Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

Non-exponential tolerance to infection in epidemic systems--modeling, inference, and assessment

The transmission dynamics of infectious diseases have been traditionally described through a time-inhomogeneous Poisson process, thus assuming exponentially distributed levels of disease tolerance following the Sellke construction. Here we focus on a generalization using Weibull individual tolerance thresholds under the susceptible-exposed-infectious-removed class of models which is widely employed in epidemics. Applications with experimental foot-and-mouth disease and historical smallpox data are discussed, and simulation results are presented. Inference is carried out using Markov chain Monte Carlo methods following a Bayesian approach. Model evaluation is performed, where the adequacy of the models is assessed using methodology based on the properties of Bayesian latent residuals, and comparison between 2 candidate models is also considered using a latent likelihood ratio-type test that avoids problems encountered with relevant methods based on Bayes factors.     

Effects of temperature, pH, water activity and CO2 concentration on growth of Rhizopus oligosporus NRRL 2710

AIMS: To investigate the effects of temperature, pH, water activity (aw) and CO2 concentration on the growth of Rhizopus oligosporus NRRL 2710. METHODS AND RESULTS: Hyphal extension rates from mycelial and spore inocula were measured on media with different aw (approximately 1.0, 0.98 and 0.96) and pH (3.5, 5.5 and 7.5) incubated at 30, 37 or 42 degrees C in atmospheres containing 0.03, 12.5 or 25% (v/v) CO2. The effects of environmental conditions on hyphal extension rate were modelled using surface response methodology. The rate of hyphal extension was very sensitive to pH, exhibiting a pronounced optimum at pH 5.5-5.8. The hyphal extension rate was less sensitive to temperature, aw or CO2, exhibiting maximum rates at 42 degrees C, a(w) approximately 1.0 and 0.03% (v/v) CO2. CONCLUSIONS: The fastest hyphal extension rate (1.7 mm h(-1)) was predicted to occur at 42 degrees C, pH 5.85, a(w) approximately 1.0 and 0.03% CO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is the first to model the simultaneous effects of temperature, pH, aw and CO2 concentration on mould growth. The information relates to tempe fermentation and to possible control of the microflora in Tanzanian cassava heap fermentations.     

Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.     

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F₁ families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F₂ mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F₂ family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F₂ family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.     

A Global Review of Causes of Morbidity and Mortality in Free-Living Vultures

Vulture species worldwide play a key role in ecosystems as obligate scavengers, and several populations have had precipitous declines. Research on vulture health is critical to conservation efforts including free-living vultures and captive breeding programs, but is limited to date. In this systematic review, we determined the reported causes of free-living vulture species morbidity and mortality worldwide. The most commonly reported cause of mortality was from toxins (60%), especially lead and pesticides, followed by traumatic injury (49%), including collisions with urban infrastructure and gunshot. Neglected areas of research in free-living vulture health include infectious diseases (16%), endocrine and nutritional disorders (6%), and neoplasia (<?1%). Almost half of the studies included in the review were conducted in either Spain or the USA, with a paucity of studies conducted in South America and sub-Saharan Africa. The highest number of studies was on Griffon (Gyps fulvus) (24%) and Egyptian vultures (Neophron percnopterus) (19%), while half of all vulture species had five or fewer studies. Future investigations on free-living vulture health should focus on neglected areas of research, such as infectious diseases, and areas with gaps in the current literature, such as South America, sub-Saharan Africa, and under-studied vulture species.

     

Host Associations Between Fungal Root Endophytes and Boreal Trees

Fungal root endophytes colonize root tissue concomitantly with mycorrhizal fungi, but their identities and host preferences are largely unknown. We cultured fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern Canada. Isolates were initially grouped on the basis of cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR restriction fragment length polymorphism. Phylogenetic analysis of the sequence data revealed 31 distinct phylotypes among the isolates, comprising mainly members of the ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host tree species. Some phylotypes were evenly distributed across the roots of all three host species, some were found preferentially on particular hosts, and others were isolated from single hosts only. The results indicate that fungal root endophytes of boreal trees are not randomly distributed, but instead form relatively distinct assemblages on different host tree species.     

