Acquired pellicle and biofilm engineering with CaneCPI-5: Insights from proteomic and microbiomics analysis.

OBJECTIVE: In this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DESIGN: After prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). RESULTS: Treatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. CONCLUSION: Cane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.

Ecological transcriptomics reveals stress response pathways of a ground-herb species in a waterlogging gradient of Amazonian riparian forests.

Environmental stress is a fundamental facet of life and a significant driver of natural selection in the wild. Gene expression diversity may facilitate adaptation to environmental changes, without necessary genetic change, but its role in adaptive divergence remains largely understudied in Neotropical systems. In Amazonian riparian forests, species distribution is predominantly influenced by species' waterlogging tolerance. The flooding gradient delineates distinct wetland forest types, shaping habitats and species characteristics. Here we investigated the molecular basis of environmental stress response in a tropical ground-herb species (Ischnosiphon puberulus) to environmental variation in Amazonian riparian forests. We compared environmental variables and gene expression profiles from individuals collected in two forest types: Igapó and Terra firme in the Amazonian riparian forests. Predictable seasonal flooding poses a significant challenge in Igapó compared to Terra firme environments, with the former presenting higher water column height and longer flooding duration. Our findings suggest that contrasting environmental conditions related to flooding regimes are important drivers of population genetic differentiation and differential gene expression in I. puberulus. Enriched gene ontology terms highlight associations with environmental stresses, such as defence response, water transport, phosphorylation, root development, response to auxin, salicylic acid and oxidative stress. By uncovering key environmental stress response pathways conserved across populations, I. puberulus offers novel genetic insights into the molecular basis of plant reactions to environmental constraints found in flooded areas of this highly biodiverse neotropical ecosystem.

Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Acquired Pellicle and Biofilm engineering by rinsing with hemoglobin solution.

INTRODUCTION: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. OBJECTIVE: Combining in vivo and in vitro proof-of-concept protocols we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. METHODS: In the in vivo studies, 10-volunteers, in 2 independent experiments, rinsed (10mL,1min) with: deionized water-negative control or 1.0mg/mL Hb. The AEP and biofilm formed along 2 or 3h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA Next-Generation-Sequencing (NGS). In vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS, 2)0.12% Chlorhexidine, 3)500ppm NaF; 4)1.0mg/mL Hb; 5)2.0mg/mL Hb, and 6)4.0mg/mL Hb. The biofilm was cultivated for 5-days. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU and enamel demineralization compared to PBS. CONCLUSION: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.

Antimicrobial Peptide Arsenal Predicted from the Venom Gland Transcriptome of the Tropical Trap-Jaw Ant Odontomachus chelifer.

With about 13,000 known species, ants are the most abundant venomous insects. Their venom consists of polypeptides, enzymes, alkaloids, biogenic amines, formic acid, and hydrocarbons. In this study, we investigated, using in silico techniques, the peptides composing a putative antimicrobial arsenal from the venom gland of the neotropical trap-jaw ant Odontomachus chelifer. Focusing on transcripts from the body and venom gland of this insect, it was possible to determine the gland secretome, which contained about 1022 peptides with putative signal peptides. The majority of these peptides (75.5%) were unknown, not matching any reference database, motivating us to extract functional insights via machine learning-based techniques. With several complementary methodologies, we investigated the existence of antimicrobial peptides (AMPs) in the venom gland of O. chelifer, finding 112 non-redundant candidates. Candidate AMPs were predicted to be more globular and hemolytic than the remaining peptides in the secretome. There is evidence of transcription for 97% of AMP candidates across the same ant genus, with one of them also verified as translated, thus supporting our findings. Most of these potential antimicrobial sequences (94.8%) matched transcripts from the ant's body, indicating their role not solely as venom toxins.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

Transcriptomic and biochemical analysis from the venom gland of the neotropical ant Odontomachus chelifer.

The genus Odontomachus is widely distributed in neotropical areas throughout Central and South America. It is a stinging ant that subdues its prey (insects) by injecting them a cocktail of toxic molecules (venom). Ant venoms are generally composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Odontomachus chelifer is an ant that inhabits neotropical regions from Mexico to Argentina. Unlike the venom of other animals such as scorpions, spiders and snakes, this ant venom has seldom been analyzed comprehensively, and their compositions are not yet completely known. In the present study, we performed a partial investigation of enzymatic and functional activities of O. chelifer ant venom, and we provide a global insight on the transcripts expressed in the venom gland to better understand their properties. The crude venom showed phospholipase A2 and antiparasitic activities. RNA sequencing (Illumina platform) of the venom gland of O. chelifer generated 61, 422, 898 reads and de novo assembly Trinity generated 50,220 contigs. BUSCO analysis against Arthropoda_db10 showed that 92.89% of the BUSCO groups have complete gene representation (single-copy or duplicated), while 4.05% are only partially recovered, and 3.06% are missing. The 30 most expressed genes in O. chelifer venom gland transcriptome included important transcripts involved in venom function such as U-poneritoxin (01)-Om1a-like (pilosulin), chitinase 2, venom allergen 3, chymotrypsin 1 and 2 and glutathione S-transferase. Analysis of the molecular function revealed that the largest number of transcripts were related to catalytic activity, including phospholipases. These data emphasize the potential of O. chelifer venom for prospection of molecules with biotechnological application.

