Plasmid Crosstalk in Cell-Free Expression Systems.

Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex applications. One such application is the prototyping or implementation of complex genetic networks involving the expression of multiple proteins at precise ratios, often from different plasmids. However, expression of multiple proteins from multiple plasmids may inadvertently result in unexpected, off-target changes to the levels of the proteins being expressed, a phenomenon termed plasmid crosstalk. Here, we show that the effects of plasmid crosstalk─even at the qualitative level of increases vs decreases in protein expression─depend on the concentration of plasmids in the reaction and the type of transcriptional machinery involved in the expression. This crosstalk can have a significant impact on genetic circuitry function and even interpretation of simple experimental results and thus should be taken into consideration during the development of cell-free applications.

Rapid and Finely-Tuned Expression for Deployable Sensing Applications.

Organisms from across the tree of life have evolved highly efficient mechanisms for sensing molecules of interest using biomolecular machinery that can in turn be quite valuable for the development of biosensors. However, purification of such machinery for use in in vitro biosensors is costly, while the use of whole cells as in vivo biosensors often leads to long sensor response times and unacceptable sensitivity to the chemical makeup of the sample. Cell-free expression systems overcome these weaknesses by removing the requirements associated with maintaining living sensor cells, allowing for increased function in toxic environments and rapid sensor readout at a production cost that is often more reasonable than purification. Here, we focus on the challenge of implementing cell-free protein expression systems that meet the stringent criteria required for them to serve as the basis for field-deployable biosensors. Fine-tuning expression to meet these requirements can be achieved through careful selection of the sensing and output elements, as well as through optimization of reaction conditions via tuning of DNA/RNA concentrations, lysate preparation methods, and buffer conditions. Through careful sensor engineering, cell-free systems can continue to be successfully used for the production of tightly regulated, rapidly expressing genetic circuits for biosensors.

A Trauma Center-Based Study for Victims of Domestic Violence: Protecting Women of Richmond and Surrounding Counties.

INTRODUCTION: Domestic violence (DV) is a major health issue on both a national and global scale. In Richmond County, Georgia, there are 18.1 calls per 1000 women for DV, exceeding the national average of 5.9 and Georgia average of 15.3 calls per 1000 women. The goal of the study is to map epidemiologic and spatial trends in communities of DV survivors thereby assessing the feasibility of a prospective intervention initiative for high-risk areas. METHODS: In partnership with "SafeHomes", a local women's shelter, a retrospective review of physical addresses was compiled from first-time residential clients 18 and older from January 1, 2019 to December 31, 2019. Hot zones were overlaid with Census tracts to assess sociodemographic correlates including race, ethnicity, age, poverty, education, and employment status. RESULTS: From all records (n = 85), 5 hot zones were identified. Analysis of census tract data revealed minority predominance in all hot zones, in addition to an unemployment rate above the state average (23%, 12%, 8%, 5%, and 19%, respectively, vs 3.4%). CONCLUSION: DV affects both the inner city and suburban areas of Richmond County. Hot zone frequency was disproportionately increased in the urban center where community demographics showed minority predominance, especially of African-Americans. These hot zones also have more individuals living below the federal poverty line and experience unemployment rates greater than the state and national average of 3.4 and 3.5, respectively. This study demonstrated that mapping domestic violence epidemiologic and spatial trends is possible, allowing for targeted support and intervention to reduce the rate of domestic violence.

Low-cost, point-of-care biomarker quantification.

Low-cost, point-of-care (POC) devices that allow fast, on-site disease diagnosis could have a major global health impact, particularly if they can provide quantitative measurement of molecules indicative of a diseased state (biomarkers). Accurate quantification of biomarkers in patient samples is already challenging when research-grade, sophisticated equipment is available; it is even more difficult when constrained to simple, cost-effective POC platforms. Here, we summarize the main challenges to accurate, low-cost POC biomarker quantification. We also review recent efforts to develop and implement POC tools beyond qualitative readouts, and we conclude by identifying important future research directions.

Bridged Piperidine Analogues of a High Affinity Naphthalene-Based P2Y14R Antagonist.

High affinity phenyl-piperidine P2Y14R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y14R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21-24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC50 15.6 nM) and isonortropanol 30 (IC50 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y14R molecular modeling based on a P2Y12R structure showed stable and persistent key interactions for compound 15.

Modulation of adipocyte size and fat pad weight via resveratrol releasing scaffolds implanted into the epididymal adipose tissue.

Lipid overload of the adipose tissue, which can be caused by overnutrition, underlies metabolic disease. We hypothesized that increasing the energy demand of adipose tissue is a promising strategy to combat excessive lipid accumulation. Resveratrol, a natural polyphenol, activates lipid catabolism in fat tissue; however, its clinical success is hindered by poor bioavailability. Here, we implanted resveratrol releasing poly(lactide-co-glycolide) scaffolds into epididymal fat to overcome its poor bioavailability with the goal of enhancing local lipid catabolism. In lean mice, resveratrol scaffolds decreased adipocyte size relative to scaffolds with no drug, a response that correlated with AMP kinase activation. Immunohistochemistry indicated that macrophages and multinucleated giant cells within the scaffold expressed carnitine palmitoyltransferase 1 (CPT1) at higher levels than other cells in the adipose tissue. Furthermore, resveratrol increased CPT1 levels in cultured macrophages. Taken together, we propose that resveratrol scaffolds decrease adipocyte size because resveratrol increases lipid utilization in scaffold-infiltrating immune cells, possibly through elevating CPT1 levels or activity. In a follow-up study, mice that received resveratrol scaffolds 28-day prior to a high-fat diet exhibited decreased weight gain, adipose tissue expansion, and adipocyte hypertrophy compared to mice with control scaffolds. Notably, this scaffold-based strategy required a single resveratrol administration compared to the daily regiment generally needed for oral administration. These results indicate that localized delivery of metabolism modulating agents to the adipose tissue may overcome issues with bioavailability and that the role of biomaterials should be further investigated in this therapeutic strategy for metabolic disease.