Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

Structural and Functional Insight into the Carbohydrate Receptor Binding of F4 Fimbriae-producing Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4_ab, F4_ac, and F4_ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeG_ad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D′-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe¹⁵⁰–Glu¹⁵² and Val¹⁶⁶–Glu¹⁷⁰ of FaeG_ad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4_ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D′-α1 loop that borders the FaeG_ad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes.     

Fine‐scale spatial genetic structure analysis of the black truffle Tuber aestivum and its link to aroma variability

Truffles are symbiotic fungi in high demand by food connoisseurs. Improving yield and product quality requires a better understanding of truffle genetics and aroma biosynthesis. One aim here was to investigate the diversity and fine‐scale spatial genetic structure of the Burgundy truffle Tuber aestivum. The second aim was to assess how genetic structuring along with fruiting body maturation and geographical origin influenced single constituents of truffle aroma. A total of 39 Burgundy truffles collected in two orchards were characterized in terms of aroma profile (SPME‐GC/MS) and genotype (microsatellites). A moderate genetic differentiation was observed between the populations of the two orchards. An important seasonal and spatial genetic structuring was detected. Within one orchard, individuals belonging to the same genet were generally collected during a single season and in the close vicinity from each other. Maximum genet size nevertheless ranged from 46 to 92 m. Geographical origin or maturity only had minor effects on aroma profiles but genetic structuring, specifically clonal identity, had a pronounced influence on the concentrations of C₈‐ and C₄‐VOCs. Our results highlight a high seasonal genetic turnover and indicate that the aroma of Burgundy truffle is influenced by the identity of single clones/genets.     

Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation

Critical events in the life cycle of malaria parasites are controlled by calcium‐dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito‐derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide‐specific phospholipase C (PI‐PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP₂ and the production of the secondary messenger IP₃ in gametocytes. Both processes were selectively blocked by a PI‐PLC inhibitor, which also reduced the early Ca²⁺ signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI‐PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI‐PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP₂/IP₃ probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca²⁺ and PI‐PLC activity, with PI‐PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.     

Genetic determinants of serum testosterone concentrations in men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone''s high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10⁻⁴¹ and rs6258, p = 2.3×10⁻²²). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10⁻¹⁶). The rs6258 polymorphism in exon 4 of SHBG affected SHBG''s affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.     

Universal definition of loss to follow-up in HIV treatment programs: a statistical analysis of 111 facilities in Africa, Asia, and Latin America

Background

Although patient attrition is recognized as a threat to the long-term success of antiretroviral therapy programs worldwide, there is no universal definition for classifying patients as lost to follow-up (LTFU). We analyzed data from health facilities across Africa, Asia, and Latin America to empirically determine a standard LTFU definition.

Methods and Findings

At a set “status classification” date, patients were categorized as either “active” or “LTFU” according to different intervals from time of last clinic encounter. For each threshold, we looked forward 365 d to assess the performance and accuracy of this initial classification. The best-performing definition for LTFU had the lowest proportion of patients misclassified as active or LTFU. Observational data from 111 health facilities—representing 180,718 patients from 19 countries—were included in this study. In the primary analysis, for which data from all facilities were pooled, an interval of 180 d (95% confidence interval [CI]: 173–181 d) since last patient encounter resulted in the fewest misclassifications (7.7%, 95% CI: 7.6%–7.8%). A secondary analysis that gave equal weight to cohorts and to regions generated a similar result (175 d); however, an alternate approach that used inverse weighting for cohorts based on variance and equal weighting for regions produced a slightly lower summary measure (150 d). When examined at the facility level, the best-performing definition varied from 58 to 383 d (mean = 150 d), but when a standard definition of 180 d was applied to each facility, only slight increases in misclassification (mean = 1.2%, 95% CI: 1.0%–1.5%) were observed. Using this definition, the proportion of patients classified as LTFU by facility ranged from 3.1% to 45.1% (mean = 19.9%, 95% CI: 19.1%–21.7%).

Conclusions

Based on this evaluation, we recommend the adoption of ≥180 d since the last clinic visit as a standard LTFU definition. Such standardization is an important step to understanding the reasons that underlie patient attrition and establishing more reliable and comparable program evaluation worldwide. Please see later in the article for the Editors'' Summary     

Development of [Ile40]HTLV‐I protease inhibition assay using novel fluorogenic and chromogenic substrate

HTLV‐I is a debilitating and/or lethal retrovirus that causes HTLV‐I‐associated myelopathy/tropical spastic paraparesis, adult T‐cell leukemia and several inflammatory diseases. HTLV‐I protease is an aspartic retropepsin involved in HTLV‐I replication and its inhibition could treatHTLV‐I infection. A recombinant L40I mutant HTLV‐I protease was designed and obtained from Escherichia coli, self‐processingand purification by ion‐exchange chromatography. The protease was refolded by a one‐step dialysis and recovered activity. The cleavage efficiency of the [Ile⁴⁰]HTLV‐I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His‐tagged non‐mutated HTLV‐I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p‐nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV‐I protease inhibition potency assay. The HTLV‐I protease inhibition assay with the [Ile⁴⁰]HTLV‐I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost‐effective and more time‐efficient while being reproducible and less labor‐intensive. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.