Enhancing microbial superoxide generation and conversion to hydroxyl radicals for enhanced bioremediation using iron-binding ligands.

Harnessing bacteria for superoxide production in bioremediation holds immense promise, yet its practical application is hindered by slow production rates and the relatively weak redox potential of superoxide. This study delves into a cost-effective approach to amplify superoxide production using an Arthrobacter strain, a prevalent soil bacterial genus. Our research reveals that introducing a carbon source along with specific iron-binding ligands, including deferoxamine (DFO), diethylenetriamine pentaacetate (DTPA), citrate, and oxalate, robustly augments microbial superoxide generation. Moreover, our findings suggest that these iron-binding ligands play a pivotal role in converting superoxide into hydroxyl radicals by modulating the electron transfer rate between Fe(III)/Fe(II) and superoxide. Remarkably, among the tested ligands, only DTPA emerges as a potent promoter of this conversion process when complexed with Fe(III). We identify an optimal Fe(III) to DTPA ratio of approximately 1:1 for enhancing hydroxyl radical production within the Arthrobacter culture. This research underscores the efficacy of simultaneously introducing carbon sources and DTPA in facilitating superoxide production and its subsequent conversion to hydroxyl radicals, significantly elevating bioremediation performance. Furthermore, our study reveals that DTPA augments superoxide production in cultures of diverse soils, with various soil microorganisms beyond Arthrobacter identified as contributors to superoxide generation. This emphasizes the universal applicability of DTPA across multiple bacterial genera. In conclusion, our study introduces a promising methodology for enhancing microbial superoxide production and its conversion into hydroxyl radicals. These findings hold substantial implications for the deployment of microbial reactive oxygen species in bioremediation, offering innovative solutions for addressing environmental contamination challenges.

[Effect of sodium alginate-g-deferoxamine/chitosan microspheres on osteogenic differentiation of rat bone mesenchymal stem cells].

PURPOSE: To explore the effect of sodium alginate-g-deferoxamine/chitosan (SA-g-DFO/CS) microspheres on proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs). METHODS: A kind of SA-g-DFO/CS microsphere was developed through electrostatic interaction between porous chitosan microspheres and sodium alginate chemically grafted on the surface of DFO. Its morphology, porosity rate, pore size and sustained release of DFO in vitro were examined. Rat BMSCs were isolated and co-cultured with microspheres in osteogenic differentiation medium. MTT assay was used to study the influence of cell proliferation, and Calcein-AM/PI staining was used to observe the cell viability. Alkaline phosphatase (ALP) activity assay was conducted. PCR was used to detect the expression of genes related to angiogenesis and osteogenesis. Statistical analysis was performed using SPSS 22.0 software package. RESULTS: The SA-g-DFO/CS porous microspheres were successfully prepared with a sustained re6lease of DFO. Compared with SA/CS microspheres, the SA-g-DFO/CS microspheres were conducive to cell proliferation and differentiation, with the increases in expression level of ALP, related angiogenesis genes HIF-1α, VEGF and osteogenesis genes COLI, OCN. CONCLUSIONS: The SA-g-DFO/CS porous microspheres can provide a new choice for the development of alveolar bone regeneration.

Effects of iron concentration and DFB (Desferrioxamine-B) on transcriptional profiles of an ecologically relevant marine bacterium.

Research into marine iron cycles and biogeochemistry has commonly relied on the use of chelators (including siderophores) to manipulate iron bioavailability. To test whether a commonly used chelator, desferrioxamine B (DFB) caused effects beyond changing the iron-status of cells, cultures of the environmentally relevant marine heterotrophic bacterium, Ruegeria pomeroyii, were grown in media with different concentrations of iron and/or DFB, resulting in a gradient of iron availability. To determine how cells responded, transcriptomes were generated for cells from the different treatments and analyzed to determine how cells reacted to these to perturbations. Analyses were also performed to look for cellular responses specific to the presence of DFB in the culture medium. As expected, cells experiencing different levels of iron availability had different transcriptomic profiles. While many genes related to iron acquisition were differentially expressed between treatments, there were many other genes that were also differentially expressed between different sample types, including those related to the uptake and metabolism of other metals as well as genes related to metabolism of other types of molecules like amino acids and carbohydrates. We conclude that while DFB certainly altered iron availability to cells, it also appears to have had a general effect on the homeostasis of other metals as well as influenced metabolic processes outside of metal acquisition.

