Phenogenetic profile and agronomic contribution of Azospirillum argentinense Az39T, a reference strain for the South American inoculant industry.

Azospirillum sp. is a plant growth-promoting rhizobacteria largely recognized for its potential to increase the yield of different important crops. In this work, we present a thorough genomic and phenotypic analysis of A. argentinense Az39T to provide new insights into the beneficial mechanisms of this microorganism. Phenotypic analyses revealed the following in vitro abilities: growth at 20-38 °C (optimum, 28 °C), pH 6.0-8.0 (optimum, pH 6.8), and in the presence of 1% (w/v) NaCl; production of variable amounts of PHB as intracellular granules; nitrogen fixation under microaerophilic conditions; IAA synthesis in the presence of L-tryptophan. Through biochemical (API 20NE) and carbon utilization profiling (Biolog) assays, we proved that A. argentinense Az39T is able to use 15 substrates and metabolize 19 different carbon substrates. Lipid composition indicated a predominance of medium and long-chain saturated fatty acids. A total of 6 replicons classified as one main chromosome, three chromids, and two plasmids, according to their tRNA and core essential genes contents, were identified. Az39T genome includes genes associated with multiple plant growth-promoting (PGP) traits such as nitrogen fixation and production of auxins, cytokinin, abscisic acid, ethylene, and polyamines. In addition, Az39T genome harbor genetic elements associated with physiological features that facilitate its survival in the soil and competence for rhizospheric colonization; this includes motility, secretion system, and quorum sensing genetic determinants. A metadata analysis of Az39T agronomic performance in the pampas region, Argentina, demonstrated significant grain yield increases in wheat and maize, proving its potential to provide better growth conditions for dryland cereals. In conclusion, our data provide a detailed insight into the metabolic profile of A. argentinense Az39T, the strain most widely used to formulate non-legume inoculants in Argentina, and allow a better understanding of the mechanisms behind its field performance.

Unraveling Azospirillum's colonization ability through microbiological and molecular evidence.

It is known that members of the bacterial genus Azospirillum can promote the growth of a great variety of plants, an ability harnessed by the industry to create bioproducts aimed to enhance the yield of economically relevant crops. Its versatile metabolism allows this bacterium to adapt to numerous environments, from optimal to extreme or highly polluted. The fact of having been isolated from soil and rhizosphere samples collected worldwide and many other habitats proves its remarkable ubiquity. Azospirillum rhizospheric and endophytic lifestyles are governed by several mechanisms, leading to efficient niche colonization. These mechanisms include cell aggregation and biofilm formation, motility, chemotaxis, phytohormone and other signaling molecules production, and cell-to-cell communication, in turn, involved in regulating Azospirillum interactions with the surrounding microbial community. Despite being infrequently mentioned in metagenomics studies after its introduction as an inoculant, an increasing number of studies detected Azospirillum through molecular tools (mostly 16S rRNA sequencing) as part of diverse, even unexpected, microbiomes. This review focuses on Azospirillum traceability and the performance of the available methods, both classical and molecular. An overview of Azospirillum occurrence in diverse microbiomes and the less-known features explaining its notorious ability to colonize niches and prevail in multiple environments is provided.

Experimental studies of boron neutron capture therapy (BNCT) using histone deacetylase inhibitor (HDACI) sodium butyrate, as a complementary drug for the treatment of poorly differentiated thyroid cancer (PDTC).

PURPOSE: The present study analyzed different protocols of administration of boronophenylalanine (BPA) and sodium butyrate (NaB) to increase the BNCT efficacy for poorly differentiated thyroid cancer (PDTC). MATERIALS AND METHODS: Nude mice implanted with human PDTC cells (WRO) were distributed into four protocols: 1) BPA; 2) BPA + ip NaB; 3) BPA + oral NaB; 4) Control. Biodistribution and histologic studies were performed. LAT (BPA transporter) isoforms gene expression was assessed by RT-PCR. RESULTS: Tumor growth delay was observed in animals of the Protocol #3 (p < 0.05). NaB (Protocol #2) increased tumor boron uptake 2-h post BPA injection (p < 0.05). On the other hand, NaB upregulated the expression of all the isoforms of the LAT transporter in vitro. Histologic studies showed a significant decrease of mitotic activity and an increase of vacuoles in tumors of Protocol #3. Neutrons alone or combined with NaB caused some tumor growth delay (p < 0.05), while in the BNCT and BNCT + NaB groups, there was a halt in tumor growth in 70 and 80% of the animals, respectively. CONCLUSIONS: Intraperitoneally administration of NaB increased boron uptake while oral administration for a longer period of time induced tumor growth delay previous to BPA administration. The use of NaB via ip would optimize the irradiation results.

