Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

In Vitro Interactions of Amphotericin B Combined with Non-antifungal Agents Against Rhodotorula mucilaginosa Strains

Rhodotorula species are emerging as opportunistic pathogens, causing catheter-associated fungemia in patients with compromised immunity. R. mucilaginosa is considered the most common species involved in human infections. Correct identification and susceptibility testing of Rhodotorula isolates recovered from the blood stream or central nervous system are essential to determine the best management of this unusual infection. The antifungal susceptibility tests showed that Rhodotorula was susceptible to low concentrations of amphotericin B (AMB) but was less susceptible to voriconazole. Combinations of AMB plus several non-antifungal medications were evaluated against 35 susceptible (Rm AMB-S) and resistant (Rm AMB-R) clinical Rhodotorula isolates using the broth microdilution checkerboard technique. We showed that in vitro exposure to increasing concentrations of AMB changed the susceptibility profile to these strains, which were named the Rm AMB-R group. The most synergistic interactions were AMB?+?simvastatin, followed by AMB?+?amlodipine and AMB?+?warfarin. Synergism and antagonism were observed in both groups for the combination AMB?+?cyclosporine A. AMB combined with a fluoroquinolone (AMB?+?levofloxacin) also demonstrated antagonism for the Rm AMB-S strains, but a high percentage of synergistic interactions was observed for the Rm AMB-R group. A combination drug approach can provide a different strategy to treat infections caused by AMB-resistant R. mucilaginosa.

     

Lip anatomy and its implications for the pollination mechanisms of Bulbophyllum species (Orchidaceae)

BACKGROUND AND AIMS: The lip structure of six Brazilian and one Asiatic species of Bulbophyllum with wind-assisted fly pollination (B. involutum, B. ipanemense and B. weddellii) and non-wind-assisted fly pollination (B. epiphytum, B. glutinosum, B. regnellii and B. rothschildianum) was studied to investigate the presence of secretory tissues related to these pollination mechanisms. METHODS: The lip study was carried out through scanning electron microscopy (lip surface) and light microscopy (anatomical features). KEY RESULTS: In most of the species studied, the osmophores (odour glands) were located in the lobes and in the upper surface of the lip callus. Differences in the lip structure were observed between the two groups (the presence of a nectary and the extent of osmophore surface), depending on the mechanism of pollination. Nectaries were found in the cavity callus in B. ipanemense, B. involutum and B. weddellii, even though their pollinators were presumably attracted by the instinct to oviposit. CONCLUSIONS: These findings corroborate the hypothesis that, because pollination in these species is dependent on an unpredictable external factor (wind), nectar is necessary to keep the insect in the flower for a long period. Despite the occurrence of a liquid-like nectar in the flowers of B. epiphytum, B. glutinosum, B. regnelli and B. rothschildianum, no anatomical evidence for nectaries was found in the lips of these species, although a similar structure may occur in another region of the flowers. This observation agrees with the fact that pollination by lip movement in the latter species requires only gravity, with no additional mechanism being needed to keep the flies in the flower.     

Floral and vegetative morphometrics of five Pleurothallis (Orchidaceae) species: correlation with taxonomy,phylogeny, genetic variability and pollination systems

Morphometric analyses of vegetative and floral characters were conducted in 21 populations of five Pleurothallis (Orchidaceae) species occurring in Brazilian 'campo rupestre' vegetation. A phylogenetic analysis of this species group was also carried out using nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2). Results of the ordination and cluster analyses agree with species' delimitation revealed by taxonomic and allozyme studies. The groups formed in ordination analysis correspond to the pollinator groups determined in a previous pollination study. Relationships among the species in the cluster analysis using only vegetative characters are similar to those found in a previous allozyme study, but those indicated by cluster analysis using only floral characters differ. These results support the hypothesis that floral similarities are due to convergence driven by similar pollination mechanisms, and therefore floral traits may not be good indicators of phylogenetic relationships in this group. The results of the phylogenetic analysis support this conclusion to some extent. There is no correlation between genetic (allozyme) and morphological variability in the populations nor in the way this variability is distributed among conspecific populations. We describe a new subspecies of Pleurothallis ochreata based on differences in vegetative and chemical characters as well as geographic distribution. Absence of differentiation in floral characters, attraction of the same pollinator species, interfertility and genetic similarity support the argument for subspecific rather than specific status.     

Application of an aqueous two‐phase micellar system to extract bromelain from pineapple (Ananas comosus) peel waste and analysis of bromelain stability in cosmetic formulations

Bromelain is a set of proteolytic enzymes found in pineapple (Ananas comosus) tissues such as stem, fruit and leaves. Because of its proteolytic activity, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. The present study focused on the recovery of bromelain from pineapple peel by liquid–liquid extraction in aqueous two‐phase micellar systems (ATPMS), using Triton X‐114 (TX‐114) and McIlvaine buffer, in the absence and presence of electrolytes CaCl₂ and KI; the cloud points of the generated extraction systems were studied by plotting binodal curves. Based on the cloud points, three temperatures were selected for extraction: 30, 33, and 36°C for systems in the absence of salts; 40, 43, and 46°C in the presence of KI; 24, 27, and 30°C in the presence of CaCl₂. Total protein and enzymatic activities were analyzed to monitor bromelain. Employing the ATPMS chosen for extraction (0.5 M KI with 3% TX‐114, at pH 6.0, at 40°C), the bromelain extract stability was assessed after incorporation into three cosmetic bases: an anhydrous gel, a cream, and a cream‐gel formulation. The cream‐gel formulation presented as the most appropriate base to convey bromelain, and its optimal storage conditions were found to be 4.0 ± 0.5°C. The selected ATPMS enabled the extraction of a biomolecule with high added value from waste lined‐up in a cosmetic formulation, allowing for exploration of further cosmetic potential. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:937–945, 2015     

