The modification of plant genomes is an essential step towards the development of new crops to increase food production. Initially, genome modification relied on conventional plant breeding and the introduction of genetic traits using crossing techniques to generate new varieties. In the 1980s, trans and cis modification allowed the incorporation of specific traits into plant genomes, largely due to the development of DNA delivery systems such as Agrobacterium and biolistics. This technological breakthrough has boosted a second generation of genetically modified crops that have had a major impact on agriculture. However, the stochastic nature of the DNA delivery systems, with little control over where the genome modification occurred, required regulatory measures to ensure environmental, human, and animal safety, which has hindered the ability and speed to generate new and more adapted varieties. New technological advances have made it possible to increase the precision of genome modifications leading to a third generation of agricultural products. These technological advances rely on enzymes targeting specific genomic regions, making it possible to introduce mutations and new traits into the plant genome. Meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas9 are valuable tools that allow for specific genomic modifications. These tools have made it possible to develop new, safer varieties in a shorter timeframe. In this review, we explore the functional mechanisms of these new breeding tools, their advantages and drawbacks, and their potential to explore metabolic engineering and synthetic biology in plants.

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).

Skin wound healing is coordinated by a delicate balance between proinflammatory and anti-inflammatory responses, which can be affected by opportunistic pathogens and metabolic or vascular diseases. Several antimicrobial peptides (AMPs) possess immunomodulatory properties, suggesting their potential to support skin wound healing. Here, we evaluated the proregenerative activity of three recently described AMPs (Clavanin A, Clavanin-MO, and Mastoparan-MO). Human primary dermal fibroblasts (hFibs) were used to determine peptide toxicity and their capacity to induce cell proliferation and migration. Furthermore, mRNA analysis was used to investigate the modulation of genes associated with skin regeneration. Subsequently, the regenerative potential of the peptides was further confirmed using an ex vivo organotypic model of human skin (hOSEC)-based lesion. Our results indicate that the three molecules evaluated in this study have regenerative potential at nontoxic doses (i.e., 200 μM for Clavanin-A and Clavanin-MO, and 6.25 μM for Mastoparan-MO). At these concentrations, all peptides promoted the proliferation and migration of hFibs during in vitro assays. Such processes were accompanied by gene expression signatures related to skin regenerative processes, including significantly higher KI67, HAS2 and CXCR4 mRNA levels induced by Clavanin A and Mastoparan-MO. Such findings translated into significantly accelerated wound healing promoted by both Clavanin A and Mastoparan-MO in hOSEC-based lesions. Overall, the data demonstrate the proregenerative properties of these peptides using human experimental skin models, with Mastoparan-MO and Clavanin A showing much greater potential for inducing wound healing compared to Clavanin-MO.

The study of wasp venoms has captured attention due to the presence of a wide variety of active compounds, revealing a diverse array of biological effects. Among these compounds, certain antimicrobial peptides (AMPs) such as mastoparans and chemotactic peptides have emerged as significant players, characterized by their unique amphipathic short linear alpha-helical structure. These peptides exhibit not only antibiotic properties but also a range of other biological activities, which are related to their ability to interact with biological membranes to varying degrees. This review article aims to provide updated insights into the structure/function relationships of AMPs derived from wasp venoms, linking this knowledge to the potential development of innovative treatments against infections.

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 μM. 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 μM against A. aegypti, and LC50 1.22 μM 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.

Synoeca-MP is a 14-residue amidated peptide, belongs to the mastoparan family and it is found in the venom of the wasp Synoeca surinama and has antibacterial and antifungal activity. The low cytotoxicity of the peptide also makes it an excellent candidate for drug development. To better understand its selectivity and interaction with the membrane, the peptide behavior in membrane-like environments was studied here and the peptide structure in SDS micelles was determined by NMR spectroscopy. The behavior of the peptide in hydrophobic media and in different pH ranges was studied by CD spectroscopy. The incorporation of residues into the anionic micelles was studied by hydrogen-deuterium exchange. The peptide stability and insertion in the micelles was studied by molecular dynamics simulations. Synoeca-MP, bound to SDS micelles, exhibits a partial alpha-helix conformation, with the first five residues and the last two unfolded. H/D exchange showed that the peptide has a slow exchange rate. After 164 h, four residues had not yet completed H/D substitution, suggesting parallel alignment of the peptide with the micelle, mainly due to the hydrophobic interface. This may indicate a carpet interaction model of the peptide with micelles. The molecular simulation study of peptide showed that the peptide consists of a well-folded alpha-helix core and unfolded extremities, which are responsible for the nature of the peptide interaction. The biophysical analyses can improve the atomic understanding of the mode of action of the peptide and help in future improvements of the peptide for clinical usage.

