Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress.

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

Functional response of Acinetobacter guillouiae SFC 500-1A to tannery wastewater as revealed by a complementary proteomic approach.

Acinetobacter guillouiae SFC 500-1 A is a promising candidate for the bioremediation of tannery wastewater. In this study, we applied shotgun proteomic technology in conjunction with a gel-based assay (Gel-LC) to explore the strain's intracellular protein profile when grown in tannery wastewater as opposed to normal culture conditions. A total of 1775 proteins were identified, 52 of which were unique to the tannery wastewater treatment. Many of them were connected to the degradation of aromatic compounds and siderophore biosynthesis. On the other hand, 1598 proteins overlapped both conditions but were differentially expressed in each. Those that were upregulated in wastewater (109) were involved in the processes mentioned above, as well as in oxidative stress mitigation and intracellular redox state regulation. Particularly interesting were the downregulated proteins under the same treatment (318), which were diverse but mainly linked to the regulation of basic cellular functions (replication, transcription, translation, cell cycle, and wall biogenesis); metabolism (amino acids, lipids, sulphate, energetic processes); and other more complex responses (cell motility, exopolysaccharide production, biofilm formation, and quorum sensing). The findings suggest that SFC 500-1 A engages in survival and stress management strategies to cope with the toxic effects of tannery wastewater, and that such strategies may be mostly oriented at keeping metabolic processes to a minimum. Altogether, the results might be useful in the near future to improve the strain's effectiveness if it will be applied for bioremediation.

Biohybrid Adsorbent for the Preconcentration of Lead and Its Determination in Fruit Juices by Electrothermal Atomic Absorption Spectrometry.

BACKGROUND: Fruit juices are one of the most non-alcoholic beverages consumed in the world. Essential elements and other nutrients present in fruit juices play an important role in human well-being. However, fruit juices may also contain potentially toxic elements at trace levels, causing health risks. OBJECTIVE: The objective of this work was to develop an analytical methodology based on the preconcentration of lead using a new biodegradable hybrid material (BHM) composed of Rhodococcus erythropolis AW3 bacteria and Brassica napus hairy roots. METHODS: The BHM was implemented in an online solid-phase extraction (SPE) system for the determination of lead in fruit juices by electrothermal atomic absorption spectrometry (ETAAS). RESULTS: Effects of critical parameters on lead retention were studied. Under optimal experimental conditions, extraction efficiency higher than 99.9% and an enrichment factor of 62.5 were achieved. The dynamic capacity of the BHM was 36 mg/g, which favored the reuse of the column for at least eight biosorption-desorption cycles. The LOD and LOQ for preconcentration of 5 mL of sample were 5.0 and 16.5 ng/L lead, respectively. The RSD was 4.8% (at 1 µg/L lead and n = 10). CONCLUSION: The developed method was suitable for application to lead determination in different types of fruit juice. HIGHLIGHTS: A novel microextraction procedure based on the use of a biohybrid adsorbent. Highly sensitive determination of Pb at trace levels. Analysis of Pb in fruit juices samples. An eco-friendly microextraction technique for Pb determination.

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress.

Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10 mg/L) and Cr(VI) + phenol (10 and 300 mg/L), through two complementary proteomic approaches: gel-based (Gel-LC) and gel-free (shotgun) nanoUHPLC-ESI-MS/MS. A total of 400 differentially expressed proteins were identified, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI) + phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was also added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stress-associated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.

Promising co-inoculation strategies to reduce arsenic toxicity in soybean.

