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Aims: Evaluation if foliar spray of destructed cells of Spirulina platensis along with urea fertilizer can contribute to cotton (Gossypium barbadense L. cultivar Giza-94) plant growth, contents of photosynthetic pigments, seed cotton yield, seed index, earliness, lint percent, fineness (Micronair) and strength (Pressely). Study Design: Application of ascending rates of N fertilizer in 3 mainplot treatments and foliar spray with destructed cells of Spirulina platensis in 4 subplot treatments (Contained a non-treated control), with 3 replications. Place and Duration of Study: The Departments of Microbiology and Cotton Research, Sakha Agricultural Research Station, Kafr Elsheikh, Egypt (31º18′30″N & 30º48′14″E) during the two cotton cultivation seasons of years 2016 and 2017. Methodology: Urea (46% N) at 96, 144 or 192 kg N/ha was applied in two equal doses as main-plot treatments. Spirulina platensis was grown and total proteins, contents of 16 amino acids and Fe, K, Mn and P of dried destructed cells were determined and used for foliar application in the rates of 6, 12 or 18 g cells/ha, used as sub-plot treatments. Area of each replicated sub-plot was 12.5 m2. Calcium super phosphate (15% P2O5), potassium sulphate (48% K2O) and Insecticides (when necessary) were supplemented as common treatments. Results: Application of destructed Spirulina increased the plant leaf area, chlorophyll a, b, carotenoids and gibberellin contents, plant height, fruiting branches, open bolls/plant, seed cotton yield and seed index after ginning, fiber length, fineness (Micronair) and strength (Pressely) comparing to the corresponding non-sprayed plants. Urea at 192 kg N/ha along with destructed Spirulina exhibited superiority as enhancers of plant growth, yield and fiber qualities. Conclusion: The dual N/Spirulina combination expressed enhanced cotton growth, yield and qualities of fibers. The tested fertilization strategy is promising for enhanced cotton production economy.

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.)

We examined the bacterial endophyte-enriched root-associated microbiome within rice (Oryza sativa) 55 days after growth in soil with and without urea fertilizer and/or biofertilization with a growth-promotive bacterial strain (Rhizobium leguminosarum bv. trifolii E11). After treatment to deplete rhizosphere/rhizoplane communities, washed roots were macerated and their endophyte-enriched communities were analyzed by 16S ribosomal DNA 454 amplicon pyrosequencing. This analysis clustered 99,990 valid sequence reads into 1105 operational taxonomic units (OTUs) with 97% sequence identity, 133 of which represented a consolidated core assemblage representing 12.04% of the fully detected OTU richness. Taxonomic affiliations indicated Proteobacteria as the most abundant phylum (especially α- and γ-Proteobacteria classes), followed by Firmicutes, Bacteroidetes, Verrucomicrobia, Actinobacteria, and several other phyla. Dominant genera included Rheinheimera, unclassified Rhodospirillaceae, Pseudomonas, Asticcacaulis, Sphingomonas, and Rhizobium. Several OTUs had close taxonomic affiliation to genera of diazotrophic rhizobacteria, including Rhizobium, unclassified Rhizobiales, Azospirillum, Azoarcus, unclassified Rhizobiaceae, Bradyrhizobium, Azonexus, Mesorhizobium, Devosia, Azovibrio, Azospira, Azomonas, and Azotobacter. The endophyte-enriched microbiome was restructured within roots receiving growth-promoting treatments. Compared to the untreated control, endophyte-enriched communities receiving urea and/or biofertilizer treatments were significantly reduced in OTU richness and relative read abundances. Several unique OTUs were enriched in each of the treatment communities. These alterations in structure of root-associated communities suggest dynamic interactions in the host plant microbiome, some of which may influence the well-documented positive synergistic impact of rhizobial biofertilizer inoculation plus low doses of urea-N fertilizer on growth promotion of rice, considered as one of the world's most important food crops.

This study isolated and examined the performance of four selected strains of Rhizobium as growth enhancer inoculants of common bean (Phaseolus vulgaris) in saline- and drought-stressed fields located to the east and west of the Egypt Nile delta. Indigenous bean rhizobia were tested for salt tolerance by culturing and for taxonomic status by DNA analysis. Their nodulation and N2-fixation abilities under drought stress and persistence in biofertilizer formulations were evaluated, followed by assessment of their agronomic performance with common bean in 16 salt-affected, drought-stressed fields in combination with different doses of N-fertilizer applications in 2007 and 2008. Population dynamics studies with one model strain indicated good persistence in biofertilizer preparation. Inoculation with a test strain increased plant weight in the greenhouse from 2.718 to 3.314 g and four strains increased seed yield in saline/drought-stressed fields by 2.848⿿3.218 t ha⿿1 during seasons 2007 and 2008, respectively. Inoculation also increased straw production, harvest indices and the agronomic fertilizer N-use efficiency. The corresponding mean seed yields in adjacent uninoculated farmers⿿ fields exposed to the same intensity of aridification were 2.425 and 2.230 t ha⿿1, respectively. The study shows that locally-selected strains of rhizobia can be formulated into biofertilizers that significantly enhances seed yield and the agronomic N-use efficiency while providing a nature-based alleviation of the abiotic water deficit stress of intense aridification in saline- and drought-stressed fields.

