Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.     

From waste to resource: mycoremediation of contaminated marine sediments in the SEDITERRA Project

Purpose

Port-dredging activities produce large volumes of contaminated sediments. Dredged sediments are considered a waste by national laws, but recently the desire to consider them a resource has become widespread and remedies for their contamination are being searched to allow their reuse. In this work, we studied, developed, and tested a method for remediate marine-dredged sediments contaminated by heavy metals using native fungi and a microporous membrane, in order to achieve the sediment quality and allow their reuse.

Materials and methods

Activity was carried out on port sediments from Genoa, Leghorn, Pisa, and Cagliari (Italy). Autochthonous fungi were isolated from each sediment and employed in mycoremediation tests. Two plastic boxes were prepared (for each Port) with 5 kg of sediment in each box, employed for metal bioaccumulation using a sterile polyester membrane inoculated with fungi. Membranes were analyzed at 15, 30, and 60 days after inoculums, and sediments were analyzed after 60 days at the end of the experiment to verify metal contamination degree. Recovery efficiency (RE%) and difference recovery efficiency (DRE%) were calculated for each experiment: the first shows the absorption capability of the membrane-fungi consortium; the second evidences only the fungal contribution to the metal absorption. To assess sediment contamination before and after the mycoremediation treatment, we considered chemical levels of reference L1 (the lowest chemical level of reference) and L2 (the highest chemical level of reference), and the evaluation of chemical hazard (HQ) for the chemical contaminants defined by the Italian Ministerial Decree 173/2016.

Results and discussion

Fungi from Genoa sediments increase the membrane absorption of Cu and Zn. Regarding Leghorn results, RE (%) increases and reaches the maximum value after 60 days of treatment for each considered metal. Cr tot, Ni, and Mn appear to be hyper-bioaccumulated. DRE values of Pisa sediments show that Mn is excluded by fungi and it does not bioaccumulate, while other metals and in particular Cd, Cr tot, Zn, and Sb are bioaccumulated. Cagliari DREs show that fungi are not able to bioaccumulate Cr tot, Ni, and Mn and their accumulation is due to the membrane, while As and Cd are bioaccumulated.

Conclusions

Our work evidenced that selected fungi are able to grow on a microporous support and actively reduce metal concentrations in the sediments, achieving their quality. This biomembrane system may represent an important instrument for the remediation of the residual metal contamination of port sediments.

     

Occurrence of Emerging Contaminants in Brazilian Drinking Waters: A Sewage-To-Tap Issue

The goal of this work was to investigate the occurrence of emerging contaminants in drinking water of the city of Campinas, Brazil. Tap water samples were analyzed using SPE-GC-MS for 11 contaminants of recent environmental concern. Six emerging contaminants (stigmasterol, cholesterol, bisphenol A, caffeine, estrone, and 17β-estradiol) were found in the samples. The latter two were detected only during the dry season, with concentrations below quantification limits. Stigmasterol showed the highest average concentration (0.34?±?0.13?µg L^?1), followed by cholesterol (0.27?±?0.07?µg L^?1), caffeine (0.22?±?0.06?µg L^?1), and bisphenol A (0.16?±?0.03?µg L^?1). In Campinas, where surface drinking water supplies receive large amounts of raw sewage inputs, the emerging contaminants levels in drinking waters were higher than median values compiled for drinking and finished water samples around the world.     

Fractal dimension and shape parameters of asexual Fusarium spores from selected species: Which species can be distinguished?

Journal of Plant Diseases and Protection - Fungi of the genus Fusarium are prominent plant pathogens producing various toxic secondary metabolites. Digital microscopic images were taken from the...     

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

Considering the efficiency of steroidal anti-inflammatory drugs (SAIDs) in the immunomodulation of breeding-induced endometritis and the possibility of using these drugs by intrauterine route instead of the parenteral application, the present study aimed at evaluating the effect of SAIDs added to the semen extender on equine sperm viability and fertility. In experiment 1, 15 SAIDs were individually added to a skim milk-based extender and, based on the results of sperm motility, dexamethasone was the drug of choice for the subsequent trials. The effect of dexamethasone on the viability of fresh and 24-hour cooled semen was investigated in experiment 2. In experiment 3, fertility rate was measured in both post-breeding endometritis-resistant and susceptible mares. Although dexamethasone supplementation caused a premature decrease in sperm total and progressive motility and in sperm velocities (P < .05), no difference was observed for sperm membrane integrities and fertility (P > .05). Based on these results, we can conclude that dexamethasone can be added to equine semen at the time of insemination or before cooling, although its use was not able to increase fertility.     

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.     

Assessment of the impact of collection temperature and sampler design on the measurement of exhaled breath condensate pH in healthy horses

The pH measurement of exhaled breath condensate (EBC) may provide a non-invasive method of assessing the lower airways of horses but the methodology used may influence findings. The aim of this study was to investigate the effect of two sampling devices and three methods of condensation surface cooling (ethanol slush, -100°C; dry ice, -75°C; water ice, 0°C) on EBC pH. Each method was tested 30 times using six healthy ponies. Sample pH was determined before and after de-aeration with argon for 10 min. Sampler design was found to significantly affect pH. Samples collected as a liquid had a significantly higher pH than samples frozen during collection (P<0.05). De-aeration resulted in significantly higher pH (P<0.05) with less variation. This study has shown that device design and condensation surface temperature will influence EBC pH, which will prevent a direct comparison of results when different methodologies are used.     

Conjunctival dacryops in two golden retrievers

Two 5-month-old golden retriever dogs were referred for the evaluation of a mass arising from the left ventrolateral conjunctiva of the lower eyelid. The masses were externally located, deemed to be cystic in nature, and were surgically removed, and in one case, 0.15 mL of clear fluid was collected. Cytological examination of the aspirated fluid revealed proteinaceous, basophilic amorphous material, and few epithelial cells with foamy cytoplasm and small nuclei. No micro-organisms were detected. Histological examination of the cysts showed a wall composed of single to double cell layers. The cells were cuboidal to flattened, nonciliated, lined with mild submucosal connective tissues and slight inflammation composed of lymphocytes, plasma cells, and rare macrophages. In some areas, lobules of acinar glandular tissue were observed. To the authors' knowledge, this is the first case report of conjunctival dacryops in dogs. The complete surgical removal of the cysts was curative in both cases.     

Validation of DNA barcoding as an efficient tool for taxon identification and detection of species diversity in Italian conifers

DNA barcoding may be particularly important in impacting the current trends of world’s biodiversity crisis, public economic issues and science. Forests play a crucial role in self-sustaining natural ecosystems and human environment at both local and regional scales. Over 39% of the world’s forests are formed by conifers; most species are highly exposed to threats and exploited at high frequency. Conifers have an urgent need to be identified through DNA barcodes, but received little attention up to date. The application of standard barcoding protocols appears to be hampered by a lack of consensus on the selection of markers and low rates of successful species identification. We examined whether four marker regions (trnh-psba, rbcL, rpoc1 and matK) matched conifer species taxonomy in a practical biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co-occurrence of the species. Twenty-five conifer species were included in a floristic study based on regional samplings. Genetic distance, similarity and phylogeny approaches were used to assess identification of taxa and species monophyly. We obtained 100% total discrimination success, with rbcL?+?trnH-psbA as the suggested two-locus barcode in terms of universality and efficacy. Consistent barcoding gaps, supported monophyly and regional patterns of diversity, were evidenced. Species requiring further investigation to be fully assessed at a broader context are indicated. We conclude that the time seems now ripe for coherent barcoding campaigns of entire forest tree regional floras.