Arsenic (As) is the cause for concern worldwide due to its high toxicity. Its presence in agricultural soils and groundwater adversely affects soybean (Glycine max L.) growth and yield and also endangers food safety. Plant growth-promoting rhizobacteria (PGPR) could be used as part of cost-effective and eco-friendly strategies to mitigate As phytotoxicity. However, simple inoculation of soybean with PGPR Bradyrhizobium japonicum E109 (E109), a common practice in Argentina, is not effective in counteracting the effects of As exposure. Our aim was to assess whether the response of soybean to arsenate (AsV) and arsenite (AsIII) could be helpfully modulated by co-inoculating E109 with the free-living PGPRs Azospirillum brasilense Cd (Cd) or Bacillus pumilus SF5 (SF5). Co-inoculation with E109 + SF5 alleviated As-induced depletion of chlorophyll a and b, and carotenoid content, reaching an increase of 26, 28 y 31%, respectively. It also enhanced nodulation (15-19%) under As exposure. E109 + Cd and E109 + SF5 induced changes in the antioxidant system, which could be related to the maintenance of redox homeostasis. Moreover, As accumulation was reduced by 53% in aerial parts of plants inoculated with E109 + Cd, and by 16% in the roots of those inoculated with E109 + SF5. The strains selected show interesting potential for the development of biotechnological schemes to improve soybean yield while guaranteeing safer food production.

Groundwater quality and vulnerability in farms from agricultural-dairy basin of the Argentine Pampas.

Agricultural and livestock activities strongly influence groundwater quality and conditioning its use as water supply in rural areas. The aim was to determine the quality and suitability of the groundwater supply used in dairy farms of an agricultural area of Pampa plain of Córdoba (Argentina). Piper's diagram showed that the groundwater types were sodium bicarbonate, sodium bicarbonate-chloride, sodium chloride-sulfate, and sodium sulfate. Physicochemical parameters revealed that cations and anions showed a high and significant correlation in water samples, indicating a strong water-rock interaction. Nitrate (NO3-) content was significantly correlated with pH, water well depth, and distance from contamination sources. A high positive correlation between arsenic (As) and bicarbonate, sulfate, sodium, and chloride (p < 0.05) indicates a similar origin. Among the pesticides monitored, 2,4-D was detected in 25% of groundwater samples (0.4 to 0.8 µg/L) coinciding with the ordinary application practices. In general, most of the groundwater samples did not comply with national and international regulations for drinking water and dairy hygiene, due to the high content of As, NO3-, bacteria, and the presence of 2,4-D herbicide. However, the quality of water was suitable for livestock drink. The data obtained in this study contribute to a better understanding of the contamination processes taking place and improve the agricultural and livestock management for an efficient use of this resource by dairy farmers.

Tobacco hairy root's peroxidases are rhythmically controlled by phenol exposure.

Plants like almost all living organisms, have developed a biological clock or circadian clock (CC) capable of synchronizing and adjusting various metabolic and physiological processes at certain times of the day and in a period of 24 h. This endogenous timekeeping is able to predict the environmental changes providing adaptive advantages against stressful conditions. Therefore, the aim of this work was to analyze the possible link between metabolism of xenobiotic compounds (MXC) and the CC. Synchronized Nicotiana tabacum hairy roots (HRs) were used as a validated plant model system, and peroxidases (PODs), key enzymes of the phase I in the MCX, were evaluated after phenol treatment. Two POD genes were selected and their temporal expression profiles as well as the total POD activity were analyzed in order to find circadian oscillations either under control conditions or phenol treatment. It was demonstrated that these PODs genes showed oscillatory profiles with an ultradian period (period length shorter than the circadian period), and preserving the same phases and expression peaks still under phenol treatment. The total PODs activity showed also a marked oscillatory behavior mainly in phenol-treated HRs with the highest levels at ZT23. Untreated HRs showed decrease and increase in the intensity of some basic isoforms at light and dark phase, respectively, while in phenol- treated HRs, an increase in the intensity of almost all isoforms was observed, mainly during the dark phase, being coincident with the high PODs activity detected at ZT23. The periodic analysis determined an ultradian period either in total POD activity or in the POD activity of isoform VI, being 18.7 and 15.3 h, respectively. Curiously, in phenol treated HRs, the period length of total POD activity was longer than in untreated HRs, suggesting that phenol could induce a marked oscillatory behavior in the POD activity with better performance during the dark phase, which explain the higher phenol removal efficiencies at ZT23. These findings showed novel information about the performance of PODs, which would be rhythmically controlled at biochemical level, by phenol exposure.