Azospirillum brasilense Az39, a model rhizobacterium with AHL quorum-quenching capacity.

AIMS: The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. METHODS AND RESULTS: We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500 nmol l-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium. CONCLUSIONS: Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism.

Improvement of the boron neutron capture therapy (BNCT) by the previous administration of the histone deacetylase inhibitor sodium butyrate for the treatment of thyroid carcinoma.

We have shown that boron neutron capture therapy (BNCT) could be an alternative for the treatment of poorly differentiated thyroid carcinoma (PDTC). Histone deacetylase inhibitors (HDACI) like sodium butyrate (NaB) cause hyperacetylation of histone proteins and show capacity to increase the gamma irradiation effect. The purpose of these studies was to investigate the use of the NaB as a radiosensitizer of the BNCT for PDTC. Follicular thyroid carcinoma cells (WRO) and rat thyroid epithelial cells (FRTL-5) were incubated with 1 mM NaB and then treated with boronophenylalanine ¹°BPA (10 µg ¹°B ml⁻¹) + neutrons, or with 2, 4-bis (α,ß-dihydroxyethyl)-deutero-porphyrin IX ¹°BOPP (10 µg ¹°B ml⁻¹) + neutrons, or with a neutron beam alone. The cells were irradiated in the thermal column facility of the RA-3 reactor (flux = (1.0 ± 0.1) × 10¹° n cm⁻² s⁻¹). Cell survival decreased as a function of the physical absorbed dose in both cell lines. Moreover, the addition of NaB decreased cell survival (p < 0.05) in WRO cells incubated with both boron compounds. NaB increased the percentage of necrotic and apoptotic cells in both BNCT groups (p < 0.05). An accumulation of cells in G2/M phase at 24 h was observed for all the irradiated groups and the addition of NaB increased this percentage. Biodistribution studies of BPA (350 mg kg⁻¹ body weight) 24 h after NaB injection were performed. The in vivo studies showed that NaB treatment increases the amount of boron in the tumor at 2-h post-BPA injection (p < 0.01). We conclude that NaB could be used as a radiosensitizer for the treatment of thyroid carcinoma by BNCT.

Studies for the application of boron neutron capture therapy to the treatment of differentiated thyroid cancer.

The aim of these studies was to evaluate the possibility of treating differentiated thyroid cancer by BNCT. These carcinomas are well controlled with surgery followed by therapy with (131)I; however, some patients do not respond to this treatment. BPA uptake was analyzed both in vitro and in nude mice implanted with cell lines of differentiated thyroid carcinoma. The boron intracellular concentration in the different cell lines and the biodistribution studies showed the selectivity of the BPA uptake by this kind of tumor.

Reference systems for the determination of 10B through autoradiography images: application to a melanoma experimental model.

The amount of (10)B in tissue samples may be determined by measuring the track density in the autoradiography image produced on a nuclear track detector. Different systems were evaluated as reference standards to be used for a quantitative evaluation of boron concentration. The obtained calibration curves were applied to evaluate the concentration of (10)B in melanoma tumour of NIH nude mice after a biodistribution study. The histological features observed in the tissue sections were accurately reproduced by the autoradiography images.

BNCT for skin melanoma in extremities: updated Argentine clinical results.

As part of phase I/II melanoma BNCT clinical trial conducted in Argentina in a cooperative effort of the Argentine Atomic Energy Commission (CNEA) and the Oncology Institute Angel H. Roffo (IOAHR), 7 patients (6 female-1 male) received eight treatment sessions covering ten anatomical areas located in extremities. Mean age of the patients was 64 years (51-74). The treatments were performed between October 2003 and June 2007. All patients presented multiple subcutaneous skin metastases of melanoma and received an infusion containing approximately 14 gr/m(2) of (10)borophenyl-alanine (BPA) followed by the exposition of the area to a mixed thermal-epithermal neutron beam at the RA-6 reactor. The maximum prescribed dose to normal skin ranged from 16.5 to 24 Gy-Eq and normal tissue administered dose varied from 15.8 to 27.5 Gy-Eq. Considering evaluable nodules, 69.3% of overall response and 30.7% of no changes were seen. The toxicity was acceptable, with 3 out of 10 evaluable areas showing ulceration (30% toxicity grade 3).