This study examined whether rhizobia naturally associate with wheat (Triticum aestivum) and can enhance its production. Field-grown, surface-sterilized, macerated wheat roots were inoculated on legume roots to isolate rhizobial endophytes. Nodule occupants were purified, genotypically differentiated, and tested gnotobiotically for legume symbiotic effectiveness and wheat growth-promotion. Biofertilizer performance of 13 selected strains was evaluated on 8 wheat varieties in 24 field experiments in 10 counties of the Kafr El-Sheikh governorate in the Egypt Nile delta. Only inoculated clover (Trifolium alexandrinum) produced root nodules hosting Rhizobium leguminosarum bv. trifolii. Some were symbiotically effective on clover and promoted wheat growth under gnotobiotic conditions. Inoculation significantly increased wheat grain yield in 21, 23, and 20 field experiments under N-application of 60, 120, and 180 kg N/ha, respectively, with corresponding increases of 9.2, 18.9, and 22.5 % higher grain yield over the mean of farmers’ yields using the same varieties in adjacent fields. Unlike the harvest indices, straw yield and agronomic fertilizer N-use efficiency benefitted from inoculation. Rhizobial mixed-strain inocula frequently outperformed single-strain inocula. Inoculation did not adversely affect endomycorrhizal infection of field-grown wheat roots. Rhizobium naturally develops an endophytic association with wheat. Selected biofertilizer strains can enhance wheat production with acceptable input/output economy.

Colonization of the rhizoplane habitat is an important activity that enables certain microorganisms to promote plant growth. Here we describe various types of computer-assisted microscopy that reveal important ecological insights of early microbial colonization behavior within biofilms on plant root surfaces grown in soil. Examples of the primary data are obtained by analysis of processed images of rhizoplane biofilm landscapes analyzed at single-cell resolution using the emerging technology of CMEIAS bioimage informatics software. Included are various quantitative analyses of the in situ biofilm landscape ecology of microbes during their pioneer colonization of white clover roots, and of a rhizobial biofertilizer strain colonized on rice roots where it significantly enhances the productivity of this important crop plant. The results show that spatial patterns of immature biofilms developed on rhizoplanes that interface rhizosphere soil are highly structured (rather than distributed randomly) when analyzed at the appropriate spatial scale, indicating that regionalized microbial cell-cell interactions and the local environment can significantly affect their cooperative and competitive colonization behaviors.

This paper describes the utility of CMEIAS (Center for Microbial Ecology Image Analysis System) computer-assisted microscopy to extract data from accurately segmented images that provide 63 different insights into the ecophysiology of microbial populations and communities within biofilms and other habitats. Topics include quantitative assessments of: (i) morphological diversity as an indicator of impacts that substratum physicochemistries have on biofilm community structure and dominance-rarity relationships among populations; (ii) morphotype-specific distributions of biovolume body size that relate microbial allometric scaling, metabolic activity and growth physiology; (iii) fractal geometry of optimal cellular positioning for efficient utilization of allocated nutrient resources; (iv) morphotype-specific stress responses to starvation, environmental disturbance and bacteriovory predation; (v) patterns of spatial distribution indicating positive and negative cell–cell interactions affecting their colonization behavior; and (vi) significant methodological improvements to increase the accuracy of color-discriminated ecophysiology, e.g., differentiation of cell viability based on cell membrane integrity, cellular respiratory activity, phylogenetically differentiated substrate utilization, and N-acyl homoserine lactone-mediated cell–cell communication by bacteria while colonizing plant roots. The intensity of these ecophysiological attributes commonly varies at the individual cell level, emphasizing the importance of analyzing them at single-cell resolution and the proper spatial scale at which they occur in situ.

This study assessed the ability of biofertilizer inoculants containing Rhizobium leguminosarum bv. trifolii to enhance production of rice (Oryza sativa L.) under actual agricultural conditions in the Nile delta. Large-scale field experiments evaluated 5 rice varieties inoculated with 7 endophytic rhizobial strains during 5 growing seasons, including at sites ranked as the world’s highest in rice production. Inoculation with single strains or multi-strain consortia significantly increased grain yield in 19 of the 24 trials. By combining superior rhizobial inoculants with agricultural extension training, grain yield increased up to 47% in farmers’ fields, with an average increase of 19.5%. Data on rice straw production, harvest index and the agronomic fertilizer N-use efficiency also indicated positive agronomic benefits of rhizobial inoculation. These results establish the merit of deploying our biofertilization strategy using selected rhizobial strains to promote rice production capacity while reducing the need for additional chemical N-fertilizer inputs to maintain agricultural sustainability and acceptable production economy. Technology transfer of this important translational research can significantly help to alleviate hunger and meet the nutritional needs of many people in developing countries.

