Structural and biochemical insights into 7β‐hydroxysteroid dehydrogenase stereoselectivity

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo‐ and regio‐selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β‐hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active‐site accessibility, the bases of the specificity for NADP⁺, and the general architecture of the steroid binding site. Comparison with 7α‐hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C‐terminal extension reshapes the substrate site of the β‐selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859–865. © 2016 Wiley Periodicals, Inc.     

Functional architecture of higher plant photosystem II supercomplexes

Photosystem II (PSII) is a large multiprotein complex, which catalyses water splitting and plastoquinone reduction necessary to transform sunlight into chemical energy. Detailed functional and structural studies of the complex from higher plants have been hampered by the impossibility to purify it to homogeneity. In this work, homogeneous preparations ranging from a newly identified particle composed by a monomeric core and antenna proteins to the largest C₂S₂M₂ supercomplex were isolated. Characterization by biochemical methods and single particle electron microscopy allowed to relate for the first time the supramolecular organization to the protein content. A projection map of C₂S₂M₂ at 12 Å resolution was obtained, which allowed determining the location and the orientation of the antenna proteins. Comparison of the supercomplexes obtained from WT and Lhcb‐deficient plants reveals the importance of the individual subunits for the supramolecular organization. The functional implications of these findings are discussed and allow redefining previous suggestions on PSII energy transfer, assembly, photoinhibition, state transition and non‐photochemical quenching.     

Distribution of Serotonin Receptor of Type 6 (5-HT6) in Human Brain Post-mortem. A Pharmacology, Autoradiography and Immunohistochemistry Study

The aim of this study was to investigate the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in postmortem human prefrontal cortex, striatum and hippocampus. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by the pharmacological characterization, where possible, and by autoradiographic, immunohistochemical and immunofluorescence evaluations. A specific and saturable [(125)I]SB-258585 binding was detected in striatum only, with a pharmacological characterization consistent with that of a 5-HT(6) receptor. The autoradiography showed the presence of a specific [(125)I]SB-258585 binding distributed homogeneously in caudate, putamen and accumbens. The immunohistochemistry, carried out in the striatum only, coupled with the immunofluorescence with glial fibrillary acidic protein (GFAP) and parvalbumin (PV) showed the co-localization of 5-HT(6) receptor with PV, while indicating that this receptor subtype was expressed in neurons and not in astrocytes. Taken together, the present findings showed the presence of a higher density of 5-HT(6) receptors, as labeled by [(125)I]SB-258585, in striatum than in hippocampus and prefrontal cortex, and specifically within the neuronal body. In addition, they would suggest that striatum is one of the major potential CNS targets linked to 5-HT(6) receptor modulation.     

Climate change decreases the cooling effect from postfire albedo in boreal North America

Fire is a primary disturbance in boreal forests and generates both positive and negative climate forcings. The influence of fire on surface albedo is a predominantly negative forcing in boreal forests, and one of the strongest overall, due to increased snow exposure in the winter and spring months. Albedo forcings are spatially and temporally heterogeneous and depend on a variety of factors related to soils, topography, climate, land cover/vegetation type, successional dynamics, time since fire, season, and fire severity. However, how these variables interact to influence albedo is not well understood, and quantifying these relationships and predicting postfire albedo becomes increasingly important as the climate changes and management frameworks evolve to consider climate impacts. Here we developed a MODIS‐derived ‘blue sky’ albedo product and a novel machine learning modeling framework to predict fire‐driven changes in albedo under historical and future climate scenarios across boreal North America. Converted to radiative forcing (RF), we estimated that fires generate an annual mean cooling of ?1.77 ± 1.35 W/m² from albedo under historical climate conditions (1971–2000) integrated over 70 years postfire. Increasing postfire albedo along a south–north climatic gradient was offset by a nearly opposite gradient in solar insolation, such that large‐scale spatial patterns in RF were minimal. Our models suggest that climate change will lead to decreases in mean annual postfire albedo, and hence a decreasing strength of the negative RF, a trend dominated by decreased snow cover in spring months. Considering the range of future climate scenarios and model uncertainties, we estimate that for fires burning in the current era (2016) the cooling effect from long‐term postfire albedo will be reduced by 15%–28% due to climate change.     

