Retrospective study of a TTR FAP cohort to modify NIS+7 for therapeutic trials.

Protein stabilization and oligonucleotide therapies are being tested in transthyretin amyloid polyneuropathy (TTR FAP) trials. From retrospective analysis of 97 untreated TTR FAP patients, we test the adequacy of Neuropathy Impairment Score+7 tests (NIS+7) and modifications to comprehensively score impairments for use in such therapeutic trials. Our data confirms that TTR FAP usually is a sensorimotor polyneuropathy with autonomic features which usually is symmetric, length dependent, lower limb predominant and progressive. NIS+7 adequately assesses weakness and muscle stretch reflexes without ceiling effects but not sensation loss, autonomic dysfunction or nerve conduction abnormalities. Three modifications of NIS+7 are suggested: 1) use of Smart Somatotopic Quantitative Sensation Testing (S ST QSTing); 2) choice of new autonomic assessments, e.g., sudomotor testing of distributed anatomical sites; and 3) use of only compound muscle action potential amplitudes (of ulnar, peroneal and tibial nerves) and sensory nerve action potentials of ulnar and sural nerve - than the previously recommended attributes suggested for the sensitive detection of diabetic sensorimotor polyneuropathy. These modifications of NIS+7 if used in therapeutic trials should improve characterization and quantification of sensation and autonomic impairment in TTR FAP and provide better nerve conduction tests.

Genetic evidence for adaptation-driven incipient speciation of Drosophila melanogaster along a microclimatic contrast in "Evolution Canyon," Israel.

Substantial genetic differentiation, as great as among species, exists between populations of Drosophila melanogaster inhabiting opposite slopes of a small canyon. Previous work has shown that prezygotic sexual isolation and numerous differences in stress-related phenotypes have evolved between D. melanogaster populations in "Evolution Canyon," Israel, in which slopes 100-400 m apart differ dramatically in aridity, solar radiation, and associated vegetation. Because the canyon's width is well within flies' dispersal capabilities, we examined genetic changes associated with local adaptation and incipient speciation in the absence of geographical isolation. Here we report remarkable genetic differentiation of microsatellites and divergence in the regulatory region of hsp70Ba which encodes the major inducible heat shock protein of Drosophila, in the two populations. Additionally, an analysis of microsatellites suggests a limited exchange of migrants and lack of recent population bottlenecks. We hypothesize that adaptation to the contrasting microclimates overwhelms gene flow and is responsible for the genetic and phenotypic divergence between the populations.

A Drosophila melanogaster strain from sub-equatorial Africa has exceptional thermotolerance but decreased Hsp70 expression.

Drosophila melanogaster collected in sub-equatorial Africa in the 1970s are remarkably tolerant of sustained laboratory culture above 30 degrees C and of acute exposure to much warmer temperatures. Inducible thermotolerance of high temperatures, which in Drosophila melanogaster is due in part to the inducible molecular chaperone Hsp70, is only modest in this strain. Expression of Hsp70 protein and hsp70 mRNA is likewise reduced and has slower kinetics in this strain (T) than in a standard wild-type strain (Oregon R). These strains also differed in constitutive and heat-inducible levels of other molecular chaperones. The lower Hsp70 expression in the T strain apparently has no basis in the activation of the heat-shock transcription factor HSF, which is similar in T and Oregon R flies. Rather, the reduced expression may stem from insertion of two transposable elements, H.M.S. Beagle in the intergenic region of the 87A7 hsp70 gene cluster and Jockey in the hsp70Ba gene promoter. We hypothesize that the reduced Hsp70 expression in a Drosophila melanogaster strain living chronically at intermediate temperatures may represent an evolved suppression of the deleterious phenotypes of Hsp70.

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.

Changes in thermotolerance and Hsp70 expression with domestication in Drosophila melanogaster.

To examine how the duration of laboratory domestication may affect Drosophila stocks used in studies of thermotolerance, we measured expression of the inducible heat-shock protein Hsp70 and survival after heat shock in D. melanogaster strains recently collected from nature and maintained in laboratory culture for up to 50 or more generations. After an initial increase in both Hsp70 expression and thermotolerance immediately after transfer to laboratory medium, both traits remained fairly constant over time and variation among strains persisted through laboratory domestication. Furthermore, variation in heat tolerance and Hsp70 expression did not correlate with the length of time populations evolved in the laboratory. Therefore, while environmental variation likely contributed most to early shifts in strain tolerance and Hsp70 expression, other population parameters, for example genetic drift, inbreeding, and selection likely affected these traits little. As long as populations are maintained with large numbers of individuals, the culture of insects in the laboratory may have little effect on the tolerance of different strains to thermal stress.