Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation.

Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

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Genetically Engineered Phages: a Review of Advances over the Last Decade

In recent years, nanoparticles (NPs) have increasingly found practical applications in technology, research, and medicine. The small particle size coupled with their unique chemical and physical properties is thought to underline their exploitable biomedical activities. Its form may be latex body, polymer, ceramic particle, metal particles, and the carbon particles. Due to their small size and physical resemblance to physiological molecules such as proteins, NPs possess the capacity to revolutionise medical imaging, diagnostics, therapeutics, as well as carry out functional biological processes. But these features may also underline their toxicity. Indeed, a detailed assessment of the factors that influence the biocompatibility and toxicity of NPs is crucial for the safe and sustainable development of the emerging NPs. Due to the unique structure, size, and shape, much effort has been dedicated to analyzing biomedical applications of nanotubes. This paper focuses on the current understanding of the biocompatibility and toxicity of NPs with an emphasis on nanotubes.

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Biocompatibility and toxicity of nanoparticles and nanotubes

Summary
Escherichia coli biofilm consists of a bacterial colony embedded in a matrix of extracellular polymeric substances (EPS) which protects the microbes from adverse environmental conditions and results in infection. Besides being the major causative agent for recurrent urinary tract infections, E. coli biofilm is also responsible for indwelling medical device-related infectivity. The cell-to-cell communication within the biofilm occurs due to quorum sensors that can modulate the key biochemical players enabling the bacteria to proliferate and intensify the resultant infections. The diversity in structural components of biofilm gets compounded due to the development of antibiotic resistance, hampering its eradication. Conventionally used antimicrobial agents have a restricted range of cellular targets and limited efficacy on biofilms. This emphasizes the need to explore the alternate therapeuticals like anti-adhesion compounds, phytochemicals, nanomaterials for effective drug delivery to restrict the growth of biofilm. The current review focuses on various aspects of E. coli biofilm development and the possible therapeutic approaches for prevention and treatment of biofilm-related infections.

Escherichia coli biofilm: development and therapeutic strategies

Engineered nanomaterials offer numerous and tantalizing opportunities in many sectors of society, including medicine. Needless to say, attention should also be paid to the potential for unexpected hazardous effects of these novel materials. To date, much of the nanotoxicology literature has focused on the assessment of cell viability or cell death using primitive assays for the detection of plasma membrane integrity or mitochondrial function or assessment of cellular morphology. However, when assessing the cytotoxic effects of engineered nanomaterials, researchers need not only to consider whether cells are dead or alive but also to assess which of the numerous, highly specific pathways of cell death might be involved. Moreover, it is important to diagnose cell death based not only on morphological markers but on the assessment and quantification of biochemical alterations specific to each form of cell death. In this Account, we provide a description of the three major forms of programmed cell death in mammalian cells: apoptosis, autophagic cell death, and regulated necrosis, sometimes referred to as necroptosis. Apoptosis can be activated via the extrinsic (death receptor-dependent) or via the intrinsic (mitochondria-dependent) route. Apoptotic cell death may or may not require the activation of cytosolic proteases known as caspases. Autophagy (self-eating) has an important homeostatic role in the cell, mediating the removal of dysfunctional or damaged organelles thereby allowing the recycling of cellular building blocks. However, unrestrained autophagy can kill cells. Studies in recent years have revealed that necrosis that depends on activation of the kinases RIP1 and RIP3 is a major form of programmed cell death with roles in development and immunity. We also discuss recent examples of the impact of engineered nanoparticles on the three different pathways of programmed cell death. For example, acute exposure of cells to carbon nanotubes (CNTs) can induce apoptosis whereas chronic exposure to CNTs may yield an apoptosis-resistant and tumorigenic phenotype in lung epithelial cells. Several reports show that nanoparticles, including polystyrene particles, are routed to the lysosomal compartment and trigger cell death through the destabilization of lysosomal membranes with engagement of the intrinsic apoptosis pathway. In addition, a number of studies have demonstrated that nanomaterials such as CNTs, quantum dots, and gold nanoparticles can affect cellular autophagy. An improved understanding of the complexities of the nanomaterial-induced perturbation of different cell death pathways may allow for a better prediction of the consequences of human exposure.

Programmed Cell Death: Molecular Mechanisms and Implications for Safety Assessment of Nanomaterials

Nanosized polyamidoamine (PAMAM) dendrimer-induced apoptosis mediated by mitochondrial dysfunction

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.

Phage-Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherichia coli Strain

Escherichia coli O157:H7 is a human pathogen. We isolated a novel bacteriophage infecting this bacterium from a sewage water treatment facility. Phage PBECO4 belongs to the family Myoviridae, having an isometric head and a contractile tail. It has a linear double-stranded DNA genome of 348,113 base pairs in length with a GC content of 34.09 %. Whole-genome sequencing revealed that PBECO4 is distantly related to enterobacteria phage vB_KleM_RaK2, with 10 % similarity, and Cronobacter phage vB_CsaM_GAP32 with 6 % similarity. Five hundred fifty-one putative open reading frames (ORFs) and six tRNA genes were found. Eight ORFs are related to genes encoding structural proteins, nine to DNA packaging, two to DNA lysis activity, and 42 to replication and regulation. Four hundred ninety ORFs have not been functionally annotated.

Genomic analysis of bacteriophage PBECO4 infecting Escherichia coli O157:H7.

A novel lytic bacteriophage, SA11, infecting Staphylococcus aureus was isolated, and the whole genome was sequenced. It belongs to the siphoviridae based on electron microscopic observation. It has a linear double-stranded DNA genome of 136,326 bp. Genomic analysis showed that it is distantly related to Staphylococcus phages A5W, K, ISP, Sb-1, and G1.

Complete Genome of Staphylococcus aureus Phage SA11

Hepatitis C virus (HCV) causes chronic hepatitis leading to liver fibrosis and autoimmune diseases. AIMP1/p43 is a multifunctional protein initially known as a cofactor of aminoacyl tRNA synthetase complex. Its function includes negative regulation of TGF-β signaling and suppression of Lupus-like autoimmune disease by inhibition of surface expression of gp96. HCV E2 was shown to directly interact with AIMP1/p43 by GST pulldown assay and coimmunoprecipitation. Their subcellular colocalization was observed in an immunofluorescence confocal microscopy. We showed that HCV E2 led to degradation of AIMP1/p43 in two ways. First, in the presence of HCV E2, endogenous AIMP1/p43 was shown to be degraded in an ubiquitin-dependent proteasome pathway. Second, grp78, an ER chaperone, was shown to interact with and stabilize AIMP1/p43. And HCV E2 inhibited this interaction leading to reduction of cellular AIMP1/p43. The degradation of AIMP1/p43 by HCV E2 resulted in increase of TGF-β signaling and cell surface expression of gp96. Thus we suggest that these are novel mechanisms responsible for liver fibrosis and autoimmune diseases caused by HCV.

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Min Soo Kim

Papers

Nanosized polyamidoamine (PAMAM) dendrimer-induced apoptosis mediated by mitochondrial dysfunction

Phage-Encoded Colanic Acid-Degrading Enzyme Permits Lytic Phage Infection of a Capsule-Forming Resistant Mutant Escherichia coli Strain

Genomic analysis of bacteriophage PBECO4 infecting Escherichia coli O157:H7.

Complete Genome of Staphylococcus aureus Phage SA11

Degradation of AIMP1/p43 Induced by Hepatitis C Virus E2 Leads to Upregulation of TGF-β Signaling and Increase in Surface Expression of gp96