Transcriptomic analysis of Malpighian tubules from the stingless bee Melipona scutellaris reveals thiamethoxam-induced damages.

The concern about pesticide exposure to neotropical bees has been increasing in the last few years, and knowledge gaps have been identified. Although stingless bees, (e.g.: Melipona scutellaris), are more diverse than honeybees and they stand out in the pollination of several valuable economical crops, toxicity assessments with stingless bees are still scarce. Nowadays new approaches in ecotoxicological studies, such as omic analysis, were pointed out as a strategy to reveal mechanisms of how bees deal with these stressors. To date, no molecular techniques have been applied for the evaluation of target and/or non-target organs in stingless bees, such as the Malpighian tubules (Mt). Therefore, in the present study, we evaluated the differentially expressed genes (DEGs) in the Mt of M. scutellaris after one and eight days of exposure to LC50/100 (0.000543 ng a.i./µL) of thiamethoxam (TMX). Through functional annotation analysis of four transcriptome libraries, the time course line approach revealed 237 DEGs (nine clusters) associated with carbon/energy metabolism and cellular processes (lysosomes, autophagy, and glycan degradation). The expression profiles of Mt were altered by TMX in processes, such as detoxification, excretion, tissue regeneration, oxidative stress, apoptosis, and DNA repair. Transcriptome analysis showed that cell metabolism in Mt was mainly affected after 8 days of exposure. Nine genes were selected from different clusters and validated by RT-qPCR. According to our findings, TMX promotes several types of damage in Mt cells at the molecular level. Therefore, interference of different cellular processes directly affects the health of M. scutellaris by compromising the function of Mt.

Speciation with gene flow between two Neotropical sympatric species (Pitcairnia spp.: Bromeliaceae).

The study of mechanisms that generate new species is considered fundamental for broad areas of ecology and evolution. Speciation is a continuous process in which reproductive isolation is established, and it is of fundamental importance to understand the origins of the adaptations that contribute to this process. Hybrid zones are considered natural laboratories for the study of speciation and represent ideal systems for such studies. Here, we investigated genomic differentiation between hybridizing Neotropical species Pitcairnia staminea (G. Lodd.) and P. albiflos (Herb.). Using thousands of SNPs genotyped through RAD-seq, we estimate effective population sizes, interspecific gene flow, as well as time of divergence between these two sister species and identify candidate genomic regions for positive selection that may be related to reproductive isolation. We selected different scenarios of speciation and tested them by using approximate Bayesian computation (ABC); we found evidence of divergence with gradual reduction in gene flow between these species over time, compatible with the hypothesis of speciation with gene flow between these Pitcairnia species. The parameter estimates obtained through ABC suggested that the effective population size of P. albiflos was around three times larger than that of P. staminea. Our divergence date estimates showed that these two species diverged during the Pliocene (4.7 Mya; CI = 1.3-8.5 Mya), which has likely allowed this species to accumulate genome-wide differences. We also detected a total of 17 of 4165 loci which showed signatures of selection with high genetic differentiation (F ST > 0.85), 12 of these loci were annotated in de novo assembled transcriptomes of both species, and 4 candidate genes were identified to be putatively involved in reproductive isolation. These four candidate genes were previously associated with the function of pollen development, pollen tube germination and orientation, abiotic stress, and flower scent in plants, suggesting an interplay between pre- and postpollination barriers in the evolution of reproductive isolation between such species.

Identification and Functional Analysis of a Pseudo-Cysteine Protease from the Midgut Transcriptome of Sphenophorus levis.

The Sphenophorus levis (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (Vmax = 1017.60 ± 135.55, Km = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (Ki = 38.52 ± 1.20 µM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.

Marine associated microbial consortium applied to RBBR textile dye detoxification and decolorization: Combined approach and metatranscriptomic analysis.