Ferroptosis inhibitor improves cardiac function more effectively than inhibitors of apoptosis and necroptosis through cardiac mitochondrial protection in rats with iron-overloaded cardiomyopathy.

Iron overload cardiomyopathy (IOC) is the leading cause of death in cases of iron overload in patients. Previous studies demonstrated that iron overload led to cardiomyocyte dysfunction and death through multiple pathways including apoptosis, necroptosis and ferroptosis. However, the dominant cell death pathway in the iron-overloaded heart needs clarification. We tested the hypothesis that ferroptosis, an iron-dependent cell death, plays a dominant role in IOC, and ferroptosis inhibitor exerts greater efficacy than inhibitors of apoptosis and necroptosis on improving cardiac function in iron-overloaded rats. Iron dextran was injected intraperitoneally into male Wistar rats for four weeks to induce iron overload. Then, the rats were divided into 5 groups: treated with vehicle, apoptosis inhibitor (z-VAD-FMK), necroptosis inhibitor (Necrostatin-1), ferroptosis inhibitor (Ferrostatin-1) or iron chelator (deferoxamine) for 2 weeks. Cardiac function, mitochondrial function, apoptosis, necroptosis and ferroptosis were determined. The increased expression of apoptosis-, necroptosis- and ferroptosis-related proteins, were associated with impaired cardiac and mitochondrial function in iron-overloaded rats. All cell death inhibitors attenuated cardiac apoptosis, necroptosis and ferroptosis in iron-overloaded rats. Ferrostatin-1 was more effective than the other drugs in diminishing mitochondrial dysfunction and Bax/Bcl-2 ratio. Moreover, both Ferrostatin-1 and deferoxamine reversed iron overload-induced cardiac dysfunction as indicated by restored left ventricular ejection fraction and E/A ratio, whereas z-VAD-FMK and Necrostatin-1 only partially improved this parameter. These results indicated that ferroptosis could be the predominant form of cardiomyocyte death in IOC, and that inhibiting ferroptosis might be a potential novel treatment for IOC.

Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells.

Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence.

Transcriptomic analysis of the effect of deferoxamine exposure on the growth, photosynthetic activity and iron transfer of Microcystis aeruginosa.

Deferoxamine (DFB) is a trihydroxamic acid siderophore that chelates with iron (Fe) to form iron-siderophore complexes. The existence of siderophores in nature changes the form of iron and affects the absorption and utilization of iron by organisms. However, the relationship between siderophores and the growth of Cyanobacteria is largely unknown. In this study, the cellular and transcriptomic responses to the addition of DFB were investigated. A high concentration of DFB (12 mg/L) significantly inhibited the growth of Cyanobacteria cells, reduced photosynthetic activity, and induced the production of peroxidase, with the highest inhibition rate of algal growth of 74.82%. These indexes were also affected for the low (3 mg/L) and medium concentration (6 mg/L) groups, but this difference is closely related to the growth stage of Cyanobacteria cells. This may be due to competition between the cell-associated iron-binding part/system and the extracellular Fe (Ⅲ)-DFB ligand. Transcriptome results showed that most of the genes involved in iron uptake and transport were down-regulated, and only the fur gene encoding the iron uptake regulator protein was significantly up-regulated. Most genes related to photosynthesis, glycolysis, and fatty acid metabolism were also down-regulated, while the obvious up-regulation of a few genes may be a complex regulation in response to the down-regulation of most genes. These findings will provide important insights into the effects of siderophores on iron bioavailability in algae.

Heat-Stable Enterotoxin Secretions Assessed via ICP-MS Reveal Iron-Mediated Regulation of Virulence in CFA/I- and CS6-Expressing ETEC Isolates.

