How plants communicate using the underground information superhighway

The rhizosphere is a densely populated area in which plant roots must compete with invading root systems of neighboring plants for space, water, and mineral nutrients, and with other soil-borne organisms, including bacteria and fungi. Root-root and root-microbe communications are continuous occurrences in this biologically active soil zone. How do roots manage to simultaneously communicate with neighboring plants, and with symbiotic and pathogenic organisms within this crowded rhizosphere? Increasing evidence suggests that root exudates might initiate and manipulate biological and physical interactions between roots and soil organisms, and thus play an active role in root-root and root-microbe communication.     

Nordexfenfluramine causes more severe pulmonary vasoconstriction than dexfenfluramine

The anorectic agent dexfenfluramine (dex) causes the development of primary pulmonary hypertension in susceptible patients by an unknown mechanism. We compared the effects of dex with those of its major metabolite, nordexfenfluamine (nordex), in the isolated perfused rat lung and in isolated rings of resistance pulmonary arteries. Nordex caused a dose-dependent and more intense vasoconstriction, which can be inhibited by the nonspecific 5-hydroxytryptamine type 2 (5-HT(2)) blocker ketanserin. Similarly a rise in cytosolic calcium concentration ([Ca(2+)](i)) in dispersed pulmonary artery smooth muscle cells (PASMCs) induced by nordex could be prevented by ketanserin. Unlike prior observations with dex, nordex did not inhibit K(+) current or cause depolarization in PASMCs. Removal of Ca(2+) from the tissue bath or addition of nifedipine (1 microM) reduced ring contraction to nordex by 60 +/- 9 and 63 +/- 4%, respectively. The addition of 2-aminoethoxydiphenyl borate (2-APB), a blocker of store-operated channels and the inositol 1,4,5-trisphosphate receptor, caused a dose-dependent decrease in the ring contraction elicited by nordex. The combination of 2-APB (10 microM) and nifedipine (1 microM) completely ablated the nordex contraction. Likewise the release of Ca(2+) from the sarcoplasmic reticulum by cyclopiazonic acid markedly reduced the nordex contraction while leaving the KCl contraction unchanged. We conclude that nordex may be responsible for much of the vasoconstriction stimulated by dex, through the activation of 5-HT(2) receptors and that the [Ca(2+)](i) increase in rat PASMCs caused by dex/nordex is due to both influx of extracellular Ca(2+) and release of Ca(2+) from the sarcoplasmic reticulum.     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

The enzymatic basis for pesticide bioremediation

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.     

Comparison of Microscopy and Alamar Blue Reduction in a Larval Based Assay for Schistosome Drug Screening

Background

In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS) to identify hits amongst large compound libraries.

Methodology/Principal Findings

We have been carrying out low throughput (24-well plate) in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB), a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes.

Conclusion

We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.     

