Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Risk assessment and bioburden evaluation of Agrobacterium tumefaciens-mediated transient protein expression in plants using the CaMV35S promoter.

Large-scale transient expression of recombinant proteins in plants is increasingly used and requires the multi-liter cultivation of Agrobacterium tumefaciens transformed with an expression vector, which is often cloned in Escherichia coli first. Depending on the promoter, unintentional activity can occur in both bacteria, which could pose a safety risk to the environment and operators if the protein is toxic. To assess the risk associated with transient expression, we first tested expression vectors containing the CaMV35S promoter known to be active in plants and bacteria, along with controls to measure the accumulation of the corresponding recombinant proteins. We found that, in both bacteria, even the stable model protein DsRed accumulated at levels near the detection limit of the sandwich ELISA (3.8 µg L-1). Higher levels were detected in short cultivations (< 12 h) but never exceeded 10 µg L-1. We determined the abundance of A. tumefaciens throughout the process, including infiltration. We detected few bacteria in the clarified extract and found none after blanching. Finally, we combined protein accumulation and bacterial abundance data with the known effects of toxic proteins to estimate critical exposures for operators. We found that unintended toxin production in bacteria is negligible. Furthermore, the intravenous uptake of multiple milliliters of fermentation broth or infiltration suspension would be required to reach acute toxicity even when handling the most toxic products (LD50 ~ 1 ng kg-1). The unintentional uptake of such quantities is unlikely and we therefore regard transient expression as safe in terms of the bacterial handling procedure.

Microorganisms for Bioremediation of Soils Contaminated with Heavy Metals.

Heavy-metal contaminants are one of the most relevant problems of contemporary agriculture. High toxicity and the ability to accumulate in soils and crops pose a serious threat to food security. To solve this problem, it is necessary to accelerate the pace of restoration of disturbed agricultural lands. Bioremediation is an effective treatment for agricultural soil pollution. It relies on the ability of microorganisms to remove pollutants. The purpose of this study is to create a consortium based on microorganisms isolated from technogenic sites for further development in the field of soil restoration in agriculture. In the study, promising strains that can remove heavy metals from experimental media were selected: Pantoea sp., Achromobacter denitrificans, Klebsiella oxytoca, Rhizobium radiobacter, and Pseudomonas fluorescens. On their basis, consortiums were compiled, which were investigated for the ability to remove heavy metals from nutrient media, as well as to produce phytohormones. The most effective was Consortium D, which included Achromobacter denitrificans, Klebsiella oxytoca, and Rhizobium radiobacter in a ratio of 1:1:2, respectively. The ability of this consortium to produce indole-3-acetic acid and indole-3-butyric acid was 18.03 µg/L and 2.02 µg/L, respectively; the absorption capacity for heavy metals from the experimental media was Cd (56.39 mg/L), Hg (58.03 mg/L), As (61.17 mg/L), Pb (91.13 mg/L), and Ni (98.22 mg/L). Consortium D has also been found to be effective in conditions of mixed heavy-metal contamination. Due to the fact that the further use of the consortium will be focused on the soil of agricultural land cleanup, its ability to intensify the process of phytoremediation has been studied. The combined use of Trifolium pratense L. and the developed consortium ensured the removal of about 32% Pb, 15% As, 13% Hg, 31% Ni, and 25% Cd from the soil. Further research will be aimed at developing a biological product to improve the efficiency of remediation of lands withdrawn from agricultural use.

New two-stage pH combined with dissolved oxygen control strategy for cyclic ß-1,2 glucans synthesis.

