Developing a taxonomic identification system of Phytophthora species based on microsatellites

BackgroundPhytophthora is the most important genus of the Oomycete plant pathogens. Nowadays, there are 117 described species in this genus, most of them being primary invaders of plant tissues. The different species are causal agents of diseases in a wide range of crops and plants in natural environments. In order to develop control strategies against Phytophthoraspecies, it is important to know the biology, ecology and evolutionary processes of these important pathogens.AimsThe aim of this study was to propose and validate a low cost identification system for Phytophthora species based on a set of polymorphic microsatellite (SSRs) markers.MethodsThirty-three isolates representing Phytophthora infestans, Phytophthora andina, Phytophthora sojae, Phytophthora cryptogea, Phytophthora nicotianae, Phytophthora capsici and Phytophthora cinnamomi species were obtained, and 13 SSRs were selected as potentially transferable markers between these species. Amplification conditions, including annealing temperatures, were standardized for several markers.ResultsA subset of these markers amplified in all species, showing species-specific alleles.ConclusionsThe adaptability and impact of the identification system in Colombia, an Andean agricultural country where different Phytophthora species co-exist in the same or in several hosts grown together, are discussed.     

Three binding sites in protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase tetramer assembly

Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a'. It is a ubiquitous protein folding catalyst that in addition functions as the beta-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) alpha(2)beta(2) tetramers. We report here that point mutations in the primary peptide substrate binding site in the b' domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a' domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a' domain mutations were combined with the b' domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b', and a', contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H alphabeta dimer and human alpha(2)beta(2) tetramer formation.     

Prioritizing emerging zoonoses in the Netherlands

Background

To support the development of early warning and surveillance systems of emerging zoonoses, we present a general method to prioritize pathogens using a quantitative, stochastic multi-criteria model, parameterized for the Netherlands.

Methodology/Principal Findings

A risk score was based on seven criteria, reflecting assessments of the epidemiology and impact of these pathogens on society. Criteria were weighed, based on the preferences of a panel of judges with a background in infectious disease control.

Conclusions/Significance

Pathogens with the highest risk for the Netherlands included pathogens in the livestock reservoir with a high actual human disease burden (e.g. Campylobacter spp., Toxoplasma gondii, Coxiella burnetii) or a low current but higher historic burden (e.g. Mycobacterium bovis), rare zoonotic pathogens in domestic animals with severe disease manifestations in humans (e.g. BSE prion, Capnocytophaga canimorsus) as well as arthropod-borne and wildlife associated pathogens which may pose a severe risk in future (e.g. Japanese encephalitis virus and West-Nile virus). These agents are key targets for development of early warning and surveillance.     

Caspase inhibition in apoptotic T cells triggers necrotic cell death depending on the cell type and the proapoptotic stimulus

CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.     

When can we measure stress noninvasively? Postdeposition effects on a fecal stress metric confound a multiregional assessment

Measurement of stress hormone metabolites in fecal samples has become a common method to assess physiological stress in wildlife populations. Glucocorticoid metabolite (GCM) measurements can be collected noninvasively, and studies relating this stress metric to anthropogenic disturbance are increasing. However, environmental characteristics (e.g., temperature) can alter measured GCM concentration when fecal samples cannot be collected immediately after defecation. This effect can confound efforts to separate environmental factors causing predeposition physiological stress in an individual from those acting on a fecal sample postdeposition. We used fecal samples from American pikas (Ochotona princeps) to examine the influence of environmental conditions on GCM concentration by (1) comparing GCM concentration measured in freshly collected control samples to those placed in natural habitats for timed exposure, and (2) relating GCM concentration in samples collected noninvasively throughout the western United States to local environmental characteristics measured before and after deposition. Our timed‐exposure trials clarified the spatial scale at which exposure to environmental factors postdeposition influences GCM concentration in pika feces. Also, fecal samples collected from occupied pika habitats throughout the species' range revealed significant relationships between GCM and metrics of climate during the postdeposition period (maximum temperature, minimum temperature, and precipitation during the month of sample collection). Conversely, we found no such relationships between GCM and metrics of climate during the predeposition period (prior to the month of sample collection). Together, these results indicate that noninvasive measurement of physiological stress in pikas across the western US may be confounded by climatic conditions in the postdeposition environment when samples cannot be collected immediately after defecation. Our results reiterate the importance of considering postdeposition environmental influences on this stress metric, especially in multiregional comparisons. However, measurements of fecal GCM concentration should prove useful for population monitoring within an eco‐region or when postdeposition exposure can be minimized.     

