Preserving US microbe collections sparks future discoveries.

Collections of micro-organisms are a crucial element of life science research infrastructure but are vulnerable to loss and damage caused by natural or man-made disasters, the untimely death or retirement of personnel, or the loss of research funding. Preservation of biological collections has risen in priority due to a new appreciation for discoveries linked to preserved specimens, emerging hurdles to international collecting and decreased funding for new collecting. While many historic collections have been lost, several have been preserved, some with dramatic rescue stories. Rescued microbes have been used for discoveries in areas of health, biotechnology and basic life science. Suggestions for long-term planning for microbial stocks are listed, as well as inducements for long-term preservation.

Fungal biological resources to support international development: challenges and opportunities.

Exploitation of microbes, especially fungi, has the potential to help humankind meet the UN's sustainable development goals, help feed the worlds growing population and improve bioeconomies of poorer nations. The majority of the world's fungal genetic resources are held in collections in developed countries, primarily within the USA, Europe and Japan. Very little capacity exists in low to middle income countries, which are often rich in biodiversity but lack resources to be able to conserve and exploit their own microbial resources. In this paper we review the current challenges facing culture collections and the challenges of integrating new approaches, the worth of collaborative networks, and the importance of technology, taxonomy and data handling. We address the need to underpin research and development in developing countries through the need to build 'in country' infrastructure to address these challenges, whilst tackling the global challenges to meet the requirements of the research community through the impacts of legislation and the Nagoya protocol on access to biological resources.

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters.

Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.

Ureteroarterial Fistula: A Diagnosis Which Is Not Always Black and White.

Ureteroiliac artery fistulas are a rare, life-threatening condition that requires a high index of suspicion for prompt diagnosis. Presurgical diagnosis is challenging as this condition can lie hidden despite advanced imaging modalities. We present two cases of patients presenting with gross hematuria and exsanguination in the setting of a ureteroiliac artery fistula. These cases highlight the difficulties in timely diagnosis and treatment in a multidisciplinary team.

Evidence for dominant suppression of repeat-induced point mutation (RIP) in crosses with the wild-isolated Neurospora crassa strains Sugartown and Adiopodoume-7.

A convenient assay to score repeat-induced point mutation (RIP) in Neurospora employs the erg-3 locus as a mutagenesis target. Using this assay we screened 132 wild-isolated Neurospora crassa strains for ability to dominantly suppress RIP. RIP was exceptionally inefficient in crosses with the wild isolates Sugartown (P0854) and Adiopodoume-7 (P4305), thereby suggesting the presence of dominant RIP suppressors in these strains. In other experiments, we found no evidence for dominant RIP suppression by the Spore killer haplotypes Sk-2 and Sk-3.

Visual comparison by preschool children: effects of age, sex, and cognitive style.

Differences in visual scanning by reflective and impulsive children can be detected as young as 3 years of age. Such differences are indicative of differences in information. Such differences emerge rapidly between the ages of 3 and 5 years, and females and reflective males develop faster than impulsive males.

Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa.

Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.

Molecular analysis of mutants of the Neurospora adenylosuccinate synthetase locus.

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.

The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research.

The Fungal Genetics Stock Center (FGSC) was established in 1960 to ensure that important strains used in early genetics research were available to subsequent generations of fungal geneticists. Originally, only mutant strains were held. At present, any organism that has had its genome sequenced is a genetic system and so the FGSC has added many new organisms. The FGSC is well integrated in its core community and, as research came to depend on cloned genes, vectors and gene libraries, the FGSC included these materials. When the community expanded to include plant and human pathogens, the FGSC adopted these systems as well. Wild isolates from around the world have also proven instrumental in answering important questions. The FGSC holds tremendous diversity of the Neurospora species, which form the core of the collection. The growth in the number of strains distributed illustrates the growth in research on fungi. Because of its position near the centre of the fungal genetics effort, the FGSC is also the first to see trends in research directions. One recent example is the 300% jump in requests for strains of Neurospora crassa carrying a mutation that makes them sensitive to high salt concentration. These strains were seldom requested over many years, but became among our most popular resources following the demonstration of their utility in studying fungicide resistance. This exemplifies why materials need to be preserved without regard to their immediate perceived value and reinforces the need for long-term support for preservation of a broad variety of genetic resources.

