Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences

PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl₂), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.     

Lipolysis: pathway under construction

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.     

Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background

Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF.

Methods/Principal Findings

Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7^th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels.

Conclusions

Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.     

Cytotoxic effects of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCHQ) on liver cells are modulated by antioxidants

The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.     

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae)

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 μg As/L). A control group was exposed to tap water (pH 8.0; 26 °C; 7.20 mg O₂/L). As exposure resulted in (1) an increase (p < 0.05) of glutathione (GSH) levels after exposure to 10 and 100 μg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p < 0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p < 0.05) oxygen consumption after exposure to 100 μg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p > 0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.     

Membrane inlet—ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.     

iBench: A ground truth approach for advanced validation of mass spectrometry identification method

The discovery of many noncanonical peptides detectable with sensitive mass spectrometry inside, outside, and on cells shepherded the development of novel methods for their identification, often not supported by a systematic benchmarking with other methods. We here propose iBench, a bioinformatic tool that can construct ground truth proteomics datasets and cognate databases, thereby generating a training court wherein methods, search engines, and proteomics strategies can be tested, and their performances estimated by the same tool. iBench can be coupled to the main database search engines, allows the selection of customized features of mass spectrometry spectra and peptides, provides standard benchmarking outputs, and is open source. The proof-of-concept application to tryptic proteome digestions, immunopeptidomes, and synthetic peptide libraries dissected the impact that noncanonical peptides could have on the identification of canonical peptides by Mascot search with rescoring via Percolator (Mascot+Percolator).     

Signaling by IL-31 and functional consequences

Cytokines are key to control cellular communication. Interleukin-31 (IL-31) was recently discovered as a new member of the IL-6 family of cytokines. IL-31 signals through a heterodimeric receptor composed of OSMR and IL-31RA, a complex that stimulates the JAK-STAT, the RAS/ERK and the PI3K/AKT signal transduction pathways. The available data suggests that IL-31 is important for both innate and adaptive immunity in tissues that are in close contact with the environment, i.e. the skin, the airways and the lung, and the lining of the intestine. Enhanced expression of IL-31 is associated with a number of diseases, including pruritic diseases such as atopic dermatitis, but also in allergy and inflammatory bowel disease. In these tissues IL-31 coordinates the interaction of different immune cells, including T-cells, mast cells, and eosinophils, with epithelial cells. In this review we have summarized the available data on IL-31 and its receptor, their expression pattern and how they are regulated. We describe the current state of knowledge of the involvement of IL-31 in diseases, both in humans and in mouse models. From these studies it is becoming clear that IL-31 plays an important role in the proper functioning of the skin and of airway and intestinal epithelia. The findings available suggest that IL-31 might be an interesting target for directed drug therapy.