Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Functional imaging in radiation therapy planning for head and neck cancer

Functional imaging and its application to radiotherapy (RT) is a rapidly expanding field with new modalities and techniques constantly developing and evolving. As technologies improve, it will be important to pay attention to their implementation. This review describes the main achievements in the field of head and neck cancer (HNC) with particular remarks on the unsolved problems.     

Associations of physical activity with gut microbiota in pre-adolescent children

[Purpose] To determine whether physical activity (PA), primarily the recommended 60 minutes of moderate-to-vigorous PA, is associated with gut bacterial microbiota in 10-year-old children.[Methods] The Block Physical Activity Screener, which provides minutes/day PA variables, was used to determine whether the child met the PA recommendations. 16S rRNA sequencing was performed on stool samples from the children to profile the composition of their gut bacterial microbiota. Differences in alpha diversity metrics (richness, Pielou’s evenness, and Faith’s phylogenetic diversity) by PA were determined using linear regression, whereas beta diversity (unweighted and weighted UniFrac) relationships were assessed using PERMANOVA. Taxon relative abundance differentials were determined using DESeq2.[Results] The analytic sample included 321 children with both PA and 16S rRNA sequencing data (mean age [SD] =10.2 [0.8] years; 54.2% male; 62.9% African American), where 189 (58.9%) met the PA recommendations. After adjusting for covariates, meeting the PA recommendations as well as minutes/day PA variables were not significantly associated with gut richness, evenness, or diversity (p ≥ 0.19). However, meeting the PA recommendations (weighted UniFrac R² = 0.014, p = 0.001) was significantly associated with distinct gut bacterial composition. These compositional differences were partly characterized by increased abundance of Megamonas and Anaerovorax as well as specific Christensenellaceae_R-7_group taxa in children with higher PA.[Conclusion] Children who met the recommendations of PA had altered gut microbiota compositions. Whether this translates to a reduced risk of obesity or associated metabolic diseases is still unclear.     

Plasticity of retrovirus-labelled myotubes in the newt limb regeneration blastema

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.     

Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3′-UTR of an α-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.     

Targeted disruption of iNOS prevents LPS-induced S-nitrosation of IRbeta/IRS-1 and Akt and insulin resistance in muscle of mice

We have previously demonstrated that the insulin resistance associated with inducible nitric oxide synthase (iNOS) induction in two different models of obesity, diet-induced obesity and the ob/ob mice, is mediated by S-nitrosation of proteins involved in insulin signal transduction: insulin receptor beta-subunit (IRbeta), insulin receptor substrate 1(IRS-1), and Akt. S-nitrosation of IRbeta and Akt impairs their kinase activities, and S-nitrosation of IRS-1 reduces its tissue expression. In this study, we observed that LPS-induced insulin resistance in the muscle of wild-type mice, as demonstrated by reduced insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, reduced IRS-1 expression and reduced insulin-induced serine phosphorylation of Akt. This resistance occurred in parallel with enhanced iNOS expression, which was accompanied by S-nitrosation of IRbeta/IRS-1 and Akt. In the muscle of iNOS(-/-) mice, we did not observe enhanced iNOS expression or any S-nitrosation of IRbeta/IRS-1 and Akt after LPS treatment. Moreover, insulin resistance was not present. The preservation of insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, of IRS-1 protein expression, and of insulin-induced serine phosphorylation of Akt observed in LPS-treated iNOS(-/-) mice strongly suggests that the insulin resistance induced by LPS is iNOS mediated, probably through S-nitrosation of proteins of early steps of insulin signaling.