Characterization of genome plasticity in Ustilago hordei.

Southern-blot hybridization analysis was used to identify and quantify chromosome-length polymorphisms for ten linkage groups of 14 races of Ustilago hordei. The bands identified by the probes were shown to vary as much as hundreds of kilobase pairs, but the magnitude of the variability was typically 5-15% of the average size of all bands to which a particular probe hybridized. A filamentous morphology mutant, recovered following heat-shock treatment of a strain with the greatest number of chromosome bands, was shown to have suffered a 50-kb deletion in a 940-kb chromosome. The mutation to filamentous morphology, designated fil1-1, and the deletion, were shown to invariably cosegregate 2:2 with the wild-type (sporidial) morphology in an ordered tetrad. Genetic and physical analyses place the Fil1 locus and the deletion near the terminus of one arm of the 940-kb chromosome. These results suggest that deletions of this type may be one of the causes of chromosome-length polymorphisms observed in field isolates of U. hordei.

"A change of pace": an investigation of the salience of maternal temporal style in mother-infant play.

The salience of maternal temporal style of playful stimulation to 3- and 5-month-old infants during free play was investigated. 32 mother-infant dyads were videotaped for 8 min of interaction which consisted of 4 2-min phases of differing maternal temporal styles: natural (usual), slower-than-usual, return-to-usual, and faster-than-usual temporal styles. Multivariate analyses revealed that changes in the pacing of playful stimulation significantly affected several infant, maternal, and interactive variables. For both the 3- and 5-month-olds, the initial phase of natural temporal style emerged as the period of most positive interactions, and the faster play phase included more positive behaviors than the slower play phase. While most maternal behaviors were very similar in the 2 phases of natural temporal style, infant and some interactive variables failed to demonstrate a return-to-baseline effect. Patterns of differential responding suggested the contributions of possible sequencing effects, infant temporal estimation capacities, and expectancies for the appropriateness of pacing in playful exchanges.

Repeat-induced point mutation (RIP) in crosses with wild-isolated strains of Neurospora crassa: evidence for dominant reduction of RIP.

Seventy-one wild-isolated strains of Neurospora crassa were examined for their ability to support repeat-induced point mutation (RIP) in the erg-3 locus. RIP was exceptionally inefficient but detectable in crosses with the strain FGSC 430 from Adiopodoume, Ivory Coast. We could find no consistent differences in ascospore yields when wild isolates identified as "low-RIP" or "high-RIP" strains were crossed with strains bearing the segmental duplication Dp(IIIR > [I; II])AR17. This suggested that RIP may not be responsible for the barren phenotype of crosses involving segmental duplication strains.

Location of pathogenicity genes on dispensable chromosomes in Nectria haematococca MPVI.

Nectria haematococca MPVI can be found in many different biological habitats but has been most studied as a pathogen of pea (Pisum sativum). Genetic analyses of isolates obtained from a variety of biological sources has indicated that a number of genes control pathogenicity on pea but that one important PEa Pathogenicity (PEP) gene is PDA, which confers the ability to detoxify the pea phytoalexin pisatin. In these studies, all naturally occurring isolates that lacked PDA (i.e. Pda- isolates) and all Pda- progeny were essentially non-pathogenic on pea. However, we have demonstrated recently that Pda- mutants created by transformation-mediated gene disruptions, while having a modest reduction in virulence, and more virulent than any naturally occurring Pda- isolates. In addition we know that PDA genes are on dispensable (DS) chromosomes in this fungus. We believed that the gene disruption mutants have allowed the detection of other PEP genes that are present on the DS chromosomes along with PDA and that naturally occurring Pda- isolates usually lack this DS chromosome. This would explain why naturally occurring Pda- isolates are always low in virulence. We propose that the DS chromosomes in fungi are analogous to bacterial plasmids which allow those microorganisms to colonise different habitats, i.e. the DS chromosomes of Nectria haematococca contain genes that allow individual isolates of this broad host range pathogen to occupy different biological niches.