Field trials and laboratory experiments were undertaken for monitoring the relative susceptibility of four potato varieties namely; Atlas, Spunta, Simone and Nicola to potato tuber moth (PTM) Phthorimaea operculella (Zeller) infestations. Ex-periments were carried out for two summer planta-tions during 2006 and 2007. Results obtained indi-cate that Nicola potato variety was the least sus-ceptible one among the four tested varieties. The least infestation (5.5 & 7.17) as average number of larvae/100 leaves, percentages of tuber infestation (5% & 18%) and number of larve/100 tubers (5 & 20) was recorded for Nicola variety in the two sea-sons, respectively. The analysis of variance yield-ed significant differences among the four tested varieties. Positive and highly significant relation-ships were recorded between plant age and the corresponding infestation (r = +0.91 for 2006 and +0.93 for 2007). Chemical analysis for tubers showed a positive relationship between lowest sap content of free phenol and the least level of PTM infestations. That will explain why Nicola variety is considered as least susceptible to PTM infestation where it had the least concentration of free phe-nols (19.507 ppm.). The other verities which demonstrated high concentrations of free phenols harboured the highest levels of infestation with PTM. Positive and highly significant correlation were detected between free phenols content and both tuber infestation & larval content (r=+0.80 and +0.96) respectively.

A workshop titled "Application of Genotyping Methods to Assess Pathogen Risks from Cryptosporidium in Drinking Water Catchments" was held at the International Water Association biennial conference, Marrakech, Morocco, 23 September 2004. The workshop presented and discussed the findings of an interlaboratory trial that compared methods for genotyping Cryptosporidium oocysts isolated from feces. The primary goal of the trial and workshop was to assess the utility of current Cryptosporidium genotyping methods for determining the public health significance of oocysts isolated from feces in potable-water-supply watersheds. An expert panel of 16 watershed managers, public health practitioners, and molecular parasitologists was assembled for the workshop. A subordinate goal of the workshop was to educate watershed management and public health practitioners. An open invitation was extended to all conference delegates to attend the workshop, which drew approximately 50 interested delegates. In this report we summarize the peer consensus emerging from the workshop. Recommendations on the use of current methods by watershed managers and public health practitioners were proposed. Importantly, all the methods that were reported in the trial were mutually supporting and found to be valuable and worthy of further utility and development. Where there were choices as to which method to apply, the small-subunit ribosomal RNA gene was considered to be the optimum genetic locus to target. The single-strand conformational polymorphism method was considered potentially the most valuable for discriminating to the subtype level and where a large number of samples were to be analyzed. A research agenda for protozoan geneticists was proposed to improve the utility of methods into the future. Standardization of methods and nomenclature was promoted.

A non-isotopic single-strand conformation polymorphism ('cold' SSCP) technique has been assessed for the analysis of sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. Data are consistent with the ability of cold SSCP to identify intra-specific as well as inter-specific variability among Trichinella genotypes. The cold SSCP approach should be applicable to a range of other genetic markers for comparative studies of Trichinella populations globally.

In the present study, PCR-based single-strand conformation polymorphism (SSCP) analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was applied to the genetic characterisation of Dictyocaulus from red deer from New Zealand and to its differentiation from species of lungworm from cattle and other hosts. Based on SSCP profiles, Dictyocaulus individuals from red deer from different geographical localities in New Zealand could be readily distinguished from those representing other lungworms examined, irrespective of low-level sequence variability in the ITS-2 (0.4-2.6%) detectable among individuals. The ITS-2 of Dictyocaulus from red deer differed in sequence by approximately 7-35% from congeners from other cervid hosts, demonstrating that this parasite is genetically distinct from other species of Dictyocaulus for which ITS-2 sequence data are presently available. The results emphasize the need for a large-scale molecular systematic study of Dictyocaulus specimens from various species of cervid and other ruminant hosts and the usefulness of mutation scanning for taxonomic, epidemiological and population genetic investigations.

Nucleotide variation in a portion of the mitochondrial cytochrome c oxidase subunit1 (cox1) gene from asexual stages of bucephalids of southern Australian scallops (Chlamys asperrima, Chlamys bifrons and Pecten fumatus) was investigated using a mutation scanning-sequencing approach. Single-strand conformation polymorphism (SSCP) analysis revealed three main profile types (A, B and C) for parasites isolated from scallops. Sequence analysis revealed that samples represented by profiles B and C had a high degree (97.3%) of sequence similarity, whereas they were approximately 21% different in sequence from those represented by profile A. These findings suggested that at least two types or species (represented by profile A, or profile B or C) of bucephalid infect scallops, of which both were detected in South Australia, while only one was found in Victoria. The prevalence of bucephalids (and their SSCP haplotypes) appeared to differ among the three species of scallop in South Australia as well as between the two scallop species in Victoria, indicating a degree of host specificity. Adult bucephalids were collected from Eastern Australian Salmon (Arripis trutta), in an attempt to match them with the asexual stages from the scallop hosts. Neither of the two taxa of adult bucephalid (Telorhynchus arripidis and an un-named Telorhynchus species) shared SSCP profiles with the bucephalids from scallops, but were genetically similar, suggesting that the asexual stages from scallops may represent the genus Telorhynchus. This study, which assessed nucleotide sequence variation in a portion of the mitochondrial cox1 gene for bucephalids found in scallops and arripid fish, illustrates the usefulness of the mutation scanning approach to elucidate complex life-cycles of marine parasites.