The reliability and validity of fatigue measures during short-duration maximal-intensity intermittent cycling

The purpose of the present study was to assess the reliability and validity of fatigue measures, as derived from 4 separate formulae, during tests of repeat sprint ability. On separate days over a 3-week period, 2 groups of 7 recreationally active men completed 6 trials of 1 of 2 maximal (20 x 5 seconds) intermittent cycling tests with contrasting recovery periods (10 or 30 seconds). All trials were conducted on a friction-braked cycle ergometer, and fatigue scores were derived from measures of mean power output for each sprint. Apart from formula 1, which calculated fatigue from the percentage difference in mean power output between the first and last sprint, all remaining formulae produced fatigue scores that showed a reasonably good level of test-retest reliability in both intermittent test protocols (intraclass correlation range: 0.78-0.86; 95% likely range of true values: 0.54-0.97). Although between-protocol differences in the magnitude of the fatigue scores suggested good construct validity, within-protocol differences highlighted limitations with each formula. Overall, the results support the use of the percentage decrement score as the most valid and reliable measure of fatigue during brief maximal intermittent work.     

Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts,Determined by Multilocus Variable-Number Tandem-Repeat Analysis

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.Vibrio cholerae is the etiologic agent of cholera, a secretory diarrheal disease with a high mortality rate in humans if untreated (25). Serogroups of V. cholerae, a motile, Gram-negative, curved rod, can be defined serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane (9). V. cholerae is found in a variety of forms in aquatic ecosystems (41, 42), and more than 200 different serogroups have been isolated, mostly from environmental sources (45). However, the vast majority of V. cholerae strains that cause the clinical disease cholera belong to serogroup O1 or O139 (37, 42). V. cholerae O1, the historical agent of epidemic and pandemic cholera and the current leading cause of cholera both globally and in Bangladesh (42), is classified into two major biotypes, classical and El Tor (44), and two major serotypes, Ogawa and Inaba (48). The current global pandemic is caused by V. cholerae O1 El Tor. A second pathogenic serogroup, O139, emerged in the Bengal region in 1992 by horizontal transfer of new LPS biosynthesis-encoding genes into the El Tor biotype (1, 4). This new serogroup continues to cocirculate with El Tor V. cholerae O1 serotypes Ogawa and Inaba as a cause of disease in humans, although it accounts for a smaller proportion of all cholera now than in its first years of circulation (16, 20). Recently, comparative genomics has revealed an extensive amount of lateral gene transfer between strains, suggesting that genomic classification may be an alternative to serogrouping for classifying pathogenic V. cholerae strains (11).Toxigenic V. cholerae may be present in environmental sources in regions of endemicity and emerge, often seasonally, to cause cholera in humans (12, 18). Once an outbreak has begun, organisms from one infected individual are more infectious for the next individual, a property termed hyperinfectivity, and these forms may be able to pass directly from human to human through fecal-oral contamination (35). However, because vibrio organisms are difficult to isolate from implicated environmental or domestic water sources (28, 29), little is known about the diversity of V. cholerae in inocula that cause human infection.Established laboratory methods for differentiating V. cholerae strains, apart from serogrouping and serotyping, include rRNA restriction fragment length polymorphism (ribotyping), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). These methods, however, have a limited capacity to differentiate between pathogenic V. cholerae strains, as clinical isolates are relatively genetically monomorphic. For instance, V. cholerae O1 comprises approximately 30 ribotypes (39); however, only a few ribotypes are common in clinical isolates, ribotypes evolve slowly, and all isolates of a given pathogenic V. cholerae serotype in a local area over a period of multiple years often belong to a single ribotype (8, 14, 17). In a broad sampling of 154 V. cholerae isolates from Bangladesh and worldwide over several decades, only 15 ribotypes were identified, and of these, many were found in nonpathogenic environmental isolates only; only five ribotypes were associated with the