On the basis of a novel two-stage pH combined with dissolved oxygen (DO) control strategy in fed-batch fermentation, this research addresses the influence of pH on cyclic ß-1,2-glucans (CßGs) biosynthesis and melanin accumulation during the production of CßGs by Rhizobium radiobacter ATCC 13,333. Under these optimal fermentation conditions, the maximum cell concentration and CßGs concentration in a 7-L stirred-tank fermenter were 7.94 g L-1 and 3.12 g L-1, which were the maximum production reported for R. radiobacter. The melanin concentration of the fermentation broth was maintained at a low level, which was beneficial to the subsequent separation and purification of the CßGs. In addition, a neutral extracellular oligosaccharide (COGs-1) purified by the two-stage pH combined with DO control strategy fermentation medium was structurally characterized. Structural analyses indicated that COGs-1 was a family of unbranched cyclic oligosaccharides composed of only ß-1,2-linked D-glucopyranose residues with degree of polymerization between 17 and 23, namely CßGs. This research provides a reliable source of CßGs and structural basis for further studies of biological activity and function. KEY POINTS: • A two-stage pH combined with DO control strategy was proposed for CßGs production and melanin biosynthesis by Rhizobium radiobacter. • The final extracellular CßGs production reached 3.12 g L-1, which was the highest achieved by Rhizobium radiobacter. • The existence of CßGs could be detected by TLC quickly and accurately.

Central line-associated Rhizobium radiobacter bloodstream infection in two allogeneic hematopoietic cell transplant recipients.

INTRODUCTION: Rhizobium radiobacter is a gram-negative, opportunistic phytopathogen that rarely causes human infections. We report two cases of Rhizobium radiobacter central line-associated bloodstream infection (CLABSI) in allogeneic hematopoietic cell transplantation (alloHCT) recipients. We review previous reports and common microbiological characteristics associated with this organism. CASE REPORTS: Two adult males developed R. radiobacter CLABSIs at day +81 and day +77 post-alloHCT. Patient one was asymptomatic on presentation while patient two was febrile. One patient had a polymicrobial infection, which has not been previously described. The presence of high-level ceftazidime resistance in both patients suggests third-generation cephalosporin resistance may be more common than previously recognized. MANAGEMENT AND OUTCOME: For both patients, microbiologic clearance was achieved through peripherally inserted central catheter removal and initiation of intravenous cefepime. Antibiotic therapy was narrowed to oral levofloxacin for a total 14-day course from the time of first negative blood culture. There has been no subsequent recurrence of R. radiobacter infection at 12 and 5 months of follow-up for patients one and two, respectively. DISCUSSION: These two cases add to the scant literature characterizing R. radiobacter infection following alloHCT. Immunosuppressive agents for graft-versus-host disease prophylaxis may have predisposed these patients to R. radiobacter infection. Our reports, and previously reported cases, suggest R. radiobacter exhibits low virulence, mild symptom burden, and does not confer a high mortality risk. In the alloHCT setting, further accumulation of cases is needed to aid in understanding clinical features and characteristics of R. radiobacter infection.

A simple method for creating transgenic pea hairy roots using a Japanese pea cultivar and a Japanese Rhizobium radiobacter strain.

Pea (Pisum sativum) is an agriculturally important leguminous crop cultivated worldwide. It is also the plant from which phytoalexin was isolated for the first time. Several studies have investigated gene functions using pea hairy root culture systems. However, the procedures for producing hairy roots are relatively complicated and only a few pea cultivars and Rhizobium strains have been used. In this study, we established a simple method for generating transgenic hairy roots using a pea cultivar and a Rhizobium strain available in Japan. The transformation efficiency for the transgenic hairy roots (approximately 14%) was calculated on the basis of GFP fluorescence because the binary vector used in this study carried a GFP cassette as a marker. Furthermore, we confirmed that the production of the phytoalexin (+)-pisatin was induced by a copper dichloride treatment, indicating that this system can be used to characterize the biosynthesis of (+)-pisatin, which is a compound with a unique pterocarpan structure. Interestingly, some of the hairy roots turned into crown galls during the culture period. In summary, our simple method enables the production of transgenic pea hairy roots using biological materials accessible in Japan. The generated hairy roots can be used to elucidate the molecular mechanisms underlying (+)-pisatin biosynthesis as well as hairy root/crown gall formation.

Rhizobium radiobacter-Induced Peritonitis: A Case Report and Literature Analysis.

Rhizobium radiobacter (R. radiobacter) is a gram-negative bacterium, primarily a soil contaminant and rarely pathogenic to humans. Only a few cases of peritonitis secondary to R. radiobacter have been reported worldwide. A 66-year-old male with end-stage renal disease who was on peritoneal dialysis (PD) developed R. radiobacter-induced peritonitis. We have treated the infection successfully with intraperitoneal antibiotics and managed to keep his PD catheter intact without interruption in PD treatment. More prolonged antibiotic therapy and frequent clinical follow-up is required to treat this infection. Better clinician awareness is needed to prevent this rare infection.

