River water is a small percentage of the total freshwater on Earth but represents an essential resource for mankind. Microbes in rivers perform essential ecosystem roles including the mineralization ...of significant quantities of organic matter originating from terrestrial habitats. The Amazon river in particular is famous for its size and importance in the mobilization of both water and carbon out of its enormous basin. Here we present the first metagenomic study on the microbiota of this river. It presents many features in common with the other freshwater metagenome available (Lake Gatun in Panama) and much less similarity with marine samples. Among the microbial taxa found, the cosmopolitan freshwater acI lineage of the actinobacteria was clearly dominant. Group I Crenarchaea and the freshwater sister group of the marine SAR11 clade, LD12, were found alongside more exclusive and well known freshwater taxa such as Polynucleobacter. A metabolism-centric analysis revealed a disproportionate representation of pathways involved in heterotrophic carbon processing, as compared to those found in marine samples. In particular, these river microbes appear to be specialized in taking up and mineralizing allochthonous carbon derived from plant material.
Leishmaniasis is a neglected disease that affects 12 million people living mainly in developing countries. Herein, 24 new N-oxide-containing compounds were synthesized followed by in vitro and in ...vivo evaluation of their antileishmanial activity. Compound 4f, a furoxan derivative, was particularly remarkable in this regard, with EC50 value of 3.6 μM against L. infantum amastigote forms and CC50 value superior to 500 μM against murine peritoneal macrophages. In vitro studies suggested that 4f may act by a dual effect, by releasing nitric oxide after biotransformation and by inhibiting cysteine protease CPB (IC50: 4.5 μM). In vivo studies using an acute model of infection showed that compound 4f at 7.7 mg/Kg reduced ~90% of parasite burden in the liver and spleen of L. infantum-infected BALB/c mice. Altogether, these outcomes highlight furoxan 4f as a promising compound for further evaluation as an antileishmanial agent.
Key message
Transgenic sugarcane expressing
V-ATPase
subunit
E
dsRNA affects growth and survival of
Sphenophorus levis
.
Plants being sessile organisms are constantly confronted with several biotic ...and abiotic stresses. Sugarcane (
Saccharum
spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to
Sphenophorus levis
(sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with
V-ATPase E
dsRNA in
S. levis
larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the
V-ATPase E
gene of
S. levis
in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.
SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by
Diatraea saccharalis
larvae. These proteins play an ...important role in plant defense, in particular against fungal pathogens, such as
Colletothricum falcatum
(Went) and
Fusarium verticillioides
. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) H44 and H146, SUGARWINs in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.
The Amazon Basin is the largest hydrographic basin on the planet, and the dynamics of its aquatic microorganisms strongly impact global biogeochemical cycles. However, it remains poorly studied. This ...metagenome project was performed to obtain a snapshot of prokaryotic microbiota from four important lakes in the Amazon Basin.
SUGARWIN1 and 2 are defense proteins from sugarcane. Their gene expression is known to be induced in response to wound and
damage. Although the recombinant SUGARWIN protein does not affect insect ...development, it promotes significant morphological and physiological changes in
and
, which lead to fungal cell death via apoptosis. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that
are induced by
, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of
induction exhibited a considerable reduction in
infection. Furthermore, SUGARWIN1 exhibited ribonuclease, chitosanase, and chitinase activity, whereas SUGARWIN2 exhibited only chitosanase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site.
Tropical freshwater environments, like rivers, are important reservoirs of microbial life. This study employed metagenomic sequencing to survey prokaryotic microbiota in the Solimões, Purus, and ...Urucu Rivers of the Amazon Basin in Brazil. We report a rich and diverse microbial community.
•Recombinant CaneCPI-5 inhibits inflammation in sponge implants.•Results highlight the effects of recombinant CaneCPI-5 on promoting angiogenesis and collagen deposition.•CaneCPI-5 holds a potential ...therapeutic use to treatment inflammatory conditions and for wound healing.
Cystatins are natural inhibitors of cysteine peptidases that are found practically in all living organisms. CaneCPI-5 is a sugarcane cystatin with inhibitory activity against human cathepsins B, K and L, which are cysteine proteases highly expressed in a variety of pathological conditions, usually marked by persistent inflammation and processing of the extracellular matrix. This work evaluated the effects of daily administration of the recombinant cystatin CaneCPI-5 0.01, 0.1 or 1.0 μg in 10 μL of Phosphate-Buffered Saline (PBS) on the inflammatory, angiogenic and fibrogenic components during chronic inflammatory response induced by subcutaneous sponge implants. The anti-inflammatory effect of treatment with CaneCPI-5 was confirmed by reduction of the levels of the pro-inflammatory mediators TNF-α, CXCL1 and CCL2/JE/MCP-1, as well as the activity of the myeloperoxidase and n-acetyl-β-D-glucosaminidase. Treatment with CaneCPI-5 promoted angiogenesis in the implants, increasing the production of cytokines VEGF and FGF and the formation of new blood vessels. Finally, the administration of the recombinant cystatin favored the production of the pro-fibrogenic cytokine TGF-β1 and collagen deposition next to the implants. Together, these results show the potential therapeutic application of CaneCPI-5 as an anti-inflammatory agent, capable of favoring angiogenesis and fibrogenesis processes, necessary for tissue repair.
Display omitted
•CaneCPI-5, statherin and hemoglobin protect native enamel against dental erosion.•The combination group provides the best protection against dental erosion.•Mechanism of action of ...how the proteins/peptides act on the salivary pellicle.•Acquired pellicle engineering protects enamel demineralization.
This study investigated the mechanism of action of different proteins/peptides (separately or in combination), focusing on how they act directly on the native enamel surface and on modifying the salivary pellicle.
A total of 170 native human enamel specimens were prepared and submitted to different treatments (2 h; 37 °C): with deionized water, CaneCPI-5, Hemoglobin, Statherin, or a combination of all three proteins/peptides. The groups were subdivided into treatment acting on the enamel surface (NoP – absence of salivary pellicle), and treatment modifying the salivary pellicle (P). Treatment was made (2 h; 37 °C) in all specimens, and later, for P, the specimens were incubated in human saliva (2 h; 37 °C). In both cases, the specimens were immersed in 1% citric acid (pH 3.6; 2 min; 25 °C). Calcium released from enamel (CaR) and its relative surface reflection intensity (%SRI) was measured after 5 cycles. Between-group differences were verified with two-way ANOVA, with “presence of pellicle” and “treatment” as factors (α = 0.05).
The presence of pellicle provided better protection regarding %SRI (p < 0.01), but not regarding CaR (p = 0.201). In relation to treatment, when compared to the control group, all proteins/peptides provided significantly better protection (p < 0.01 for %SRI and Car). The combination of all three proteins/peptides demonstrated the best protective effect (p < 0.01 for %SRI). Conclusion: Depending on the protein or peptide, its erosion-inhibiting effect derives from their interaction with the enamel surface or from modifying the pellicle, so a combination of proteins and peptides provides the best protection.
The present study opens a new direction for a possible treatment with a combination of proteins for native human enamel, which can act directly on the enamel surface as well on the modification of the salivary pellicle, for the prevention of dental erosion.
Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant ...species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.
PDF
