Exploring the specialized metabolome of the plant pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are notable for producing chemically diverse specialized metabolites that exhibit various bioactivities and mediate interactions with different organisms. Streptomyces sp. 11-1-2 is a plant pathogen that produces nigericin and geldanamycin, both of which display toxic effects against various plants. Here, the 'One Strain Many Compounds' approach was used to characterize the metabolic potential of Streptomyces sp. 11-1-2. Organic extracts were prepared from 11-1-2 cultures grown on six different agar media, and the extracts were tested in antimicrobial and plant bioassays and were subjected to untargeted metabolomics and molecular networking. Most extracts displayed strong bioactivity against Gram-positive bacteria and yeast, and they exhibited phytotoxic activity against potato tuber tissue and radish seedlings. Several known specialized metabolites, including musacin D, galbonolide B, guanidylfungin A, meridamycins and elaiophylin, were predicted to be present in the extracts along with closely related compounds with unknown structure and bioactivity. Targeted detection confirmed the presence of elaiophylin in the extracts, and bioassays using pure elaiophylin revealed that it enhances the phytotoxic effects of geldanamycin and nigericin on potato tuber tissue. Overall, this study reveals novel insights into the specialized metabolites that may mediate interactions between Streptomyces sp. 11-1-2 and other bacteria and eukaryotic organisms.

Use of venovenous (VV) extracorporeal membrane oxygenation (ECMO) in near-fatal asthma: a case series.

Introduction: Status asthmaticus (SA) and near-fatal asthma (NFA) are life-threatening conditions that continue to present a management challenge for physicians. Extracorporeal Membrane Oxygenation (ECMO) has been employed as a last resort in treating these patients. Case presentation: We described six patients who were admitted to the ICU for NFA and received ECMO treatment at a high-complexity institution in Cali, Colombia, between 2015 and 2019. All patients are registered in the ELSO registry. Baseline patient characteristics, arterial blood gases (ABG), ventilatory parameters, and complications were collected as specified in the ELSO registry form. Efficacy was analyzed in terms of the improvement in respiratory acidosis, the number of ventilator-free days (VFD), and a reduction in mechanical power (MP). MP, which refers to the energy associated with the mechanical forces involved in breathing and the functioning of the respiratory system, was calculated using a mathematical formula. Safety was evaluated based on the incidence of complications. After 12 hours of ECMO, we achieved a correction of respiratory acidosis, a significant decrease in all ventilatory parameters, and a reduction in MP ranging from 52.8% to 89%. There was one mortality. Among the five surviving patients, all except one, who required a tracheostomy, had a high VFD score, with a mode of 26 days, demonstrating a reduction in ventilation time. Conclusion: Further randomized controlled trials are needed to fully understand the efficacy and safety profiles of ECMO in SA/NFA. MP is being widely used to achieve safer ventilation, and although more data is required, it appears to be a promising option for evaluating the risk of developing VILI and the success of the therapy.

Molecular Level Sucrose Quantification: A Critical Review.

Sucrose is a primary metabolite in plants, a source of energy, a source of carbon atoms for growth and development, and a regulator of biochemical processes. Most of the traditional analytical chemistry methods for sucrose quantification in plants require sample treatment (with consequent tissue destruction) and complex facilities, that do not allow real-time sucrose quantification at ultra-low concentrations (nM to pM range) under in vivo conditions, limiting our understanding of sucrose roles in plant physiology across different plant tissues and cellular compartments. Some of the above-mentioned problems may be circumvented with the use of bio-compatible ligands for molecular recognition of sucrose. Nevertheless, problems such as the signal-noise ratio, stability, and selectivity are some of the main challenges limiting the use of molecular recognition methods for the in vivo quantification of sucrose. In this review, we provide a critical analysis of the existing analytical chemistry tools, biosensors, and synthetic ligands, for sucrose quantification and discuss the most promising paths to improve upon its limits of detection. Our goal is to highlight the criteria design need for real-time, in vivo, highly sensitive and selective sucrose sensing capabilities to enable further our understanding of living organisms, the development of new plant breeding strategies for increased crop productivity and sustainability, and ultimately to contribute to the overarching need for food security.