A Rare Case of Endophthalmitis with Rhizobium radiobacter, Soon after a Resolved Keratitis: Case Report.

BACKGROUND: Rhizobium (Agrobacterium) species are plant aerobic bacteria, which in some cases can produce endophthalmitis in humans after corneal trauma. CASE PRESENTATION: A 42-year-old female patient presented in the Emergency Department of the Emergency County Hospital of Craiova, Romania, reporting pain, epiphora, and blurry vision in her right eye for about five days. This initial infectious keratitis episode was successfully resolved, but after 20 days she presented again after trauma with a leaf with corneal abscess. In the conjunctival secretion, R. radiobacter was identified. Despite antibiotherapy, the patient's state did not improve, and ultimately the eye was eviscerated. METHODS: A search was performed in the ProQuest, PubMed, and ScienceDirect databases for the terms Agrobacterium, Rhizobium, radiobacter, and eye. We eliminated non-human studies, editorials and commentaries, and non-relevant content, and excluded the duplicates. RESULTS: In total, 138 studies were initially obtained, and then we selected 26 studies for retrieval. After the selection process, we ended up including 17 studies in our analysis. Most studies reported R. radiobacter endophthalmitis after ocular surgical procedures or outdoor activities that involve exposure to soil. CONCLUSION: R. radiobacter is a rare cause of endophthalmitis after eye trauma that generally responds well to usual antibiotherapy, but occasionally can evolve to severe, leading to the loss of the eye.

Triple-valve replacement for Rhizobium radiobacter endocarditis with septic shock in an adult with ventricular septal defect. A case report.

INTRODUCTION AND IMPORTANCE: Triple-valve replacement in active infective endocarditis has rarely been reported. This paper is the first report of a triple-valve replacement performed in endocarditis with septic shock and the first presentation of multivalvular endocarditis due to Rhizobium radiobacter. CASE PRESENTATION: A 26-year-old patient with a neglected ventricular septal defect referred to us in septic shock, with multiple organ failure, severe biventricular dysfunction, and pulmonary hypertension, due to Rhizobium radiobacter infective endocarditis affecting the aortic, tricuspid and pulmonary valves. Initially, he was deemed unfit for surgery. However, after clinical stabilization, triple-valve replacement, aortic annular abscess repair, membranous septum aneurysm resection, and ventricular septal defect patch closure were performed. The postoperative evolution was good; both ventricles showed functional recovery after six months. CLINICAL DISCUSSION: Although surgery provides the best chances of survival in endocarditis with septic shock, reportedly, most cases are considered inoperable. Clinical stabilization under intensive care using specific therapies to manage septic shock, myocardial dysfunction, and pulmonary hypertension was crucial for surgery success. Custodiol® cardioplegia, and replacement of the right-sided valves using a beating-heart technique were used to reduce the myocardial ischemic time. CONCLUSION: Rhizobium radiobacter, an opportunistic gram-negative bacterium, potentially may cause multiple valve endocarditis. Patients with endocarditis and septic shock initially considered inoperable can still benefit from surgery after tenacious intensive care (cytokine hemoadsorption and levosimendan are helpful in this process). In complex multivalvular procedures, a beating heart technique to replace the right-sided valves should be considered to minimize the duration of myocardial ischemia.

Three enzymes of Rhizobium radiobacter involved in the novel metabolism of two naturally occurring bioactive oxidative derivatives of l-isoleucine.

Rhizobium radiobacter C58 was found to convert 4-hydroxyisoleucine (HIL) and 2-amino-3-methyl-4-ketopentanoate (AMKP), bioactive oxidative derivatives of l-isoleucine, in both cases producing 2-aminobutyrate. Three native enzymes involved in these metabolisms were purified by column chromatography and successfully identified. In this strain, HIL was converted to acetaldehyde and 2-aminobutyrate by coupling action of the transaminase rrIlvE and the aldolase HkpA. AMKP was also converted to acetate and 2-aminobutyrate by coupling action of rrIlvE and a hydrolase DkhA. In the multi-enzymatic reactions, HkpA catalyzes the retro-aldol reaction of 4-hydroxy-3-methyl-2-ketopentanoate into acetaldehyde and 2-ketobutyrate, and DkhA catalyzes hydrolytic cleavage of the carbon-carbon bond of 2,4-diketo-3-methylpentanoate into acetate and 2-ketobutyrate. rrIlvE catalyzes reversible transamination between HIL and 4-hydroxy-3-methyl-2-ketopentanoate, AMKP and 2,4-diketo-3-methylpentanoate, and 2-ketobutyrate and 2-aminobutyrate. The results suggested that the conversion activity of Rhizobium bacteria plays an important role in the complex biological metabolic networks associated with HIL and AMKP.

