Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis,Immaturity and Oxidative Stress

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.     

Performance of a mobile mechanical screen to improve the commercial quality of wood chips for energy

The study analyzed the performance of a mobile screening device for upgrading coarse wood chips to residential user standards, by removing oversize particles and fines. The machine was designed for transportation to forest landings, logistic terminals and plant chip yards. Average productivity was 1.9 oven-dry tons (odt) h⁻¹, corresponding to a screening cost of 28.5 € odt⁻¹. This figure was lower than the price increase obtained by upgrading industrial chips to residential user standards. Hence, screening offered a profit of 4.7 € odt⁻¹, or 16% of the original screening cost. The screening process was capable of upgrading chips from industrial to residential specifications, by reducing the incidence of oversize particles below the 1% critical threshold. Screening also allowed a substantial reduction in the content of fines. A similar effect was not verified for crushed wood, which failed to meet the specifications for residential fuel.     

An unexpected evolution of symptomatic mild middle cerebral artery (MCA) stenosis: asymptomatic occlusion

Background  

The intracranial localization of large artery disease is recognized as the main cause of ischemic stroke in the world, considering all countries, although its global burden is widely underestimated. Indeed it has been reported more frequently in Asians and African-American people, but the finding of intracranial stenosis as a cause of ischemic stroke is relatively common also in Caucasians. The prognosis of patients with stroke due to intracranial steno-occlusion is strictly dependent on the time of recanalization. Moreover, the course of the vessel involvement is highly dynamic in both directions, improvement or worsening, although several data are derived from the atherosclerotic subtype, compared to other causes.     

Influence of Water Activity and System Mobility on Peroxidase Activity in Maltodextrin Solutions

Maltodextrins influenced the enzymatic activity in aqueous solutions by affecting the water activity (a_w) and mobility as described by viscosity and T’g. In diluted solutions, viscosity being equal, (1) maltodextrin with dextrose equivalents (DE) of 33 was more effective than glucose in limiting horseradish peroxidase (HRP) activity; (2) an increase in the maltodextrin chain length from DE 33 to DE 8.7 did not further limited enzymatic activity; (3) the maltodextrin with the highest chain length (DE 2.5) determined the highest enzyme inhibition. In general, the increase of molecular weight negatively affected the HRP activity by increasing the viscosity and T’g (decreasing molecular mobility) but it positively affected the a_w and, in some cases, this compensated the HRP activity inhibition. In concentrated solutions (apparent viscosity ≈ 40 mPa s) the HRP activity decreased with the increase of the maltodextrin molecular weight, and it showed a dependence on T’g which could be described by a William, Landel and Ferry (WLF)-type equation. On the contrary, in the solution added with the maltodextrin with the highest chain length (DE 2.5) the HRP activity was much higher than that predicted by the WLF-type equation. The maltodextrin with DE 2.5 contains intact starch fragments and in water forms a suspension. In such a discontinuous system, the viscosity in the vicinity of the protein is lower than the bulk viscosity, and thus, the enzyme activity is higher than expected. Moreover, since T’g is a property of the soluble phase, it does not explain the mobility in discontinuous systems.     

Glandular trichomes and essential oil composition of endemic Sideritis italica (Mill.) Greuter et Burdet from central Italy

Sideritis italica (Mill.) Greuter et Burdet belongs to the Lamiaceae family and is endemic to Italy. The glandular trichomes (morphology, distribution, histochemistry, and ultrastructure) of the plant were studied for the first time, along with the chemical composition of the essential oils. Abundant non-glandular hairs and peltate (type A) and capitate (types B, C(1), and C(x)) glandular trichomes were observed both on the vegetative and reproductive organs. The histochemical procedures and the ultrastructural investigation enabled specific location of the main site of essential oil production mainly in type-A peltate hairs. Particular emphasis is given to the release mechanism of the secreted material in all of the types of glands, and the potential taxonomic value of the indumentum in the Lamiaceae family is briefly discussed. Essential oils were hydrodistilled from flowering aerial parts of S. italica, and 136 compounds (112 in flowerheads, 79 in vegetative parts) were identified. The quantitative prevalence of diterpenoids (43.4% in flowerheads and 22.3% in vegetative parts) was the most significant characteristic of the essential oil of S. italica that could be classified as a diterpene-rich essential oil according to the classification of Kirimer.     

Volatile components of whole and different plant parts of bastard balm (Melittis melissophyllum L., Lamiaceae) collected in Central Italy and Slovakia

The aim of this work was to trap the volatiles released from whole frozen and dry aerial parts, and, separately, from different organs (leaves, stems, corolla and calyx) of bastard balm (Melittis melissophyllum L., Lamiaceae) populations collected in Italy and Slovakia by HS-SPME, and to identify the headspace constituents responsible for the characteristic aroma impression by GC/FID and GC/MS techniques. Among more than 100 volatile components detected, the C(8) alcohol oct-1-en-3-ol, responsible for the typical mushroom-like odor, and the phenolic coumarin, with a characteristic sweet and creamy vanilla bean odor, played a major role in the aroma of whole aerial parts and different plant organ samples. In particular, dry calyx parts could be proposed as flavoring agent in food products as mushroom aroma enhancer. Multivariate chemometric techniques, such as cluster analysis and principal component analysis, were used to characterize the sample populations according to the geographical origin and processing of plant material.     

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Background

Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/Principal Findings

Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s⁻¹ over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/Significant Findings

We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.     

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.     

Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol

The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose. However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product. In this study, the growth characteristics of F. oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations. Under aerobic conditions the maximum specific growth rate was found to be 0.043 h(-1), and the highest ethanol yield (1.66 mol/mol) was found under anaerobic conditions. During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions. The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden-Meyerhof-Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions. The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation. On the other hand, the presence of high levels of gamma-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions.     

Ghrelin regulates mitochondrial-lipid metabolism gene expression and tissue fat distribution in liver and skeletal muscle

Ghrelin is a gastric hormone increased during caloric restriction and fat depletion. A role of ghrelin in the regulation of lipid and energy metabolism is suggested by fat gain independent of changes in food intake during exogenous ghrelin administration in rodents. We investigated the potential effects of peripheral ghrelin administration (two times daily 200-micrograms [DOSAGE ERROR CORRECTED] sc injection for 4 days) on triglyceride content and mitochondrial and lipid metabolism gene expression in rat liver and muscles. Compared with vehicle, ghrelin increased body weight but not food intake and circulating insulin. In liver, ghrelin induced a lipogenic and glucogenic pattern of gene expression and increased triglyceride content while reducing activated (phosphorylated) stimulator of fatty acid oxidation, AMP-activated protein kinase (AMPK, all P < 0.05), with unchanged mitochondrial oxidative enzyme activities. In contrast, triglyceride content was reduced (P < 0.05) after ghrelin administration in mixed (gastrocnemius) and unchanged in oxidative (soleus) muscle. In mixed muscle, ghrelin increased (P < 0.05) mitochondrial oxidative enzyme activities independent of changes in expression of fat metabolism genes and phosphorylated AMPK. Expression of peroxisome proliferator-activated receptor-gamma, the activation of which reduces muscle fat content, was selectively increased in mixed muscle where it paralleled changes in oxidative capacities (P < 0.05). Thus ghrelin induces tissue-specific changes in mitochondrial and lipid metabolism gene expression and favors triglyceride deposition in liver over skeletal muscle. These novel effects of ghrelin in the regulation of lean tissue fat distribution and metabolism could contribute to metabolic adaptation to caloric restriction and loss of body fat.