Characterization of two radical S-adenosyl-L-methionine enzymes in the biosynthesis of aminoglycosides

Biosynthetic studies of natural products are essential to the discovery and development of new drugs, because by understanding biosynthetic pathways and the enzymes that characterize them, new pathways can be engineered for the production of new compounds with improved clinical properties. Aminoglycosides have traditionally been used as important antibiotics, but resistance against aminoglycosides is well known. This has resulted in a need to better understand the biosynthesis of aminoglycosides. Besides the practical value, the discovery of new enzymes with unprecedented functions in the biosynthesis of aminoglycosides can expand our knowledge and advance our understanding of enzyme catalysis. The work described in this dissertation focuses on the in vitro characterization of two radical S-adenosyl-L-methionine (SAM) enzymes in the biosynthesis of aminoglycosides. First, GenK is a cobalamin (Cbl)-dependent radical SAM enzyme that is responsible for catalyzing the methylation of gentamicin X₂ to produce G418. In vitro assays of purified and reconstituted GenK from Micromonospora echinospora showed that this enzyme is a radical SAM enzyme with one [4Fe-4S] cluster. Assays of GenK with SAM, gentamicin X₂ and Cbl confirmed that the methylation reaction occurs at an unactivated carbon during gentamicin biosynthesis. Isotope labeling experiments strongly suggested that SAM is the preliminary methyl donor to cobalamin, followed by secondary transfer from Me-Cbl to gentamicin X₂. It was demonstrated that GenK also accepts alternative aminoglycoside substrates besides gentamicin X₂. Second, three possible mechanisms for the GenK reaction were suggested and tested. Experiments investigating the stoichiometry of the GenK reaction revealed 5'-deoxyadenosine (5'-dAdo), S-adenoxylhomocysteine (SAH), and G418 were produced in equal proportion and one-to-one with each methylation reaction catalyzed by GenK. The experiment with labeled substrates indicated that the 6'-pro-R-hydrogen atom of gentamicin X₂ is abstracted by 5'-dAdo• and that methylation occurs with retention of configuration at C6'. Several substrate derivatives were synthesized to investigate the manner of methyl transfer from cobalamin to the substrate. Finally, the in vitro characterization of AprD4 and AprD3 in the biosynthesis of C3'-deoxyaminoglycoside was also conducted. Purified and reconstituted AprD4 from Streptomyces tenebrarius is a radical SAM enzyme, catalyzing homolysis of SAM to 5'-dAdo in the presence of paromamine to produce 4-oxolividamine. AprD3 from the same strain is shown to be a dehydrogenase acting as the reductase counterpart to AprD4 to catalyze the reduction of 4-oxolividamine to generate lividamine. The experiments with labeled compounds confirmed the regiochemistry of hydrogen atom abstraction by 5'-dAdo• and the stereochemical course of C3'-deoxygenation of paromamine.Chemistr

EFFECT OF LOW DOSE RADIATION ON DIFFERENTIATION OF BONE MARROW CELLS INTO DENDRITIC CELLS

Low dose radiation has been shown to be beneficial to living organisms using several biological systems, including immune and hematopoietic systems. Chronic low dose radiation was shown to stimulate immune systems, resulting in controlling the proliferation of cancer cells, maintain immune balance and induce hematopoietic hormesis. Since dendritic cells are differentiated from bone marrow cells and are key players in maintaining the balance between immune activation and tolerance, it may be important to further characterize whether low dose radiation can influence the capacity of bone marrow cells to differentiate into dendritic cells. We have shown that bone marrow cells from low dose- irradiated (γ-radiation, 0.2Gy, 15.44mGy/h) mice can differentiate into dendritic cells that have several different characteristics, such as expression of surface molecules, cytokine secretion and antigen uptake capacity, when compared to dentritic cells differentiated from the control bone marrow cells. These differences observed in the low dose radiation group can be beneficial to living organisms either by activation of immune responses to foreign antigens or tumors, or maintenance of self-tolerance. To the best of our knowledge, this is the first report showing that total-body low dose radiation can modulate the capacity of bone marrow cells to differentiate into dendritic cells