The combination of different microorganisms and their metabolisms makes the use of microbial consortia in bioremediation processes a useful approach. In this sense, this study aimed at structuring and selecting a marine microbial consortium for Remazol Brilliant Blue R (RBBR) detoxification and decolorization. Experimental design was applied to improve the culture conditions, and metatranscriptomic analysis to understand the enzymatic pathways. A promising consortium composed of Mucor racemosus CBMAI 847, Marasmiellus sp. CBMAI 1062, Bacillus subtilis CBMAI 707, and Dietzia maris CBMAI 705 was selected. This consortium showed 52% of detoxification and 86% of decolorization in the validation assays after seven days of incubation in the presence of 500 ppm of RBBR. Reduction in RBBR color and toxicity were achieved by biosorption and microbial metabolisms. Metatranscriptomic data indicate that the consortium was able to decolorize and breakdown the RBBR molecule using a coordinated action of oxidases, oxygenases, and hydrolases. Epoxide hydrolases and glyoxalases expression could be associated with the decrease in toxicity. The efficiency of this marine microbial consortium suggests their use in bioremediation processes of textile effluents.

Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants.

The obligate mutualistic basidiomycete fungus, Leucocoprinus gongylophorus, mediates nutrition of leaf-cutting ants with carbons from vegetal matter. In addition, diazotrophic Enterobacteriales in the fungus garden and intestinal Rhizobiales supposedly mediate assimilation of atmospheric nitrogen, and Entomoplasmatales in the genus Mesoplasma, as well as other yet unidentified strains, supposedly mediate ant assimilation of other compounds from vegetal matter, such as citrate, fructose, and amino acids. Together, these nutritional partners would support the production of high yields of leafcutter biomass. In the present investigation, we propose that three phylogenetically distinct and culturable diazotrophs in the genera Ralstonia, Methylobacterium, and Pseudomonas integrate this symbiotic nutrition network, facilitating ant nutrition on nitrogen. Strains in these genera were often isolated and directly sequenced in 16S rRNA libraries from the ant abdomen, together with the nondiazotrophs Acinetobacter and Brachybacterium. These five isolates were underrepresented in libraries, suggesting that none of them is dominant in vivo. Libraries have been dominated by four uncultured Rhizobiales strains in the genera Liberibacter, Terasakiella, and Bartonella and, only in Acromyrmex ants, by the Entomoplasmatales in the genus Mesoplasma. Acromyrmex also presented small amounts of two other uncultured Entomoplasmatales strains, Entomoplasma and Spiroplasma. The absence of Entomoplasmatales in Atta workers implicates that the association with these bacteria is not mandatory for ant biomass production. Most of the strains that we detected in South American ants were genetically similar with strains previously described in association with leafcutters from Central and North America, indicating wide geographic dispersion, and suggesting fixed ecological services.

Fungus-growing insects host a distinctive microbiota apparently adapted to the fungiculture environment.

Some lineages of ants, termites, and beetles independently evolved a symbiotic association with lignocellulolytic fungi cultivated for food, in a lifestyle known as fungiculture. Fungus-growing insects' symbiosis also hosts a bacterial community thought to integrate their physiology. Similarities in taxonomic composition support the microbiota of fungus-growing insects as convergent, despite differences in fungus-rearing by these insects. Here, by comparing fungus-growing insects to several hosts ranging diverse dietary patterns, we investigate whether the microbiota taxonomic and functional profiles are characteristic of the fungiculture environment. Compared to other hosts, the microbiota associated with fungus-growing insects presents a distinctive taxonomic profile, dominated by Gammaproteobacteria at class level and by Pseudomonas at genera level. Even with a functional profile presenting similarities with the gut microbiota of herbivorous and omnivorous hosts, some differentially abundant features codified by the microbiota of fungus-growing insects suggest these communities occupying microhabitats that are characteristic of fungiculture. These features include metabolic pathways involved in lignocellulose breakdown, detoxification of plant secondary metabolites, metabolism of simple sugars, fungal cell wall deconstruction, biofilm formation, antimicrobials biosynthesis, and metabolism of diverse nutrients. Our results suggest that the microbiota could be functionally adapted to the fungiculture environment, codifying metabolic pathways potentially relevant to the fungus-growing insects' ecosystems functioning.

Revealing the Venomous Secrets of the Spider's Web.

Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins, and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomic and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web, and the presence of fatty acids in the web may be a responsible mechanism opening the way to the web toxins for accessing the interior of prey's body, as shown here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups, Araneomorphae and Mygalomorphae, and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems and also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins.

A proteotranscriptomic study of silk-producing glands from the orb-weaving spiders.

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.

Pyrene degradation by marine-derived ascomycete: process optimization, toxicity, and metabolic analyses.

