Composition and antimicrobial activity of the essential oil of six Hypericum species from Serbia

The volatile composition of six Hypericum species has been studied. The essential oils were obtained by steam distillation in 500 mL H₂O for 2 h in a modified Clevenger apparatus with a water-cooled oil receiver to reduce hydrodistillation over-heating artifacts, and their analyses were performed by GC and GC–MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. A total of 100 different compounds were identified. The main constituents of the investigated populations of each taxon have been revealed as follows: Hypericum alpinum: (−)-β-pinene, γ-terpinene, (−)-(E)-caryophyllene; Hypericum barbatum: (−)-α-pinene, (−)-β-pinene, (−)-limonene, (−)-(E)-caryophyllene, (−)-caryophyllene oxide; Hypericum rumeliacum: (−)-α-pinene, (−)-β-pinene, (−)-limonene, Hypericum hirsutum: nonane, undecane, (−)-(E)-caryophyllene, (−)-caryophyllene oxide; Hypericum maculatum: spathulenol, globulol; Hypericum perforatum: (−)-α-pinene, (Z)-β-farnesene, germacrene D; Monoterpene hydrocarbons were shown to be the main group of the taxa belonging to the section Drosocarpium, while the taxa of section Hypericum were more rich in sesquiterpene hydrocarbons.     

Circadian Regulation of Cochlear Sensitivity to Noise by Circulating Glucocorticoids

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Amphiphilic helical peptide enhances the uptake of single-walled carbon nanotubes by living cells

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that approximately 6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.     

Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, alpha-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, alpha-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation.     

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest

Two thermophilic extracellular proteases, designated Lmm-protease-Lh (29 kDa) and Hmm-protease-Lh (62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK₂. Both novel proteases maintained full activity at 60 °C after 1-h incubation at 10 °C as well as at 80 °C, showing optimum k_cat/K_m values at pH 7.00 and 60 °C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis–Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis–Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k_cat/K_m) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis–Menten parameters.     

Mesenchymal stromal cell delivery as a potential therapeutic strategy against COVID-19: Promising evidence from in vitro results

BACKGROUNDSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic, which was initiated in December 2019. COVID-19 is characterized by a low mortality rate (< 6%); however, this percentage is higher in elderly people and patients with underlying disorders. COVID-19 is characterized by mild to severe outcomes. Currently, several therapeutic strategies are evaluated, such as the use of anti-viral drugs, prophylactic treatment, monoclonal antibodies, and vaccination. Advanced cellular therapies are also investigated, thus representing an additional therapeutic tool for clinicians. Mesenchymal stromal cells (MSCs), which are known for their immunoregulatory properties, may halt the induced cytokine release syndrome mediated by SARS-CoV-2, and can be considered as a potential stem cell therapy.AIMTo evaluate the immunoregulatory properties of MSCs, upon stimulation with COVID-19 patient serum. METHODSMSCs derived from the human Wharton’s Jelly (WJ) tissue and bone marrow (BM) were isolated, cryopreserved, expanded, and defined according to the criteria outlined by the International Society for Cellular Therapies. Then, WJ and BM-MSCs were stimulated with a culture medium containing 15% COVID-19 patient serum, 1% penicillin-streptomycin, and 1% L-glutamine for 48 h. The quantification of interleukin (IL)-1 receptor a (Ra), IL-6, IL-10, IL-13, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-a, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and indoleamine-2,3-dioxygenase (IDO) was performed using commercial ELISA kits. The expression of HLA-G1, G5, and G7 was evaluated in unstimulated and stimulated WJ and BM-MSCs. Finally, the interactions between MSCs and patients’ macrophages were established using co-culture experiments.RESULTSThawed WJ and BM-MSCs exhibited a spindle-shaped morphology, successfully differentiated to “osteocytes”, “adipocytes”, and “chondrocytes”, and in flow cytometric analysis were characterized by positivity for CD73, CD90, and CD105 (> 95%) and negativity for CD34, CD45, and HLA-DR (< 2%). Moreover, stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation, in comparison to unstimulated cells. The HLA-G isoforms (G1, G5, and G7) were successfully expressed by the unstimulated and stimulated WJ-MSCs. On the other hand, only weak expression of HLA-G1 was identified in BM-MSCs. Stimulated MSCs secreted high levels of IL-1Ra, IL-6, IL-10, IL-13, TGF-β1, FGF, VEGF, PDGF, and IDO in comparison to unstimulated cells (P < 0.05) after 12 and 24 h. Finally, macrophages derived from COVID-19 patients successfully adapted the M2 phenotype after co-culturing with stimulated WJ and BM-MSCs.CONCLUSIONWJ and BM-MSCs successfully produced high levels of immunoregulatory agents, which may efficiently modulate the over-activated immune responses of critically ill COVID-19 patients.     

