Rosiane Andrade da Costa

Bacterial extracellular vesicles (bEVs) are membrane-enclosed nanoparticles that transport bioactive cargo and mediate interactions between bacteria and their environment. Although bEVs are increasingly recognized as natural delivery systems, their potential application in plant pest biocontrol remains poorly explored. Here, we provide proof-of-concept evidence that isolated bEVs from two entomopathogenic bacteria, Chromobacterium subtsugae and Bacillus thuringiensis var. kurstaki , exert insecticidal activity against the soybean pest Euschistus heros . Isolated bEVs were characterized by tunable resistive pulse sensing, nano-flow cytometry, transmission electron microscopy, SDS-PAGE, MALDI-TOF mass spectrometry, and label-free quantitative proteomics. C. subtsugae bEVs displayed a proteome clearly remodeled relative to the soluble protein fraction, with enrichment of outer- membrane, secretion-associated, proteolytic, and membrane-active proteins. MALDI-TOF analysis detected a violacein-associated ion selectively in the C. subtsugae bEV fraction, supporting vesicular association of this hydrophobic bioactive metabolite. In survival assays, C. subtsugae bEVs strongly reduced E. heros nymph survival (HR = 4.0, p < 0.0001), whereas the corresponding soluble protein fraction was inactive (HR = 1.2, p = 0.50). In contrast, B. thuringiensis bEVs and soluble protein fractions produced similar moderate activity (both HR = 2.1), consistent with their largely overlapping proteomic profiles. Cry1Ab was detected mainly in the B. thuringiensis soluble fraction rather than selectively enriched in bEVs. Together, these findings support a multi-component cargo model in which C. subtsugae bEVs combine vesicle-associated violacein with enriched protein cargo, establishing bacterial EVs as promising natural nanocarriers for next-generation, cell-free bioinsecticides against Cry-resistant hemipteran pests such as E. heros .

Inonotus is a hymenochaetoid genus with worldwide distribution, known for its wood-decaying species. Since Karsten proposed it in 1879, significant revisions have been made to its circumscription, and several species complexes are currently being reevaluated through integrative taxonomic approaches. Although rare, some species in the genus have a described anamorphic stage. Historically, dense, ferruginous, pulvinate masses of chlamydospores attached to trunks and branches have been attributed to Ptychogaster cubensis , the anamorph of Inonotus rickii . However, during a survey of wood-inhabiting fungi in Central Brazil, similar structures were found on living and dead trees. Long-term observations revealed that the fungus grew annually on the same host and adjacent trees and exhibited an ecological relationship with beetles that fed on its structures and dispersed chlamydospores to nearby hosts. The collected sporomata were isolated and characterized morphologically and phylogenetically using ITS, LSU, RPB2 , and TEF1-α partial sequences. Based on these analyses, we describe a new species represented in its anamorphic stage, Inonotus cerradensis sp. nov. The new species is characterized by sporomata formed by beard-like rhizomorphs bearing dense clusters of globose, soft, fragile chlamydospores that easily disintegrate upon touch, rhizomorphs cylindrical to flattened, with superficial outward-projecting and hyphal setae. We also conducted a detailed review of specimens representing both stages of I. rickii to refine its morphological circumscription. Both species form distinct and well-defined clades within Inonotus s. str. and differ mainly in sporomata organization: loose, rhizomorph-supported structures characterize the new species, whereas compact, stratified, and concentrically organized sporomata correspond to the anamorph of I. rickii . This study highlights how integrative taxonomy clarifies species boundaries within Inonotus and advances the understanding of the genus and its ecology in Neotropical environments.