Population Structure With Localized Haplotype Clusters

We propose a multilocus version of F_ST and a measure of haplotype diversity using localized haplotype clusters. Specifically, we use haplotype clusters identified with BEAGLE, which is a program implementing a hidden Markov model for localized haplotype clustering and performing several functions including inference of haplotype phase. We apply this methodology to HapMap phase 3 data. With this haplotype-cluster approach, African populations have highest diversity and lowest divergence from the ancestral population, East Asian populations have lowest diversity and highest divergence, and other populations (European, Indian, and Mexican) have intermediate levels of diversity and divergence. These relationships accord with expectation based on other studies and accepted models of human history. In contrast, the population-specific F_ST estimates obtained directly from single-nucleotide polymorphisms (SNPs) do not reflect such expected relationships. We show that ascertainment bias of SNPs has less impact on the proposed haplotype-cluster-based F_ST than on the SNP-based version, which provides a potential explanation for these results. Thus, these new measures of F_ST and haplotype-cluster diversity provide an important new tool for population genetic analysis of high-density SNP data.GENOME-WIDE data sets from worldwide panels of individuals provide an outstanding opportunity to investigate the genetic structure of human populations (Conrad et al. 2006; International Hapmap Consortium 2007; Jakobsson et al. 2008; Auton et al. 2009). Populations around the globe form a continuum rather than discrete units (Serre and Paabo 2004; Weiss and Long 2009). However, notions of discrete populations can be appropriate when, for example, ancestral populations were separated by geographic distance or barriers such that little gene flow occurred.F_ST (Wright 1951; Weir and Cockerham 1984; Holsinger and Weir 2009) is a measure of population divergence. It measures variation between populations vs. within populations. One can calculate a global measure, assuming that all populations are equally diverged from an ancestral population, or one can calculate F_ST for specific populations or for pairs of populations while utilizing data from all populations (Weir and Hill 2002). One use of F_ST is to test for signatures of selection (reviewed in Oleksyk et al. 2010).F_ST may be calculated for single genetic markers. For multiallelic markers, such as microsatellites, this is useful, but single-nucleotide polymorphisms (SNPs) contain much less information when taken one at a time, and thus it is advantageous to calculate averages over windows of markers (Weir et al. 2005) or even over the whole genome. The advantage of windowed F_ST is that it can be used to find regions of the genome that show different patterns of divergence, indicative of selective forces at work during human history.Another measure of human evolutionary history is haplotype diversity. Haplotype diversity may be measured using a count of the number of observed haplotypes in a region or by the expected haplotype heterozygosity based on haplotype frequencies in a region. Application of this regional measure to chromosomal data can be achieved by a haplotype block strategy (Patil et al. 2001) or by windowing (Conrad et al. 2006; Auton et al. 2009).One problem with the analysis of population structure based on genome-wide panels of SNPs is that a large proportion of the SNPs were ascertained in Caucasians, potentially biasing the results of the analyses. Analysis based on haplotypes is less susceptible to such bias (Conrad et al. 2006). This is because haplotypes can be represented by multiple patterns of SNPs; thus lack of ascertainment of a particular SNP does not usually prevent observation of the haplotype. On a chromosome-wide scale, one cannot directly use entire haplotypes, because all the haplotypes in the sample will almost certainly be unique, thus providing no information on population structure. Instead one can use haplotypes on a local basis, either by using windows of adjacent markers or by using localized haplotype clusters, for example those obtained from fastPHASE (Scheet and Stephens 2006) or BEAGLE (Browning 2006; Browning and Browning 2007a).Localized haplotype clusters are a clustering of haplotypes on a localized basis. At the position of each genetic marker, haplotypes are clustered according to their similarity in the vicinity of the position. Both fastPHASE and BEAGLE use hidden Markov modeling to perform the clustering, although the specific models used by the two programs differ.Localized haplotype clusters derived from fastPHASE have been used to investigate haplotype diversity, to create neighbor-joining trees of populations, and to create multidimensional scaling (MDS) plots (Jakobsson et al. 2008). It was found that haplotype clusters showed different patterns of diversity to SNPs, while the neighbor-joining and MDS plots were similar between haplotype clusters and SNPs.In this work, we apply windowed F_ST methods to localized haplotype clusters derived from the BEAGLE program (Browning and Browning 2007a,b, 2009). We consider population-average, population-specific, and pairwise F_ST estimates (Weir and Hill 2002). Population-average F_ST''s either assume that all the populations are equally diverged from a common ancestor, which is not realistic, or represent the average of a set of population-specific values. This can be convenient in that the results are summarized by a single statistic; however, information is lost. A common procedure is to calculate F_ST for each pair of populations, and these values reflect the degree of divergence between the two populations. Different levels of divergence are allowed for each pair of populations but each estimate uses data from only that pair of populations. On the other hand, population-specific F_ST''s allow unequal levels of divergence in a single analysis that makes use of all the data.We compare results from the localized haplotype clusters to those using SNPs directly. The results of applying localized haplotype clusters to population-specific F_ST estimation are very striking, showing better separation of populations and a more realistic pattern of divergence than for population-specific F_ST estimation using SNPs directly. We also use BEAGLE''s haplotype clusters in a haplotype diversity measure and investigate the relationship between this measure of haplotype-cluster diversity and the recombination rate.     

Effect of normoxia and hypoxia on K(+) current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.     

Recent emergence of serogroup C meningococcal disease in Greece

The number of cases of meningococcal disease reported to the Meningitis Reference Laboratory in Athens rose dramatically in 1996-1997. The aims were (1) to determine if the increase was due to introduction of new strains, (2) to assess the geographic and age distribution of the cases, (3) to compare antibiotic sensitivity patterns of the current isolates with strains from the early 1990s. In 1993-1994, 15/19 (74%) of the cases for which information on age was available were in children < or = 5 years; in 1995-1997, 80/179 (45%) of cases were in children < or = 5 years and 99 (55%) in the older age range (P < 0.02). From 593 cases in 1993 1997, 214 (36%) isolates were available for characterisation. Serogroup B was predominant in the early 1990s, but by 1997, serogroup C accounted for 46/72 (64%) of isolates and serogroup B for 25/72 (35%). Serogroup B was predominant in children < or = 5 years (44/78, 56%) but only 19/99 (18%) of older children and adults (P=0.0000005). Sulfonamide resistance decreased from 10/22 (45%) in 1993-1994 to 27/192 (14%) in 1995-1997 (P<0.01). Multilocus enzyme electrophoresis of 70 strains obtained during this period identified the epidemic ET-15 clone in 24 (34.3)%. The profiles of the Greek ET-15 isolates were identical to C:2a:P1.2(P1.5) strains responsible for the epidemic in the Czech Republic which began in 1993. This genotype was not found in Greek strains isolated prior to 1993. We conclude that the increase in meningococcal disease is due to introduction of the epidemic serogroup C:2a:P1.2(P1.5) strain responsible for disease in the Czech Republic and Canada.