Differential regulation of monocyte matrix metalloproteinase and TIMP-1 production by TNF-alpha, granulocyte-macrophage CSF, and IL-1 beta through prostaglandin-dependent and -independent mechanisms.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) produced by monocytes are believed to be involved in the migration of these cells through the basement membrane and the ensuing destruction of connective tissue in chronic inflammatory lesions. Because monocytes encounter a variety of cytokines at these sites, we examined the effect of cytokines either alone or in combination on the production of monocyte MMPs and TIMP-1. TNF-alpha, granulocyte-macrophage-CSF (GM-CSF), or IL-1 beta when added individually enhanced the endogenous levels of 92-kDa gelatinase (MMP-9) and TIMP-1 but failed to induce interstitial collagenase (MMP-1). However, GM-CSF, when added with either TNF-alpha or IL-1 beta, induced MMP-1 and synergistically enhanced MMP-9 and TIMP-1. Th2 cytokines, such as IL-4, inhibited the induction of MMPs and TIMP-1 by TNF-alpha, GM-CSF, and IL-1. Cytokine stimulation of MMP-1 was due, at least in part, to an increase in the release of arachidonic acid and PG E2 (PGE2), because inhibition of MMP-1 by indomethacin could be reversed by exogenous PGE2. In contrast to MMP-1, cytokine stimulation of MMP-9 and TIMP-1 was unaffected by indomethacin. The PGE2-independent induction of monocyte MMP-9 and TIMP-1 by these cytokines differed from stimulation of MMP-9 and TIMP-1 by LPS, which is in large part PG-dependent. In addition, LPS stimulated higher levels of MMP-1 whereas cytokines induced higher levels of MMP-9 and TIMP-1. This is the first demonstration that monocyte MMP-1 can be induced by cytokines and that MMP-1, MMP-9, and TIMP-1 are differentially regulated by cytokines through PG-dependent and -independent mechanisms.

Electrophoretic karyotyping without the need for generating protoplasts.

Chromosome samples for pulsed-field electrophoresis have been prepared without first generating protoplasts. The technique involves treatment of intact, agarose-solidified cell material with protease in the presence of EDTA and SDS. Saccharomyces cerevisiae, Ustilago hordei, Tilletia caries, and T. controversa karyotypes are clearly resolved with this technique. Colonies of U. hordei and S. cerevisiae removed from the surface of agar-solidified media and prepared for PFGE by this abbreviated method also yield well resolved karyotypes.

Suppression of prostaglandin H synthase-2 induction in human monocytes by in vitro or in vivo administration of interleukin 4.

IL-4 is a potent modulator of monocyte function. Our previous studies demonstrated that the suppression of monocyte matrix metalloproteinase production by IL-4 is a result of its inhibition of PGE2 synthesis, which was attributed to an effect on prostaglandin synthase. Here we report on the in vitro and in vivo effects of IL-4 on monocyte prostaglandin H synthase-2 (PGHS-2) and its regulation by second messengers. Stimulation of monocytes with either LPS or Con A resulted in the induction of PGHS-2 which was significantly inhibited by IL-4. Inhibition of PGHS-2 mRNA and protein was detected at 0.05 to 0.1 ng/ml of IL-4 with substantial suppression at 10 to 20 ng/ml. If added later than 2 hr after LPS, IL-4 failed to suppress PGHS-2, indicating that IL-4 acted early in the signaling cascade. Moreover, the ability of exogenously added PGE2 or Bt2cAMP to restore PGHS-2 production in IL-4-treated monocytes further suggested early disruption of the pathway. The early event inhibited by IL-4 did not involve suppression of phospholipase activity, because LPS-induced arachidonic acid release was relatively unaffected by IL-4. Unlike PGHS-2, PGHS-1, the constitutively expressed PGHS, was not modulated by IL-4. Thus, IL-4 appears to selectively block PGHS-2 synthesis, thereby blocking subsequent steps in the pathway leading to the production of matrix metalloproteinases. In an extension of these findings, we examined peripheral blood monocytes from cancer patients undergoing IL-4 therapy. In these cells the induction of PGHS-2 expression by LPS was significantly reduced compared to that of monocytes obtained prior to IL-4 therapy. Although perhaps not relevant as an antitumor mechanism, these findings have important implications in defining the potent anti-inflammatory activities of IL-4 in vitro and in vivo.