Introduction This study aimed to evaluate the antimicrobial, cytotoxic, proliferative, migratory, and immunomodulatory potential of Bio-C Sealer and Bio-C Sealer ion + extracts compared to AH Plus, using human periodontal ligament stem cells (hPDLSCs). Materials and methods Initially, the minimum concentrations of sealer extracts required to inhibit the growth of Enterococcus faecalis and Candida albicans were determined. hPDLSCs were then obtained from extracted third molars of young donors and cultured under standard conditions. Cells were characterized by flow cytometry using mesenchymal stem cell markers (CD44, CD73, CD90, CD105) and a negative cocktail of hematopoietic markers, following the minimal criteria of the International Society for Cellular Therapy. Subsequently, the cytotoxic, proliferative, and migratory effects on hPDLSCs were assessed using MTT, Trypan Blue exclusion and scratch assays. Finally, the gene expression profile of hPDLSCs exposed to sealer extracts was analyzed by qPCR. Statistical differences were evaluated using one-way and two-way ANOVA followed by Tukey’s post hoc tests. Results AH Plus and Bio-C Sealer ion + inhibited 100% of E. faecalis growth at extract dilutions of 1:1, 1:2, and 1:4. However, only undiluted extracts of AH Plus, Bio-C Sealer, and Bio-C Sealer ion + completely inhibited C. albicans growth. The cytotoxicity of all sealers was dose-dependent in hPDLSC cultures, with lower cell viability observed in undiluted extracts: AH Plus (55%), Bio-C Sealer (69%), and Bio-C Sealer ion + (72%) (p < 0.0001). Bio-C Sealer ion + extract (1:4) promoted the highest rate of cell migration and proliferation after 48 h (p < 0.0001). Regarding gene expression, bioceramic sealers-maintained levels of TNF-α, IL-6, and IL-10 similar to the control condition, whereas AH Plus upregulated these cytokines. Conclusions: Bioceramic sealers exhibited satisfactory antimicrobial activity with moderate cytotoxicity. Moreover, they enhanced wound closure and maintained cytokine expression at levels comparable to the control condition in periodontal ligament stem cells. Clinical relevance Bioceramic sealers such as Bio-C Sealer and Bio-C Sealer ion + demonstrated superior biological compatibility compared with AH Plus, including lower cytotoxicity, enhanced cell migration and proliferation, and effective control of inflammatory cytokine induction. These properties suggest greater safety for endodontic use and potential to promote tissue healing and long-term clinical success.

Kefir is a probiotic and a rich source of bioactive compounds including antimicrobial peptides. Due to its natural versatility and characteristics, peptides prospected from kefir may become an affordable and sustainable tool against microorganisms. Here, we investigated the peptide profile and evaluate the antimicrobial, membrane permeabilization, and antibiofilm properties of a peptide-rich fraction obtained from a bovine milk-based kefir from Brazil. The milk-fermented drink was centrifuged using a three-step ultrafiltration process. The peptide profile of the resulting fraction (< 3 kDa) was analyzed by uHPLC and MALDI-TOF MS-Spectra. The antimicrobial activity of the peptide fraction was assessed using the broth microdilution technique. Quantitative assays and qualitative fluorescence microscopy images were used to assess its effects on bacterial membrane and antibiofilm formation. The peptide profile analysis of the fraction led to the discovery of 32 peptide molecules, with sizes ranging from 700 to 1400 Da. The combination of these peptides exhibited broad-spectrum bacteriostatic and bactericidal activity. The Sytox Green assay suggested that these peptides may exert their antimicrobial mechanisms via membrane damage. The peptide fraction also inhibited the biofilm formation of Staphylococcus aureus MRSA up to 69.9 to 81.4%. This study unraveled the antimicrobial potential of a peptide fraction from a Brazilian milk-based kefir. It is important to note that, to our knowledge, this is the first report that identifies peptides from kefir beverage as inhibitors of bacterial biofilm formation. These results emphasize this product as a source of new biomolecules that may be explored as biotechnological alternatives to combat microorganisms.

Concerns over the replicability and reproducibility of published research have grown in many research fields, but empirical data to inform policies are still scarce. Biomedical research in Brazil expanded rapidly over the last three decades, with no systematic assessment of the replicability of its findings. With this in mind, we set up the Brazilian Reproducibility Initiative, a multicenter replication of published experiments from Brazilian science using three common experimental methods: the MTT assay, the reverse transcription polymerase chain reaction (RT-PCR) and the elevated plus maze (EPM). A total of 56 laboratories performed 143 replications of 56 experiments; of these, 90 replications of 45 experiments were considered valid by an independent committee. Replication rates for these experiments varied between 20 and 44% according to five predefined criteria. In median terms, ratios between group means were 58% lower in replications than in original experiments, while coefficients of variation were 82% higher. Effect size decrease was smaller for MTT experiments, original results with less variability and those considered more challenging to replicate, while t values for replications were positively correlated with researcher predictions about replicability, and negatively correlated with the rate of publications by the original article’s last author. Deviations from preregistered protocols were very common in replications, most frequently due to reasons inherent to the experimental model or related to infrastructure and logistics. Our results highlight factors that limit the replicability of results published by researchers in Brazil and suggest ways by which this scenario can be improved.