Some species of Lactobacillus are of major industrial and health significance as fermenting agents in the manufacturing of food products, as food preservatives, as "probiotic" bacteria or as vaccine delivery vehicles. In spite of their importance, there is a paucity of published information on their genome organization and structure. In this study, a combination of pulsed field gel electrophoresis (PFGE) and hybridization approaches was used to investigate the genome of L. gasseri neotype strain ATCC33323. PFGE analysis of chromosomal DNA (after digestion with the rare-cutting restriction enzymes I-CeuI, CspI, SmaI, ApaI, and SgrAI) allowed the chromosome size of L. gasseri to be estimated at 1.96 Mbp, and also revealed the presence of a linear plasmid of 48.5 kbp. A physical map of the L. gasseri chromosome, containing 6 sites for the enzymes I-CeuI and 12 for CspI, was constructed. Placed on the map were the genes dnaA and gyrB (usually located close to the origin of replication on the bacterial chromosome) and 18 ribosomal RNA (rrn) genes. Mapping analysis also revealed that the chromosome contained six rrn operons, and that one of them was inverted in orientation with respect to the others. Each rrn operon contained a single copy of each of the three rrn genes, 23S rRNA (rrl), 16S rRNA (rrs) and 55 rRNA (rrf) gene. The constructed physical map should be a useful foundation for genomic and genetic studies of the lactobacilli and provides a platform for applied research, such as the engineering of Lactobacillus strains with improved characteristics for industrial and probiotic applications.

Rice (Oryza sativa L.) is one of the world's most important crops. The present investigation was designed to assess the range of growth-promoting activities of various diazotrophic bacteria on rice seedling vigor, its carryover effect on straw and grain yield, and the persistence of an inoculant strain on rice roots under greenhouse conditions. Growth responses to inoculation exhibited bacterial strain-rice variety specificity that were either stimulatory or inhibitory. Growth responses included changes in rates of seedling emergence, radical elongation, height and dry matter, plumule length, cumulative leaf and root areas, and grain and straw yields. Most notable were the inoculation responses to Rhizobium leguminosarum bv. trifolii E11 and Rhizobium sp. IRBG74, which stimulated early rice growth resulting in a carryover effect of significantly (P = 0.05) increased grain and straw yields at maturity, even though their culturable populations on roots diminished to below detectable values at 60 d after planting. The test strains were positive for indole-3-acetic acid production in vitro, but only some reduced acetylene to ethylene in association with rice under laboratory growth conditions. These studies indicate that certain strains of nonphotosynthetic diazotrophs, including rhizobia, can promote growth and vigor of rice seedlings, and this benefit of early seedling development can carryover to significantly increased grain yield at maturity.

Summary: Purpose: Proton magnetic resonance spectroscopy (MRS) was used to identify specific in situ metabolic markers for seizures and seizure-induced neuronal damage. Kainic acid (KA)-induced seizures lead to histopathologic changes in rat brain. The protective effect of cycloheximide treatment against neuronal damage caused by KA-induced seizures was studied, using in situ proton MRS imaging technique. Methods: Rats were pretreated with placebo or cycloheximide 1 h before KA injection. Rat brains (n = 25) were scanned at the level of the hippocampus before, during, and 24 h after seizures. Spectra were recorded and the relative ratios of N-acetylaspartate (NAA), choline (cho), and lactate (Lac) to creatine (Cr) were calculated and compared between groups. Results: A significant increase in Lac ratios was observed in KA-treated rats during and 24 h after seizure onset and this increase was prevented by cycloheximide pretreatment. NAA ratios were significantly higher during the ictal phase following KA treatment and this effect was not affected by cycloheximide pretreatment. Nissl staining confirmed previously reported prevention of KA-induced neuronal loss in CA1 and CA areas of the hippocampus by cycloheximide pretreatment. Conclusions: Our results suggest that in situ Lac increase is a marker of seizure-induced neuronal damage, whereas N-acetylaspartate (NAA) changes during and after status epilepticus may be a reflection of neuronal activity and damage, respectively.

Gangliogliomas are generally low grade neoplasms composed of mixtures of neoplastic glial and neuronal elements whose origin and exact nature are still controversial. We studied a series of 60 intracranial gangliogliomas looking for coexistent cortical architectural abnormalities (cortical dysplasia, microdysgenesis) and to determine if tumor behavior correlates with MIB1 (marker of cellular proliferation) labeling. The patients included 34 males and 26 females who ranged in age from 6 months to 55 years (mean 20 years). Thirty-eight tumors (63%) were located in the temporal lobe and 6 (10%) in the frontal lobe. Fifty-four patients (90%) presented with seizures (most with intractable epilepsy) and the duration of seizures ranged from 1 to 38 years (mean 14 years). In all cases, the predominant glioma component resembled a low grade fibrillary astrocytoma. In 14 tumors (23%), an oligodendroglial component was present. In one case, the glial component resembled an anaplastic astrocytoma. The tumors were characterized variously by perivascular chronic inflammation (N = 45, 75%), vascular proliferation (N = 36, 60%), granular bodies (N = 54, 90%), binucleated neurons (N = 36, 60%), calcification (N = 28, 47%), and cystic degeneration (N = 26, 43%). Meningeal involvement by tumor was observed in five (8%) cases. In 38 patients, sufficient tissue was resected to evaluate for the presence of concomitant cortical architectural abnormalities. Cortical architectural abnormalities were identified near to but clearly separate from the tumor in 19 (50%) patients. Only four patients including the anaplastic tumor died with tumor progression. MIB1 indices (positive tumor cells/1,000 tumor cells counted) in 54 cases ranged from 0 to 10.2 (mean 1.1 ± 1.0). Mortality did not reliably correlate with the MIB1 index; however, the highest index was observed in the anaplastic tumor. The high incidence of associated architectural abnormalities suggests that gangliogliomas may arise on a maldevelopmental basis. Although generally benign and slow-growing tumors, as indicated by the generally low MIB1 indices, gangliogliomas can rarely behave in a malignant fashion. It is not possible to reliably predict prognosis on the basis of histologic features or MIB1 immunoreactivity.

