Introduction and expression of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis and its use in comobilizing the Staphylococcus aureus resistance plasmid pC194

The Staphylococcus aureus plasmid, pC194, was introduced into Bacillus thuringiensis subsp. israelensis using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. The conjugative S. faecalis transposon Tn916 was introduced into B. thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline were detected at a frequency of approximately 7.0 x10[superscript] -7 per recipient cell when filter matings were performed, but tetracycline-resistant transconjugants were not observed when a broth-mating protocol was used. The tetracycline resistance phenotype was not lost during serial passage of subsp. israelensis without antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome. Conjugation-like movement of Tn916 was demonstrated when tetracycline-resistant B. thuringiensis transconjugants were mated with isogenic recipients. Transfer of pC194 only occurred when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor containing pC194 and Tn916. B. thuringiensis transconjugants resistant to chloramphenicol and tetracycline were detected at a frequency of 1.96 x 10[superscript] -6 per recipient cell when filter matings were performed. Transconjugants resistant to chloramphenicol were not observed when a broth mating protocol was used. The tetracycline resistance phenotype was maintained during serial passage of B. thuringiensis without antibiotic selection, whereas the chloramphenicol resistance phenotype was not. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis chromosome, and pC194 was introduced as an autonomous plasmid into the B. thuringiensis genome

Next generation sequencing analysis of nine Corynebacterium ulcerans isolates reveals zoonotic transmission and a novel putative diphtheria toxin-encoding pathogenicity island

Background: Toxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries. Methods: To gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics. Results: We provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene. Conclusion: The novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans

Identification of host cell factors required for intoxication through use of modified cholera toxin

We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.Fundação para a Ciência e a Tecnologia (Fellowship

Targeted Toxins in Brain Tumor Therapy

Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins

Role of Matrix Metalloproteinases and Therapeutic Benefits of Their Inhibition in Spinal Cord Injury

This review will focus on matrix metalloproteinases (MMPs) and their inhibitors in the context of spinal cord injury (SCI). MMPs have a specific cellular and temporal pattern of expression in the injured spinal cord. Here we consider their diverse functions in the acutely injured cord and during wound healing. Excessive activity of MMPs, and in particular gelatinase B (MMP-9), in the acutely injured cord contributes to disruption of the blood-spinal cord barrier, and the influx of leukocytes into the injured cord, as well as apoptosis. MMP-9 and MMP-2 regulate inflammation and neuropathic pain after peripheral nerve injury and may contribute to SCI-induced pain. Early pharmacologic inhibition of MMPs or the gelatinases (MMP-2 and MMP-9) results in an improvement in long-term neurological recovery and is associated with reduced glial scarring and neuropathic pain. During wound healing, gelatinase A (MMP-2) plays a critical role in limiting the formation of an inhibitory glial scar, and mice that are genetically deficient in this protease showed impaired recovery. Together, these findings illustrate complex, temporally distinct roles of MMPs in SCIs. As early gelatinase activity is detrimental, there is an emerging interest in developing gelatinase-targeted therapeutics that would be specifically tailored to the acute injured spinal cord. Thus, we focus this review on the development of selective gelatinase inhibitors

Intracellular Trafficking Considerations in the Development of Natural Ligand-Drug Molecular Conjugates for Cancer

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates

Introduction and expression of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis and its use in comobilizing the Staphylococcus aureus resistance plasmid pC194

The Staphylococcus aureus plasmid, pC194, was introduced into Bacillus thuringiensis subsp. israelensis using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. The conjugative S. faecalis transposon Tn916 was introduced into B. thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline were detected at a frequency of approximately 7.0 x10[superscript] -7 per recipient cell when filter matings were performed, but tetracycline-resistant transconjugants were not observed when a broth-mating protocol was used. The tetracycline resistance phenotype was not lost during serial passage of subsp. israelensis without antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome. Conjugation-like movement of Tn916 was demonstrated when tetracycline-resistant B. thuringiensis transconjugants were mated with isogenic recipients. Transfer of pC194 only occurred when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor containing pC194 and Tn916. B. thuringiensis transconjugants resistant to chloramphenicol and tetracycline were detected at a frequency of 1.96 x 10[superscript] -6 per recipient cell when filter matings were performed. Transconjugants resistant to chloramphenicol were not observed when a broth mating protocol was used. The tetracycline resistance phenotype was maintained during serial passage of B. thuringiensis without antibiotic selection, whereas the chloramphenicol resistance phenotype was not. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis chromosome, and pC194 was introduced as an autonomous plasmid into the B. thuringiensis genome.</p

Filter synthesis and design techniques for highly adaptable systems

Software defined radio and cognitive radio (SDR/CR) are promising concepts toward more optimally using the electromagnetic spectrum for communications and data transfer. These systems are highly agile in terms of modulation technique and frequency of operation due to early digitization and software processing of received radio frequency signals. However, the front ends of SDR/CR systems often use static antennas and switched banks of static, wide bandwidth filters before the digitization process. These components limit the ability of SDR/CR systems to operate in environments with high levels of interference and are therefore a bottleneck in the path to achieving optimal adaptation of wireless systems. This dissertation focuses on tunable and reconfigurable filter synthesis and design techniques that have the potential to alleviate the hardware adaptation bottleneck currently experienced by SDR/CR systems. The presented filter techniques and structures promise the capability to dynamically adapt the center frequency, order, response shape, bandwidth, group delay, passband insertion loss-stopband attenuation tradeoff, and transmission zero spectral location of a filter response in the field. Such reconfiguration capability is made possible through the use of new coupling relationships and structures that enable dynamic reconfiguration between electric, magnetic, and zero coupling values