Monitoring of Atrazine Pollution and its Spatial-Seasonal Variation on Surface Water Sources of an Agricultural River Basin.

Surface water sources are greatly impacted in areas with major agricultural land use. The atrazine quantification in surface waters as well as the spatial-temporal patterns of this herbicide was studied to detect pollution hotspots and to understand the putative factors responsible of its occurrence at the Ctalamochita river basin. The samples were collected on the aeolian fluvial plain of the river basin during five consecutive years. The results showed the high ubiquity of this compound and several sites with hazardous concentration (exceeding 0.1 µg/L international guidelines). The frequencies of quantification range from 67 to 100% in spring and 33%-67% in autumn. The atrazine content in surface water increased during the warm-rainy season, as consequence of atrazine application events (coinciding to the prevalent crop type). Overall, the study highlights the factors that could have favored atrazine pollution in the river basin such as land use, transport by runoff processes and atmospheric deposition.

Vicia villosa Roth: a cover crop to phytoremediate arsenic polluted environments.

Vicia villosa Roth is a legume species with a growing application in Argentina as a cover crop (CC), a practice that favors the sustainable development of agricultural systems. However, several areas where the use of this CC provides numerous advantages are affected by high concentrations of arsenic (As). Thus, in the present work we studied hairy vetch ability to cope with arsenate [As(V)], arsenite [As(III)], and the mixture of both along with oxidative stress indexes [chlorophyll content, malondialdehyde (MDA) equivalents] as well as anatomical and histological changes in the root structure. The results obtained suggested a different behavior of hairy vetch depending on its growth stage and on metal(oid) concentration. The roots treated with the contaminant showed less turgidity, thickening of the epidermal and subepidermal parenchymal outer layers, and the presence of dark deposits. The morpho-anatomic parameters (cortex length, vascular cylinder diameter, total diameter, and vascular cylinder area) were altered in plants treated with As(V) and As(V)/As(III) whereas the roots of plants treated with As(III) did not show significant differences respect to the control. Moreover V. villosa could tolerate and remove As from soil, thus the use of this legume species seems an attractive approach to remediate As while protecting contaminated soils.

Biohybrid membranes for effective bacterial vehiculation and simultaneous removal of hexavalent chromium (CrVI) and phenol.

The aim of the present study was to obtain an effective vehiculation system in which bacterial agents could maintain viability improving their removal capacity. Herein, we present a novel biohybrid membrane of polymeric nanofibers and free-living bacteria for the simultaneous removal of pollutants. In this system, bacteria are free within the pores between the nanofibers and adsorbed to the surface of the membranes. Association between bacteria and the membranes was performed through a self-formulated medium, and the presence of the bacteria in the polymeric matrix was evidenced through atomic force microscopy (AFM). Biohybrid membranes associated with the remediation agents Bacillus toyonensis SFC 500-1E and Acinetobacter guillouiae SFC 500-1A promoted a reduction of up to 2.5 mg/L of hexavalent chromium and up to 200 mg/L of phenol after 24 h of treatment in synthetic medium containing the contaminants. Similarly, more than 46% of the hexavalent chromium and all of the phenol content were removed after treatment of a tannery effluent with initial concentrations of 7 mg/L of Cr(VI) and 305 mg/L of phenol. Counts of the remediation agents from the membranes were always above 1.107 CFU/g, also in the reutilization assays performed without reinoculation. Biohybrid membranes were hydrolysis-resistant, reusable, and effective in the simultaneous removal of contaminants for more than 5 cycles. Viability of the microorganisms was maintained after long-term storage of the membranes at 4 °C, without the use of microbiological media or the addition of cryoprotectants. Graphical abstract KEY POINTS: • Polymeric membranes were effectively associated with the SFC 500-1 remediation consortium • Biohybrid membranes removed hexavalent chromium and phenol from different matrices • Removal of contaminants was achieved in many successive cycles without reinoculation.