The vision of self-fertilizing crops contributed to the euphoria created by the emergence of biotechnology and the Green Revolution. In his 1970 Nobel Peace Prize lecture, Norman Borlaug highlighted the need to extend the range of symbioses to include nitrogen-fixing bacteria, such as rhizobia, and cereals to sustain the Green Revolution. He went on to acknowledge that even though high-yielding dwarf rice and wheat varieties were the catalysts that had ignited the Green Revolution, chemical fertilizers were the fuel that gave it thrust [11]. Since then, there have been extensive discussions on the prospects of establishing such novel symbiotic systems including research plans for their implementation. However, it is now clear that the energy required for the reduction of nitrogen to ammonia in nitrogen fixation is not greater than that required for the production of ammonia by reduction of nitrate, the main form of nitrogen assimilated by plants. Consequently, cereals such as rice would not likely suffer any significant energy penalty if they were supporting nitrogen fixation [12]. A huge amount of natural gas is consumed in the synthesis of nitrogenous fertilizer as anhydrous ammonia. In addition, this industrial process produces carbon dioxide, the main cause of greenhouse effect and global warming. The industrial production of nitrogenous fertilizer is also expensive, and in developing countries, the additional costs often exceed the means of low-income farmers, limiting the yield potential of their crops. Once chemical fertilizers are applied, additional residual problems can arise. Roughly one third of the nitrogen fertilizer applied is actually used by the crop. The nonassimilated nitrogen may result in nitrate (NO3 ) contamination of groundwater [13], posing a serious health hazard. In addition, excess nitrogen can also lead to soil acidification [14] and increased denitrification resulting in higher emission of nitrous oxide (N2O), another potent greenhouse gas that may exacerbate global warming [15]. Therefore, cropping j165

Full-length genes representing different isoforms of the ubiquitin-conjugating enzyme UBC-2 were isolated from Oesophagostomum dentatum, cloned and sequenced. The alignment of their sequences (designated Od-ubc-2.1 to Od-ubc-2.3) revealed nucleotide variation at three positions within the predicted open reading frame of 444 bp. Substitutions were at positions 141 (A--G), 142 (A--G) and 296 (T--C). Both former substitutions resulted in amino acid changes from a glycine residue to an arginine residue, whereas the latter resulted in a change from isoleucine to threonine. Comparison of predicted OD-UBC-2 with UBC-2 (protein) homologues/orthologues from 12 other species representing nematodes, Drosophila melanogaster, Saccharomyces cerevisiae, mice and humans revealed identities between species varying from 77 to 100% at the amino acid level, and motifs associated with protein conformation and function were identified. While the function of a representative ubc-2 gene from O. dentatum could not be established in C. elegans, it is likely to play a key role in the catabolism of proteins and in the development of O. dentatum.

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp- tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 10 10 rhizobia per cm 3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp -tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.

Cryptosporidium oocyst DNA samples (n=80) from humans with cryptosporidiosis in Australia and the UK were characterized genetically and categorized by capillary electrophoretic (CE) analysis of part of the small subunit gene (pSSU; approximately 300bp) and second internal transcribed spacer (pITS-2; approximately 230bp) of nuclear ribosomal DNA. The amplicons were heat denatured and subjected to capillary electrophoresis in LPA matrix (Amersham) in a MegaBACEtrade mark 1000 system (Amersham). The chromatograms captured were stored electronically and then analysed using MegaBACEtrade mark Fragment Profiler software. Using reference DNA control samples representing Cryptosporidium hominis and Cryptosporidium parvum, particular peaks in the profiles were defined for their specific identification and differentiation. The two species could be readily differentiated based on their profile in the pSSU, the peak differences being associated with a nucleotide difference of1.7%. While no variation was detectable in the pSSU profiles within each species, significant intraspecific variability in the positions of peaks in the pITS-2 chromatograms was displayed. For the 80 samples subjected to CE analysis of the pITS-2, four different genetic variants (genotypes) were detected within C. hominis and seven within C. parvum. Based on CE analysis of either pSSU and pITS-2 amplicons, it was readily possible to detect both species in 'mixed samples'. This CE method is time- and cost-effective, and may find applicability as a tool for the high throughput analysis of oocyst DNA samples for epidemiological surveys and for the monitoring of cryptosporidiosis outbreaks.

The combination of PFGE and hybridization approaches was used to study the genome ofLactobacillus acidophilusneotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI,NotI,CspI,SmaI,ApaI andSgrAI allowed the size of the circular chromosome ofL. acidophilusto be estimated at 2·061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (∼0·05–4·3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses ofL. acidophilusDNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of fourrrnoperons (rrnA–D) on the chromosome, each containing a single copy of each of the threerrngenes 16S (rrl), 23S (rrs) and 5S (rrf). OperonrrnDwas inverted in orientation with respect to the others and contained a long 16S–23S intergenic spacer region with tRNAIleand tRNAAlagenes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies ofLactobacillusspecies and for improving industrial and probiotic strains.