Nigericin and Geldanamycin Are Phytotoxic Specialized Metabolites Produced by the Plant Pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are a key source of microbial specialized metabolites with useful applications in medicine and agriculture. In addition, some species are important plant pathogens and cause diseases such as potato scab, which reduces the quality and market value of affected potato crops. Most scab-associated Streptomyces spp. produce the phytotoxic metabolite thaxtomin A as the principal pathogenicity factor. However, recent reports have described scab-causing strains that do not produce thaxtomin A, but instead produce other phytotoxins that are thought to contribute to plant host infection and symptom development. Streptomyces sp. 11-1-2 is a highly pathogenic strain that was originally isolated from a scab symptomatic potato tuber in Newfoundland, Canada. The strain secretes one or more phytotoxic compounds of unknown identity, and it is hypothesized that these compounds serve as virulence factors for this organism. We analyzed the genome sequence of Streptomyces sp. 11-1-2 and found biosynthetic gene clusters for producing the known herbicidal compounds nigericin and geldanamycin. Phytotoxic culture extracts were analyzed using liquid chromatography-coupled tandem mass spectrometry and molecular networking, and this confirmed the production of both compounds by Streptomyces sp. 11-1-2 along with other, potentially related metabolites. The biosynthesis of both metabolites was found to be suppressed by the addition of N-acetylglucosamine to the culture medium, and pure nigericin and geldanamycin were able to exhibit phytotoxic effects against both radish seedlings and potato tuber tissue. Furthermore, the coadministration of the two compounds produced greater phytotoxic effects against potato tuber tissue than administration of each compound alone. IMPORTANCE Plant pathogens use a variety of mechanisms, including the production of phytotoxic specialized metabolites, to establish an infection of host tissue. Although thaxtomin A is considered the key phytotoxin involved in the development of potato scab disease, there is increasing evidence that other phytotoxins can play a role in disease development in some instances. In this study, we show that the highly pathogenic Streptomyces sp. 11-1-2 is capable of producing nigericin and geldanamycin, which individually and combined can cause significant damage to potato tuber tissue and radish seedlings. Our results suggest that the pathogenic phenotype of Streptomyces sp. 11-1-2 is due in part to the production of these specialized metabolites. As the biological activity of nigericin and geldanamycin is vastly different from the proposed activity of thaxtomin A against plants, the secretion of these compounds may represent a novel mechanism of plant pathogenicity exhibited by some Streptomyces species.

Changes in the phyllosphere and rhizosphere microbial communities of soybean in the presence of pathogens.

Soybean (Glycine max L.) is host to an array of foliar- and root-infecting pathogens that can cause significant yield losses. To provide insights into the roles of microorganisms in disease development, we evaluated the bacterial and fungal communities associated with the soybean rhizosphere and phyllosphere. For this, leaf and soil samples of healthy, Phytophthora sojae-infected and Septoria glycines-infected plants were sampled at three stages during the production cycle, and then subjected to 16S and Internal Transcribed Spacer (ITS) amplicon sequencing. The results indicated that biotic stresses did not have a significant impact on species richness and evenness regardless of growth stage. However, the structure and composition of soybean microbial communities were dramatically altered by biotic stresses, particularly for the fungal phyllosphere. Additionally, we cataloged a variety of microbial genera that were altered by biotic stresses and their associations with other genera, which could serve as biological indicators for disease development. In terms of soybean development, the rhizosphere and phyllosphere had distinct microbial communities, with the fungal phyllosphere most influenced by growth stage. Overall, this study characterized the phyllosphere and rhizosphere microbial communities of soybean, and described the impact of pathogen infection and plant development in shaping these bacterial and fungal communities.

Amplicon Sequencing Reveals Extensive Coinfections of Foliar Pathogens in Soybean.