Enhanced solubility of curcumin by complexation with fermented cyclic ß-1,2-glucans.

Curcumin (CUR) is a low-solubility polyphenolic compound with many physiological functions. Cyclic ß-1,2-glucans (cyclosophoraoses [Cys]), which contain rings of different sizes with degrees of polymerization ranging from 17 to 23, were obtained from Rhizobium radiobacter ATCC 1333, a soil microorganism. The complexation ability and solubility enhancement of cyclic ß-1,2-glucans with insoluble curcumin were investigated. Phase-solubility analysis revealed that the stoichiometric ratio of the inclusion complexes was 1:1. The stability constant of Cys was 930 M-1, which was 7.68 times that of α-cyclodextrin (α-CD) and 2.09 times that of ß-cyclodextrin (ß-CD). The characteristics of the curcumin/Cys inclusion complexes were successfully determined by using Fourier transform infrared (FTIR) spectrometry, differential scanning calorimetry (DSC), nuclear magnetic resonance (1H NMR) spectroscopy, and scanning electron microscopy (SEM). Moreover, a 1:1 molecular model of the curcumin/Cys inclusion complexes was established through molecular docking analysis. These findings indicated that cyclic ß-1,2-glucans successfully formed complexes with curcumin, which suggested that they could be used as solubility-increasing agents. To the best of our knowledge, this is the first report in which curcumin has been embedded into cyclic ß-1,2-glucans resulting in an increase in its aqueous solubility.

A rare pathogen causing pulmonary infection and liver dysfunction in a 46-day-old infant: Rhizobium radiobacter.

Rhizobium radiobacter is an aerobic, motile, oxidase-positive, non-spore-forming Gram-negative tumorigenic plant pathogen which rarely infects humans. A 46-day-old girl was admitted to hospital with a 10-day history of fever and cough. She had pneumonia and liver dysfunction owing to infection by R. radiobacter. After 3 days of treatment with ceftriaxone, (compound glycyrrhizin and ambroxol), her body temperature returned to normal and the pneumonia improved, but liver enzyme levels continued to rise. After treatment with meropenem (glycyrrhizin and reduced glutathione), her condition stabilised and she recovered with no liver damage, and was discharged after 15 days. R. radiobacter has low virulence and antibiotics are highly sensitive, but, rarely, severe organ dysfunction can occur and result in multi-system damage in vulnerable children.

Genetic Transformation of Trichoderma spp.

The production of biofuels from plant biomass is dependent on the availability of enzymes that can hydrolyze the plant cell wall polysaccharides to their monosaccharides. These enzyme mixtures are formed by microorganisms but their native compositions and properties are often not ideal for application. Genetic engineering of these microorganisms is therefore necessary, in which introduction of DNA is an essential precondition. The filamentous fungus Trichoderma reesei-the main producer of plant-cell-wall-degrading enzymes for biofuels and other industries-has been subjected to intensive genetic engineering toward this goal and has become one of the iconic examples of the successful genetic improvement of fungi. However, the genetic manipulation of other enzyme-producing Trichoderma species is frequently less efficient and, therefore, rarely managed. In this chapter, we therefore describe the two potent methods of Trichoderma transformation mediated by either (a) polyethylene glycol (PEG) or (b) Agrobacterium. The methods are optimized for T. reesei but can also be applied for such transformation-resilient species as T. harzianum and T. guizhouense, which are putative upcoming alternatives for T. reesei in this field. The protocols are simple, do not require extensive training or special equipment, and can be further adjusted for T. reesei mutants with particular properties.