A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets.

A simple, non-isotopic PCR-based single-strand conformation polymorphism (‘cold SSCP’) method is described which allows the efficient detection of genetic variation among and within genotypes of Cryptosporidium parvum . This low cost approach has important advantages over other ‘genotyping’ methods and is applicable to a wide range of genetic loci and organisms.

A mutation scanning-selective sequencing approach was employed for the genotypic identification and differentiation of Cryptosporidium parvum isolates. Genomic DNA samples (n=158) from Cryptosporidium oocysts from humans with clinical cryptosporidiosis (following recent foreign travel or during different outbreaks) in the UK were subjected to PCR-based single-strand conformation polymorphism (SSCP) analysis of a heat shock protein 70 gene region (p-hsp70; 448 bp). Samples representing different SSCP profiles were then subjected to sequencing. The analysis allowed the classification of 149 of the 158 samples as type-1 ( approximately 49%) or type-2 ( approximately 46%); amplicons from the remaining nine samples were consistent in size with p-hsp70 but represented non-specific, faecal contaminants. The percentages reflected those of a previous study in the UK for autochtonous, sporadic cases of cryptosporidiosis ( approximately 49% for type-1 and approximately 47% for type-2; n approximately 4000), but contrasted another survey of sporadic cases where type-2 dominated ( approximately 62%; n approximately 1000). The ability of the present SSCP-sequencing approach to accurately screen for C. parvum genotypes and to reliably discern erroneous amplicons has significant implications for the accurate diagnosis and monitoring of cryptosporidiosis and for population genetic studies.

Using a single-strand conformation polymorphism-based approach, we investigated nucleotide variation in part of the first internal transcribed spacer (pITS-1) of nuclear ribosomal DNA within and among a large number of Ascaris individuals from humans and pigs from six endemic regions in China, and examined the frequency of the different genotypes of Ascaris in relation to host species and geographical origin. Five different SSCP genotypes (G1-G5) were recorded for human Ascaris (n = 486), of which three (genotypes G1-G3) were detected for pig Ascaris (n = 329). Of the five Ascaris genotypes detected, genotype G1 predominantly infected humans (approximately 63-74%) whereas genotype G3 infected mainly pigs (approximately 79-86%), indicating that each of these genotypes has a particular host affiliation. In contrast, the frequencies of the other three genotypes was substantially lower for each of the two host species. The findings also suggested that the rate of cross infection of Ascaris between humans and pigs is relatively low and that gene flow between the predominant genotypes is limited, consistent with previous proposals for endemic regions in other countries. While the nature and extent of nucleotide variation in the pITS-1 (and the proposal of host affiliated Ascaris populations) may relate to "introgression" or "lineage sorting and retention of ancentral polymorphism", other explanations are possible. Evidence of multiple pITS-1 sequence types in some Ascaris individuals representing particular genotypes (e.g., G2 and G5) may suggest hybridization between human- and pig-affiliated Ascaris. This aspect and the species status of Ascaris (from each host species) warrant future experimental testing, employing the pig/Ascaris model and the present electrophoretic approach.*

Recent studies indicate that rhizobia can associate intimately with various Gramineae. We describe a natural, beneficial association of R. leguminosarum by. trifolii and rice grown in Egyptian fields. A diversity of rice-adapted rhizobia was isolated from nodules of berseem clover inoculated with macerates of field-grown, surface-sterilized rice roots. Selected isolates exhibited plant growth-promoting activities on some rice varieties in gnotobiotic culture, and significant grain yield of rice in two cycles of field inoculation trials. These findings indicate their potential ability to enhance rice production in sustainable agriculture, and current studies should help to define how beneficial rhizobia can adapt to cereals and improve productivity in agronomic cropping systems.

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of 'excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P

Rhizobia can associate with various Gramineae. However, in order to provide a real-world benefit to agronomic cereal production, rhizobia must be able to successfully colonize grass roots amidst competition with other rhizosphere microorganisms under field conditions, and reduce fertilizer requirements while promoting plant growth without imposing pathological stress on the graminaceous host plant.

Fast-scan cyclic voltammetry at carbon fiber microelectrodes allows rapid (sub-second) measurements of dopamine release in behaving animals. Herein, we report the modification of existing technology and demonstrate the feasibility of making sub-second measurements of dopamine release in the caudate nucleus of a human subject during brain surgery. First, we describe the modification of our electrodes that allow for measurements to be made in a human brain. Next, we demonstrate in vitro and in vivo, that our modified electrodes can measure stimulated dopamine release in a rat brain equivalently to previously determined rodent electrodes. Finally, we demonstrate acute measurements of dopamine release in the caudate of a human patient during DBS electrode implantation surgery. The data generated are highly amenable for future work investigating the relationship between dopamine levels and important decision variables in human decision-making tasks.