V. cholerae O1 El Tor biotype that currently predominates as the cause of clinical disease, while pathogenic isolates of serogroup O139 were indistinguishable from each other by ribotype (19).PFGE, in which restriction endonuclease digestion of genomic DNA generates mutation-sensitive banding patterns, is often more sensitive than ribotyping in detecting strain variation (7, 34, 51) and detects extensive genetic variation within nonpathogenic V. cholerae serogroups (3, 46). However, PFGE types change slowly and are useful primarily for distinguishing between strains in different pandemics or between different continental branches of those pandemics. In an analysis of 180 mostly western-hemisphere isolates (7), PFGE differences had developed from a prior pandemic strain over the 30 years since its arrival in Latin America, but a new strain that had been causing disease for 2 years still had only a single PFGE type across the 64 isolates analyzed. Similarly, in a Japanese study (2), although 19 PFGE types were identified among O1 isolates, the majority of the domestic isolates, along with several imported isolates, belonged to a single PFGE type.Further differentiation between V. cholerae isolates is achievable by MLST, which characterizes isolates by internal DNA sequences in selected housekeeping genes (32). Nevertheless, epidemic strains also cluster tightly in this typing scheme (5, 32) and the method has been useful primarily for determining relationships between nontoxigenic strains (36) or for linking regional outbreaks (which typically appear monoclonal by these methods) with the pandemic strain responsible (5, 33).Although these methods have distinguished major pandemic clones from other nonpathogenic human and environmental isolates of V. cholerae, the near clonality of pathogenic O1 and O139 strains means that established methods may not provide sufficiently robust differentiation of these genetically similar pathogenic strains to answer important epidemiological questions. Therefore, there is a need for other methods that can distinguish among clinical O1 and O139 isolates and track the epidemiology of outbreaks in a restricted geographic area on a shorter time scale.Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is one method that may be useful for differentiating between pathogenic V. cholerae O1 and O139 strains that would be indistinguishable by other techniques (15). This method examines short repeating DNA segments at various locations in the genome that can vary in number at each location and uses the number of repeats at each varying locus as a fingerprint to distinguish between isolates.Escherichia coli is the paradigm organism for demonstrating the value of the MLVA method. Noller et al. (38) showed that E. coli O157 isolates that were indistinguishable by MLST could be distinguished to some extent by PFGE but that MLVA distinguished between isolates that had the same PFGE type and did so in a manner consistent with the known epidemiology of the isolates (38a). In addition, machine-scored VNTR assays have been demonstrated to be robust and portable and to discriminate clearly between isolates by using relatively few loci, therefore limiting the effect of compounding genotyping errors (6).For V. cholerae, five VNTR loci have been identified (15), and the initial application of MLVA at those loci has demonstrated distinct populations of clinical isolates of V. cholerae in different geographic regions within Bangladesh and India (23, 47). Predominant isolates in each of two rural Bangladeshi regions varied gradually over a time scale of months to years (47), and isolates collected from India over a 15-year period varied widely, with individual MLVA types clustering in time and place—some with widespread dissemination and others with limited local occurrence only (23). MLVA has also been used to classify hybrid and altered V. cholerae variants and to demonstrate their genetic distance from the pandemic El Tor strain (10). Use of the MLVA method for epidemiologic study of cholera requires that V. cholerae VNTR alleles remain reasonably stable during bacterial replication in patients or in laboratory culture after isolation. Some degree of stability of two of the five loci used in V. cholerae MLVA has been demonstrated previously by serial passage in vitro through four overnight cultures (15). In this study, we used MLVA to examine V. cholerae O1 and O139 isolates obtained from infected patients and their household contacts—including multiple isolates from the same individual and isolates from multiple individuals within the same household—in a large city where cholera is endemic.