Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.     

Collection of semen by manual stimulation and ejaculate characteristics of the black flying‐fox (Pteropus alecto)

Semen collection and preservation is the first step toward the development of an artificial insemination program in endangered Pteropus spp. Semen was collected by manual stimulation from a single “human‐habituated” P. alecto. Manual stimulation resulted in the successful collection of motile spermatozoa on 17 of 34 attempts. The semen had a pH of 8.2 (n=2). With the exception of volume, seminal characteristics (concentration, motility, acrosome and plasma membrane status) were similar to those collected previously by electro‐ejaculation. Zoo Biol 27:159–164, 2008. © 2008 Wiley‐Liss, Inc.     

Nutritional Interventions in Depression and Perinatal Depression

Depression is the leading cause of mental disability worldwide. Women who are depressed during pregnancy are at a higher risk for preterm delivery, preeclampsia, birth difficulties, and postpartum depression. The treatment of depression in conventional medicine has focused on physiological factors that lead to impaired neurotransmitter function and treatments to improve neurotransmitter function. Pharmaceutical substances pose risks for pregnant and lactating women, and lower risk options are preferred. Micronutrients, including certain B vitamins, folate, and docosahexaenoic acid (DHA), play a role in the synthesis and absorption of neurotransmitters. Experimental studies suggest that supplementation with specific micronutrients may alleviate depressive symptoms and improve birth outcomes in patients with perinatal depression. Alternative treatments for depression, including nutritional supplements, are an important treatment option for depressive symptoms while limiting potential side effects and treatment costs. This article explores the biological basis of perinatal depression and reviews the potential benefits of non-pharmacological interventions.     

Cryopreservation of marine thraustochytrids (Labyrinthulomycetes)

In this research, the viability of three marine thraustochytrid isolates (fungoid protists) (WSG05, W15 and WH3) were investigated after freezing in liquid nitrogen. Five cryopreservative combinations containing horse serum, glycerol and dimethylsulfide (Me₂SO) were used. The thraustochytrids were assessed directly after removal from liquid nitrogen and cell concentration measured for 10 days post-thawing. Results indicated that a combination of horse serum and Me₂SO were the most effective cryoprotectants for each of the strains tested. Glycerol was only successful in producing growth in one of the strains once thawed.The protocols developed and tested in this study may have further application for cryopreserving other isolates in this class.     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Effects of UVB on interactions between Schistosoma mansoni and Biomphalaria glabrata

Ultraviolet B (UVB, 280-315 nm) radiation is detrimental to both of larvae of the digenetic trematode Schistosoma mansoni and its snail intermediate host, Biomphalaria glabrata. We explored effects of UVB on three aspects of the interaction between host and parasite: survival of infected snails, innate susceptibility and resistance of snails to infection, and acquired resistance induced by irradiated miracidia. Snails infected for 1 week showed significantly lower survival than uninfected snails following irradiation with a range of UVB intensities. In contrast to known immunomodulatory effects in vertebrates, an effect of UVB on susceptibility or resistance of snails to infection could not be conclusively demonstrated. Finally, exposure of susceptible snails to UVB-irradiated miracidia failed to induce resistance to a subsequent challenge with nonirradiated miracidia, a result similar to that reported previously with ionizing radiation.     

A simple enzyme immunoassay for detecting brucellosis antibodies

Abstract A simple enzyme immunoassay was developed and evaluated for serological diagnosis of brucellosis in 25 patients with various forms of brucellosis and 292 control patients with other conditions and disorders. All brucellosis patients gave a positive test with the initial sample. In 3 acute, febrile brucellosis patients with follow-up sera taken during therapy a sharp drop in specific antibody was noted. There was a less pronounced antibody reduction in 1 chronic and 2 relapse patients and an antibody increase in 1 chronic and 1 relapse case. All control samples gave negative results. In addition, the assay was evaluated as a screening test with 315 sera from 'healthy' individuals living in a brucellosis focus and representing 15% of that population. 11.5% (34/293) of subjects with no reported history of brucellosis and 45% (10/22) of cases treated in the past gave a positive test result. The agreement in those samples between the assay and the serum agglutination test was 95.5%.     

Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin

A coordinated study of water chemistry, sediment mineralogy, and sediment microbial community was conducted on four >73°C springs in the northwestern Great Basin. Despite generally similar chemistry and mineralogy, springs with short residence time (~5–20 min) were rich in reduced chemistry, whereas springs with long residence time (>1 day) accumulated oxygen and oxidized nitrogen species. The presence of oxygen suggested that aerobic metabolisms prevail in the water and surface sediment. However, Gibbs free energy calculations using empirical chemistry data suggested that several inorganic electron donors were similarly favorable. Analysis of 298 bacterial 16S rDNAs identified 36 species-level phylotypes, 14 of which failed to affiliate with cultivated phyla. Highly represented phylotypes included Thermus, Thermotoga, a member of candidate phylum OP1, and two deeply branching Chloroflexi. The 276 archaeal 16S rDNAs represented 28 phylotypes, most of which were Crenarchaeota unrelated to the Thermoprotei. The most abundant archaeal phylotype was closely related to “Candidatus Nitrosocaldus yellowstonii”, suggesting a role for ammonia oxidation in primary production; however, few other phylotypes could be linked with energy calculations because phylotypes were either related to chemoorganotrophs or were unrelated to known organisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.