The oxidized breakdown products of hemoglobin are important in the pathogenesis of cerebral vasospasm because of their effects on the endothelium and the smooth muscle of the arterial wall. Cytoskeletal changes in cultured vascular cells are sensitive indicators of oxidative injury. Cultured endothelial cells and smooth muscle cells showed a dose-related disruption of the cytoskeleton, particularly the F-actin and vimentin filaments, when exposed to 10(-5) M oxyhemoglobin. The cytoskeletal injury was prevented by the addition of 10(-3) M deferoxamine or 1% albumin. These experiments support a role for deferoxamine in the pharmacological treatment of vasospasm. Furthermore, cytoskeletal studies of cultured arterial endothelial and smooth muscle cells provide a novel in vitro approach by which to study the cellular mechanisms of oxidant injury initiated by the breakdown products of hemoglobin.

Extensive surgical resections of neocortical cerebral tissue (including hemispherectomies) from 13 infants and children with infantile spasms showed that 12 of 13 specimens contained either malformative and dysplastic lesions of the cortex and white matter (sometimes with associated hamartomatous proliferation of globular cells), or destructive lesions possibly acquired as a result of anoxic-ischemic injury, or a combination of the two. In brain tissue from 4 patients, coarse neuronal cytoplasmic fibrils resembling neurofibrillary tangles were seen in areas of dysplastic brain on silver-stained (Bielschowsky technique) sections. Immunohistochemical (immunoperoxidase) study of cortical lesions containing globular cells employing primary antibodies to glial fibrillary acidic protein and synaptophysin as markers of astrocytic and neuronal differentiation, respectively, revealed that many cells showed astrocytic and/or neuronal features, suggesting the local proliferation of primitive or multipotential neuroectodermal cells as one substrate for this seizure disorder. Morphological abnormalities of a severe degree and wide extent in the resected tissue (e.g., in one patient with hemimegalencephaly) often showed features to suggest that they may represent variants of tuberous sclerosis. These most likely result from abnormal movement and/or local proliferation of neuroectodermal precursors that have migrated from the germinal matrix to the cortical mantle. Cellular, molecular and neurophysiological study of these abnormalities is likely to yield information about basic molecular mechanisms of brain malformation and injury important in the pathogenesis of infantile spasms and other forms of focal or generalized epilepsy.

In a prospective, open, pilot study, the authors evaluated the effects of intraocular injection of streptokinase on intraocular fibrin. The study was composed of nine patients in whom fibrinous exudate developed after pars plana vitrectomy. There were seven male and two female patients with a mean age of 44 years (range, 27 to 65 years). Fibrinous exudates were seen 1 to 22 days after surgery with obstruction of the view of the posterior segment. Each patient received a single intraocular injection of 1000 IU of purified streptokinase. Four hours after the streptokinase injection, complete dissolution of the fibrin was noted in each patient There were no clinically detectable adverse effects from the intraocular administration of streptokinase. Streptokinase may be considered in the dissolution of intractable intraocular fibrinous exudates after vitrectomy.

Immunocytochemistry was used to study the expressions of glutamate receptor subunit proteins for NMDAR2A/B, NMDAR1 splice variants, and AMPA Glu-R2/3 in human brain resected for intractable epilepsy associated with cortical dysplasia. NMDAR2A/B intensely labeled dysplastic neurons showing staining in both the cell bodies and dendritic profiles. However, nondysplastic neurons were not immunoreactive to NMDAR2A/B. The antibody selective to NMDAR1 splice variants of NR1-1a, -1b, -2a, and -2b labeled dysplastic neurons, but few nondysplastic neurons. In contrast, the antibody to splice variants of NR1-1a, -1b, 2a, -2b, -3a, -3b, -4a, and -4b labeled both dysplastic and nondysplastic neurons. The different labeling patterns by these two antibodies indicate that variants of NMDAR1-3a, -3b, -4a, and -4b are present in nondysplastic neurons. Both dysplastic neurons and nondysplastic neurons were immunoreactive to AMPA GluR2/3, but denser immuno-reactivity was observed in dysplastic neurons. We also found that the locations of dysplastic neurons labeled by NMDAR2A/B were related to focal epileptic EEG seizure onsets or spiking and to focal behavioral seizure types. Our results suggest that there is hyperexcitability of dysplastic cortical regions, at least in part, from the presence of NMDAR2 subunits and selectively expressed NMDAR1 splice variants in dysplastic neurons.