Enterotoxigenic Escherichia coli (ETEC) are a significant cause of childhood diarrhea in low-resource settings. ETEC are defined by the production of heat-stable enterotoxin (ST) and/or heat-labile enterotoxin (LT), which alter intracellular cyclic nucleotide signaling and cause the secretion of water and electrolytes into the intestinal lumen. ETEC take cues from chemicals (e.g., glycans, bile salts, and solutes) that may be liberated following enterotoxin activity to recognize entrance into the host. ETEC then alter the expression of surface adhesins called colonization factors (CFs) to attach to the intestinal epithelium, proliferate, and cause disease. Here, we used an in vivo model of oral ST intoxication to determine its impact on luminal ion concentrations via ICP-MS. We also used functional assays, including Western blots, qPCR, and toxin activity assays, to assess the impact of luminal ion flux on CF and toxin expression. Finally, we assessed ETEC strains with CFs CFA/I or CS6 in a streptomycin mouse model of ETEC colonization. ST causes rapid and significant increases in luminal chloride but significant decreases in luminal magnesium and iron. We confirmed that increased sodium chloride suppresses CFA/I production in ETEC H10407 but does not affect CS6 production in ETEC 214-4. CFA/I production in ETEC H10407 is increased when magnesium becomes limiting, although it does not affect CS6 production in ETEC 214-4. Iron restriction via deferoxamine induces CFA/I expression in ETEC H10407 but not CS6 expression in ETEC 214-4. We demonstrate that ST production is suppressed via iron restriction in H10407, 214-4, and over 50 other ETEC clinical isolates. Lastly, we demonstrate that the iron restriction of mice using oral deferoxamine pre-treatment extends the duration of ETEC H10407 (CFA/I+) fecal shedding while accelerating ETEC 214-4 (CS6+) fecal shedding. Combined, these data suggest that enterotoxins modulate luminal ion flux to influence ETEC virulence including toxin and CF production.

Amelioration effects of the soybean lecithin-gallic acid complex on iron-overload-induced oxidative stress and liver damage in C57BL/6J mice.

CONTEXT: Gallic acid (GA) and lecithin showed important roles in antioxidant and drug delivery, respectively. A complex synthesized from GA and soybean lecithin (SL-GAC), significantly improved bioavailability of GA and pharmacological activities. However, the antioxidant activity of SL-GAC and its effect on iron-overload-induced liver injury remains unexplored. OBJECTIVE: This study investigates the antioxidant properties of SL-GAC in vitro and in mice, and its remediating effects against liver injury by iron-overloaded. MATERIALS AND METHODS: In vitro, free radical scavenging activity, lipid peroxidation inhibition, and ferric reducing power of SL-GAC were measured by absorbance photometry. In vivo, C57BL/6J mice were randomized into 4 groups: control, iron-overloaded, iron-overloaded + deferoxamine, and iron-overloaded + SL-GAC. Treatments with deferoxamine (150 mg/kg/intraperitioneally) and SL-GAC (200 mg/kg/orally) were given to the desired groups for 12 weeks, daily. Iron levels, oxidative stress, and biochemical parameters were determined by histopathological examination and molecular biological techniques. RESULTS: In vitro, SL-GAC showed DPPH and ABTS free radicals scavenging activity with IC50 values equal to 24.92 and 128.36 µg/mL, respectively. In C57BL/6J mice, SL-GAC significantly reduced the levels of serum iron (22.82%), liver iron (50.29%), aspartate transaminase (25.97%), alanine transaminase (38.07%), gamma glutamyl transferase (42.11%), malondialdehyde (19.82%), total cholesterol (45.96%), triglyceride (34.90%), ferritin light chain (18.51%) and transferrin receptor (27.39%), while up-regulated the levels of superoxide dismutase (24.69%), and glutathione (11.91%). CONCLUSIONS: These findings encourage the use of SL-GAC to treat liver injury induced by iron-overloaded. Further in vivo and in vitro studies are needed to validate its potential in clinical medicine.

High Glucose and Carbonyl Stress Impair HIF-1-Regulated Responses and the Control of Mycobacterium tuberculosis in Macrophages.

