High-throughput detection of pathogenic yeasts of the genus trichosporon

The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10(2) genome molecules in PCRs and 10(7) to 10(8) molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.     

Anthrone and oxanthrone C-glycosides from Picramnia latifolia collected in Peru

Cytotoxicity-based, bioassay-guided fractionation of the chloroform-soluble extracts of both the roots and leaves of Picramnia latifolia led to the isolation of two new anthrone C-glycosides, picramniosides G (1) and H (2), two new oxanthrone C-glycosides, mayosides D (3) and E (4), and a new benzanthrone natural product, 6,8-dihydroxy-10-methyl-7H-benz[de]anthracen-7-one (5), together with 10 known compounds, 6,8-dihydroxy-4-methyl-7H-benz[de]anthracen-7-one (6), nataloe-emodin (7), chrysophanein, chrysophanol, 1,5-dihydroxy-7-methoxy-3-methylanthraquinone, pulmatin, 7-hydroxycoumarin, 7-hydroxy-6-methoxycoumarin, beta-sitosterol, and beta-sitosterol glucoside. The structures of 1-5 were established by spectroscopic methods, including 1D and 2D NMR, HRMS, and CD data interpretation. The cytotoxic activity of all isolates was evaluated in a small panel of human cancer cell lines. Compound 7 exhibited significant in vitro cytotoxic activity in the tested cell lines, but no significant activity was observed with an in vivo hollow fiber model at doses of 6.25, 12.5, 25, and 50 mg/kg/injection.     

Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells.

T lymphocytes from patients with acute EBV-induced infectious mononucleosis rapidly die by apoptosis in vitro. Because human and viral IL-10 are likely to be induced during acute EBV infection and display a variety of functions on human T cells, we examined IL-10 effects on infectious mononucleosis T cell death. After 12 h of incubation in medium alone, only 35.6 (+/- 8.2%) of the originally seeded infectious mononucleosis T cells were viable. Addition of human IL-10 (100 U/ml) to T cell cultures significantly improved recovery of viable cells (71.3 +/- 6.2%, P = 0.0156). Viral IL-10 had comparable effects to human IL-10 in this system. Protection from death by human and viral IL-10 (100 U/ml) was dose dependent and continued over a 6-d culture period. The human IL-10 effect was neutralized by the anti-human IL-10 mAb 19F1. Morphology and analysis of DNA after separation on agarose gels showed that IL-10 inhibits loss of cell volume, chromatin condensation, and DNA fragmentation, characteristics of death by apoptosis. As assessed by [3H]thymidine incorporation, the T cells were not induced to proliferate by IL-10 above the level exhibited when first removed from blood. T cells protected from death by IL-10 proliferated to IL-2 and spontaneously killed sensitive targets as effectively as medium-precultured T cells. Thus, IL-10 promotes the survival of infectious mononucleosis T cells otherwise destined to die by apoptosis and may be critical for the establishment of immunologic memory after resolution of the illness.     

Significance of tilt table testing in patients with suspected arrhythmic syncope and negative electrophysiologic study

BACKGROUND: The diagnostic significance of a tilt table test (TTT) in patients with a suspected arrhythmic etiology for syncope and negative electrophysiologic study (EPS) has not been previously assessed comparing the TTT results with the findings of prolonged monitoring using an implantable loop recorder (ILR). We sought to assess the diagnostic yielding of TTT in patients with suspected arrhythmic syncope and negative EPS. METHODS AND RESULTS: In 81 patients with suspected arrhythmic etiology for syncope and negative EPS, TTT was performed and an ILR implanted regardless the results of TTT. TTT was positive in 38 patients. During follow-up, syncope or presyncope recurred in 32 patients (39.5%). No differences were found in recurrence rates in patients with positive and negative TTT (31.5% vs 46.5%, P = ns). According to rhythm registered during ILR activation, mechanisms of syncopal events were classified as: arrhythmic (atrioventricular [AV] block and ventricular tachycardia; n = 18), neurally mediated (sinus bradycardia and sinus pause; n = 9), and indeterminate (normal sinus rhythm; n = 5). There was no statistical association between the results of TTT and the mechanism of syncope. CONCLUSIONS: In patients with a suspected arrhythmic etiology for syncope and a negative EPS, TTT is of little value to predict the mechanism of syncope and the ILR implantation seems to be a useful and safe diagnostic strategy.     

Characterization of three monoclonal antibodies that recognize the interferon alpha 2 receptor.

The interferon system plays an important role in the control of viral infections and cell proliferation. These effects are mediated through the interaction of interferons with specific cell surface receptors. We report here the development of monoclonal antibodies against one of the subunits of the interferon alpha receptor. These antibodies detect a 110-kDa protein in surface-labeled cells and in Western blots, and 130- and 210-kDa bands after crosslinking to iodinated interferon alpha 2. No other subunits are disulfide-linked to the 130-kDa subunit or are coprecipitated by these antibodies. Analysis by two-dimensional gel electrophoresis revealed that the pI of this subunit is 3.5-5.0. We suggest that the protein recognized by these monoclonal antibodies be named the alpha subunit of the interferon alpha receptor.     

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.     

Racial differences in oxidative stress and inflammation: in vitro and in vivo

African American race is an independent risk factor for enhanced oxidative stress and inflammation. We sought to examine whether oxidative-stress and inflammatory markers that are typically measured in humans also differ by race in cell culture. We compared levels between African American and Caucasian young adults and then separately in human umbilical vein endothelial cells (HUVECs) from both races. We found heightened oxidative stress and inflammation in the African Americans both in vitro and in vivo. African American HUVECs showed higher nitric oxide (NO) levels (10.8 ± 0.4 vs. 8.8 ± 0.7 μmol/L/mg, p = 0.03), Interleukin-6 (IL-6) levels (61.7 ± 4.2 vs. 23.9 ± 9.0 pg/mg, p = 0.02), and lower superoxide dismutase activity (15.6 ± 3.3 vs. 25.4 ± 2.8 U/mg, p = 0.04), and also higher protein expression (p < 0.05) of NADPH oxidase subunit p47phox, isoforms NOX2 and NOX4, endothelial nitric oxide synthase (NOS), inducible NOS, as well as IL-6. African American adults had higher plasma protein carbonyls (1.1 ± 0.1 vs. 0.8 ± 0.1 nmol/mg, p = 0.01) and antioxidant capacity (2.3 ± 0.2 vs. 1.1 ± 0.3 mM, p = 0.01). These preliminary translational data demonstrate a racial difference in HUVECs much like that in humans, but should be interpreted with caution given its preliminary nature. It is known that racial differences exist in how humans respond to development and progression of disease, therefore these data suggest that ethnicity of cell model may be important to consider with in vitro clinical research.     

Endoplasmic reticulum protein BI-1 regulates Ca²⁺-mediated bioenergetics to promote autophagy

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²⁺ mediated by inositol triphosphate receptors (IP₃Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP₃R-dependent manner. By reducing steady-state levels of ER Ca²⁺ via IP₃Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1''s ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²⁺ signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.