Comparison of Surgical Outcomes in Thoracolumbar Fractures Operated with Posterior Constructs Having Varying Fixation Length with Selective Anterior Fusion

PURPOSE: Surgical treatment in the case of thoracolumbar burst fractures is very controversial. Posterior instrumentation is most frequently used, however, but the number of levels to be instrumented still remains a matter of debate. MATERIALS AND METHODS: A total of 94 patients who had a single burst fracture between T11 and L2 were selected and were managed using posterior instrumentation with anterior fusion when necessary. They were divided into three groups as follows; Group I (n = 28) included patients who were operated by intermediate segment fixation, Group II (n = 32) included patients operated by long segment fixation, and Group III (n = 34) included those operated by intermediate segment fixation with a pair of additional screws in the fractured vertebra. The mean follow-up period was twenty one months. The outcomes were analyzed in terms of kyphosis angle (KA), regional kyphosis angle (RA), sagittal index (SI), anterior height compression rate, Frankel classification, and Oswestry Disability Index questionnaire. RESULTS: In Groups II and III, the correction values of KA, RA, and SI were much better than in Group I. At the final follow up, the correction values of KA (6.3 and 12.1, respectively) and SI (6.2 and 12.0, respectively) were in Groups II and III found to be better in the latter. CONCLUSION: The intermediate segment fixation with an additional pair of screws at the fracture level vertebra gives results that are comparable or even better than long segment fixation and gives an advantage of preserving an extra mobile segment.ope

Stent Thrombosis, Clinical Events, and Influence of Prolonged Clopidogrel Use After Placement of Drug-Eluting Stent Data From an Observational Cohort Study of Drug-Eluting Versus Bare-Metal Stents

ObjectivesThe purpose of this study was to evaluate the risk of stent thrombosis (ST), clinical outcomes, and the benefits of extended clopidogrel use after drug-eluting stent (DES) implantation.BackgroundData are limited regarding uniform evaluation of ST and the influence of clopidogrel continuation beyond 12 months on late events after DES treatment.MethodsWe identified 7,221 patients who received DES implantation (n = 3,160) or bare-metal stent (BMS) implantation (n = 4,061), and compared long-term adverse outcomes. Additionally, 2,851 patients with DES surviving 12 months without major events were analyzed according to clopidogrel continuation.ResultsThe adjusted-risk of overall ST was similar in the 2 groups. After 1 year, however, DES patients showed a higher risk of ST; definite/probable (hazard ratio [HR]: 3.55, 95% confidence interval [CI]: 1.26 to 9.99). The adjusted-risk of death (HR: 0.60, 95% CI: 0.46 to 0.79), death/myocardial infarction (HR: 0.63, 95% CI: 0.49 to 0.81), and target lesion revascularization (HR: 0.32, 95% CI: 0.24 to 0.43) were significantly lower in the DES group than in the BMS group. Continuing clopidogrel beyond 12 months was not associated with a reduced risk for ST (HR: 0.54, 95% CI: 0.07 to 4.23), death (HR: 1.20, 95% CI: 0.55 to 2.66), or death/myocardial infarction (HR: 1.16, 95% CI: 0.56 to 2.42) after DES implantation.ConclusionsAs compared with BMS, DES showed a similar risk of overall ST, but a higher risk of very late ST. The rates of death, death/myocardial infarction, and target lesion revasuclarization were significantly lower in the DES group. Clopidogrel continuation beyond 1 year did not appear to reduce ST and clinical events after DES implantation

A polyyne toxin produced by an antagonistic bacterium blinds and lyses a Chlamydomonad alga