Diabetes mellitus (DM) increases the risk of developing tuberculosis (TB), but the mechanisms behind diabetes-TB comorbidity are still undefined. Here, we studied the role of hypoxia-inducible factor-1 (HIF-1), a main regulator of metabolic and inflammatory responses, in the outcome of Mycobacterium tuberculosis infection of bone marrow-derived macrophages (BMM). We observed that M. tuberculosis infection of BMM increased the expression of HIF-1α and HIF-1-regulated genes. Treatment with the hypoxia mimetic deferoxamine (DFO) further increased levels of HIF-1-regulated immune and metabolic molecules and diminished the intracellular bacterial load in BMM and in the lungs of infected mice. The expression of HIF-1-regulated immunometabolic genes was reduced, and the intracellular M. tuberculosis levels were increased in BMM incubated with high-glucose levels or with methylglyoxal (MGO), a reactive carbonyl compound elevated in DM. In line with the in vitro findings, high M. tuberculosis levels and low HIF-1-regulated transcript levels were found in the lungs from hyperglycemic Leprdb/db compared with wild-type mice. The increased intracellular M. tuberculosis growth and the reduced expression of HIF-1-regulated metabolic and inflammatory genes in BMM incubated with MGO or high glucose were reverted by additional treatment with DFO. Hif1a-deficient BMM showed ablated responses of immunometabolic transcripts after mycobacterial infection at normal or high-glucose levels. We propose that HIF-1 may be targeted for the control of M. tuberculosis during DM. IMPORTANCE People living with diabetes who are also infected with M. tuberculosis are more likely to develop tuberculosis disease (TB). Why diabetic patients have an increased risk for developing TB is not well understood. Macrophages, the cell niche for M. tuberculosis, can express microbicidal mechanisms or be permissive to mycobacterial persistence and growth. Here, we showed that high glucose and carbonyl stress, which mediate diabetes pathogenesis, impair the control of intracellular M. tuberculosis in macrophages. Infection with M. tuberculosis stimulated the expression of genes regulated by the transcription factor HIF-1, a major controller of the responses to hypoxia, resulting in macrophage activation. High glucose and carbonyl compounds inhibited HIF-1 responses by macrophages. Mycobacterial control in the presence of glucose or carbonyl stress was restored by DFO, a compound that stabilizes HIF-1. We propose that HIF-1 can be targeted to reduce the risk of developing TB in people with diabetes.

Norepinephrine induces growth of Desulfovibrio vulgaris in an iron dependent manner.

Desulfovibrio spp. is a commensal sulfate reducing bacterium that is present in small numbers in the gastrointestinal tract. Increased concentrations of Desulfovibrio spp. (blooms) have been reported in patients with inflammatory bowel disease and irritable bowel syndrome. Since stress has been reported to exacerbate symptoms of these chronic diseases, this study examined whether the stress catecholamine norepinephrine (NE) promotes Desulfovibrio growth. Norepinephrine-stimulated growth has been reported in other bacterial taxa, and this effect may depend on the availability of the micronutrient iron. OBJECTIVES: This study tested whether norepinephrine exposure affects the in vitro growth of Desulfovibrio vulgaris in an iron dependent manner. METHODS: DSV was incubated in a growth medium with and without 1 µm of norepinephrine. An additional growth assay added the iron chelator deferoxamine in NE exposed DSV. Iron regulatory genes were assessed with and without the treatment of NE and Deferoxamine. RESULTS: We found that norepinephrine significantly increased growth of D. vulgaris. Norepinephrine also increased bacterial production of hydrogen sulfide. Additionally, norepinephrine significantly increased bacterial expression in three of the four tested iron regulatory genes. The iron chelator deferoxamine inhibited growth of D. vulgaris in a dose-dependent manner and reversed the effect of norepinephrine on proliferation of D. vulgaris and on bacterial expression of iron regulatory genes. CONCLUSION: The data presented in this work suggests that promotion of D. vulgaris growth by norepinephrine is iron dependent.

Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation, oxidative stress, angiogenesis, and collagen deposition in diabetic rats.