The CD27L and CTP1L Endolysins Targeting Clostridia Contain a Built-in Trigger and Release Factor

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.     

Population Trend of the World’s Monitored Seabirds, 1950-2010

Seabird population changes are good indicators of long-term and large-scale change in marine ecosystems, and important because of their many impacts on marine ecosystems. We assessed the population trend of the world’s monitored seabirds (1950–2010) by compiling a global database of seabird population size records and applying multivariate autoregressive state-space (MARSS) modeling to estimate the overall population trend of the portion of the population with sufficient data (i.e., at least five records). This monitored population represented approximately 19% of the global seabird population. We found the monitored portion of the global seabird population to have declined overall by 69.7% between 1950 and 2010. This declining trend may reflect the global seabird population trend, given the large and apparently representative sample. Furthermore, the largest declines were observed in families containing wide-ranging pelagic species, suggesting that pan-global populations may be more at risk than shorter-ranging coastal populations.     

A probabilistic model that determines the social ecological system (SES) attributes that lead to successful discard management

The social ecological system (SES) framework is used to investigate the success of discard management, by gathering knowledge from worldwide examples of discard management applications. The literature search confirmed that most efforts aiming to reduce discards were implemented under top-down management schemes. For about half of the case studies where management tools were applied, a reduction of discards was achieved. To determine the attributes that influence the discard management success, a probabilistic classifier model (Naive Bayes Classifier) was used. This revealed that commonly used interventions to reduce discards can be ineffective unless advice is given in alternative forms that have societal acceptance. The role of social cohesion and stakeholder enforcement mechanisms is underlined and increases the odds of meeting successfully the recommendations for achieving sustainable fisheries. A successful combination of attributes that can lead to the minimization of discards requires robust social capital along with top-down measures. Overall, the classification algorithm proves the usefulness of the SES framework to describe the fishery response to managerial efforts that aim to reduce discarding. Establishment of links between fishing communities and scientists are the key for enhancing stakeholder engagement, increasing compliance and minimizing discards.

     

Presence of Cicadomorpha in olive orchards of Greece with special reference to Xylella fastidiosa vectors

Insects of the infraorder Cicadomorpha cause serious economic impact on agriculture because of pathogens’ transmission. The vector-borne bacterium Xylella fastidiosa, which is associated with olive quick decline syndrome, is exclusively transmitted by insects of this infraorder and more specific by sharpshooters and spittlebugs. Efficient control practices of the insect vectors are necessary for the control of the disease in cases of outbreaks. Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is of crucial importance. In the present study, extensive sampling was conducted in 28 olive orchards of Greece during the years 2017 and 2018 aiming to investigate which species of this infraorder are present, with special interest to those which are considered as vectors of the bacterium. During the surveys, 4,350 Cicadomorpha insects were collected. Forty-two, five and one species of the families Cicadellidae, Aphrophoridae and Cercopidae, respectively, were identified. The results of the surveys revealed that the main potential vectors of X. fastidiosa in Greek olive orchards are the spittlebugs Philaenus spumarius and Neophilaenus campestris. Those species have wide distribution in Greece and are present for a long period during the year, and they inhabit both the ground vegetation and the olive trees' canopy. A representative portion of the captured spittlebugs was analysed with molecular methods, and the analysis confirmed the absence of X. fastidiosa in the tested individuals.