Algae are key contributors to global carbon fixation and form the basis of many food webs. In nature, their growth is often supported or suppressed by microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green unicellular alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology [P. Aiyar et al., Nat. Commun. 8, 1756 (2017)]. Using a combination of Raman microspectroscopy, genome mining, and mutational analysis, we discovered a polyyne toxin, protegencin, which is secreted by P. protegens, penetrates the algal cells, and causes destruction of the carotenoids of their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin thus prevents the phototactic behavior of C. reinhardtii. A mutant of P. protegens deficient in protegencin production does not affect growth or eyespot carotenoids of C. reinhardtii. Protegencin acts in a direct and destructive way by lysing and killing the algal cells. The toxic effect of protegencin is also observed in an eyeless mutant and with the colony-forming Chlorophyte alga Gonium pectorale. These data reveal a two-pronged molecular strategy involving a cyclic lipopeptide and a conjugated tetrayne used by bacteria to attack select Chlamydomonad algae. In conjunction with the bloom-forming activity of several chlorophytes and the presence of the protegencin gene cluster in over 50 different Pseudomonas genomes [A. J. Mullins et al., bioRxiv [Preprint] (2021). https://www.biorxiv.org/content/10.1101/2021.03.05.433886v1 (Accessed 17 April 2021)], these data are highly relevant to ecological interactions between Chlorophyte algae and Pseudomonadales bacteria

A Multi-institutional Study on Histopathological Characteristics of Surgically Treated Renal Tumors: the Importance of Tumor Size

PURPOSE: The incidence of accidentally detected small renal tumors is increasing throughout the world. In this multi-institutional study performed in Korea, histopathological characteristics of contemporarily surgically removed renal tumors were reviewed with emphasis on tumor size. MATERIALS and METHODS: Between January 1995 and May 2005, 1,702 patients with a mean age of 55 years underwent surgical treatment at 14 training hospitals in Korea for radiologically suspected malignant renal tumors. Clinicopathological factors and patient survival were analyzed. RESULTS: Of the 1,702 tumors, 91.7% were malignant and 8.3% were benign. The percentage of benign tumors was significantly greater among those 4cm (4.5%) (p or = T3 was significantly less among tumors 4cm (26.8%) (p or = 3 was also significantly less among tumors 4cm (50.9%) (p < 0.001). The 5-year cancer-specific survival rate was 82.7%, and T stage (p < 0.001), N stage (p < 0.001), M stage (p = 0.025), and Fuhrman's nuclear (p < 0.001) grade were the only independent predictors of cancer-specific survival. CONCLUSION: In renal tumors, small tumor size is prognostic for favorable postsurgical histopathologies such as benign tumors, low T stages, and low Fuhrman's nuclear grades. Our observations are expected to facilitate urologists to adopt function-preserving approach in the planning of surgery for small renal tumors with favorable predicted outcomes.ope

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control. © 2021 The Authors. ChemBioChem published by Wiley-VCH Gmb

Inflammatory and Remodeling Events in Asthma with Chronic Exposure to House Dust Mites: A Murine Model

Although animal models with ovalbumin have been used to study chronic asthma, there are difficulties in inducing recurrence as well as in maintaining chronic inflammation in this system. Using a murine model of house dust mite (HDM)-induced bronchial asthma, we examined the airway remodeling process in response to the chronic exposure to HDM. During the seventh and twelfth weeks of study, HDM were inhaled through the nose for three consecutive days and airway responsiveness was measured. Twenty-four hours later, bronchoalveolar lavage and histological examination were performed. The degree of overproduction of mucus, subepithelial fibrosis, and the thickness of the peribronchial smooth muscle in the experimental group was clearly increased compared to the control group. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was increased; during the twelfth week, the number of neutrophils increased in the experimental group. With regard to changes in cytokines, the concentrations of IL-4, IL-13, and transforming growth factor-beta (TGF-β) were increased in the experimental group. The data suggest that eosinophils, IL-4, IL-13, and TGF-β might play an important role in the airway remodeling process and that neutrophils may be involved with increased exposure time