Piaroa Manioc Varietals: Hyperdiversity or Social Currency?

The maintenance of exceptionally high numbers of folk varieties by the Piaroa people of the Venezuelan Amazon is considered. We cataloged 113 manioc folk varieties, their nomenclature, use and relevant characters, revealing significant insights into the role of manioc in Piaroa social life. Through a qualitative investigation of the cultivation, processing and symbolic significance of manioc (Manihot esculenta) in two Piaroa regions over a period of 18 years, we have found that such agrobiodiversity can only be fully explained by a combination of multiple factors, including pragmatic and ecological considerations, the subtle and complex diversity of Piaroa manioc preparations and a variety of sociocultural factors, such as manioc’s role as a mediator of social relationships and as a marker of cultural and social heritage.

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.     

Kinetic and structural studies of phosphodiesterase-8A and implication on the inhibitor selectivity

Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 A resolutions, respectively. The PDE8A1 catalytic domain has a K(M) of 1.8 microM, V(max) of 6.1 micromol/min/mg, a k(cat) of 4.0 s(-1) for cAMP, and a K(M) of 1.6 mM, V(max) of 2.5 micromol/min/mg, a k(cat) of 1.6 s(-1) for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by K(M). The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC(50) = 700 microM). The unfavorable interaction of IBMX in the PDE8A1-IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several nonselective or family selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties but also guidelines for design of PDE8 selective inhibitors.     

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.