Advanced age increases frequencies of de novo mitochondrial mutations in macaque oocytes and somatic tissues

Mutations in mitochondrial DNA (mtDNA) contribute to multiple diseases. However, how new mtDNA mutations arise and accumulate with age remains understudied because of the high error rates of current sequencing technologies. Duplex sequencing reduces error rates by several orders of magnitude via independently tagging and analyzing each of the two template DNA strands. Here, using duplex sequencing, we obtained high-quality mtDNA sequences for somatic tissues (liver and skeletal muscle) and single oocytes of 30 unrelated rhesus macaques, from 1 to 23 y of age. Sequencing single oocytes minimized effects of natural selection on germline mutations. In total, we identified 17,637 tissue-specific de novo mutations. Their frequency increased ∼3.5-fold in liver and ∼2.8-fold in muscle over the ∼20 y assessed. Mutation frequency in oocytes increased ∼2.5-fold until the age of 9 y, but did not increase after that, suggesting that oocytes of older animals maintain the quality of their mtDNA. We found the light-strand origin of replication (OriL) to be a hotspot for mutation accumulation with aging in liver. Indeed, the 33-nucleotide-long OriL harbored 12 variant hotspots, 10 of which likely disrupt its hairpin structure and affect replication efficiency. Moreover, in somatic tissues, protein-coding variants were subject to positive selection (potentially mitigating toxic effects of mitochondrial activity), the strength of which increased with the number of macaques harboring variants. Our work illuminates the origins and accumulation of somatic and germline mtDNA mutations with aging in primates and has implications for delayed reproduction in modern human societies.

Mitochondria produce energy and are involved in myriad other cellular functions (reviewed in ref. 1). The mammalian mitochondrial DNA (mtDNA) is a small (∼16.6 kb in humans), circular, maternally transmitted molecule, which harbors 37 genes encoding 13 proteins (which form oxidative phosphorylation subunits), 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs; reviewed in ref. 2). mtDNA is present in hundreds to thousands of copies per somatic cell and in >100,000 copies in an oocyte (3).The germline nucleotide substitution rate of mtDNA is an order of magnitude higher than that of nuclear DNA (4, 5). Germline mutations increase in frequency with paternal and maternal age in nuclear DNA of humans (6) and macaques (7); however, whether they accumulate with maternal age in mtDNA of primates has been understudied. Such age-related accumulation was suggested based on the analysis of human pedigrees (4, 8) without the direct examination of germline cells and, thus, might have been influenced by selection. An investigation of mutation accumulation in the oocytes of females of different ages is needed to settle this question unequivocally.The direct examination of mtDNA mutations in oocytes has been challenging due to methodological limitations. Most studies either focused on a limited number of mtDNA sites (e.g., refs. 9, 10) or used sequencing methods with high error rates (e.g., refs. 11, 12). Recently, an age-related increase of mtDNA mutations in mouse oocytes was demonstrated with duplex sequencing (13). However, we still do not know definitively whether the frequency of mtDNA mutations increases with age in primate oocytes. Answering this question is critical due to the association of mtDNA mutations with human genetic diseases (reviewed in ref. 14) and because of frequently delayed reproduction in modern human societies. Examining mutations in human oocytes presents multiple logistical and ethical challenges, requiring one to turn to a primate model.The rhesus macaque is an excellent model organism to study mtDNA mutations in relation to aging due to 1) the high similarity between macaque and human mtDNA, innate defenses against oxidative damage (15), and age-related decline in metabolic rate (16); and 2) the possibility of collecting oocytes from macaques starting at a young age. For humans, oocyte collection is mainly restricted to the reproductive lifespan, when in vitro fertilization procedures are performed.Here, we analyzed mutations in single oocytes and somatic tissues of rhesus macaques over an age span of >20 y, including samples from animals who have not reached sexual maturity (occurring at ∼3 y; ref. 17), as well as from animals up to the age of 23 y, covering the whole reproductive lifespan (macaques reach menopause at the age of ∼25 y; ref. 18). To measure de novo mutations, we used highly accurate duplex sequencing (19), allowing one to distinguish bona fide DNA variants from artifacts (sequencing and PCR errors, or DNA lesions) by barcoding double-stranded sequencing templates and achieving error rates <10⁻⁷. With this method, first, single-strand consensus sequences (SSCSs) are formed for reads originating from each of the two template strands separately. Next, a duplex consensus sequence (DCS) is formed from the two SSCSs. True DNA variants are expected to be present in both SSCSs and, thus, in the DCS. Using this method, we directly measured the frequency of de novo germline and somatic mutations across the whole mtDNA in macaques, demonstrating their accumulation with age. We identified variant hotspots, analyzed the effect of selection, and examined the dependence of allele frequencies of inheritable mtDNA heteroplasmies on age.     

