Changes in fatty acid composition as a response to glyphosate toxicity in Pseudomonas fluorescens.

Excessive use of herbicides decreases soil biodiversity and fertility. The literature on the xenobiotic response by microorganisms is focused on herbicide biodegradation as a selective event. Non-degradation systems independent of selection could allow the survival of tolerant bacteria in contaminated environments, impacting xenobiotic turnover and, consequently, bioremediation strategies. However, it is uncertain whether the response based on these systems requires selective pressure to be effective. The objective here was to analyze non-degradation phenotypes, enzymatic and structural response systems, of Pseudomonas fluorescens CMA-55 strain, already investigated the production pattern of quorum sensing molecules in response to glyphosate, not present at the isolation site. One mode of response was associated with decrease in membrane permeability and effective antioxidative response for 0-2.30 mM glyphosate, at the mid-log growing phase, with higher activities of Mn-SOD, KatA, and KatB, and presence of fatty acids as nonadecylic acid, margaric and lauric acid. The second response system was characterized by lower antioxidative enzymes activity, presence of KatC isoform, and pelargonic, capric, myristic, stearic, palmitoleic and palmitic acid as principal fatty acids, allowing the strain to face stressful conditions in 9.20-11.50 mM glyphosate at the stationary phase. Therefore, the bacterial strain could modify the fatty acid composition and the permeability of membranes in two response modes according to the herbicide concentration, even glyphosate was not previously selective for P. fluorescens, featuring a generalist system based on physiological plasticity.

Probiotics and live biotherapeutic products aiming at cancer mitigation and patient recover.

Molecular biology techniques allowed access to non-culturable microorganisms, while studies using analytical chemistry, as Liquid Chromatography and Tandem Mass Spectrometry, showed the existence of a complex communication system among bacteria, signaled by quorum sensing molecules. These approaches also allowed the understanding of dysbiosis, in which imbalances in the microbiome diversity, caused by antibiotics, environmental toxins and processed foods, lead to the constitution of different diseases, as cancer. Colorectal cancer, for example, can originate by a dysbiosis configuration, which leads to biofilm formation, production of toxic metabolites, DNA damage in intestinal epithelial cells through the secretion of genotoxins, and epigenetic regulation of oncogenes. However, probiotic strains can also act in epigenetic processes, and so be use for recovering important intestinal functions and controlling dysbiosis and cancer mitigation through the metabolism of drugs used in chemotherapy, controlling the proliferation of cancer cells, improving the immune response of the host, regulation of cell differentiation and apoptosis, among others. There are still gaps in studies on the effectiveness of the use of probiotics, therefore omics and analytical chemistry are important approaches to understand the role of bacterial communication, formation of biofilms, and the effects of probiotics and microbiome on chemotherapy. The use of probiotics, prebiotics, synbiotics, and metabiotics should be considered as a complement to other more invasive and hazard therapies, such chemotherapy, surgery, and radiotherapy. The study of potential bacteria for cancer treatment, as the next-generation probiotics and Live Biotherapeutic Products, can have a controlling action in epigenetic processes, enabling the use of these bacteria for the mitigation of specific diseases through changes in the regulation of genes of microbiome and host. Thus, it is possible that a path of medicine in the times to come will be more patient-specific treatments, depending on the environmental, genetic, epigenetic and microbiome characteristics of the host.

Tolerance of Pseudomonas strain to the 2,4-D herbicide through a peroxidase system.

Herbicides are widely used in agricultural practices for preventing the proliferation of weeds. Upon reaching soil and water, herbicides can harm nontarget organisms, such as bacteria, which need an efficient defense mechanism to tolerate stress induced by herbicides. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide that exerts increased oxidative stress among bacterial communities. Bacterial isolates were obtained from the biofilm of tanks containing washing water from the packaging of different pesticides, including 2,4-D. The Pseudomonas sp. CMA-7.3 was selected because of its tolerance against 2,4-D toxicity, among several sensitive isolates from the biofilm collection. This study aimed to evaluate the antioxidative response system of the selected strain to 2,4-D. It was analyzed the activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase GPX enzymes, that are poorly known in the literature for bacterial systems. The Pseudomonas sp. CMA-7.3 presented an efficient response system in balancing the production of hydrogen peroxide, even at 25x the dose of 2,4-D used in agriculture. The antioxidative system was composed of Fe-SOD enzymes, less common than Mn-SOD in bacteria, and through the activities of KatA and KatB isoforms, working together with APX and GPX, having their activities coordinated possibly by quorum sensing molecules. The peroxide control is poorly documented for bacteria, and this work is unprecedented for Pseudomonas and 2,4-D. Not all bacteria harbor efficient response system to herbicides, therefore they could affect the diversity and functionality of microbiome in contaminated soils, thereby impacting agricultural production, environment sustainability and human health.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.

