Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.     

Origin of Fish Biomass in a Diverse Subtropical River: An Allochthonic-Supported Biomass Increase Following Flood Pulses

Ecosystems - The origin of resources supporting metazoan biomass in rivers has long been a subject of debate. The river wave concept (RWC) postulates that the energetic basis of food webs varies...     

Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7‐positive cells and in a mTOR‐independent mechanism

β‐Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that β‐agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of β‐adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a β‐adrenergic receptor agonist, to activate the β‐adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7‐positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of β‐adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy.     

Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.     

Attraction of Fruit-Eating Bats with Essential Oils of Fruits: A Potential Tool for Forest Restoration

Previous tests with essential oils from ripe chiropterochoric fruits suggested they can be used to attract and capture fruit-eating bats inside forest remnants. Here we evaluated the efficiency of these oils to attract frugivorous bats to open areas. We performed field tests with artificial fruits impregnated with essential oils of the genera Piper or Ficus that were attached to two groups of mist-nets set 50 m outside the border of a forest remnant. One group of artificial fruits received the corresponding oil isolated through hydrodistillation and the other received water only. Fruits with oils attracted significantly more fruit-eating bats, especially Artibeus lituratus that regularly crosses open habitats to reach other forest remnants. The highly significant attraction of A. lituratus by the oil of Piper was unexpected, since this bat is a specialist on Ficus fruits. We hypothesize that in habitats with no fruit available it is possible to attract frugivorous bats with the odor of several ripe fruit species. Furthermore, we verified that almost half of the individuals captured defecated seeds, indicating that the oils also attract recently fed bats, even when their preferred food is available nearby. This technique potentially may increase seed rain at specific locations, being particularly promising to restoration projects.     

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.     

Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clusters

DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S](2+) clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.     

Regulation of autophagy as a therapy for immunosenescence-driven cancer and neurodegenerative diseases: The role of exercise

Aging is one of the risk factors for the development of low-grade inflammation morbidities, such as several types of cancer and neurodegenerative diseases, due to changes in the metabolism, hormonal secretion, and immunosenescence. The senescence of the immune system leads to improper control of infections and tissue damage increasing age-related diseases. One of the mechanisms that maintain cellular homeostasis is autophagy, a cell-survival mechanism, and it has been proposed as one of the most powerful antiaging therapies. Regular exercise can reestablish autophagy, probably through AMP-activated protein kinase activation, and help in reducing the age-related senescence diseases. Therefore, in this study, we discuss the effects of exercise training in immunosenescence and autophagy, preventing the two main age-related disease, cancer and neurodegeneration.