The role of two Trichoderma reesei xylanases in the bleaching of pine kraft pulp

Summary The two major xylanases of Trichoderma reesei with different pI values and pH optima were compared for increasing the bleachability of pine kraft pulp. The efficiencies of the two enzymes acting on pulp substrate were very similar in hydrolysis yield, extraction kappa number or final brightness value. Only slight synergism between the two enzymes was observed in both hydrolysis and bleaching tests. The pH optimum of the pI 5.5 xylanase was similar in pulp treatment and in the hydrolysis of isolated substrates, and the bleaching result also correlated well with the hydrolysis of pulp xylan. By contrast, the pI 9.0 xylanase acted differently on pulp than on isolated xylans at different pH values and the pH optimum on pulp was increased. The bleachability of pulp by the pI 9.0 xylanase was improved more than expected at pH 7.0, although the hydrolysis of pulp xylan was substantially decreased. A similar phenomenon was also observed when the hydrolysis was performed in water instead of buffer. It thus appears that the degree of hydrolysis needed to obtain improved bleachability with pI 9.0 xylanase can be minimized by proper adjustment of the hydrolysis conditions. Correspondence to: J. Buchert     

Decomposition of cellulose in soils

1. Cellulose decomposition in forest and orchard soils was investigated by studying the breakdown of boiled and washed cellophane in the soils and in vitro. Decomposition occurred from quick to slow in the order: orchard on clay soil, forest on clay soil, forest on sandy loam, and in the latter in the order: calcareous mull, acid mull and mor. 2. In the different forest soils which were investigated the rate of decomposition was parallel to their water capacity. It slowed down considerably when the water content of the soil decreased, especially after the wilting point was reached. 3. Of the fungi isolated from these soils, those from orchard soil — 5% to 50%Fusarium spp. — were among the fastest decomposers of cellulose. This agrees with, and may explain the high rate of decomposition in orchard soil. 4. Decomposition in pure culture is quicker than in soil. As filtersterilized soil extract checked the decomposition in pure culture, but heat-sterilized soil extract did not, an extractable but heat-sensitive substance may be one retarding factor.     

Do Characteristics of Parental Child Homicide in Sweden Fit Evolutionary Predictions?

Evolutionary models have been used to explain parental child homicide. One idea is that children with low fitness value to their parents will be less loved and cared for and therefore more at risk in conflict situations. It is then important to investigate if conflicts with the children are the major pattern in cases of parental child homicide. The aim of this study is to survey the background circumstances of parental child homicide in Sweden and relate them to the evolutionary model suggested. We more specifically investigate if the homicides occur in conflict situations with the child, the frequency of several victims (including the partner or former partner) and if there are differences in characteristics of homicides between stepparents and genetic parents. Our results show that parental child homicide is a heterogeneous phenomenon, where relatively few cases were the result of a conflict with the child‐victims. Instead severe conflicts between parents were the most common circumstance in which children were killed. Many children were victims of an extended suicide, which often included several members of the family. Step‐parents were more likely to kill children aggressively in conflicts with them than genetic parents. The complexity of the phenomenon suggests that an evolutionary model based upon a mechanism related to conflicts with the child‐victim has limited explanatory value on parental child homicide in general.     

Biotransformations in microstructured reactors: more than flowing with the stream?

The state of the art in the application of microstructured flow reactors for biocatalytic process research is reviewed. A microstructured reactor that is fully automated and analytically equipped presents a powerful screening tool with which to perform biocatalyst selection and optimization of process conditions at intermediary stages of process development. Enhanced mass transfer provided by the microstructured reactor can be exploited for process intensification, particularly during multiphase biocatalytic processing where mass transfer across phase boundaries is often limiting. Reversible immobilization of enzymes in microchannels remains a challenge for flexible realization of biotransformations in microstructured reactors. Compartmentalization in microstructured reactors could be useful in performing multistep chemoenzymatic conversions.     

siRNA release from gold nanoparticles by nanosecond pulsed laser irradiation and analysis of the involved temperature increase

Nanosecond pulsed laser irradiation can trigger a release of nucleic acids from gold nanoparticles, but the involved nanoeffects are not fully understood yet. Here we investigate the release of coumarin labeled siRNA from 15 to 30 nm gold particles after nanosecond pulsed laser irradiation. Temperatures in the particle and near the surface were calculated for the different radiant exposures. Upon irradiation with laser pulses of 4 nanosecond duration release started for both particle sizes at a calculated temperature increase of approximately 500 K. Maximum coumarin release was observed for 15 nm particles after irradiation with radiant exposure of 80 mJ cm^?2 and with 32 mJ cm^?2 for 30 nm particles. This corresponds to a temperature increase of 815 and 900 K, respectively. Our results show that the molecular release by nanosecond pulsed irradiation is based on a different mechanism compared to continuous or femtosecond irradiation. Local temperatures are considerably higher and it is expected that bubble formation plays a crucial role in release and damage to cellular structures.