Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

Fe₃O₄ nanoparticles have been extensively studied for biomedical applications including magnetic resonance imaging, chemotherapeutic delivery and gene delivery due to their advantageous properties of superparamagnetism, biocompatibility and biodegradability. However, ultrafine, monodisperse and sized controlled nanoparticles synthesized by thermal decomposition are commonly encapsulated by hydrophobic layers and can’t be dispersed in aqueous solutions. Besides, during the surface modification of nanoparticles, it is still difficult to obtain a highly stable and dense polymeric surface layer. To this end, a robust multi-step polyethylene glycol(PEG)ylation approach is presented to produce biocompatible multilayer surface coated magnetic iron oxide nanoparticles that are hydrophilic and display long-term stability in biological media. Ultrafine, monodisperse and 12nm hydrophobic nanoparticles were first synthesized by elevated temperature decomposition of nontoxic iron oleate complex in the presence of 1-octadecene and oleic acid. Subsequently, the oleic acid layer of nanoparticles was replaced by 3-(triethoxysilyl)propyl succinic anhydride(SSA) through ligand exchange to form SSA layer coated nanoparticles. Then, during the first step of PEGylation, to prevent the aggregation of nanoparticles, adequate amount of methyl PEG-NH₂ was attached to the part of SSA layer through the coupling reaction of functional groups. And in the second step of PEGylation, NH₂-PEG-NH₂ was attached to SSA to achieve a SSA/PEG dense multilayer surface with an adequate number of functional groups for further conjugation of therapeutics or targeting agents. The tools such as transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) were utilized to characterize the structure and properties of surface modified nanoparticles. The excellent dispensability and stability of surface modified nanoparticles in biological media was confirmed by no significant size change after at least 30 day’s incubation in Dulbecco’s phosphate buffered saline (PBS) and dulbecco’s modified Eagle’s medium (DMEM) with 10% of fetal bovine serum (FBS), demonstrating a huge potential for biomedical applications.

Glioblastoma multiforms (GBMs) are malignant brain tumors and are among the most lethal cancers in the United States. Current clinical treatments involve surgical resection followed by chemo radiation therapy. Temozolomide (TMZ) is a DNA methylating agent that has shown promise in post-operative chemo radiation therapy, yet its effects are mediated greatly by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The addition of DNA repair inhibitor O6-benzylguanine (BG) as part of a concurrent treatment with TMZ has shown promise, but suffers from unacceptable toxicity due to BGs poor pharmacokinetics. Here we report the development of nanoparticles (NPs) loaded with a BG payload that can efficiently cross the blood brain barrier (BBB) and with the addition of targeting agents may be a more effective method for drug delivery by reducing toxicity in healthy tissue. These NPs are composed of a magnetic core and biocompatible polymer surface coating. The NPs are then conjugated with BG (NP-BG) by attachment to the polymer surface. NP-BG is further modified with chlorotoxin (NP-BG-CTX), a tumor targeting peptide, through a polyethylene glycol linker. NP-BG-CTX had a mean hydrodynamic size of 75nm with a surface charge of about +4mV and have demonstrated specific targeting in vitro of GBM cell line SF767. Finally, we have shown that NP-BG substantially reduces MGMT activity in SF767 cells in vitro. These results suggest that NP-BG-CTX have a higher specificity to tumor cells than normal healthy cells in cancer patients. This would decrease drug toxicity in patients and reduce tumor resistance to TMZ, potentially providing a more effective treatment and improve clinical outcome.