Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.

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Shape effects of filaments versus spherical particles in flow and drug delivery.

The finding that total viral abundance is higher than total prokaryotic abundance and that a significant fraction of the prokaryotic community is infected with phages in aquatic systems has stimulated research on the ecology of prokaryotic viruses and their role in ecosystems. This review treats the ecology of prokaryotic viruses ('phages') in marine, freshwater and soil systems from a 'virus point of view'. The abundance of viruses varies strongly in different environments and is related to bacterial abundance or activity suggesting that the majority of the viruses found in the environment are typically phages. Data on phage diversity are sparse but indicate that phages are extremely diverse in natural systems. Lytic phages are predators of prokaryotes, whereas lysogenic and chronic infections represent a parasitic interaction. Some forms of lysogeny might be described best as mutualism. The little existing ecological data on phage populations indicate a large variety of environmental niches and survival strategies. The host cell is the main resource for phages and the resource quality, i.e., the metabolic state of the host cell, is a critical factor in all steps of the phage life cycle. Virus-induced mortality of prokaryotes varies strongly on a temporal and spatial scale and shows that phages can be important predators of bacterioplankton. This mortality and the release of cell lysis products into the environment can strongly influence microbial food web processes and biogeochemical cycles. Phages can also affect host diversity, e.g., by 'killing the winner' and keeping in check competitively dominant species or populations. Moreover, they mediate gene transfer between prokaryotes, but this remains largely unknown in the environment. Genomics or proteomics are providing us now with powerful tools in phage ecology, but final testing will have to be performed in the environment.

Ecology of prokaryotic viruses

Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by ≈4.5 orders of magnitude (≈99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem.

https://www.pnas.org/content/pnas/104/27/11197.full.pdf

Dispersing biofilms with engineered enzymatic bacteriophage

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth.

Bacteriophage T4 Genome

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

https://www.pnas.org/content/pnas/93/8/3188.full.pdf

Long-circulating bacteriophage as antibacterial agents

A maternal line study investigating the 4977-bp mitochondrial DNA deletion.

SummaryMany of the acute and chronic diseases affecting the central nervous system (CNS) are still of unknown etiology. Given that brain cells are the most aerobic and metabolically active cells in the body, deficits in aerobic energy metabolism may play an important role in many of these diseases. Over the past decade numerous diseases affecting highly active metabolic tissues including the brain and skeletal musculature have been shown to be associated with alterations in the mitochondria, the subcellular organelle responsible for aerobic energy metabolism. In addition, a number of maternally inherited diseases have been linked to mutations in the circular, 16,569 nucleotide pair mitochondrial genome. Mendelian inherited genetic variations and diseases have also been found to affect the mitochondrial function, structure, and genome in certain tissues. Specific regions of the brain appear to be more prone to the occurrence of mitochondrial DNA (mtDNA) deletion mutations. For example, mtDNA extracted from...

Mitochondrial genome deletions in the brain and their role in neurodegenerative diseases

The central nervous system (CNS) is extremely sensitive to hypoxia, as neuronal cells require a high rate of energy metabolism to maintain transmembrane potentials. As these cells normally rely on aerobic mitochondrial metabolism to generate the required energy, a lack of oxygen results in a shift toward the anaerobic utilization of glucose. Such a metabolic shift may result in a cascade of potentially deleterious events beginning with an increased production of lactic acid and a decreased rate of ATP production.1 Increased lactic acid concentrations may cause a decrease in cellular pH, followed by the release of iron and the reaction of ferrous ions with hydrogen peroxide to produce hydroxyl radicals. A decrease in ATP may adversely affect DNA replication, transcription,2 and mRNA translation. In addition, the levels of inter-cellular calcium will increase as the ATP dependent calcium ion pump mechanism fails. Increased calcium levels can activate: endonucleases, nitric oxide synthetase, phospholipases and proteases. All of the above alterations in cellular metabolism may result in damage to the cellular DNA, particularly the mitochondrial DNA (Figure 1).

https://link.springer.com/content/pdf/10.1007%2F978-1-4899-0209-2_23.pdf

Is there a Relationship between Conditions Associated with Chronic Hypoxia, the Mitochondria, and Neurodegenerative Diseases, Such as Alzheimer’s Disease?

The increased prevalence of multidrug- resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The ther- apeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, partic- ularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial- passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Esche- richia coli phage l and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating l mutants also have greater capability as antibacterial agents than the cor- responding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant l capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria- specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

Steve Zullo

Papers

Long-circulating bacteriophage as antibacterial agents

A maternal line study investigating the 4977-bp mitochondrial DNA deletion.

Mitochondrial genome deletions in the brain and their role in neurodegenerative diseases

Is there a Relationship between Conditions Associated with Chronic Hypoxia, the Mitochondria, and Neurodegenerative Diseases, Such as Alzheimer’s Disease?

Long-circulating bacteriophage as antibacterial agents (phageybacteriayreticuloendothelial systemytoxinsyantibiotic resistance)