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BACKGROUND: Sugarcane is a crop of global importance and has been expanding to areas with soils containing high levels of exchangeable aluminum (Al), which is a limiting factor for crop development in acidic soils. The study of the sugarcane physiological and nutritional behavior together with patterns of gene expression in response to Al stress may provide a basis for effective strategies to increase crop productivity in acidic soils. METHODS: Sugarcane cultivars were evaluated for physiological parameters (photosynthesis, stomatal conductance, and transpiration), nutrient (N, P, K, Ca, Mg, and S) and Al contents in leaves and roots and gene expression, of the genes MDH, SDH by qPCR, both related to the production of organic acids, and SOD, related to oxidative stress. RESULTS: Brazilian sugarcane RB867515, RB928064, and RB935744 cultivars exhibited very different responses to induced stress by Al. Exposure to Al caused up-regulation (SOD and MDH) or down-regulation (SDH, MDH, and SOD), depending on the cultivar, Al level, and plant tissue. The RB867515 cultivar was the most Al-tolerant, showing no decline of nutrient content in plant tissue, photosynthesis, transpiration, and stomatal conductance after exposure to Al; it exhibited the highest Al content in the roots, and showed important MDH and SOD gene expression in the roots. RB928064 only showed low expression of SOD in roots and leaves, while RB935744 showed important expression of the SOD gene only in the leaves. Sugarcane cultivars were classified in the following descending Al-tolerance order: RB867515 > RB928064 = RB935744. These results may contribute to the obtention of Al-tolerant cultivars that can play their genetic potential in soils of low fertility and with low demand for agricultural inputs; the selection of potential plants for breeding programs; the elucidation of Al detoxification mechanisms employed by sugarcane cultivars.

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Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.

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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.

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Huanglongbing (HLB) is a devastating citrus disease associated with Candidatus Liberibacter asiaticus (CLas) and is transmitted by the psyllid Diaphorina citri Kuwayama. Diaphorina citri belongs to Hemiptera order, which has cysteine peptidases as the most abundant proteolytic enzymes present in digestive tract. As cysteine peptidases are involved in different insect development processes, this class of enzymes has acquired biotechnological importance. In this context, we identified a cathepsin L-like (DCcathL1) from the Diaphorina citri transcriptome database and expressed the enzyme in E. coli. Quantitative real-time RT-PCR was conducted to determine DCcathL1 gene expression in different parts and developmental phases of the insect. We observed that DCcathL1 expression in the gut was 2.59 and 2.87-fold higher than in the head and carcass, respectively. Furthermore, DCcathL1 expression was greater in eggs than in nymphs and adults, suggesting a putative role of the enzyme in the embryonic development. In addition, enzymatic inhibitory activity using four recombinant Citrus cystatins were performed. Among them, CsinCPI-2 was the strongest DCcathL1 inhibitor with a Ki value of 0.005 nM. Our results may contribute in the development of strategies for D. citri control, such as silencing the DCcathL1 gene and the use of transgenic plants that overexpress peptidase inhibitors.

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Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.

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Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.

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Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.

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Sugarcane, a major crop grown in the tropical and subtropical areas of the world, is produced mainly for sucrose, which is used as a sweetener or for the production of bioethanol. Among the numerous pests that significantly affect the yield of sugarcane, the sugarcane rhizome borer (Migdolus fryanus, a cerambycidae beetle) is known to cause severe damage to the crops in Brazil. The absence of molecular information about this insect reinforces the need for studies and an effective method to control this pest. In this study, RNA-Seq technology was employed to study different parts of M. fryanus larvae. The generated data will help in further investigations about the taxonomy, development, and adaptation of this insect. RNA was extracted from six different parts (head, fat body, integument, hindgut, midgut, and foregut) using Trizol methodology. Using Illumina paired-end sequencing technology and the Trinity platform, trimming and de novo assembly was performed, resulting in 44,567 contigs longer than 200 nt for a reunion of data from all transcriptomes, with a mean length of 1,095.27 nt. Transcripts were annotated using BLAST against different protein databanks (Uniprot/Swissprot, PFAM, KEEG, SignalP 4.1, Gene Ontology, and CAZY) and were compared for similarity using a Venn diagram. Differential expression patterns were studied for select genes through qPCR and FPKM comprising important protein families (digestive peptidases, glucosyl hydrolases, serine protease inhibitors and otopetrin), which allowed a better understanding of the insect's digestion, immunity and gravity sensorial mechanisms.

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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.

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KEY MESSAGE: Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

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