AIM OF THE STUDY: The study was performed to understand the detailed mechanism of diabetic wound healing by bilirubin-deferoxamine (DFO) combination on topical application. MATERIALS AND METHODS: There were two study groups, control, and treatment. The granulation tissues collected on different days (3, 7, 14, and 19) were studied in detail for inflammatory mediators, angiogenesis markers, epithelialization, and oxidative stress parameters. RESULTS: A significant increase in wound contraction percentage was observed from day 7 in the bilirubin-DFO treatment group. The combinatorial treatment significantly reduced tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), and enhanced IL-10 levels. Upregulated mRNAs of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 α) along with CD31 immunohistochemistry showed the pro-angiogenesis potential of the combination. Hematoxylin and Eosin (H and E) staining and Masson's trichrome staining showed reduced inflammatory cell infiltration, enhanced fibroblast proliferation, well-organized collagen fibers, and the development of new blood vessels. Collagen deposition is further supported by immunohistochemistry studies and Masson's trichrome staining. Bilirubin-DFO combination also reduced lipid peroxidation and elevated antioxidative enzymes. CONCLUSION: Topical application of bilirubin-DFO showed immense potential in augmenting skin wound regeneration in diabetes by upregulating the antioxidant status as well as increasing angiogenesis, collagen deposition, and modulating cytokines.

Iron overload-induced ferroptosis of osteoblasts inhibits osteogenesis and promotes osteoporosis: An in vitro and in vivo study.

Growing evidence indicates that iron overload is an independent risk factor for osteoporosis. However, the mechanisms are not fully understood. The purpose of our study was to determine whether iron overload could lead to ferroptosis in osteoblasts and to explore whether ferroptosis of osteoblasts is involved in iron overload-induced osteoporosis in vitro and in vivo. Ferric ammonium citrate was used to mimic iron overload conditions, while deferoxamine and ferrostatin-1 were used to inhibit ferroptosis of MC3T3-E1 cells in vitro. The ferroptosis, osteogenic differentiation and mineralization of MC3T3-E1 cells were assessed in vitro. A mouse iron overload model was established using iron dextran. Immunohistochemical analysis was performed to determine ferroptosis of osteoblasts in vivo. Enzyme-linked immunosorbent assays and calcein-alizarin red S labelling were used to assess new bone formation. Dual x-ray absorptiometry, micro-computed tomography and histopathological analysis were conducted to evaluate osteoporosis. The results showed that iron overload reduced cell viability, superoxide dismutase and glutathione levels, increased reactive oxygen species generation, lipid peroxidation, malondialdehyde levels and ferroptosis-related protein expression, and induced ultrastructural changes in mitochondria. Iron overload could also inhibit osteogenic differentiation and mineralization in vitro. Inhibiting ferroptosis reversed the changes described above. Iron overload inhibited osteogenesis, promoted the ferroptosis of osteoblasts and induced osteoporosis in vivo, which could also be improved by deferoxamine and ferrostatin-1. These results demonstrate that ferroptosis of osteoblasts plays a crucial role in iron overload-induced osteoporosis. Maintaining iron homeostasis and targeting ferroptosis of osteoblasts might be potential measures of treating or preventing iron overload-induced osteoporosis.

Deferoxamine ameliorated Al(mal)3-induced neuronal ferroptosis in adult rats by chelating brain iron to attenuate oxidative damage.

Aluminum (Al), a neurotoxic element, can induce Alzheimer's disease-like (AD-like) changes by triggering neuronal death. Iron homeostasis disturbance has also been implicated in Alzheimer's disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of neuronal death induced by aluminum maltolate (Al(mal)3) in the pathogenesis of AD remains elusive. In this study, the results of three different behavioral experiments suggested that the learning and memory ability deteriorated and autonomous activity declined of these rats that exposed Al(mal)3 were alleviated by deferoxamine (DFO). Transmission electron microscope observations showed that the membrane was ruptured, and the membrane density increased and ridge disappearance (the most prominent characteristic of ferroptosis) in the perinuclear and cytoplasmic compartments of the hippocampal neurons were perceived in the exposure group, while the DFO group and 18 µM/kg Al(mal)3+DFO group were alleviated compared with 18 µM/kg Al(mal)3. In addition, DFO prevented oxidative stress, such as increased glutathione (GSH) and decreased malondialdehyde (MDA) and reactive oxygen species (ROS), while the latter two indexes had the same changing tendency as the total iron of brain tissue. These data indicated that Al(mal)3 could cause ferroptosis in Sprague-Dawley (SD) rat neurons, which was inhibited by DFO via reducing the content of iron and increasing the ability of cells to resist oxidative damage.

Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerant Streptomyces sp. S29 desferrioxamine production.

Siderophores are iron-chelating compounds that aid iron uptake, one of the key strategies for microorganisms to carve out ecological niches in microbially diverse environments. Desferrioxamines are the principal siderophores produced by Streptomyces spp. Their biosynthesis has been well studied and as a consequence, the chemical potential of the pathway continues to expand. With all of this in mind, our study aimed to explore extremotolerant and lupine rhizosphere-derived Streptomyces sp. S29 for its potential antifungal capabilities. Cocultivation of isolate S29 was carried out with Aspergillus niger and Botrytis cinerea, both costly fungal phytopathogens in the wine industry, to simulate their interaction within the rhizosphere. The results indicate that not only is Streptomyces sp. S29 extraordinary at producing hydroxamate siderophores but uses siderophore production as a means to 'starve' the fungi of iron. High resolution LC-MS/MS followed by GNPS molecular networking was used to observe the datasets for desferrioxamines and guided structure elucidation of new desferrioxamine analogues. Comparing the new chemistry, using tools like molecular networking and MS2LDA, with the known biosynthesis, we show that the chemical potential of the desferrioxamine pathway has further room for exploration.

Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. 55Fe uptake assays using foxA mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in light of these results.

Detecting TRA-1-60 in Cancer via a Novel Zr-89 Labeled ImmunoPET Imaging Agent.

TRA-1-60 (TRA) is a cell-surface antigen implicated in drug resistance, relapse, and recurrence. Its expression has been reported in breast, prostate, pancreatic, ovarian tumors, and follicular lymphoma, which paved the development of the therapeutic antibody, Bstrongomab (Bsg), and its drug conjugates. Because patient selection is critical to achieve clinical benefit, a noninvasive imaging agent to select TRA+ lesions in patients is needed. Herein, we report the development of the immunopositron emission tomography (immunoPET) radiotracer 89Zr-radiolabeled Bsg and its potential to delineate TRA+ tumors. Bsg was conjugated to the bifunctional chelator desferrioxamine (DFO) and radiolabeled with [89Zr]Zr-oxalate. [89Zr]Zr-DFO-Bsg was characterized in vitro and evaluated in vivo for uptake and specificity in high and low TRA-expressing BxPC-3 pancreatic and PC-3 prostate cancer models, respectively. Uptake was compared against [89Zr]Zr-DFO-IgG, a nonspecific control radiotracer. Immunohistochemical (IHC) staining of patient cancer tissues using Bsg was performed to explore its clinical significance. A specific activity of 0.18 ± 0.01 GBq/mg (4.8 ± 0.3 mCi/mg) was obtained for [89Zr]Zr-DFO-Bsg. BxPC-3 xenografts exhibited three-fold higher radiotracer uptake compared to [89Zr]Zr-DFO-IgG. Competitive saturation studies using BxPC-3 xenografts further confirmed tracer specificity. The TRA-specific probe had lower accumulation in PC-3 xenografts. Ex vivo autoradiographs correlated with TRA expression from the histopathology of the resected tumor xenografts. Additionally, patient cancer tissues demonstrated positive staining with Bsg with metastatic lesions exhibiting the highest staining. This study demonstrates the potential of [89Zr]Zr-DFO-Bsg as an imaging agent for noninvasive detection of TRA+ tumors.

89Zr-DFO-Cetuximab as a Molecular Imaging Agent to Identify Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma.