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 This paper summarizes a multinational collaborative project to search for natural, intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact on plant growth, and exploit those combinations that can enhance grain yield with less dependence on inputs of nitrogen (N) fertilizer. Diverse, indigenous populations of Rhizobium leguminosarum bv. trifolii (the clover root-nodule endosymbiont) intimately colonize rice roots in the Egyptian Nile delta where this cereal has been rotated successfully with berseem clover (Trifolium alexandrinum L.) since antiquity. Laboratory and greenhouse studies have shown with certain rhizobial strain–rice variety combinations that the association promotes root and shoot growth thereby significantly improving seedling vigour that carries over to significant increases in grain yield at maturity. Three field inoculation trials in the Nile delta indicated that a few strain–variety combinations significantly increased rice grain yield, agronomic fertilizer N-use efficiency and harvest index. The benefits of this association leading to greater production of vegetative and reproductive biomass more likely involve rhizobial modulation of the plant’s root architecture for more efficient acquisition of certain soil nutrients [e.g. N, phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), zinc (Zn), sodium (Na) and molybdenum (Mo)] rather than biological N2 fixation. Inoculation increased total protein quantity per hectare in field-grown grain, thereby increasing its nutritional value without altering the ratios of nutritionally important proteins. Studies using a selected rhizobial strain (E11) indicated that it produced auxin (indoleacetic acid) and gibberellin [tentatively identified as gibberellin (GA 7 )] phytohormones representing two major classes of plant growth regulators. Axenically collected rice root exudate significantly enhanced E11’s production of this auxin. This strain extensively colonized the rice root surface under gnotobiotic culture conditions, producing distributions of spatial patchiness that would favour their localized erosion of the epidermal surface, colonization of small crevices at epidermal junctions as a possible portal to enter into the root, and quorum sensing of diffusible signal molecules indicating that their nearest bacterial neighbours are in close proximity in situ. Studies of selected rhizobial endophytes of rice indicated that they produced cell-bound cellulase and polygalacturonase enzymes that can hydrolyze glycosidic bonds in plant cell walls, and non-trifolitoxin bacteriocin(s) that can inhibit other strains of clover rhizobia. Strain E11 was able to endophytically colonize rice roots of varieties commonly used by Filipino peasant farmers, and also to stimulate genotype-specific growth-promotion of corn (Zea mays, maize) under field conditions. An amalgam of these results indicate some rhizobia have evolved an additional ecological niche enabling them to form a three-component life cycle including a free-living heterotrophic phase in soil, a N2-fixing endosymbiont phase within legume root nodules, and a beneficial growth-promoting endocolonizer phase within cereal roots in the same crop rotation. Our results further indicate the potential opportunity to exploit this newly described, plant�rhizobia association by developing biofertilizer inoculants that may assist low-income farmers in increasing cereal production (especially rice) with less fertilizer N inputs, fully consistent with both sustainable agriculture and environmental safety.

The interactions between two groups of rice endophytic bacterial strains and several rice cultivars were investigated. Various strains of Rhizobium leguminosarum bv. trifolii, originally isolated from rice plants grown in Egypt, comprise one group. The second group of bacterial strains was isolated from rice cultivars grown in the Philippines. Inoculation experiments with rice seedlings showed that specific isolates of these rice-associating bacteria could either promote, inhibit, or have no influence on rice plant growth. Furthermore, these growth effects were greatly influenced by the environmental growth conditions used. Studies to examine root colonisation patterns, using Rhizobium strains into which a plasmid expressing the green fluorescent protein has been placed, showed that the bacteria preferentially colonise rice seedling surfaces mainly in clumps. This occurs along grooves on the rice root surface, or at the emerging lateral root zones and at the root tips. However, rhizobia could also colonise intercellularly in lateral roots formed on the main roots near the culm region of the seedling. Under the growth conditions used, this occurred most frequently with strain R4 which multiplied and migrated to form long lines of individual bacterial cells along the inside of growing lateral roots. A bioassay to measure bacterial multiplication in rice leaves showed that the rice-associating strains can multiply and survive at different rates within these tissues. They were not, however, detected migrating into other parts of the leaf from the original site of pressure-infiltration, indicating that the bacterial ability to migrate within the lateral roots is not matched by a similar capacity in rice leaves. We suggest that some of these rice-associating bacteria possess important genes that enhance their ability to intimately colonise niches on and within rice tissues, and promote rice plant growth.

A full-length cDNA (Tv-stp-1) encoding a serine/threonine protein phosphatase (Tv-STP-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 951 nucleotides encoded a predicted protein of 316 amino acids (aa), containing the characteristic motif [LIVMN]-[KR]-G-N-H-E. Comparison with other sequences in non-redundant databases showed that Tv-STP-1 had significant identities/similarities to those from a range of metazoans and protists. Sequence similarity was most pronounced in the central region of the protein, in which the catalytic activity is inferred to be modulated by eight conserved residues (Asp 61, His 63, Asp 92, Asp 95, Asn 121, His 171, His 246 and Tyr 270), known to coordinate the binding of two metal ions (Mn2+ and Fe2+) in various organisms. Phylogenetic analyses of selected amino acid sequence data using the neighbor-joining and maximum parsimony methods revealed Tv-STP-1 to be most closely related to the glc seven-like phosphatases inferred for genes from the free-living nematode Caenorhabditis elegans and the parasitic nematode Oesophagostomum dentatum (order Strongylida). Comparison of the genomic organization of the full-length Tv-stp-1 gene with related molecules from other nematodes revealed substantial variation in the lengths and numbers of the exons and introns. The entire genes Tv-stp-1 (5041–5362 bp; 10 exons and 9 introns) and Od-mpp-1 (10,271 bp; 8 exons and 9 introns) from the parasitic nematodes T. vitrinus and O. dentatum were considerably longer than the C. elegans genes (1222–1603 bp; 3–7 exons and 2–6 introns). Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-stp-1 was transcribed in adult males of T. vitrinus, but not in the adult female or in any larval stages of this species. In spite of considerable variation at the genomic level, the findings of the present study suggest that there is relative conservation in features and function of the serine/threonine protein phosphatase characterized among T. vitrinus, O. dentatum and C. elegans, which should have implications for exploring molecular reproductive and developmental processes in strongylid nematodes of socio-economic importance.