Silencing of Transposable Elements Mediated by 5-mC and Compensation of the Heterochromatin Content by Presence of B Chromosomes in Astyanax scabripinnis.

The way in which transcriptional activity overcomes the physical DNA structure and gene regulation mechanisms involves complex processes that are not yet fully understood. Modifications in the cytosine-guanine sequence of DNA by 5-mC are preferentially located in heterochromatic regions and are related to gene silencing. Herein, we investigate evidence of epigenetic regulation related to the B chromosome model and transposable elements in A. scabripinnis. Indirect immunofluorescence using anti-5-mC to mark methylated regions was employed along with quantitative ELISA to determine the total genomic DNA methylation level. 5-mC signals were dispersed in the chromosomes of both females and males, with preferential accumulation in the B chromosome. In addition to the heterochromatic methylated regions, our results suggest that methylation is associated with transposable elements (LINE and Tc1-Mariner). Heterochromatin content was measured based on the C-band length in relation to the size of chromosome 1. The B chromosome in A. scabripinnis comprises heterochromatin located in the pericentromeric region of both arms of this isochromosome. In this context, individuals with B chromosomes should have an increased heterochromatin content when compared to individuals that do not. Although, both heterochromatin content and genome methylation showed no significant differences between sexes or in relation to the occurrence of B chromosomes. Our evidence suggests that the B chromosome can have a compensation effect on the heterochromatin content and that methylation possibly operates to silence TEs in A. scabripinnis. This represents a sui generis compensation and gene activity buffering mechanism.

Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization.

Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.(AU)

LALLT (Loxosceles Allergen-Like Toxin) from the venom of Loxosceles intermedia: Recombinant expression in insect cells and characterization as a molecule with allergenic properties.

Bites evoked by Brown spiders (Loxosceles genus) are associated with skin injuries (cutaneous rash, itching, swelling, erythema and dermonecrosis) and systemic manifestations. Transcriptome analyses of Loxosceles venom glands showed that the venom has a complex composition containing toxins such as phospholipases-D, metalloproteases and hyaluronidases. Here, by screening the RNA from L. intermedia venom glands, we cloned a novel allergen toxin, and named LALLT (LoxoscelesAllergen-Like Toxin). Sequence analysis showed that LALLT is closely related to allergens from other spiders and RNA screening indicated the presence of LALLT orthologues in the venom of other Loxosceles spiders. Recombinant LALLT was expressed (~45 kDa) in baculovirus-infected insect cells and purified by affinity chromatography. Antibodies against different Loxosceles venoms cross-reacted with LALLT and antibodies against LALLT recognized three Loxosceles venoms, revealing epitopes identity. LALLT triggered paw edema in mice and erythema, edema and leukocyte infiltration into the dermis of rabbit skin. Also, LALLT induced vascular permeability in mice, degranulation of rat mesentery mast cells, as well as prompted degranulation and increased calcium influx in RBL-2H3 cells. Data reported suggest for the first time the existence of allergens in Loxosceles venoms and make LALLT available for clinical studies about allergenic events arisen by Loxosceles envenoming.

Dynamics of Replication and Nuclear Localization of the B Chromosome in Kidney Tissue Cells in Astyanax scabripinnis (Teleostei: Characidae).

B chromosomes are extra genomic compounds found in different taxonomic groups, including plants and animals. Obtaining patterns of resolutive chromosomal bands is necessary to understand the nuclear organization, variability and nature of B chromosome chromatin and possible transcriptional regions. In this study, we analyzed 35 Astyanax scabripinnis specimens sampled from Fazenda Lavrinha, a stream in the Paraíba do Sul river basin, Brazil. Through the incorporation of the thymidine analog 5'-bromo-2'-deoxyuridine (5-BrdU) in vivo, it was possible to recognize the replicating regions of the B chromosome at the beginning of the S phase, differentially characterized in relationship to the regions of late replication. In this perspective, it is possible to suggest that the B chromosome of this species possesses a territory and the chromatin accessible for transcription, especially in the light (i.e., early replicating) bands (p1.1; p1.3; and p2.1 and q1.1, q1.3, q2.1, and q2.2). The late-replicating regions are corresponding to the blocks of constitutive heterochromatin. They show a preferential accumulation of satellite DNA As51. By the use of the fluorochrome chromomycin A3 (CMA3), it was possible to identify GC-rich chromosomal regions, corresponding to late-replicating parts of genome, confirming the revealed data by the replication banding and C-banding. In addition, the analysis by confocal microscopy in kidney cells indicates the location of a peripheral anchorage of this chromosome in the nuclear lamina, reinforcing the idea of downregulation of the associated regions.

Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics.

Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5-40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action.