Characterization and rheological properties analysis of the succinoglycan produced by a high-yield mutant of Rhizobium radiobacter ATCC 19358.

Succinoglycan is an industrially important exopolysaccharide biosynthesized by bacteria. In this study, mutant strain 18052 N-11 was obtained from the wild type strain Rhizobium radiobacter ATCC 19358 by NTG mutagenesis. It has a high yield succinoglycan of 32.5 g/L cultured in a 15 L-fementer for 72 h. Succinoglycan SG-A from the wild type strain has two components, and the molecular weights were 1.55 × 107 Da and 1.26 × 106 Da, respectively. While, succinoglycan SG-N from the mutant strain was a homogeneous polysaccharide, and the molecular weight was 1.01 × 107 Da. The molecular weight of both succinoglycan was higher than those reported in literatures. DSC thermogram of SG-A showed a higher endothermic peak than that of SG-N due to the higher crystallinity of SG-A. The dynamic frequency sweep test of SG-A and SG-N showed that the elastic modulus G' and viscosity modulus G" curves intersected at 65 °C, indicating the thermally induced order-disorder conformation. The results of effect of concentrations (2.5-15%) and temperatures (25-75 °C) on apparent viscosity of SG-A and SG-N showed that the succinoglycan solutions exhibited non-Newtonian, shear-thinning behavior. Both SG-A and SG-N showed an excellent emulsification activity. The characterizations and rheological properties make SG-A and SG-N prominent candidates in food, cosmetics, pharmaceutical and petroleum industries.

Gene expression abundance dictated exopolysaccharide modification in Rhizobium radiobacter SZ4S7S14 as the cell's response to salt stress.

A molecular and metabolic behaviour of EPS-producing and salt-tolerant bacterium Rhizobium radiobacter SZ4S7S14 along with its practical application in salt-stress was investigated. The research target was identification and expression profiles of a large EPS biosynthesis gene cluster, possible structural modification of EPS under salt-stress effect and analysis of the gene(s) relative expression and structural modification correlation. As expected, transposons insertions were identified within or near the coding regions of exoK and exoM, previously known large gene cluster that is required for EPS I synthesis. Different expression levels of exoK and exoM in different salt-stress models resulted in structural modification of EPS, which was seen basically in monomers molar ratio. As a result of downregulation of the genes the strain produced EPS samples with monomers ratio: (1) Glu:Man:Gal:Xyl:Ara:Rha:Rib = 31.21:3.02:2.77:1:0.91:0.64:0.41 (in 0.25% NaCl); (2) Glu:Man:Gal:Xyl:Ara:Rha:Rib = 7.65:1:0.69:0.22:0.2:0.16:0.1 (in 0.5% NaCl); (3) Glu:Man:Gal:Ara:Xyl:Rha:Rib = 9.39:1.89:1:0.58:0.52:0.46:0.26 (in 1% NaCl); and (4) Glu:Man:Ara:Xyl:Rib:Gal = 7.9:2:2:1.58:1.1:1 (in 2.0% NaCl), whereas in control (without NaCl): Glc:Man:Gal:Xyl:Ara:Rha:Rib = 11.66:1:0.90:0.37:0.37:0.15:0.14. It was found that, salt-stress not only leads to downregulation of a large EPS biosynthesis gene cluster, including exoK and exoM genes, but also impacting on their relative expression degree, re-groups of the monomers within the EPS matrix and dictates molar ratio of the monosaccharides in the final metabolite.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as "A. viscosum" (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Case of isolated Rhizobium radiobacter contact lens-related infectious keratitis: A plant microbe now emerging as a human pathogen.

PURPOSE: To report a case of contact lens-related infectious keratitis caused by the Gram-negative plant pathogen Rhizobium radiobacter. OBSERVATIONS: A 26-year old lady with history of contact lens use presented with three weeks history of right eye redness and pain, with the left eye also being involved in the past week. Slit lamp examination of the right eye demonstrated multiple faint subepithelial and stromal infiltrates with no overlying epithelial defect, and no anterior chamber activity. Anterior segment optical coherence tomography demonstrated multiple hyper-reflective foci scattered at various depths of the corneal stroma. Corneal scrapings grew Rhizobium radiobacter, and culture-directed antibiotic therapy with topical gentamicin and levofloxacin resulted in slow clinical improvement of the R. radiobacter keratitis without visual sequelae. CONCLUSIONS AND IMPORTANCE: We have described the clinical features, microbial susceptibilities, and response to treatment in a case of R. radiobacter infectious keratitis.R. radiobacter has recently emerged as a source for several ocular and systemic infections and was identified in a series of polymicrobial keratitis cases. Our case report of monomicrobial R. radiobacter keratitis adds to the sparse literature on this uncommon but potentially sight-threatening infection.