Marine-derived fungi are relevant genetic resources for bioremediation of saline environments/processes. Among the five fungi recovered from marine sponges able to degrade pyrene (Py) and benzo[a]pyrene (BaP), Tolypocladium sp. strain CBMAI 1346 and Xylaria sp. CBMAI 1464 presented the best removal rates of Py and BaP, respectively. Since the decrease in BaP was related to mycelial adsorption, a combined strategy was applied for the investigation of Py degradation by the fungus Tolypocladium sp. CBMAI 1346. The selected fungus was able to degrade about 95% of Py after 7 days of incubation (optimized conditions), generating metabolites different from the ones found before optimization. Metabolites and transcriptomic data revealed that the degradation occurred mainly by the cytochrome P450 pathway. Putative monooxygenases and dioxygenases found in the transcriptome may play an important role. After 21 days of degradation, no toxicity was found in the optimized culture conditions. The findings from the present study highlight the potential of marine-derived fungi to degrade environmental pollutants and convey innovative information related to the metabolism of pyrene.

De novo transcriptome assembly: a new laccase multigene family from the marine-derived basidiomycete Peniophora sp. CBMAI 1063.

Laccases are multicopper oxidases that are able to catalyze reactions involving a range of substrates, including phenols and amines, and this ability is related to the existence of different laccases. Basidiomycetes usually have more than one gene for laccase, but until now, this feature has not been demonstrated in a marine-derived fungus. Peniophora sp. CBMAI 1063 is a basidiomycete fungus isolated from a marine sponge that exhibits the ability to secrete significant amounts of laccase in saline conditions. In the present study, we identified laccase sequences from the transcriptome of Peniophora sp. CBMAI 1063 and used them to perform different molecular in silico analyses. The results revealed the presence of at least eight putative genes, which may encode ten different laccases with peptide lengths ranging from 482 to 588 aa and molecular weights ranging from 53.5 to 64.4 kDa. These laccases seem to perform extracellular activities, with the exception of one that may represent an intracellular laccase. The 10 predicted laccases expressed by Peniophora sp. CBMAI 1063 in laccase-induced media showed different patterns of N-glycosylation and isoelectric points and are divided into two classes based on the residue associated with the regulation of the redox potential of the enzyme. None of the predicted laccases showed more than 61% similarity to other fungal laccases. Based on the differences among the laccases expressed by Peniophora sp. CBMAI 1063, this marine-derived basidiomycete represents a valuable resource with strong potential for biotechnological exploitation.

Fungal communities in pressmud composting harbour beneficial and detrimental fungi for human welfare.

Pressmud is a substrate derived from sugarcane juice filtrate, and around 26-40 kg of this residue are produced per ton of sugarcane. It is mainly used as fertilizer in crops, and its application in the field is often made without any prior treatment, but, in this research, it was studied for the risk this practice poses for human health. This research was stimulated by previous results indicating the presence of opportunistic pathogens in residues used in various composting systems and the extensive use of fresh pressmud in agriculture. Here, It was assessed the fungal diversity present in both fresh and composting pressmud using 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their enzymatic repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). A wide range of opportunistic pathogens was found among the most abundant taxa in the fresh pressmud, such as Lomentospora prolificans (43.13 %), Trichosporon sp. (10.07 %), Candida tropicalis (7.91 %), and Hormographiella aspergillata (8.19 %). This indicates that fresh pressmud might be a putative source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without any treatment. With regard to the heat-tolerant fungi found in this substrate, all the 110 isolates screened were able to produce at least one of the tested enzymes. The pressmud composting process not only effectively reduces the load of pathogenic fungi, but also creates an interesting environment for fungi able to produce thermostable hydrolytic and oxidative enzymes with biotechnological applications.

ITScan: a web-based analysis tool for Internal Transcribed Spacer (ITS) sequences.

BACKGROUND: Studies on fungal diversity and ecology aim to identify fungi and to investigate their interactions with each other and with the environment. DNA sequence-based tools are essential for these studies because they can speed up the identification process and access greater fungal diversity than traditional methods. The nucleotide sequence encoding for the internal transcribed spacer (ITS) of the nuclear ribosomal RNA has recently been proposed as a standard marker for molecular identification of fungi and evaluation of fungal diversity. However, the analysis of large sets of ITS sequences involves many programs and steps, which makes this task intensive and laborious. FINDINGS: We developed the web-based pipeline ITScan, which automates the analysis of fungal ITS sequences generated either by Sanger or Next Generation Sequencing (NGS) platforms. Validation was performed using datasets containing ca. 2,000 to 40,000 sequences each. CONCLUSIONS: ITScan is an online and user-friendly automated pipeline for fungal diversity analysis and identification based on ITS sequences. It speeds up a process which would otherwise be repetitive and time-consuming for users. The ITScan tool and documentation are available at http://evol.rc.unesp.br:8083/itscan.