Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species‐specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species‐specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species‐specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species‐specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36‐base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.     

Control of pancreatic β cell regeneration by glucose metabolism

Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand.     

The prevalence of Campylobacter amongst a free-range broiler breeder flock was primarily affected by flock age

Campylobacter successfully colonizes broiler chickens, but little is known about the longer term natural history of colonization, since most flocks are slaughtered at an immature age. In this study, the prevalence and genetic diversity of Campylobacter colonizing a single free-range broiler breeder flock was investigated over the course of a year. The age of the flock was the most important factor in determining both the prevalence and diversity of Campylobacter over time. There was no correlation with season, temperature, the amount of rain and sunshine, or the dynamics of colonization amongst geographically and temporally matched broiler flocks. The higher prevalence rates coincided with the age at which broiler chickens are typically slaughtered, but then in the absence of bio-security or other intervention methods, and despite changes in flock management, the prevalence fell to significantly lower levels for the remainder of the study. The genetic diversity of Campylobacter increased as the flock aged, implying that genotypes were accumulated within the flock and may persist for a long time. A better understanding of the ecology of Campylobacter within commercial chicken flocks will allow the design of more effective farm-based interventions.     

Reasons for ineligibility in phase 1 and 2A HIV vaccine clinical trials at Kenya AIDS vaccine initiative (KAVI), Kenya

Background

With the persistent challenges towards controlling the HIV epidemic, there is an ongoing need for research into HIV vaccines and drugs. Sub-Saharan African countries - worst affected by the HIV pandemic - have participated in the conduct of clinical trials for HIV vaccines. In Kenya, the Kenya AIDS Vaccine Initiative (KAVI) at the University of Nairobi has conducted HIV vaccine clinical trials since 2001.

Methodology

Participants were recruited after an extensive informed consent process followed by screening to determine eligibility. Screening included an assessment of risk behavior, medical history and physical examination, and if clinically healthy, laboratory testing. In the absence of locally derived laboratory reference ranges, the ranges used in these trials were derived from populations in the West.

Principal findings

Two hundred eighty-one participants were screened between 2003 and 2006 for two clinical trials. Of these, 167 (59.4%) met the inclusion/exclusion criteria. Overall, laboratory abnormalities based on the non-indigenous laboratory references used were the most frequent reasons (61.4%) for ineligibility. Medical abnormalities contributed 30.7% of the total reasons for ineligibility. Based on the laboratory reference intervals now developed from East and Southern Africa, those ineligible due to laboratory abnormalities would have been 46.3%. Of the eligible participants, 18.6% declined enrolment.

Conclusions

Participant recruitment for HIV vaccine clinical trials is a rigorous and time-consuming exercise. Over 61% of the screening exclusions in clinically healthy people were due to laboratory abnormalities. It is essential that laboratory reference ranges generated from local populations for laboratory values be used in the conduct of clinical trials to avoid unnecessary exclusion of willing participants and to avoid over-reporting of adverse events for enrolled participants.

Trial registration

Protocol IAVI VRC V001 [1]. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00124007","term_id":"NCT00124007"}}NCT00124007 Protocol IAVI 010 [2] (registration with ClincalTrials.gov is in progress) Protocols IAVI 002 and IAVI 004 are Phase 1 trials only mentioned in introductory paragraphs; details will not be reported. Registration was not required when they were conducted.     

Self-organization, layered structure, and aggregation enhance persistence of a synthetic biofilm consortium

Microbial consortia constitute a majority of the earth's biomass, but little is known about how these cooperating communities persist despite competition among community members. Theory suggests that non-random spatial structures contribute to the persistence of mixed communities; when particular structures form, they may provide associated community members with a growth advantage over unassociated members. If true, this has implications for the rise and persistence of multi-cellular organisms. However, this theory is difficult to study because we rarely observe initial instances of non-random physical structure in natural populations. Using two engineered strains of Escherichia coli that constitute a synthetic symbiotic microbial consortium, we fortuitously observed such spatial self-organization. This consortium forms a biofilm and, after several days, adopts a defined layered structure that is associated with two unexpected, measurable growth advantages. First, the consortium cannot successfully colonize a new, downstream environment until it self-organizes in the initial environment; in other words, the structure enhances the ability of the consortium to survive environmental disruptions. Second, when the layered structure forms in downstream environments the consortium accumulates significantly more biomass than it did in the initial environment; in other words, the structure enhances the global productivity of the consortium. We also observed that the layered structure only assembles in downstream environments that are colonized by aggregates from a previous, structured community. These results demonstrate roles for self-organization and aggregation in persistence of multi-cellular communities, and also illustrate a role for the techniques of synthetic biology in elucidating fundamental biological principles.     

Rapid behavioural diagnosis of domoic acid toxicosis in California sea lions

Domoic acid is a neurotoxic metabolite of widely occurring algal blooms that has caused multiple marine animal stranding events. Exposure to high doses of domoic acid, a glutamate agonist, may lead to persistent medial temporal seizures and damage to the hippocampus. California sea lions (Zalophus californianus) are among the most visible and frequent mammalian victims of domoic acid poisoning, but rapid, reliable diagnosis in a clinical setting has proved difficult owing to the fast clearance of the toxin from the blood stream. Here, we show that the behavioural orienting responses of stranded sea lions diagnosed with domoic acid toxicosis habituate more slowly to a series of non-aversive auditory stimuli than do those of sea lions with no apparent neurological deficits. A signal detection analysis based on these habituation measures was able to correctly identify 50 per cent of subjects with domoic acid toxicosis while correctly rejecting approximately 93 per cent of controls, suggesting potential diagnostic merit.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair

Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.