Boron neutron capture therapy (BNCT) inhibits tumor development from precancerous tissue: an experimental study that supports a potential new application of BNCT.

We previously demonstrated the efficacy of boron neutron capture therapy (BNCT) mediated by boronophenylalanine (BPA), GB-10 (Na(2)(10)B(10)H(10)) and (GB-10+BPA) to control tumors, with no normal tissue radiotoxicity, in the hamster cheek pouch oral cancer model. Herein we developed a novel experimental model of field-cancerization and precancerous lesions (globally termed herein precancerous tissue) in the hamster cheek pouch to explore the long-term potential inhibitory effect of the same BNCT protocols on the development of second primary tumors from precancerous tissue. Clinically, second primary tumor recurrences occur in field-cancerized tissue, causing therapeutic failure. We performed boron biodistribution studies followed by in vivo BNCT studies, with 8 months follow-up. All 3 BNCT protocols induced a statistically significant reduction in tumor development from precancerous tissue, reaching a maximum inhibition of 77-100%. The inhibitory effect of BPA-BNCT and (GB-10+BPA)-BNCT persisted at 51% at the end of follow-up (8 months), whereas for GB-10-BNCT it faded after 2 months. Likewise, beam-only elicited a significant but transient reduction in tumor development. No normal tissue radiotoxicity was observed. At 8 months post-treatment with BPA-BNCT or (GB-10+BPA)-BNCT, the precancerous pouches that did not develop tumors had regained the macroscopic and histological appearance of normal (non-cancerized) pouches. A potential new clinical application of BNCT would lie in its capacity to inhibit local regional recurrences.

Tumor control and normal tissue complications in BNCT treatment of nodular melanoma: a search for predictive quantities.

A previous work concerning tumor control and skin damage in cutaneous melanoma treatments with BNCT has been extended to include doses, volumes and responses of 104 subcutaneous lesions from all patients treated in Argentina. Acute skin reactions were also scored for these patients, and cumulative dose-area histograms and dose-based figures of merit for skin were calculated. Broadening the tumor response analysis with the latest data showed that the (minimum or mean) tumor dose is not a good predictor of the observed clinical outcome by itself. However, when the tumor volume was included in the model as second explicative variable, the dose increases its significance and becomes a critical variable jointly with the volume (p-values<0.05). A preliminary analysis to estimate control doses for two groups of tumor sizes revealed that for small tumor volumes (< 0.1cm(3)) doses greater than 20 Gy-Eq produce a high tumor control (> 80%). However, when tumor volumes are larger than 0.1cm(3), control is moderate (< 40%) even for minimum doses up to 40 Gy-Eq. Some quantities based on skin doses, areas and complication probabilities were proposed as candidates for predicting the severity of the early skin reactions. With the current data, all the evaluated figures of merit derived similar results: ulceration is present among the cases for which these quantities take the highest values.

Boron biodistribution study in colorectal liver metastases patients in Argentina.

Ex-situ BNCT for multifocal unresectable liver metastases employing whole or partial autograft techniques requires knowledge of boron concentrations in healthy liver and metastases following perfusion and immersion in Wisconsin solution (W), the procedure employed for organ preservation during ex-situ irradiation. Measurements of boron concentration in blood, liver and metastases following an intravenous infusion of BPA-F in five colorectal liver metastases patients scheduled for surgery were performed. Tissue samples were evaluated for boron content pre and post perfusion and immersion in W. Complementary histological studies were performed. The data showed a dose-dependent BPA uptake in liver, a boron concentration ratio liver/blood close to 1 and a wide spread in the metastases/liver concentration ratios in the range 0.8-3.6, partially attributable to histological variations between samples. Based on the boron concentrations and dose considerations (liver < or =15 Gy-Eq and tumor> or =40 Gy-Eq) at the RA-3 thermal neutron facility (mean flux of about (6+/-1) x 10(9) n cm(-2)s(-1)), ex-situ treatment of liver metastases at RA-3 would be feasible.