Soybean (Glycine max) is one of the most economically important crops grown in North America and in other regions worldwide. However, the plant is susceptible to a variety of foliar pathogenic microorganisms, some of which are a significant threat to production. Several molecular and serological approaches are currently available to diagnose plant pathogens, but all have limitations including their capability to accurately detect coinfections of individual plants. We therefore used 16S and internal transcribed spacer amplicon sequencing to identify the suite of bacterial and fungal organisms infecting 96 soybean leaf samples collected throughout southern Manitoba, Canada, at two growth stages (V2/3 and R6). We were able to confirm the presence of pathogens previously known to our sampling regions, such as Septoria glycines, Alternaria alternata, and Pseudomonas spp. Importantly, we found that most of plants were infected by more than one putative pathogen, with 64% of V2/3 and 26% of R6 plants infected by three or more pathogens. Amplicon sequencing also indicated the presence of residual pathogens that infect crops other than soybean, as well as nonfoliar pathogens and nonpathogenic microorganisms. We discuss some of the benefits and drawbacks of using amplicon sequencing to detect foliar pathogens of soybean.

Clinical Veterinary Boron Neutron Capture Therapy (BNCT) Studies in Dogs with Head and Neck Cancer: Bridging the Gap between Translational and Clinical Studies.

Translational Boron Neutron Capture Therapy (BNCT) studies performed by our group and clinical BNCT studies worldwide have shown the therapeutic efficacy of BNCT for head and neck cancer. The present BNCT studies in veterinary patients with head and neck cancer were performed to optimize the therapeutic efficacy of BNCT, contribute towards exploring the role of BNCT in veterinary medicine, put in place technical aspects for an upcoming clinical trial of BNCT for head and neck cancer at the RA-6 Nuclear Reactor, and assess the feasibility of employing the existing B2 beam to treat large, deep-seated tumors. Five dogs with head and neck cancer with no other therapeutic option were treated with two applications of BNCT mediated by boronophenyl-alanine (BPA) separated by 3-5 weeks. Two to three portals per BNCT application were used to achieve a potentially therapeutic dose over the tumor without exceeding normal tissue tolerance. Clinical and Computed Tomography results evidenced partial tumor control in all cases, with slight-moderate mucositis, excellent life quality, and prolongation in the survival time estimated at recruitment. These exploratory studies show the potential value of BNCT in veterinary medicine and contribute towards initiating a clinical BNCT trial for head and neck cancer at the RA-6 clinical facility.

Applications of Next-Generation Sequencing for Large-Scale Pathogen Diagnoses in Soybean.

Soybean (Glycine max) has become an important crop in Manitoba, Canada, with a 10-fold increase in dedicated acreage over the past decade. Given the rapid increase in production, scarce information about foliar diseases present in the province has been recorded. In order to describe the foliar pathogens affecting this legume, we harnessed next-generation sequencing (NGS) to carry out a comprehensive survey across Manitoba in 2016. Fields were sampled during the V2/3 (33 fields) and R6 (70 fields) growth stages, with at least three symptomatic leaves per field collected and subjected to RNA sequencing. We successfully detected several bacteria, fungi, and viruses known to infect soybean, including Pseudomonas savastanoi pv. glycinea, Septoria glycines, and Peronospora manshurica, as well as pathogens not previously identified in the province (e.g., Pseudomonas syringae pv. tabaci, Cercospora sojina, and Bean yellow mosaic virus). For some microorganisms, we were able to disentangle the different pathovars present and/or assemble their genome sequence. Since NGS generates data on the entire flora and fauna occupying a leaf sample, we also identified residual pathogens (i.e., pathogens of crops other than soybean) and multiple species of arthropod pests. Finally, the sequence information produced by NGS allowed for the development of polymerase chain reaction-based diagnostics for some of the most widespread and important pathogens. Although there are many benefits of using NGS for large-scale plant pathogen diagnoses, we also discuss some of the limitations of this technology.

Evaporation process in histological tissue sections for neutron autoradiography.

The analysis of the distribution and density of nuclear tracks forming an autoradiography in a nuclear track detector (NTD) allows the determination of 10B atoms concentration and location in tissue samples from Boron Neutron Capture Therapy (BNCT) protocols. This knowledge is of great importance for BNCT dosimetry and treatment planning. Tissue sections studied with this technique are obtained by cryosectioning frozen tissue specimens. After the slicing procedure, the tissue section is put on the NTD and the sample starts drying. The thickness varies from its original value allowing more particles to reach the detector and, as the mass of the sample decreases, the boron concentration in the sample increases. So in order to determine the concentration present in the hydrated tissue, the application of corrective coefficients is required. Evaporation mechanisms as well as various factors that could affect the process of mass variation are outlined in this work. Mass evolution for tissue samples coming from BDIX rats was registered with a semimicro analytical scale and measurements were analyzed with software developed to that end. Ambient conditions were simultaneously recorded, obtaining reproducible evaporation curves. Mathematical models found in the literature were applied for the first time to this type of samples and the best fit of the experimental data was determined. The correlation coefficients and the variability of the parameters were evaluated, pointing to Page's model as the one that best represented the evaporation curves. These studies will contribute to a more precise assessment of boron concentration in tissue samples by the Neutron Autoradiography technique.