Demography, and genetic and morphological variability of the endangered Sophronitis sincorana (Orchidaceae) in the Chapada Diamantina, Brazil

We carried out a demographic study and evaluated the genetic and morphological variability in five populations of the endangered Sophronitis sincorana (Orchidaceae) endemic to Northeastern Brazil, based on allozyme and morphometric analyses. Plant density was approximately 0.5 plants/m², and the projected total number of plants was approximately 50,000 individuals. However, fruit set and recruitment of individuals are rare. The genetic variability was very high in all populations (P = 100, A = 3.0−3.5, H _e = 0.33−0.48), and all populations presented similar values of morphological variability. Low genetic and morphological structuring were found in the species (F _ST = 0.053, A_MRPP = 0.018). The elevated coefficient of endogamy encountered in populations of S. sincorana indicates the occurrence of structuring within the populations. The lack of correlation between morphological and genetic variation in this species indicates that none of the markers examined should be used separately for either conservation purposes.     

Pollination in Brazilian Syngonanthus (Eriocaulaceae) species: evidence for entomophily instead of anemophily

BACKGROUND AND AIMS: The reproductive biology of Syngonanthus mucugensis and S. curralensis (Eriocaulaceae) was studied in areas of 'campo rupestre' vegetation in the Chapada Diamantina, north-eastern Brazil. These species are herbaceous and the individuals have a grouped distribution. Their leaves are united in a rosette, and their inflorescence is monoecious, of the capitulum type. The staminate and pistillate rings mature in a centripetal manner on the capitulum. METHODS: A field study was conducted, including observations concerning the morphology and biology of the flowers, fruit development, insect visits and anemophily, in both S. mucugensis and S. curralensis. Experimental pollinations were also carried out to study the mating systems of S. mucugensis. KEY RESULTS: Both species flower from June to August. The staminate cycle lasts approx. 7 d, and the pistillate cycle from 3 to 4 d, with no temporal overlap between them on the same capitulum. The pollen viability of S. mucugensis was 88.6%, and 92.5% for S. curralensis. The inflorescences of both species demonstrated ultraviolet absorbance, and a sweet odour was detected during both the staminate and pistillate phases. No nectar production was ever noted, although nectaries are present. Both species were visited by numerous groups of insects, with the Diptera being the principal pollinators, especially the species of Syrphidae and Bombyliidae. There were secondary pollinators among species of Coleoptera and Hymenoptera. There was no evidence of wind pollination. Syngonanthus mucugensis is a self-compatible species, and forms fruits by agamospermy at low frequencies. CONCLUSIONS: This is apparently the first report for pollination biology and mating systems of Eriocaulaceae. Conversely to that stated by some authors, entomophily, mainly effected by species of Diptera but also by species of Coleoptera and Hymenoptera, is probably the only pollination system in these species. In spite of the monoecious inflorescences without overlap of the staminate and pistillate phases, geitonogamy may occur in S. mucugensis, as the species is self-compatible and different capitula in the same plant at different phases is common.     

Geographical genetic structuring and phenotypic variation in the Vellozia hirsuta (Velloziaceae) ochlospecies complex

? Premise of the study: Vellozia hirsuta forms a complex presenting wide morphological and anatomical variation, resulting in five specific names and 14 morpho-anatomical patterns occurring in disjunct populations. We carried out a phylogeographical study to investigate the existence of correlation among the genetic and morphological patterns within this complex, and to determine whether it is composed of various species or should be treated as an ochlospecies, a species having widely polymorphic and weakly polytypic complex variation, with morphological characteristics varying independently. ? Methods: We carried out phylogeographical analyses using cpDNA rpl32F-trnL intergenic region. ? Key results: We found 20 haplotypes in 23 populations sampled. The populations are genetically structured (Φ(ST) = 0.818) into four phylogeographical groups demonstrating geographical structuring but with no correlation with morpho-anatomical patterns. Our analyses do not support recognizing any of the species now synonymized under Vellozia hirsuta. The northern populations were the most genetically differentiated and could be considered a distinct taxon, as they are also morphologically different. ? Conclusions: It is recommended that Vellozia hirsuta be considered a single enormously variable species. The patterns of variation within V. hirsuta probably are related to climatic changes that occurred during the Pleistocene Epoch in tropical Brazil when reductions in forest cover favored the expansion of V. hirsuta populations into extensive lowland areas. The expansion of forest cover at the end of the glaciations would have again restricted the occurrence of campos rupestres vegetation to high elevations, which constitute the current centers of diversity of this species.