In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.

Coral reefs are marine environments with notable biodiversity, considered rich in organisms that produce active molecules. The microorganisms present in coral tissue play different roles that are not yet fully understood. The study of the microbiota can explain how microorganisms influence the health of corals and their participation in defense mechanisms. In this study, the cultivable bacterial communities of Millepora alcicornis and Phyllogorgia dilatata corals were analyzed following the steps of isolation, morphological characterization, and molecular identification by amplification of the gene encoding 16S rRNA. Of the 135 bacterial isolates, 121 of which were from M. alcicornis and 14 from P. dilatata, most belonged to the phyla Proteobacteria and Firmicutes, with the majority from the genera Vibrio and Bacillus. Coral metagenomic DNA was extracted and the gene encoding 16S rRNA was analyzed. The isolates were evaluated, in vitro, for antimicrobial activity against ATCC strains of Escherichia coli and Staphylococcus aureus, and 29 isolates from M. alcicornis showed activity against at least one of the bacteria evaluated. Using specific pairs of primers, 27 isolates showed amplification for at least one pair of primers designed for amplification of genes involved in the synthesis of non-ribosomal peptides (NRPs) and polyketides (PKs). However, only in some cases was a relationship detected between the isolates that showed antimicrobial activity in vitro and the isolates where amplification of genes involved in the synthesis of NRPs and PKs was observed.

The root-knot nematode (RKN) Meloidogyne incognita infects a large host range causing enormous agricultural losses. Although the control of this pest is mainly based on the use of chemical pesticides, natural alternatives are currently being largely considered, especially the use of plant-derived molecules, as allelochemicals and plant extracts. Solanum stramonifolium is a plant of the Solanaceae family and has been studied for its potential to control RKN, either by the use aqueous extracts of its seeds, or due to its immunity to nematode penetration. Our studies demonstrate that S. stramonifolium has an efficient defense mechanism against RKN completely preventing its penetration into its root system. Additionally, the compounds exuded by this plant in the soil can affect nematode infection in nearby grown Nicotiana benthamiana (-89% biomass for treatment I respectively, and -61% and +57% of galls for the treatments and Solanum lycopersicum (-79% biomass for treatment I, and -83% of galls for the treatments I and II). Herein, external (ED, < 3.5 kDa) and internal (ID, > 3.5 kDa) dialysates were prepared from the extract of S. stramonifolium seeds. Arabidopsis thaliana Col-0 inoculated with RKN treated with the dialysates showed a reduced gall diameter in 45% and 35% for ED and ID respectively. Additionally, a significant reduction in biomass was observed in germinated seeds after treatment with ID. In addition, Arabidopsis roots showed changes in their ploidy levels when treated with ED and ID, compared to the untreated control, indicating its effect on endoreduplicating cells. Finally, a partial identification of glycoalkaloids present in the ED and root extracts may help to explain plant immunity to root-knot nematode infection. Results obtained shed light on the defense mechanisms of S. stramonifolium against M. incognita, its allelopathic potential in co-cultivated plants and its biotechnological potential of dialysates obtained from its seeds, representing a strong alternative to the management of RKN in conventional agriculture.

Root-knot nematodes (Meloidogyne spp.) considerably affect their plant hosts, causing extensive damage in the world agriculture. The most widely used method to control these pathogens is through the intensive application of nematicides, despite being highly toxic to humans, animals, and the environment. The urgent search for alternative forms of control based on natural resources that are effective, provide a targeted strategy that is less toxic and less harmful to the environment. The species Solanum stramonifolium Jacq. (Solanaceae) have been described as resistant to root-knot nematode infection and other diseases, such as fungi and bacteria. Nematotoxic assays here presented demonstrated that aqueous crude seed extract from S. stramonifolium is very effective against second stage juveniles (J2) of M. incognita even at very low concentrations such 100µg mL-1 during in vitro bioassays. Furthermore, this extract also demonstrated a nematicidal effect after a heating process at 50 °C, killing more than 90% of M. incognita J2. No toxic activity was observed against non-target organisms, like bacteria, and the free-living nematode Caenorhabditis elegans at concentrations varying from 25 to 512 µg mL-1. Finally, greenhouse assays showed that external dialysate (ED) can be used to control nematodes in the soil, and that the plants treated with the dialysates display a reproduction factor lower than the synthetic nematicide used as positive control.