First Complete Genome Sequence of Tobacco necrosis virus D Isolated from Soybean and from North America.

We present the first complete genome sequence of the tombusvirus Tobacco necrosis virus D (TNV-D) from North America, obtained from an infected soybean plant. Compared with the three other TNV-D genomes isolated from different geographic regions and host plants, its nucleotide identities were between 83% and 93%.

Abscopal effect of boron neutron capture therapy (BNCT): proof of principle in an experimental model of colon cancer.

The aim of the present study was to evaluate, for the first time, the abscopal effect of boron neutron capture therapy (BNCT). Twenty-six BDIX rats were inoculated subcutaneously with 1 × 106 DHD/K12/TRb syngeneic colon cancer cells in the right hind flank. Three weeks post-inoculation, the right leg of 12 rats bearing the tumor nodule was treated with BPA-BNCT (BPA-Boronophenylalanine) at the RA-3 nuclear reactor located in Buenos Aires, Argentina, at an absorbed dose of 7.5 Gy to skin as the dose-limiting tissue. The remaining group of 14 tumor-bearing rats were left untreated and used as control. Two weeks post-BNCT, 1 × 106 DHD/K12/TRb cells were injected subcutaneously in the contralateral left hind flank of each of the 26 BDIX rats. Tumor volume in both legs was measured weekly for 7 weeks to determine response to BNCT in the right leg and to assess a potential influence of BNCT in the right leg on tumor development in the left leg. Within the BNCT group, a statistically significant reduction was observed in contralateral left tumor volume in animals whose right leg tumor responded to BNCT (post-treatment/pre-treatment tumor volume <1) versus animals who failed to respond (post/pre ≥1), i.e., 13 ± 15 vs 271 ± 128 mm3. In addition, a statistically significant reduction in contralateral left leg tumor volume was observed in BNCT-responsive animals (post/pre <1) vs untreated animals, i.e., 13 ± 15 vs 254 ± 251 mm3. The present study performed in a simple animal model provides proof of principle that the positive response of a tumor to BNCT is capable of inducing an abscopal effect.

Microdosimetry: Principles and applications.

AIM: to present the most important aspects of Microdosimetry, a research field in radiation biophysics. BACKGROUND: microdosimetry is the branch of radiation biophysics that systematically studies the spatial, temporal and spectral aspects of the stochastic nature of the energy deposition processes in microscopic structures. MATERIALS AND METHODS: we briefly review its history, the people, the formalism and the theories and devices that allowed researchers to begin to understand the true nature of radiation action on living matter. RESULTS AND CONCLUSIONS: we outline some of its applications, especially to Boron Neutron Capture Therapy, attempting to explain the biological effectiveness of the boron thermal neutron capture reaction.

Experimental Studies of Boronophenylalanine ((10)BPA) Biodistribution for the Individual Application of Boron Neutron Capture Therapy (BNCT) for Malignant Melanoma Treatment.