Application of two bioassays as potential indicators of phenol phytoremediation efficiency by tobacco hairy roots.

Phenol is one of the contaminants most frequently found in the environment and it is considered a priority pollutant due to their toxic effects. Hairy roots (HR) constitute a good model tool for the removal of this contaminant. In this work, phenol removal using wild type (WT) and double transgenic (DT) Nicotiana tabacum HR was performed with high efficiency (60-80%, for 25-250 mg L-1 phenol solutions, respectively). After phytoremediation process, the toxicity of post removal solutions (PRS) was evaluated through two-toxicity test belonging to two trophic levels, Lactuca sativa test and Rhinella arenarum (AMPHITOX). Toxicity of PRS showed variable results since these solutions were less toxic to L. sativa seeds compared to R. arenarum embryos, which could be attributed to different sensitivities of the exposed organisms. Although PRS obtained using WT and DT HR reduced phenol phytotoxicity on L. sativa seeds, WT PRS were even less toxic than DT PRS according to this test. Regarding AMPHITOX, HR culture medium without phenol but incubated with HR and phenol PRS exerted a toxic effect on the embryos, which could be related to the presence of toxic products derived from HR metabolism. The results demonstrated that an efficient phenol removal is not always accompanied by a considerable reduction of the solution toxicity and therefore, the use of organisms from different trophic levels to evaluate the toxicity after the removal process gains importance.

Improving soybean growth under arsenic stress by inoculation with native arsenic-resistant bacteria.

Certain metal (loid)-resistant bacteria that inhabit the rhizosphere have shown to improve plant growth and tolerance under toxic metal stress. In this study, we tested if six native, arsenic-resistant and plant growth promoting bacteria (PGPB) were able to enhance soybean (Glycine max L.) growth and modulate arsenic (As) uptake. As a previous work, we tested all single isolates and all possible binary combinations without arsenic stress to identify the combinations that would have the greatest plant growth promoting effect. In this study, a screening assay was performed with only five inoculation options selected after first stage (Pseudomonas sp. AW4, Pseudomonas sp. AW6, AW4+AW6, Rhodococcus sp. AW3+Pseudomonas sp. AW5 and Enterobacter sp. AW1+AW6). In both stages, inoculation was implemented by imbibition of soybean seeds with bacterial suspensions, and plant growth was carried out in pots using perlite as substrate in a chamber with controlled conditions. In the third stage, we performed similar assays, under As stress, using the three most promising inoculation options (AW4, AW6 and AW3+AW5). Treatments were performed by irrigation with 25 µM arsenite (As3+), 25 µM arsenate (As5+), 25 µM equimolar As3+/As5+ solution or water (control). Biometric and biochemical parameters indicated that inoculation with Pseudomonas sp. AW4 significantly promoted soybean growth under As3+/As5+ treatment and did not modified As accumulation pattern. Further field studies are needed to determine if some of these inoculation options are useful to improve in situ soybean growth under arsenic stress and could become a tool for the development of sustainable agriculture in As-impacted environments.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Impact assessment of bioaugmented tannery effluent discharge on the microbiota of water bodies.

Effluents are commonly discharged into water bodies, and in order for the process to be as environmentally sound as possible, the potential effects on native water communities must be assessed alongside the quality parameters of the effluents themselves. In the present work, changes in the bacterial diversity of streamwater receiving a tannery effluent were monitored by high-throughput MiSeq sequencing. Physico-chemical and microbiological parameters and acute toxicity were also evaluated through different bioassays. After the discharge of treated effluents that had been either naturally attenuated or bioaugmented, bacterial diversity decreased immediately in the streamwater samples, as evidenced by the over-representation of taxa such as Brachymonas, Arcobacter, Marinobacterium, Myroides, Paludibacter and Acinetobacter, typically found in tannery effluents. However, there were no remarkable changes in diversity over time (after 1 day). In terms of the physico-chemical and microbiological parameters analyzed, chemical oxygen demand and total bacterial count increased in response to discharge of the treated effluents. No lethal effects were observed in Lactuca sativa L. seeds or Rhinella arenarum embryos exposed to the streamwater that had received the treated effluents. All of these results contribute to the growing knowledge about the environmental safety of effluent discharge procedures.

