Understanding the Lifetime and Rate of Protein Production in Cell-Free Reactions While Maximizing Energy Use

Liposomes, or vesicles, offer promising applications in fields including biofuel synthesis, drug delivery, and toxin removal. Programmable liposomes can be used for optimal protein synthesis and to prototype genetic technologies. However, one of the major challenges is the short lifetime of protein production. Here, we add metabolites and molecules to cell-free reactions at different times to interrogate their importance. Through this testing, we find that ATP only slightly enhances protein synthesis, and ADP can help a reaction reach steady state faster. We also note that the excess of particular molecules, such as NAD and 3PGA, can halt protein production. With this data, we developed a more accurate chemical reaction network-based model for cell-free reactions. We also begin to study an unexplained discrepancy in protein production between bulk and vesicle dynamics. To quantify protein synthesis, we use E. coli extract and energy buffer, often called cell-free transcription and translation (TXTL), with a chosen DNA template both within vesicles (encapsulated) and without (bulk). We have also been able to uncover fundamental properties of transcription/translation systems. We supplement this data with computational models utilizing chemical reaction networks. We established a vesicle setup with membrane pores and supplemental energy buffer on the outside which increased the efficiency of protein synthesis. By using chemical reaction network models, we have highlighted differences and similarities between models and experiments. With this setup, vesicles can be used for more complicated applications, such as drug delivery or genetic construct testing

Selective Ablation of Cancer Cells with Low Intensity Pulsed Ultrasound

Ultrasound can be focused into deep tissues with millimeter precision to perform noninvasive ablative therapy for diseases such as cancer. In most cases, this ablation uses high intensity ultrasound to deposit nonselective thermal or mechanical energy at the ultrasound focus, damaging both healthy bystander tissue and cancer cells. Here, we describe an alternative low intensity (I_(SPTA) 20 ms causes selective disruption of a panel of breast, colon, and leukemia cancer cell models in suspension without significantly damaging healthy immune or red blood cells. Mechanistic experiments reveal that the formation of acoustic standing waves and the emergence of cell-seeded cavitation lead to cytoskeletal disruption, expression of apoptotic markers, and cell death. The inherent selectivity of this low intensity pulsed ultrasound approach offers a potentially safer and thus more broadly applicable alternative to nonselective high intensity ultrasound ablation

Hepatic transcriptome signature correlated with HOMA-IR explains early nonalcoholic fatty liver disease pathogenesis

Introduction and objectives: Non-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. Materials and methods: In this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (N = 72) and non-alcoholic steatohepatitis (NASH; N = 51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. Results: Significantly higher weight (p = 0.005) and elevated levels of HbA1c (p = 0.009), FBG (p = 0.03) and HOMA-IR (p = 0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold change > 1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. Conclusions: Hepatic expression of genes associated with insulin resistance may drive NAFLD development and progression