Characterization of BASP1-mediated neurite outgrowth

The brain acid-soluble protein BASP1 (CAP-23, NAP-22) belongs to the family of growth-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite outgrowth. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite outgrowth in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite outgrowth was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the fibroblast growth factor receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite outgrowth. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite outgrowth to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite outgrowth. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite outgrowth, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite outgrowth. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite outgrowth and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.     

Divergence in the spatial pattern of gene expression between human duplicate genes

Microarray gene expression data provide a wealth of information for elucidating the mode and tempo of molecular evolution. In the present study,we analyze the spatial expression pattern of human duplicate gene pairs by using oligonucleotide microarray data,and study the relationship between coding sequence divergence and expression divergence. First,we find a strong positive correlation between the proportion of duplicate gene pairs with divergent expression (as presence or absence of expression in a tissue) and both synonymous (K(S)) and nonsynonymous divergence (K(A)). The divergence of gene expression between human duplicate genes is rapid, probably faster than that between yeast duplicates in terms of generations. Second,we compute the correlation coefficient (R) between the expression levels of duplicate genes in different tissues and find a significant negative correlation between R and K(S). There is also a negative correlation between R and K(A), when K(A) 相似文献   

Abnormal vaginal microbioma is associated with severity of localized provoked vulvodynia. Role of aerobic vaginitis and <Emphasis Type="Italic">Candida</Emphasis> in the pathogenesis of vulvodynia

Localized provoked vulvodynia (LPV) causes introital dyspareunia in up to 14% of premenopausal women. Vaginal infections like candidosis may play a initiating role. The aim of this study was to test a possible association of vaginal microbiota alternations such as Candida vaginitis (CV), aerobic vaginitis (AV) and bacterial vaginosis (BV) with severity of vulvodynia and painful intercourse. In an observational study, Q-tip touch test (score 1 (no pain) to 10 (worst possible pain)) was performed on seven vestibular locations in 231 LPV patients presenting in the Vulvovaginal Disease Clinics in Tienen, Leuven and Antwerp, Belgium. Severity of pain upon attempting sexual intercourse was recorded in a similar scale. Both scales were compared to results from fresh wet mount phase contrast microscopy on vaginal fluid smears tested for abnormal vaginal flora (AVF), BV, AV and CV according the standardized microscopy method (Femicare). Fisher’s exact test was used. Average age was 31.3?±?11.6 years, and 58.8% (n?=?132) had secondary vestibulodynia. There was an inverse relation between the presence of Candida in the vaginal smears and pain score (p?=?0.03). There was no relation of pain score, nor Q-tip score with BV. LPV patients with Q-tip score above 7 at 5 and/or 7 o’clock or at 1 and/or 11 o’clock had more often AV than women with lower pain scores (30 vs 14.5%, p?=?0.01, and 39 vs 14.7%, p?<?0.005, respectively). Detailed study of the vaginal microflora in patients demonstrates that the most severe patients suffer more from AV and less from Candida. These abnormalities need to be actively looked for and corrected before considering surgery or other therapies.     

„Der epidurale Blood Patch: ein Auftrag für den versierten Anästhesisten?“

Die Anaesthesiologie -     

Pharmacotherapy for the treatment of vaginal atrophy

Introduction: Despite its frequency, recognition and therapy of vulvovaginal atrophy (VVA) remain suboptimal. Wet mount microscopy, or vaginal pH as a proxy, allows VVA diagnosis in menopause, but also in young contraception users, after breast cancer, or postpartum. Intravaginal low dose estrogen product is the main therapy. Ultra-low-dose vaginal estriol is safe and sufficient in most cases, even in breast cancer patients, while hyaluronic acid can help women who cannot or do not want to use hormones.

Areas covered: The authors provide an overview of the current pharmaceutical treatment for vulvovaginal atrophy and provide their expert opinions on its future treatment.

Expert opinion: The basis of good treatment is a correct and complete diagnosis, using a microscope to study the maturity index of the vaginal fluid. Minimal dose of estriol intravaginally with or without lactobacilli is elegant, cheap and can safely be used after breast cancer and history of thromboembolic disease. Laser therapy requires validation and safety data, as is can potentially cause vaginal fibrosis and stenosis, and safer and cheaper alternatives are available.     

Y染色体或将在进化中消失

美国宾夕法尼亚州立大学的2位科学家在性染色体进化变异机制的研究上取得进展。研究发现,Y染色体比X染色体的演化速率快得多,这将导致Y染色体上的基因急剧丢失,如此,Y染色体将会完全消失,人类的传宗接代将受威胁。     

Review of lisdexamfetamine dimesylate in children and adolescents with attention deficit/hyperactivity disorder

Abstract

Objective

Lisdexamfetamine dimesylate is a stimulant prodrug with low abuse and diversion potential that is used in treatment of attention deficit hyperactivity disorder (ADHD) in children, adolescents and adults. This current literature review article aims to examine safety and efficacy of LDX in children and adolescents for the treatment of ADHD based on currently available data.