Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

INTRODUCTION: Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. MATERIALS AND METHODS: This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. RESULTS: A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. CONCLUSIONS: Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

S1 hydrophobic residues modulate voltage sensing phosphatase enzymatic function and voltage sensing.

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

Zombie projects, negative networks, and multigenerational science: The temporality of the International Map of the World.

The International Map of the World was a hugely ambitious scheme to create standardized maps of the entire world. It was first proposed in 1891 and remained a going concern until 1986. Over the course of the project's official life, nearly every country in the world took part, and map sheets were published showing all but a few areas of the planet. But the project ended quite unceremoniously, repudiated by cartographers and mapping institutions alike, and it is now remembered as a 'sad story' of network failure. How can we evaluate this kind of sprawling, multigenerational project? In order to move beyond practitioners' (and historians') habit of summarizing the entire endeavor using the blunt categories of success and failure, I propose a more temporally aware reading, one that both disaggregates the (persistent) project from the (always changing) network and sees project and network as invertible, with the possibility of zombie projects and negative networks that can remain robust even when disconnected from their original goals. I therefore see the abandonment of the International Map of the World as resulting from vigorous collaboration and new norms in cartography, not from lack of cooperation or other resources. New categories are required for analyzing science over the long durée.

Optical and Magnetic Properties of ZnO Nanoparticles Doped with Co, Ni and Mn and Synthesized at Low Temperature.

Zinc oxide nanomaterials were synthesized with small amounts of magnetic ions to create dilute magnetic semiconductors (DMS), by using a low temperature sol-gel method. Conditions were controlled such that a range of amounts of Co, Ni and Mn were incorporated. The incorporation could be tracked by color changes in the powders to blue for Co, green for Ni and yellow for Mn. XRD measurements showed the ZnO has the wurtzite structure with crystallites 8-12 nm in diameter. Nanoparticles were observed by SEM and TEM and TEM showed that the lattice fringes of different nanoparticles align. Nanoparticle alignment was disrupted when high concentrations of metal dopants were incorporated. Magnetic measurements showed a change in behavior from diamagnetic to paramagnetic with increasing concentration of metal dopants.

Malawi women and HIV: socio-cultural factors and barriers to prevention.

Heterosexually transmitted HIV/AIDS continues to devastate the health and economy of sub-Saharan African countries. In Malawi, 15.4% of 15-49 year olds are infected with HIV and 18-26% of pregnant women are living with HIV. Research has shown that sociocultural factors, especially gender roles and relationships, play a significant role in the transmission of HIV in Africa but little is known about Malawi women's perspective on HIV/AIDS. What do Malawi women say about the impact of HIV/AIDS on their lives, their role in prevention, and the barriers they face in trying to stem the spread of the disease? To answer these questions, three focus groups with Malawi women were conducted and analyzed for themes. The purpose of this paper is to describe one emergent theme captured in the statement, "We are just vessels for our husbands." This theme is explicated through discussions of women's and men's images, women's roles, gender/power relationships, disempowerment, role models and empowerment. Evident in this theme are interrelated messages for those involved in HIV/AIDS prevention. Health education alone is insufficient to stem the tide of HIV in Malawi. A multidisciplinary, systematic approach that includes women's education and economic empowerment as well as modifying legal and social structures that contribute to the spread of HIV/AIDS in Malawi is suggested as necessary additions to HIV and AIDS intervention programs. Only through forging partnerships between health, education, women's development groups, and political and social leaders will we be able to reduce the impact of HIV/AIDS in Malawi.

Donkey work: women, religion, and HIV/AIDS in Malawi.

Addressed in this article are the familial, cultural and religious influences on Malawi women that contribute to HIV/AIDS. Thirty-nine adult Malawi women representing voluntary assistance groups, religious groups, and university women participated in 3 focus groups in Malawi. Interview data were taped, transcribed, and analyzed using qualitative descriptive analysis. Findings revealed that multiple burdens in the lives of Malawi women resulting from poverty and responsibility for family members are made more onerous by religious institutions, sexual practices, and cultural beliefs. In conclusion, women's "donkey work" may result in at-risk sexual behavior as a means of survival, thus increasing the incidence of HIV/AIDS. Alleviating the burdens involves efforts from religious groups and restructuring of belief systems.