Corals can be considered holobiont organisms, since they have an important symbiotic relationship with microbial communities such as zooxanthellae, bacteria, Archaea, fungi and viruses. It is important to understand how those microbial communities influence the health of the corals and how environmental conditions could affect them. The present study aimed to describe the bacterial communities associated with three Brazilian coral species, Millepora alcicornis, Mussismilia harttii and Phyllogorgia dilatata, by a culture-independent method, using 16S rRNA gene sequencing. The corals were collected from two distinct coral reefs: Recife de Fora, in Bahia (BA) and Búzios, in Rio de Janeiro (RJ). The phylum Proteobacteria showed the highest relative abundance in most corals and sites. The bacterial compositions of these three corals from the two sample sites were very distinct from each other, not presenting similarities in coral species or related to sampling site. In M. alcicornes/RJ, the most abundant class was Gammaproteobacteria, order Piscirickettsiales, while the same species collected in BA showed unassigned Gammaproteobacteria, and Vibrionaceae was the second most abundant family. M. harttii/BA presented the most distinct bacterial phylum composition with 16 phyla (26% Proteobacteria, 16% Chloroflexi, 12% Acidobacteriota).

Defensins are present in many organisms and are divided into two evolutionary groups, termed cis- and trans-defensins. Cis-defensins have only recently been reported in bacteria, and knowledge of these defensins is limited, with no family classification. Here, we describe the identification of 74 cis-defensins from bacteria and propose five classes for their classification. We also report the first NMR structure determination of a Myxoccocus xanthus defensin, as well as its in silico expression analysis. Xanthusin-1 has a unique structure among the published defensins, which could indicate that the proposed class II peptides constitute a separate group of defensins. Xanthusin-1 gene expression was observed in casitone-based and Streptomyces coelicolor coculture-grown media. Our results demonstrate a wider distribution of defensins outside the Eukarya domain, shedding light on the origin and distribution of defensins. The sharing of three disulfide defensins between bacteria and eukaryotes points to a possible prokaryotic origin of the CSαβ motif. Moreover, the identification of defensins in Gram-positive and Gram-negative bacteria indicates an early origin but with many gene losses during the evolutionary process, similar to findings for eukaryotic defensins.

Pelgipeptins, tridecaptins, and elgicins are among the antimicrobials produced by Paenibacillus elgii. Growth in complex media is commonly applied to obtain lipopeptides from culture's supernatant, but it requires further purification. This study aimed to improve the yield of pelgipeptins and tridecaptins using chemically defined media. The kinetics of antimicrobial lipopeptide yield in chemically defined media was evaluated in P. elgii AC13. Pelgipeptins were detected in the supernatant and the culture pellet, but tridecaptins were mainly associated with cell debris or endospores. We investigated whether removing Ca²⁺ would impair P. elgii sporogenesis, consequently improving the yield of tridecaptin. The kinetics of both lipopeptides in the presence and absence of Ca²⁺ were quantitatively and qualitatively evaluated and further correlated with the cell cycle. The impairment of P elgii AC13 sporogenesis had no effect on tridecaptin production, which remained undetected in the supernatant of the culture. On the other hand, the yield of pelgipeptin in a Ca²⁺-free medium increased. We showed for the first time that the removal of Ca²⁺ interrupted the sporogenesis in P. elgii and improved the yield of pelgipeptins. However, Ca⁺² absence had no effect on tridecaptin yield, which is possibly degraded or associated with other cell debris component.

Aedes aegypti and Culex quinquefasciatus are vectors of numerous diseases of worldwide public importance, such as arboviruses and filariasis. The main strategy for controlling these vectors is the use of chemicals, which can induce the appearance of resistant insects. The use of Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) with larvicidal activity against arboviral-transmitting insects has been successful in many studies. In contrast, the use and knowledge of peptides with insecticidal activity are so far scarce. In this work, 25 peptides and 5 strains of each bacterial species were prospected individually or together regarding their insecticidal activity. Initially, in vitro assays of cellular cytotoxicity of the peptides against SF21 cells of Spodoptera frugiperda were performed. The peptides Polybia-MPII and pelgipeptin caused 69 and 60% of cell mortality, respectively, at the concentration of 10 mu M. Thus, they were evaluated in vivo against second-stage larvae of the two Culicidae. However, in the in vivo bioassays, only pelgipeptin showed larvicidal mortality against both larvae (LC50 6.40 mu M against A. aegypti, and LC50 1.22 mu M against C. quinquefasciatus). The toxin-producing bacterial strain that showed the lowest LC50 against A. aegypti was Bt S8 (LC50 = 0.71 ng/ mL) and against C. quinquefasciatus, it was Ls S260 (LC50 = 2.32 ng/mL). So, the synergistic activity between the association of the bacterial toxins and pelgipeptin was evaluated. A synergic effect of pelgipeptin was observed with Ls strain S260 against C. quinquefasciatus. Our results demonstrate the possibility of synergistic or individual use of both biologically active larvicides against C. quinquefasciatus and A. aegypti.