DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimized.     

The Paracoccus denitrificans NarK‐like nitrate and nitrite transporters—probing nitrate uptake and nitrate/nitrite exchange mechanisms

Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.     

Distinct Agonist Regulation of Muscarinic Acetylcholine M2-M3 Heteromers and Their Corresponding Homomers

Each subtype of the muscarinic receptor family of G protein-coupled receptors is activated by similar concentrations of the neurotransmitter acetylcholine or closely related synthetic analogs such as carbachol. However, pharmacological selectivity can be generated by the introduction of a pair of mutations to produce Receptor Activated Solely by Synthetic Ligand (RASSL) forms of muscarinic receptors. These display loss of potency for acetylcholine/carbachol alongside a concurrent gain in potency for the ligand clozapine N-oxide. Co-expression of a form of wild type human M₂ and a RASSL variant of the human M₃ receptor resulted in concurrent detection of each of M₂-M₂ and M₃-M₃ homomers alongside M₂-M₃ heteromers at the surface of stably transfected Flp-In^TM T-REx^TM 293 cells. In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers. However, selective agonist occupancy of the M₂ receptor resulted in enhanced M₂-M₂ homomer interactions but decreased M₂-M₃ heteromer interactions. By contrast, selective activation of the M₃ RASSL receptor did not significantly alter either M₃-M₃ homomer or M₂-M₃ heteromer interactions. Selectively targeting closely related receptor oligomers may provide novel therapeutic opportunities.     

Mentha spicata and Salvia fruticosa composts as soil amendments in tomato cultivation

Τhe potential use of the aromatic plants Mentha spicata L. (spearmint) and Salvia fruticosa Mill. (sage) as soil amendments was evaluated. For this purpose, tomato seeds were sown in pots that had been filled with composts made from these plants and mixed with soil collected from an organically cultivated tomato field. A 2?×?2?×?4 [two types of fertilizer (synthetic and organic), two types of compost (M. spicata and S. fruticosa) and four compost rates (0%, 2%, 4% and 8%, w/w)] factorial experiment was used; the experiment was conducted twice in a growth chamber and lasted 60 days. At 0, 20, 40 and 60 days, after the establishment of the experiment, the soil bacterial and fungal abundance, the growth of nitrifying bacteria, the number of emerging weeds and the shoot length of tomato plants were measured in all treatments; at the end of the experiment, the above and belowground biomass of tomato plants was also determined. Soil microbial density increased with increasing compost rate of both species; the highest fungal and bacterial densities were recorded at 40 and 60 days, after the establishment of the experiment, respectively. Nitrifying bacteria were present in all treatments and at all sampling times. Both composts had a stimulating effect on tomato growth, which was remarkably pronounced with M. spicata. In contrast, weed emergence was reduced, but only in soils amended with M. spicata. The results suggest that M. spicata compost added at a rate of 4% to 8% is a very promising soil amendment, since it stimulates tomato growth, increases soil bacterial and fungal abundance and inhibits weed emergence. Further research is needed to elucidate the mode of action of M. spicata compost, its effect under field conditions and its possible use in mixed crop, rotational crop or cover crop systems.     

Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis

Background

Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).

Methods

Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.

Results

A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.

Conclusion

Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.     

Oxidation state-dependent protein-protein interactions in disulfide cascades

Bacterial growth and pathogenicity depend on the correct formation of disulfide bonds, a process controlled by the Dsb system in the periplasm of Gram-negative bacteria. Proteins with a thioredoxin fold play a central role in this process. A general feature of thiol-disulfide exchange reactions is the need to avoid a long lived product complex between protein partners. We use a multidisciplinary approach, involving NMR, x-ray crystallography, surface plasmon resonance, mutagenesis, and in vivo experiments, to investigate the interaction between the two soluble domains of the transmembrane reductant conductor DsbD. Our results show oxidation state-dependent affinities between these two domains. These observations have implications for the interactions of the ubiquitous thioredoxin-like proteins with their substrates, provide insight into the key role played by a unique redox partner with an immunoglobulin fold, and are of general importance for oxidative protein-folding pathways in all organisms.     

Cytochrome c biogenesis System I

Cytochromes c are widespread respiratory proteins characterized by the covalent attachment of heme. The formation of c-type cytochromes requires, in all but a few exceptional cases, the formation of two thioether bonds between the two cysteine sulfurs in a -CXXCH- motif in the protein and the vinyl groups of heme. The vinyl groups of the heme are not particularly activated and therefore the addition reaction does not physiologically occur spontaneously in cells. There are several diverse post-translational modification systems for forming these bonds. Here, we describe the complex multiprotein cytochrome c maturation (Ccm) system (in Escherichia coli comprising the proteins CcmABCDEFGH), also called System I, that performs the heme attachment. System I is found in plant mitochondria, archaea and many Gram-negative bacteria; the systems found in other organisms and organelles are described elsewhere in this minireview series.     

The hydrobioid freshwater gastropods (Caenogastropoda,Truncatelloidea) of Greece: new?records,taxonomic re-assessments using DNA sequence data and an update of?the IUCN Red List Categories

Hydrobioid freshwater gastropods were collected from mainland and insular Greece. Several threatened taxa, such as Graecoanatolica vegorriticola, Pseudamnicola negropontina, Pseudamnicola pieperi, Pseudobithynia eubooensis and Pseudoislamia balcanica, were recorded from new localities. Trichonia trichonica, which has been considered extinct from its type locality for the last twenty eight years, was re-discovered, whereas the presence of Daphniola exigua, G. vegorriticola, Marstoniopsis graeca, P. pieperi and Pseudobithynia trichonis in their type localities was verified. The taxonomic status of P. negropontina and the newly discovered populations of G. vegorriticola was elucidated using COI sequence data. The new data recorded during this survey indicate that the IUCN status of some Greek endemic hydrobioids needs to be updated.     

Ischemia–Reperfusion Injury of Sciatic Nerve in Rats: Protective Role of Combination of Vitamin C with E and Tissue Plasminogen Activator

An ischemia/reperfusion injury of rat’s sciatic nerve was experimentally developed. In this model, we measured the in vivo production of superoxide radical, as a marker of oxidative stress and the occludin expression as an indicator of blood-nerve barrier function and we examined potential protective innervations against these abnormalities. Right sciatic nerves of the animals underwent 3 h of ischemia followed by 7 days of reperfusion and were divided into three groups: ischemic, pretreated with vitamin C in conjunction with vitamin E and treated with tissue plasminogen activator. Compared to measurements from left sciatic nerves used as sham, the ischemic group showed significantly increased superoxide radical and reduced expression of occludin in western blot and immunohistochemistry. No such differences were detected between sham and nerves in the vitamin or tissue plasminogen activator groups. It is suggested that the experimental ischemia/reperfusion model was suitable for studying the relationship between oxidative state and blood-nerve barrier. The reversion of abnormalities by the applied neuroprotective agents might prove to be a clinically important finding in view of the implication of vascular supply derangement in various neuropathies in humans.