The increase in resistance to conventional antimicrobials in recent years has boosted the search for new antibiotics to treat serious infectious diseases, especially those generated by multi-resistant bacteria. In this context, antimicrobial peptides (AMPs) are alternative molecules for use as new therapeutic agents. AMPs are small bioactive proteins commonly produced by all living organisms, and they can be part of innate immunity. Due to their broad-spectrum antibacterial potential and other activities, including immunomodulatory and antitumor, they are of great interest to the pharmaceutical industry’s production of biopharmaceuticals. Among the technological platforms applied in the process of development and manufacturing of AMPs, recombinant DNA technology has enabled the production of such molecules using bacterial and yeast cells as expression host systems on a laboratory scale and in large-scale environments. Furthermore, different bioprocessing strategies can be used for peptide industrial production, aiming to optimize the yield, make cultures more robust and significantly increase cell density. In this chapter, we will address recent developments and future directions in AMPs bioprocessing, including microbial expression systems, as well as bioprocessing and purification technologies. Here we also describe successful cases in this field and emphasize the prospects and challenges related to AMPs bioengineering.

Antimicrobial peptides (AMPs) have shown cell membrane-directed mechanisms of action. This specificity can be effective against infectious agents that have acquired resistance to conventional drugs. The AMPs’ membrane-specificity and their great potential to combat resistant microbes has brought hope to the medical/therapeutic scene. The high death rate worldwide due to antimicrobial resistance (AMR) has pushed forward the search for new molecules and product developments, mainly antibiotics. In the current scenario, other strategies including the association of two or more drugs have contributed to the treatment of difficult-to-treat infectious diseases, above all, those caused by bacteria. In this context, the synergistic action of AMPs associated with current antibiotic therapy can bring important results for the production of new and effective drugs to overcome AMR. This review presents the advances obtained in the last 5 years in medical/antibiotic therapy, with the use of products based on AMPs, as well as perspectives on the potentialized effects of current drugs combined with AMPs for the treatment of bacterial infectious diseases.

Elastin-like polypeptides (ELPs) are biopolymers formed by amino acid sequences derived from tropoelastin. These biomolecules can be soluble below critical temperatures, forming aggregates at higher temperatures, which makes them an interesting source for the design of different nanobiomaterials. These nanobiomaterials can be obtained from heterologous expression in several organisms such as bacteria, fungi, and plants. Thanks to the many advantages of ELPs, they have been used in the biomedical field to develop nanoparticles, nanofibers, and nanocomposites. These nanostructures can be used in multiple applications such as drug delivery systems, treatments of type 2 diabetes, cardiovascular diseases, tissue repair, and cancer therapy. Thus, this review aims to shed some light on the main advances in elastin-like-based nanomaterials, their possible expression forms, and importance to the medical field.

Antimicrobial peptides (AMPs) are components in the innate immune system of various organisms, and many AMPs can be found in poisons from animals such as spiders, scorpions, and snakes. The peptide Cupiennin-1a is present in the venom of the spider Cupiennius salei and belongs to a group of peptides called cupiennins. The peptide demonstrated high cytotoxic activity against mammalian cells; thus, aiming to solve this problem, seven analogs were designed (R1a, R1b, R2b, R3b, R6b, R8b, and R10b) based on the primary structure of the peptide Cupiennin 1a, reducing its size and substituting some amino acid residues. The antimicrobial results showed that all Cupiennin 1a analogs displayed antimicrobial activity against the tested bacterial and fungal strains. Cytotoxicity tests demonstrated a decrease in the cytotoxic effect of the analogs when compared to the peptide Cupiennin-1a. The antitumor activity against breast adenocarcinoma lines was observed for all the peptides, displaying a better effect against the MCF-7 and MDAMB-231 cell lines. The eight peptides have insecticidal potential, and the original peptide and analogs R6b, R8b, and R10b showed better efficiency even at low concentrations. The rational design of the analogs led to new molecules displaying activities against different cell types and reduced cytotoxicity toward healthy mammalian cells when compared to the original peptide, demonstrating that this was an interesting approach for the development of molecules with biotechnological potential.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