Long Tree-Ring Chronologies Provide Evidence of Recent Tree Growth Decrease in a Central African Tropical Forest

It is still unclear whether the exponential rise of atmospheric CO₂ concentration has produced a fertilization effect on tropical forests, thus incrementing their growth rate, in the last two centuries. As many factors affect tree growth patterns, short -term studies might be influenced by the confounding effect of several interacting environmental variables on plant growth. Long-term analyses of tree growth can elucidate long-term trends of plant growth response to dominant drivers. The study of annual rings, applied to long tree-ring chronologies in tropical forest trees enables such analysis. Long-term tree-ring chronologies of three widespread African species were measured in Central Africa to analyze the growth of trees over the last two centuries. Growth trends were correlated to changes in global atmospheric CO₂ concentration and local variations in the main climatic drivers, temperature and rainfall. Our results provided no evidence for a fertilization effect of CO₂ on tree growth. On the contrary, an overall growth decline was observed for all three species in the last century, which appears to be significantly correlated to the increase in local temperature. These findings provide additional support to the global observations of a slowing down of C sequestration in the trunks of forest trees in recent decades. Data indicate that the CO₂ increase alone has not been sufficient to obtain a tree growth increase in tropical trees. The effect of other changing environmental factors, like temperature, may have overridden the fertilization effect of CO₂.     

Carvacrol Codrugs: A New Approach in the Antimicrobial Plan

Objective

The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs – obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids via an ester bond – to develop novel compounds with improved antimicrobial and antibiofilm activities and reduced toxicity respect to carvacrol alone.

Method

All carvacrol codrugs were screened against a representative panel of Gram positive (S. aureus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C. albicans, using broth microdilution assays.

Findings

Results showed that carvacrol codrug 4 possesses the most notable enhancement in the anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and 10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8 and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, respect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also analyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans.

Conclusion

The current study presents an insight into the use of codrug strategy for developing carvacrol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity.     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

DNA SEQUENCE DATA DEMONSTRATE THE POLYPHYLY OF THE GENUS CYSTOSEIRA AND OTHER SARGASSACEAE GENERA (PHAEOPHYCEAE)1

Phylogenetic relationships in the Sargassaceae were explored using three DNA markers, and the monophyly of its genera was challenged. Nineteen out of 24 currently recognized genera were sampled, representing 63 species. The variable mt23S‐tRNA Val intergenic spacer could only be aligned within genera and could not be used to infer intergeneric relationships. The partial mt23S was also useful to delineate genera and was alignable at the family level but provided few informative characters. Analysis of mt23S DNA sequences together with chloroplast‐encoded psbA sequences resulted in a better resolved phylogeny. Hormophysa was the first genus to branch off within the Sargassaceae, followed by Myriodesma; then the three genera Caulocystis, Carpoglossum, and Scaberia in unresolved order; and then Acrocarpia. The other taxa studied here were divided over three major clades, but there was no branch support for the monophyly of two of these. The genera Bifurcaria, Cystoseira, Halidrys, and Sargassum appeared polyphyletic. The following taxonomic changes are proposed: a new genus Brassicophycus for Bifurcaria brassicaeformis (Kützing) E. S. Barton; reinstatement of the genus Sargassopsis for Sargassum decurrens (R. Brown ex Turner) C. Agardh; reinstatement of the genus Sirophysalis for Indo‐Pacific Cystoseira trinodis (Forsskål) C. Agardh; reinstatement of the genus Polycladia for the western Indian Ocean species Cystoseira indica (Thivy et Doshi) Mairh, Cystoseira myrica (S. G. Gmelin) C. Agardh, and Acystis heinii Schiffner; and reinstatement of the genus Stephanocystis for the North Pacific Cystoseira species and Halidrys dioica N. L. Gardner. The European Cystoseira species should be split into three genera, but no name changes are proposed yet, because diagnostic characters were found only for the clade including the type species. Some evolutionary trends could be discerned from the mt23S + psbA phylogeny.     

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.