Morphological and structural response of Bacillus sp. SFC 500-1E after Cr(VI) and phenol treatment.

Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.

Impact of phenol on the glycerophospholipid turnover and potential role of circadian clock in the plant response against this pollutant in tobacco hairy roots.

Glycerophospholipids (GPLs) from cell membranes (CM) are a proper source for the synthesis of lipid messengers able to activate signal pathways that will define the plant survival under changing and stressful environmental conditions. Little is known about how GPLs metabolism (GPLsM) is regulated and the effects of phenol treatment on GPLs composition. In this work, we studied the effects of phenol both on GPLs turnover and on the expression of GPLsM-related genes potentially regulated by the circadian clock, using tobacco hairy root cultures (HRC). Phenol decreased the total PC levels and increased PE, PG and CL levels in the dark phase. Different molecular species of PC and PE showed the same trend than the total PC and PE upon phenol treatment. Besides, significant differences in the expression of all studied genes related to GPLsM were found. NtCCT2 expression was affected at all analyzed times while NtPECT1 and NtAAPT1 showed similar expression patterns. NtCDS1, NtPGPS2 and NtCLS genes showed significant and differential expression profiles both in untreated and treated HRC. PECT1 and NtPGPS2 genes seem to conserve a circadian expression profile mainly in untreated HRC. However, phenol was able to modify the GPLs composition and the expression of genes related to GPLs synthesis. The GPLs modification could be explained by the up-regulation of NtPECT1, NtAAPT1 and NtCLS genes during the dark phase, suggesting for being a crucial moment for HRC to trigger an adaptive response against this organic pollutant.

Deepening the knowledge on the removal of Cr(VI) by L. minuta Kunth: removal efficiency and mechanisms, lipid signaling pathways, antioxidant response, and toxic effects.

Lemna minuta Kunth was used to remove Cr(VI) from aqueous solutions, and some of the mechanisms involved in this process were analyzed. In addition, the cellular signaling mediated by phospholipase D activity as well as antioxidant responses was also evaluated during the process. Cr(VI) removal efficiencies were 40% for 0.5 mg/L, after 24 h, and up to 18% at metal concentrations as high as 5 mg/L. Removal mechanisms displayed by these macrophytes include bioadsorption to cell surfaces and, to a greater extent, Cr internalization and bioaccumulation within cells. Inside of them, Cr(VI) was reduced to Cr(III), a less toxic form of this metal. At the first hours of Cr(VI) exposure, plants were able to sense chromium, activating membrane signal transduction pathways mediated by phospholipase D and phosphatidic acid. Moreover, an increase in the activity of antioxidant enzymes such as superoxide dismutases and peroxidases was observed in the same time. These and other components of the antioxidant defense system would help to reduce the stress generated by the metal. The toxicity of the products formed during the removal process was assessed through Lactuca sativa L. and AMPHIAGU test. It was evidenced that Cr(VI) phytoremediation process by L. minuta plants did not generate acute toxicity neither for L. sativa seeds nor for embryos of Rhinella arenarum (Hensel, 1876). Thus, L. minuta plants could be considered as valuable species for the treatment of waters contaminated with Cr(VI).

CR(VI) phytoremediation by hairy roots of Brassica napus: assessing efficiency, mechanisms involved, and post-removal toxicity.