The purpose of this article is to study the isolated and combined effect of the peptides Synoeca-MP and IDR-1018 against multi-resistant clinical isolates of K. pneumoniae (Kp2177569 - LACEN) in vitro. The bactericidal activity of the peptide Synoeca-MP in combination with three different classes of commercial antimicrobials and its immunomodulatory potential was also evaluated. Synoeca-MP showed better antimicrobial activity than IDR-1018 and presented synergistic action combined with levofloxacin. Therefore, Synoeca-MP and levofloxacin, and the combination of both, were used in subsequent analyses. In the presence of heat-killed antigens, cellular viability and TNF-α levels was maintained, the production of NO increased and a reduction in IL-10 production was observed. The synergistic antibacterial effect between Synoeca-MP and levofloxacin was effective against multidrug-resistant strains of K. pneumoniae. The association of Synoeca-MP and levofloxacin may present a low modulating action of pro and anti-inflammatory mediators, based on these results.

Sclerotinia sclerotiorum (Lib.), the causal agent of white mold, is a necrotrophic fungus with worldwide distribution. This fungus can infect more than 600 agricultural crops, causing damage worth hundreds of millions of dollars annually. The control of white mold is usually performed using integrated management practices. Nevertheless, after the establishment of the disease, chemical compounds need to be used, increasing production costs and offering environmental risks. In this study, we used Host Induced Gene Silencing (HIGS) in an attempt to control white mold. Specific vectors were constructed to express a silencing hairpin (dsRNAs) of the pathogen effector genes Ss-caF1 (putative Ca2+ binding protein), SspG1d (endopolygalacturonase) and SsiTL (integrin). The results showed a reduction in the severity of the symptoms in Arabidopsis thaliana transgenic plants and a delay in the occurrence of early symptoms. The results obtained for the control of S. sclerotiorum support the potential of HIGS in the generation of plants resistant to phytopathogenic fungi. It is also possible to suggest that the effector genes Ss-caF1, SspG1d and SsiTL are not only involved in the interaction but also play important roles during the host colonization and infection process.

Antimicrobial peptides are small molecules, up to 10 kDa, present in all kingdoms of life, including in plants. Several studies report that these molecules have a broad spectrum of activity, including antibacterial, antifungal, antiviral, and insecticidal activity. Thus, they can be employed in agriculture as alternative tools for phytopathogen and pest control. However, the application of peptides in agriculture can present challenges, such as loss of activity due to degradation of these molecules, off-target effects, and others. In this context, nanotechnology can offer versatile structures, including metallic nanoparticles, liposomes, polymeric nanoparticles, nanofibers, and others, which might act both in protection and in release of AMPs. Several polymers and biomaterials can be employed for the development of nanostructures, such as inorganic metals, natural or synthetic lipids, synthetic and hybrid polymers, and others. This review addresses the versatility of NanoAMPs (Nanoparticles in association with antimicrobial peptides), and their potential applications in agribusiness, as an alternative for the control of phytopathogens in crops.

Aims Sclerotinia sclerotiorum, the causal agent of white mold, can infect several host species, including economically important crops. In this study, we propose and validate a new in vitro system able to mimic the conditions of interaction with the host and promote the induction of S. sclerotiorum effectors. Methods and results For culture media production, we selected three plant species, common bean (Phaseolus vulgaris L, cv. Requinte.), maize (Zea mays, cv. BRS1030) and beggarticks (Bidens pilosa). To validate this system as an in vitro inducer of effectors, the qRT-PCR technique was used to investigate the expression profile of some S. sclerotiorum effector genes in each growth medium at different times after inoculation. Conclusion The results obtained in this study provide a validation of a new method to study S. sclerotiorum during mimetic interaction with different hosts. Although leaf extract does not fully represent the plant environment, the presence of plant components in the culture medium seems to induce effector genes, mimicking in planta conditions. The use of MEVM is simpler than in planta growth, bypasses problems such as the amount of mycelium produced, as well as contamination of host cells during transcriptomic and proteomic analyses. Significance and Impact of the Study We have devised MEVM media as a model mimicking the interaction of S. sclerotiorum and its hosts and used it to evaluate in vitro expression of effectors normally expressed only in planta.

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.