Background: Despite the improvement in clinical outcomes for head and neck squamous cell carcinoma (HNSCC) as the result of cetuximab, patients may present with or develop resistance that increases tumor recurrence rates and limits clinical efficacy. Therefore, identifying those patients who are or become resistant is essential to tailor the best therapeutic approach. Materials and Methods: Cetuximab was conjugated to p-NCS-Bz-DFO and labeled with 89Zr. The resistance model was developed by treating FaDu cells with cetuximab. Western blotting (WB) and specific binding assays were performed to evaluate epidermal growth factor receptor (EGFR) expression and 89Zr-DFO-cetuximab uptake in FaDu cetuximab-resistant (FCR) and FaDu cetuximab-sensitive (FCS) cells. Positron emission tomography imaging and biodistribution were conducted in NU/NU nude mice implanted with FCR or FCS cells. Results: Cetuximab was successfully radiolabeled with 89Zr (≥95%). Binding assays performed in FCR and FCS cells showed significantly lower 89Zr-DFO-cetuximab uptake in FCR (p < 0.0001). WB suggests that the resistance mechanism is associated with EGFR downregulation (p = 0.038). This result is in agreement with the low uptake of 89Zr-DFO-cetuximab in FCR cells. Tumor uptake of 89Zr-DFO-cetuximab in FCR was significantly lower than FCS tumors (p = 0.0340). Conclusions: In this work, the authors showed that 89Zr-DFO-cetuximab is suitable for identification of EGFR downregulation in vitro and in vivo. This radiopharmaceutical may be useful for monitoring resistance in HNSCC patients during cetuximab therapy.

A genome-wide analysis of desferrioxamine mediated iron uptake in Erwinia spp. reveals genes exclusive of the Rosaceae infecting strains.

Erwinia amylovora is the etiological agent of fire blight, a devastating disease which is a global threat to commercial apple and pear production. The Erwinia genus includes a wide range of different species belonging to plant pathogens, epiphytes and even opportunistic human pathogens. The aim of the present study is to understand, within the Erwinia genus, the genetic differences between phytopathogenic strains and those strains not reported to be phytopathogenic. The genes related to the hydroxamate siderophores iron uptake have been considered due to their potential druggability. In E. amylovora siderophore-mediated iron acquisition plays a relevant role in the progression of Fire blight. Here we analyzed the taxonomic relations within Erwinia genus and the relevance of the genes related to the siderophore-mediated iron uptake pathway. The results of this study highlight the presence of a well-defined sub-group of Rosaceae infecting species taxonomically and genetically related with a high number of conserved core genes. The analysis of the complete ferrioxamine transport system has led to the identification of two genes exclusively present in the Rosaceae infecting strains.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Noninvasive Trafficking of Brentuximab Vedotin and PET Imaging of CD30 in Lung Cancer Murine Models.

CD30 has been considered a unique diagnostic and therapeutic target for CD30-positive lymphomas and some lung diseases. Additionally, CD30 has shown high expression in clinical lung cancer samples. In this study, 89Zr-radiolabeled brentuximab vedotin (BV) was developed for in vivo tracking of BV and imaging CD30 expression in lung cancer models via conjugation with desferrioxamine (Df). CD30 expression in three lung cancer cell lines (H460, H358, and A549) was quantified by Western blot. Flow cytometry and saturation binding assays were used to evaluate the binding capabilities of the tracer in vitro. After longitudinal positron emission tomography (PET) imaging and quantitative analysis were performed, ex vivo biodistribution and histological studies were used to verify PET results. Finally, dosimetric extrapolation of murine data to humans was performed. At the cellular level, CD30 was found to be expressed on H460 and A549 cells with the highest and lowest levels of expression, respectively. Both Df-BV and 89Zr-Df-BV displayed high binding affinity to H460 cells. PET images and their quantification verified that BV accumulated in H460 tumor models (9.93 ± 2.70% ID/g at 24 h after injection; n = 4) at the highest level, followed by H358 and A549 tumors (8.05 ± 2.43 and 5.00 ± 1.56% ID/g; n = 4). The nonspecific 89Zr-labeled IgG showed a low tumor uptake of 5.2 ± 1.0% ID/g for H460 models. Ex vivo biodistribution and fluorescence immunohistochemistry also corroborated these findings. Dosimetric results displayed safe dose estimations. Therefore, 89Zr-Df-BV provides a potential agent for evaluating CD30 expression noninvasively in lung cancer, and also for imaging of brentuximab vedotin for better understanding of its pharmacokinetics.