Industrial activities such as leather tanning involve the use of highly toxic inorganic pollutants, like Chromium (Cr). This work evaluated Cr(VI) remediation by hairy roots (HR) of Brassica napus, paying close attention to the mechanisms involved and the toxicity of post-removal solutions. Results showed that these roots were capable of tolerating concentrations of up to 10 mg L-1 Cr(VI), while higher concentrations were toxic for HR development. Removal efficiency was assessed through the use of synthetic solutions containing different initial Cr(VI) concentrations (2, 5, or 10 mg L-1). Regardless of these initial concentrations, the highest removal efficiency values were between 80 and 90% after 24 and 48 h of treatment, using a 2.0 g inoculum. The mechanisms involved were Cr accumulation (60%) and to a lesser extent, adsorption to the root biomass (30%). A fraction of Cr(VI) was intracellularly reduced to Cr(III), which suggests reductases may have played a role. Additionally, post-removal toxicity was evaluated through two bioassays (Lactuca sativa L. and AMPHITOX test) after the removal of 10 mg L-1 Cr(VI). The treated solutions showed moderate phytotoxicity for L. sativa L. and no toxicity for R arenarum. The ability of HR to remove 10 mg L-1 Cr(VI) from real tannery effluents collected from a regional industry (Córdoba province, Argentina) was also determined. The high removal efficiency observed (98%) demonstrates this system can be successful in treating complex wastewaters.

Catanionic nanocarriers as a potential vehicle for insulin delivery.

Diabetes is a disease that affects millions of people in the World, constituting a global problem. Patients are administered insulin subcutaneous injections, resulting in high costs and frequent infections in the injection site. A possible solution to this problem may be the use of nanotechnology. Nanotransporters can act as specific release systems able to overcome the current limitations to drug delivery. Liposomes and vesicles can deliver drugs directly and efficiently to the site of action, decreasing toxicity and adverse effects. In previous studies, we demonstrated the biocompatibility and safety of catanionic benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHD-AOT) vesicles using both in vitro and in vivo tests. Thus, the aims of this work were to evaluate the ability of the BHD-AOT vesicles to encapsulate insulin; to analyze the structural properties and stability of the system, vesicle-Insulin (VIn), at different pH conditions; and to study the ability of VIn to decrease the glycemia in miceby different administration routes. Our results showed that 2 and 5 mg mL-1 of vesicles were able to encapsulate about 55 % and 73 % of insulin, respectively. The system VIn showed a significant increase in size from 120 to 350 nm, changes in the surface zeta potential value, and high stability to different pH conditions. A significant decrease of the glycemia after VIn administration was demonstrated in in vivo assays, including the oral route. Our results reveal that BHD-AOT vesicles may be an appropriate system to encapsulate and protect insulin, and may be a potential system to be administrated in different ways as an alternative strategy to conventional therapy.

Biochemical and molecular characterization of arsenic response from Azospirillum brasilense Cd, a bacterial strain used as plant inoculant.

Azospirillum brasilense Cd is a bacterial strain widely used as an inoculant of several crops due to its plant growth promoting properties. However, its beneficial effects depend on its viability and functionality under adverse environmental conditions, including the presence of arsenic (As) in agricultural soils. Therefore, the aim of this work was to evaluate the response of A. brasilense Cd to arsenate (AsV) and arsenite (AsIII). This bacterium was tolerant to As concentrations frequently found in soils. Moreover, properties related to roots colonization (motility, biofilm, and exopolymers) and plant growth promotion (auxin, siderophore production, and N2 fixation) were not significantly affected by the metalloid. In order to deepen the understanding on As responses of A. brasilense Cd, As resistance genes were sequenced and characterized for the first time in this work. These genes could mediate the redox As transformation and its extrusion outside the cell, so they could have direct association with the As tolerance observed. In addition, its As oxidation/reduction capacity could contribute to change the AsV/AsIII ratio in the environment. In conclusion, the results allowed to elucidate the As response of A. brasilense Cd and generate interest for its potential use in polluted environments.