PURPOSE: Patients with the same histopathologic diagnosis of cutaneous melanoma treated with identical protocols of boron neutron capture therapy (BNCT) have shown different clinical outcomes. The objective of the present studies was to evaluate the biodistribution of boronophenilalanina ((10)BPA) for the potential application of BNCT for the treatment of melanoma on an individual basis. METHODS AND MATERIALS: The boronophenilalanine (BPA) uptake was evaluated in 3 human melanoma cell lines: MEL-J, A375, and M8. NIH nude mice were implanted with 4 10(6) MEL-J cells, and biodistribution studies of BPA (350 mg/kg intraperitoneally) were performed. Static infrared imaging using a specially modified infrared camera adapted to measure the body infrared radiance of small animals was used. Proliferation marker, Ki-67, and endothelial marker, CD31, were analyzed in tumor samples. RESULTS: The in vitro studies demonstrated different patterns of BPA uptake for each analyzed cell line (P<.001 for MEL-J and A375 vs M8 cells). The in vivo studies showed a maximum average boron concentration of 25.9 ± 2.6 µg/g in tumor, with individual values ranging between 11.7 and 52.0 µg/g of (10)B 2 hours after the injection of BPA. Tumor temperature always decreased as the tumors increased in size, with values ranging between 37 °C and 23 °C. A significant correlation between tumor temperature and tumor-to-blood boron concentration ratio was found (R(2) = 0.7, rational function fit). The immunohistochemical studies revealed, in tumors with extensive areas of viability, a high number of positive cells for Ki-67, blood vessels of large diameter evidenced by the marker CD31, and a direct logistic correlation between proliferative status and boron concentration difference between tumor and blood (R(2) = 0.81, logistic function fit). CONCLUSION: We propose that these methods could be suitable for designing new screening protocols applied before melanoma BNCT treatment for each individual patient and lesion.

A prospective study to assess the performance of the improved Boron Neutron Capture Therapy Facility in Argentina.

From 2008 to 2011, several planned modifications were implemented at the RA-6 reactor in Argentina, leading to significant benefits for future BNCT treatments. New capabilities have been implemented in NCTPlan treatment planning system. To assess the performance of the new BNCT facility, a dosimetric reevaluation of previous clinical cases was performed, taking into account the modifications carried out in the new facility and compared the results of the original treatment plans with optimized plans that are considered as feasible patient setups.

The photon-isoeffective dose in boron neutron capture therapy.

With the aim to relate the effects observed in a clinical boron neutron capture therapy protocol to the corresponding outcomes in a standard photon radiation therapy, "RBE-weighted" doses are customarily calculated by adding the contributions of the different radiations, each one weighted by a fixed (dose and dose rate independent) relative biological effectiveness factor. In this study, the use of fixed factors is shown to have a formal inconsistency, which in practice leads to unrealistically high tumor doses. We then introduce a more realistic approach that essentially exploits all the experimental information available from survival experiments. The proposed formalism also includes first-order repair of sublethal lesions by means of the generalized Lea-Catcheside factor in the modified linear-quadratic model, and considers synergistic interactions between different radiations. This formalism is of sufficient simplicity therefore to be directly included in all BNCT treatment planning systems. In light of this formalism, the photon-isoeffective doses for two BNCT clinical targets were computed and compared with the standard dose calculation procedure. For the case of brain tumors and clinically relevant absorbed doses, the proposed approach derives isoeffective doses that are much lower than the fixed RBE method, regardless of considering synergism. Thus, for a tumor that receives a mean total absorbed dose of 15 Gy (value achievable with 50 ppm of boron concentration and typical beams used in the clinic), the photon-isoeffective doses are 28 Gy (IsoE) and 30 Gy (IsoE) (without and with synergism, respectively), in contrast to 51 Gy (RBE) for the fixed RBE method. When the clinical outcome of the Argentine cutaneous melanoma treatments is assessed with regard to the doses derived from the standard procedure, it follows that the fixed RBE approach is not suitable to understand the observed clinical results in terms of the photon radiotherapy data. Moreover, even though the assumed (10)B concentration in tumors is lowered to reduce the obtained doses with the standard procedure, the fixed RBE approach is still unsuitable to explain the observed outcomes (the model is always rejected with P values of virtually zero). Additionally, the numbers of controlled tumors predicted by the proposed approach are statistically consistent with observed outcomes. As a by-product of this work, a dose-response clinical reference for single-fraction melanoma treatments is developed.

"Sequential" boron neutron capture therapy (BNCT): a novel approach to BNCT for the treatment of oral cancer in the hamster cheek pouch model.

