Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

C. elegans Expressing Human β2-Microglobulin: A Novel Model for Studying the Relationship between the Molecular Assembly and the Toxic Phenotype

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β₂-microglobulin (β₂-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β₂-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β₂-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β₂-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β₂-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β₂-m.     

Protein profiling in F<Subscript>1</Subscript> and F<Subscript>2</Subscript> generations of two tomato genotypes differing in ripening time

Pericarp polypeptide profiles were analyzed at three ripening stages in the F₁ hybrid and the F₂ population from the cross between the accessions: LA1385 (Lycopersicon esculentum var. cerasiforme) and 804627 (L. esculentum, a homozygous genotype for the nor mutant). Six polymorphic polypeptides were observed in LA1385, while no polymorphic polypeptides among ripening stages was observed in 804627. On the other hand, some polypeptides in the F₁ hybrid were not observed in the parents whereas others were present in both parental genotypes and were unnoticeable in the hybrid genotype. From a cluster analysis on the protein profiles of the F₂ population, the differential expression of proteins allowed to distinguish mature green (MG) stage from the others two stages, while for breaker stage (BR) and red ripe stage, the genetic background was more important in forming groups. The differential expression of proteins could be associated with fruit morphology traits such as a 72 kDa polypeptide present in MG stage with fruit diameter, height and mass and a 47 kDa polypeptide found in BR with fruit shelf life.     

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

Universal Strategy with Structural and Chemical Crosslinking Interface for Efficient and Stable Perovskite Solar Cells

Due to the limited interface contact and weak interfacial interaction, planar heterojunction perovskite solar cells (PSCs) have space for further improvement. Herein, a structural and chemical crosslinking interface is proposed and constructed by introducing an extra layer, which blends tin dioxide (SnO₂) nanoparticles with chloride salts. Since the incorporated materials can be dissolved during the fabrication of perovskite, the quality of perovskite films is improved, leading to larger grain size and reduced trap-state density. Also, more chloride ions at the SnO₂/perovskite interface are observed and the interaction between Cl⁻ and Sn⁴⁺ is confirmed. It results in more pronounced n-type SnO₂ with better conductivity and deeper conduction bands, leading to preferable energy level alignment between SnO₂ and perovskite. Consequently, the open-circuit voltage and fill factor of the devices increase, and target cells present better stability, retaining 98% of initial efficiencies after >10 000 h storage in dry air (≈5% relative humidity) and maintaining 85.50% of the initial efficiency after 1000 h of operation under light. This strategy enables the achievement of 25.28% efficiency with a low bandgap (1.53 eV) perovskite composition, and it is confirmed to be universal when other related materials are utilized.     

Use of lignocellulosic wastes of pecan (Carya illinoinensis) in the cultivation of Ganoderma lucidum

Background

The wastes of pecan nut (Carya illinoinensis (Wangenh.) K. Koch) production are increasing worldwide and have high concentrations of tannins and phenols.

The Action of Ribonuclease on Fixed Tissues

To study the optimal conditions for histochemical use of ribonuclease on fixed tissues, the factors of (1) type of fixation, (2) temperature, pH, type of buffer and length of incubation, (3) concentration of enzyme, and (4) staining and dehydration of sections were observed on rabbit pancreas.

The fixing fluids studied were sublimate-alcohol, Bouin's, Zenker-acetic, Zenker-formol, Petrunkevich's cupric-paranitrophenol, 10% neutral formalin, SUSA, Carnoy, Bensley's chrom-sublimate, absolute ethyl alcohol and acetone. Formaldehyde was a satisfactory fixative, although others might be preferred for special purposes. Of the five buffers tested, McIlvaine's citric-acid-disodium-phosphate mixture was the most satisfactory, whereas veronal-acetate extracted considerable stainable cytoplasmic material. The optimum concentration of ribonuclease and length of incubation varied greatly after the 11 different types of fixation. For example, with ribonuclease buffered by Mcllvaine's fluid, the intense cytoplasmic staining of formaldehyde-fixed tissues was removed by concentrations as low as 0.001 mg./ml., whereas, with sections fixed in Zenkers fluid some cytoplasmic staining persisted even after 3 hours in 0.2 mg./ml. Under the conditions employed the temperature and hydrogen-ion concentration during incubation were less important. Examples of nonspecific action of ribonuclease were noted. Until the degree and optimum conditions of specific action have been more precisely established by further experiments, it is suggested that this histo-chemical reaction only be interpreted as a confirmatory test which is, under the best conditions, only relatively specific for ribonucleic acid and not highly quantitative.     

Land use practices and ectomycorrhizal fungal communities from oak woodlands dominated by Quercus suber L. considering drought scenarios

Oak woodlands in the Mediterranean basin have been traditionally converted into agro-silvo-pastoral systems and exemplified sustainable land use in Europe. In Portugal, in line with the trend of other European countries, profound changes in management options during the twentieth century have led to landscape simplification. Landscapes are dynamic and the knowledge of future management planning combining biological conservation and soil productivity is needed, especially under the actual scenarios of drought and increasing evidence of heavy oak mortality. We examined the ectomycorrhizal (ECM) fungal community associated with cork oak in managed oak woodlands (called montado) under different land use practices, during summer. ECM fungal richness and abundance were assessed in 15 stands established in nine montados located in the Alentejo region (southern Portugal), using morphotyping and ITS rDNA analysis. Parameters related to the montados landscape characteristics, land use history over the last 25 years, climatic and edaphic conditions were taken into account. Fifty-five ECM fungal taxa corresponding to the most abundant fungal symbionts were distinguished on cork oak roots. Cenococcum geophilum and the families Russulaceae and Thelephoraceae explained 56% of the whole ECM fungal community; other groups were represented among the community: Cortinariaceae, Boletaceae, Amanita, Genea, Pisolithus, Scleroderma, and Tuber. There were pronounced differences in ECM fungal community structure among the 15 montados stands: C. geophilum was the only species common to all stands, tomentelloid and russuloid species were detected in 87–93% of the stands, Cortinariaceae was detected in 60% of the stands, and the other groups were more unequally distributed. Ordination analysis revealed that ECM fungal richness was positively correlated with the silvo-pastoral exploitation regime and low mortality of cork oak, while ECM fungal abundance was positively correlated with extensive agro-silvo-pastoral exploitation under a traditional 9-year rotation cultivation system and recent soil tillage. The effects of land use on the ECM fungal community and its implications in different scenarios of landscape management options, oak mortality, and global warming are discussed.