heavy rainfall. Clear associations were also observed between consecutive days of wet weather and decreased water quality. The precipitation effect on increased levels of indicator bacteria was significant in all seasons. Conclusions: Rainfall elevates microbial risks year-round in this river and freshwater source and acts as the main driver of varying water quality. Heavy rainfall appears to be a better predictor of fecal pollution than water turbidity. An increase of wet weather and extreme events with climate change will lower river water quality even more, indicating greater challenges for drinking water producers, and suggesting better control of sources of pollution

Adubação orgânica na produção, rendimento e composição do óleo essencial da alfavaca quimiotipo eugenol Organic fertilization in the production, yield and chemical composition of basil chemotype eugenol

Este trabalho foi realizado na UFPR, município de Pinhais-PR, de outubro de 2005 a junho de 2006. Avaliou-se a resposta do Ocimum gratissimum à adubação orgânica com composto de esterco de carneiro, verificando a viabilidade dessa prática para o aumento no rendimento de biomassa, na quantidade e qualidade de seu óleo essencial. As mudas foram plantadas com espaçamento de 0,5 m entre plantas dentro das parcelas e 1 m entre as parcelas. Testaram-se 3 doses de adubação com composto orgânico (4; 8 e 12 kg m-2) e uma testemunha, com 5 repetições e 8 plantas por parcela. As colheitas foram realizadas aos 150 e 226 dias após o plantio, sendo avaliado o rendimento de massa fresca e massa seca total e de folhas, flores e caule, o teor de óleo essencial das folhas e flores e a sua composição por cromatógrafo a gás acoplado a um detector de massas (CG-MS). Houve diferença entre os tratamentos, quanto ao rendimento de biomassa na primeira colheita, onde o tratamento com 8 kg m-2 de composto orgânico foi superior à testemunha sem adubação, para a massa fresca de flores por planta e massa seca total e de flores por planta, mas não diferiu dos outros níveis de adubação. Na segunda colheita não houve diferença entre os tratamentos. Quanto ao rendimento de óleo essencial não houve diferença entre os tratamentos nas duas colheitas. Também não foram observadas grandes variações nos componentes do óleo essencial decorrente dos tratamentos aplicados. Entretanto, ocorreram variações na composição do óleo essencial quando extraído das folhas e das flores, sendo o teor médio de eugenol de 90,4% nas folhas e 80,8% nas flores, na colheita em março. Já na colheita em junho, houve uma redução do teor de eugenol nas folhas e uma elevação dos teores, principalmente de alfa trans trans farneseno, beta bisaboleno, beta cariofileno, germacrene D e alfa selineno.<br>This work was carried out at the Universidade Federal do Parana, Paraná State, Brazil, from October 2005 to June 2006. The effect of organic fertilization with sheep manure was evaluated on Ocimum gratissimum development and the viability of this practice was determined to increase biomass and essential oil yield and quality. The plants were spaced 0.5 m from each other in the experimental units which were separated in 1 m. Three rates of organic fertlization (4; 8 and 12 kg m-2) were compared to control (without fertilizers), with 5 replications and 8 plants by experimental unit. Plants were harvested 150 and 226 days after planting, and the total, leaves, flowers and stems fresh and dried biomass, essential oil yield from leaves and flowers and composition by GC/MS beign evaluated. There was significant difference among the treatments on biomass production at the first harvest, where the plants treated with 8 kg m-2 of organic fertilizer presented higher flowers fresh biomass, and flowers and total dry biomass than plants without fertilizer (control plants), but with no difference compared to other organic fertilization levels. At the second harvest there were no differences among treatments. Regarding the essential oil yield no differences were found comparing the organic fertilization levels in both harvest times. In addition, the essential oil composition was similar on plants from all treatments. However, some variations in composition were observed when the essential oil was extracted from flowers and leaves, where the eugenol percentage was 90.4% on leaves and 80.8% on flowers at the first harvest. At the second harvest, a reduction of eugenol content and an increase of alpha trans trans farnesene, beta bisabolene, beta cariophilene, germacrene D and alfa selineno was found

Application of Methyl-TROSY to a Large Paramagnetic Membrane Protein Without Perdeuteration: \u3csup\u3e13\u3c/sup\u3eC-MMTS-labeled NADPH-cytochrome P450 Oxidoreductase

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic 13C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction–oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized 13C-methyl probes were sufficiently mobile to allow for signal detection at 600 MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins

Online flow cytometry reveals microbial dynamics influenced by concurrent natural and operational events in groundwater used for drinking water treatment

Detailed measurements of physical, chemical and biological dynamics in groundwater are key to understanding the important processes in place and their influence on water quality - particularly when used for drinking water. Measuring temporal bacterial dynamics at high frequency is challenging due to the limitations in automation of sampling and detection of the conventional, cultivation-based microbial methods. In this study, fully automated online flow cytometry was applied in a groundwater system for the first time in order to monitor microbial dynamics in a groundwater extraction well. Measurements of bacterial concentrations every 15 minutes during 14 days revealed both aperiodic and periodic dynamics that could not be detected previously, resulting in total cell concentration (TCC) fluctuations between 120 and 280 cells mu L-1. The aperiodic dynamic was linked to river water contamination following precipitation events, while the (diurnal) periodic dynamic was attributed to changes in hydrological conditions as a consequence of intermittent groundwater extraction. Based on the high number of measurements, the two patterns could be disentangled and quantified separately. This study i) increases the understanding of system performance, ii) helps to optimize monitoring strategies, and iii) opens the possibility for more sophisticated (quantitative) microbial risk assessment of drinking water treatment systems