In the present study the therapeutic effect and potential toxicity of the novel "Sequential" boron neutron capture therapy (Seq-BNCT) for the treatment of oral cancer was evaluated in the hamster cheek pouch model at the RA-3 Nuclear Reactor. Two groups of animals were treated with "Sequential" BNCT, i.e., BNCT mediated by boronophenylalanine (BPA) followed by BNCT mediated by sodium decahydrodecaborate (GB-10) either 24 h (Seq-24h-BNCT) or 48 h (Seq-48h-BNCT) later. In an additional group of animals, BPA and GB-10 were administered concomitantly [(BPA + GB-10)-BNCT]. The single-application BNCT was to the same total physical tumor dose as the "Sequential" BNCT treatments. At 28 days post-treatment, Seq-24h-BNCT and Seq-48h-BNCT induced, respectively, overall tumor responses of 95 ± 2% and 91 ± 3%, with no statistically significant differences between protocols. Overall response for the single treatment with (BPA + GB-10)-BNCT was 75 ± 5%, significantly lower than for Seq-BNCT. Both Seq-BNCT protocols and (BPA + GB-10)-BNCT induced reversible mucositis in the dose-limiting precancerous tissue around treated tumors, reaching Grade 3/4 mucositis in 47 ± 12% and 60 ± 22% of the animals, respectively. No normal tissue toxicity was associated with tumor response for any of the protocols. "Sequential" BNCT enhanced tumor response without an increase in mucositis in dose-limiting precancerous tissue.

Voxel model in BNCT treatment planning: performance analysis and improvements.

In recent years, many efforts have been made to study the performance of treatment planning systems in deriving an accurate dosimetry of the complex radiation fields involved in boron neutron capture therapy (BNCT). The computational model of the patient's anatomy is one of the main factors involved in this subject. This work presents a detailed analysis of the performance of the 1 cm based voxel reconstruction approach. First, a new and improved material assignment algorithm implemented in NCTPlan treatment planning system for BNCT is described. Based on previous works, the performances of the 1 cm based voxel methods used in the MacNCTPlan and NCTPlan treatment planning systems are compared by standard simulation tests. In addition, the NCTPlan voxel model is benchmarked against in-phantom physical dosimetry of the RA-6 reactor of Argentina. This investigation shows the 1 cm resolution to be accurate enough for all reported tests, even in the extreme cases such as a parallelepiped phantom irradiated through one of its sharp edges. This accuracy can be degraded at very shallow depths in which, to improve the estimates, the anatomy images need to be positioned in a suitable way. Rules for this positioning are presented. The skin is considered one of the organs at risk in all BNCT treatments and, in the particular case of cutaneous melanoma of extremities, limits the delivered dose to the patient. Therefore, the performance of the voxel technique is deeply analysed in these shallow regions. A theoretical analysis is carried out to assess the distortion caused by homogenization and material percentage rounding processes. Then, a new strategy for the treatment of surface voxels is proposed and tested using two different irradiation problems. For a parallelepiped phantom perpendicularly irradiated with a 5 keV neutron source, the large thermal neutron fluence deviation present at shallow depths (from 54% at 0 mm depth to 5% at 4 mm depth) is reduced to 2% on average. Reassigning fluence values in the case of this phantom in angular position produced the maximum deviation in the thermal fluence to decrease from 140% to 23% at the surface of the phantom. Thus, even for the largest deviations, obtained by intentionally placing the phantom in the most disadvantageous position with respect to the voxel grid, the reassignment shows very good performance. Since these results substantially improve the performance of the 1 cm based voxel model in surface boundary regions, the proposed strategy will be implemented in future versions of the NCTPlan code.

The microdosimetry of the (10)B reaction in boron neutron capture therapy: a new generalized theory.

The microdosimetry of (10)B thermal neutron capture reactions should be considered as an essential step to be followed before studying the radiobiological aspects of boron neutron capture therapy. The boron dose itself is insufficient as the only quantity used to describe the biological effectiveness of the (10)B reaction for two important reasons: the specific microdistribution that the (10)B carrier compound exhibits at the cellular level and the primarily stochastic nature of the energy deposition process, which influences the biological response to the particulate radiation. In this work, these two aspects are analyzed in detail and an innovative rigorous analytical framework is developed in the microdosimetry domain. This formalism provides the necessary microdosimetric tools for more precisely describing the (10)B dose distribution deposited in sensitive microscopic structures and offers improved approaches for analyzing the biological dose--effect relationship of (10)B reactions.