Physical and functional characterization of succinoglycan exopolysaccharide produced by Rhizobium radiobacter CAS from curd sample.

Identification and rheological behaviour of succinoglycan exopolysaccharide (EPS) from Rhizobium radiobacter CAS isolated from curd was published in earlier reports. In current investigation physicochemical and functional properties of CAS EPS has been studied. SEC-MALLS revealed molecular weight (Mw), number molecular weight (Mn) and polydispersity index (PDI) of CAS EPS as 4.05×106g·mol-1, 3.82×106g·mol-1 and, 1.06 respectively. Thermogravimetric analysis showed the high thermal stability of CAS polymer where approximately 77% of CAS hydrocolloid maintain physical integrity and stability with temperature degradation (Td) at 290°C. Scanning electron microscopy and particle size analysis confirmed the porous and hygroscopic nature and 2.049µm size of CAS EPS respectively. Equally important functional properties such as water activity (0.55), water solubility (95%), water contact angle (54.83°), water binding capacity (101g/g), water holding capacity (68.19g/g), oil binding ability (soybean and groundnut oils were found 4.35g/g and 3.68g/g) and swelling index (17.5mL/g) were examined for CAS EPS. Prevention of syneresis has been studied at 1% CAS EPS concentration which significantly prohibited the degree of syneresis of starch. These physico-functional properties make CAS EPS a prominent candidate for food processing and product development sector.

Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation.

Agrobacterium tumefaciens infection of wounded plant tissues causes the formation of crown gall tumors. Upon infection, genes encoded on the A. tumefaciens tumor inducing plasmid are integrated in the plant genome to induce the biosynthesis of auxin and cytokinin, leading to uncontrolled cell division. Additional sequences present on the bacterial T-DNA encode for opine biosynthesis genes, which induce the production of opines that act as a unique carbon and nitrogen source for Agrobacterium. Crown galls therefore become a very strong sink for photosynthate. Here we found that the increased metabolic demand in crown galls causes an increase in oxygen consumption rate, which leads to a steep drop in the internal oxygen concentration. Consistent with this, plant hypoxia-responsive genes were found to be significantly upregulated in crown galls compared to uninfected stem tissue. Following this observation, we aimed at understanding whether the low-oxygen response pathway, mediated by group VII ethylene response factor (ERF-VII) transcription factors, plays a role in the development of crown galls. We found that quintuple knock-out mutants of all ERF-VII members, which are incapable of inducing the hypoxic response, show reduced crown gall symptoms. Conversely, mutant genotypes characterized by constitutively high levels of hypoxia-associated transcripts, displayed more severe crown gall symptoms. Based on these results, we concluded that uncontrolled cell proliferation of crown galls established hypoxic conditions, thereby requiring adequate anaerobic responses of the plant tissue to support tumor growth.

Fournier gangrene due to Rhizobium Radiobacter.

Fournier's gangrene (FG) is a life-threatening, rapidly progressing acute necrotizing fasciitis of perianal, genitourinary and perineal areas. The most common isolated pathogens are E.coli, Streptococcus and Enterococcus in tissue and abscess cultures. However we present the first Rhizobium radiobacter FG in this case. A 47-year-old man presented with black necrotizing area with malodorous pus drainage in the bottom of the right hemiscrotum. Intravenous imipenem and Clindamycin were started prophylactically; all necrotizing tissues were debrided and right hemiscrotectomy was performed. Rhizobium radiobacter was isolated from tissue and abscess cultures. After successful treatment patient was discharged posteroperative 18th day. The combination therapy of early and totally surgical debridement of necrotic tissues and appropriate antibiotic use are adequate to control Rhizobium radiobacter infection in FG.