Late‐in‐life treadmill training rejuvenates autophagy,protein aggregate clearance,and function in mouse hearts

Protein quality control mechanisms decline during the process of cardiac aging. This enables the accumulation of protein aggregates and damaged organelles that contribute to age‐associated cardiac dysfunction. Macroautophagy is the process by which post‐mitotic cells such as cardiomyocytes clear defective proteins and organelles. We hypothesized that late‐in‐life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs. adult mice. As expected, 24‐month‐old male C57BL/6J mice (old) exhibited repressed autophagosome formation and protein aggregate accumulation in the heart, systolic and diastolic dysfunction, and reduced exercise capacity vs. 8‐month‐old (adult) mice (all p < 0.05). To investigate the influence of late‐in‐life exercise training, additional cohorts of 21‐month‐old mice did (old‐ETR) or did not (old‐SED) complete a 3‐month progressive resistance treadmill running program. Body composition, exercise capacity, and soleus muscle citrate synthase activity improved in old‐ETR vs. old‐SED mice at 24 months (all p < 0.05). Importantly, protein expression of autophagy markers indicate trafficking of the autophagosome to the lysosome increased, protein aggregate clearance improved, and overall function was enhanced (all p < 0.05) in hearts from old‐ETR vs. old‐SED mice. These data provide the first evidence that a physiological intervention initiated late‐in‐life improves autophagic flux, protein aggregate clearance, and contractile performance in mouse hearts.     

High-throughput screening and rapid inhibitor triage using an infectious chimeric hepatitis C virus

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.     

Hyperthermophilic endoglucanase for in planta lignocellulose conversion

ABSTRACT: BACKGROUND: The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. RESULTS: Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade alpha-cellulose or even complex cell wall preparations under those pretreatment conditions. CONCLUSION: The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.     

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus–oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.     

Efficiency and allometry of seed production in grassland forbs: site and year comparative study in the Italian Eastern Pre-Alps

Forbs are important biodiversity components of grasslands and are often threatened by management intensification. As most forbs propagate predominantly by seed, knowledge of their seed regenerative traits would improve the conservation and restoration of forb-rich ecosystems. The main seed production traits of six forbs that are common in European species-rich grasslands were studied by collecting fertile shoots from different sites and over several years. Among sites and years, variability was high, particularly in the number of inflorescences per shoot, which affected ovule production more than any other trait. Relationships between inflorescence size and the number of ovules were mainly negatively allometric or almost so, with lower flower densities in larger inflorescences. The average ovule-to-seed transformation efficiency was 58%. There was significant variation among collections of the same species, and even more between species. Species with a low ovule-to-seed transformation efficiency generally exhibited compensatory, high seed viability. Large inflorescences had high ovule to seed utilization values, probably because of better nutrient conditions. Seed germinability (average, 30%) was much lower than seed viability (average, 54%); therefore, seed dormancy was an important feature of the species studied.     

Impact of food processing and detoxification treatments on mycotoxin contamination

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA)1

There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5′ ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty‐eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion.     

Recombinant near-isogenic lines: a resource for the mendelization of heterotic QTL in maize

Although heterosis is widely exploited in agriculture, a clear understanding of its genetic bases is still elusive. This work describes the development of maize recombinant near-isogenic lines (NILs) for the mendelization of six heterotic QTL previously identified based on a maize (Zea mays L.) RIL population. The efficient and inexpensive strategy adopted to generate sets of NILs starting from QTL-specific residual heterozygous lines (RHLs) is described and validated. In particular, we produced nine pairs of recombinant NILs for all six QTL starting from RHLs F_4:5 originally obtained during the production of the RIL population mentioned above. Whenever possible, two different NIL pairs were generated for each QTL. The efficiency of this procedure was tested by analyzing two segregating populations for two of the selected heterotic QTL for plant height, yield per plant and ears per plant. Both additive and dominant effects were observed, consistently with the presence of the QTL within the introgressed regions. Refinement of QTL detection was consistent with previous observations in terms of effects and position of the considered QTL. The genetic material developed in this work represents the starting point for QTL fine mapping aimed at understanding the genetic bases of hybrid vigor in maize. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.