Engineering stochasticity in gene expression

Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise

Nitric oxide and coronary vascular endothelium adaptations in hypertension

This review highlights a number of nitric oxide (NO)-related mechanisms that contribute to coronary vascular function and that are likely affected by hypertension and thus become important clinically as potential considerations in prevention, diagnosis, and treatment of coronary complications of hypertension. Coronary vascular resistance is elevated in hypertension in part due to impaired endothelium-dependent function of coronary arteries. Several lines of evidence suggest that other NO synthase isoforms and dilators other than NO may compensate for impairments in endothelial NO synthase (eNOS) to protect coronary artery function, and that NO-dependent function of coronary blood vessels depends on the position of the vessel in the vascular tree. Adaptations in NOS isoforms in the coronary circulation to hypertension are not well described so the compensatory relationship between these and eNOS in hypertensive vessels is not clear. It is important to understand potential functional consequences of these adaptations as they will impact the efficacy of treatments designed to control hypertension and coronary vascular disease. Polymorphisms of the eNOS gene result in significant associations with incidence of hypertension, although mechanistic details linking the polymorphisms with alterations in coronary vasomotor responses and adaptations to hypertension are not established. This understanding should be developed in order to better predict those individuals at the highest risk for coronary vascular complications of hypertension. Greater endothelium-dependent dilation observed in female coronary arteries is likely related to endothelial Ca2+ control and eNOS expression and activity. In hypertension models, the coronary vasculature has not been studied extensively to establish mechanisms for sex differences in NO-dependent function. Genomic and nongenomic effects of estrogen on eNOS and direct and indirect antioxidant activities of estrogen are discussed as potential mechanisms of interest in coronary circulation that could have implications for sex- and estrogen status-dependent therapy for hypertension and coronary dysfunction. The current review identifies some important basic knowledge gaps and speculates on the potential clinical relevance of hypertension adaptations in factors regulating coronary NO function

Controlled Inhibition of the Mesenchymal Stromal Cell Pro-inflammatory Secretome via Microparticle Engineering

Mesenchymal stromal cells (MSCs) are promising therapeutic candidates given their potent immunomodulatory and anti-inflammatory secretome. However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. To address this, we used a microparticle-based engineering approach to non-genetically modulate pro-inflammatory pathways in human MSCs (hMSCs) under simulated inflammatory conditions. Here we show that microparticles loaded with TPCA-1, a small-molecule NF-κB inhibitor, when delivered to hMSCs can attenuate secretion of pro-inflammatory factors for at least 6 days in vitro. Conditioned medium (CM) derived from TPCA-1-loaded hMSCs also showed reduced ability to attract human monocytes and prevented differentiation of human cardiac fibroblasts to myofibroblasts, compared with CM from untreated or TPCA-1-preconditioned hMSCs. Thus, we provide a broadly applicable bioengineering solution to facilitate intracellular sustained release of agents that modulate signaling. We propose that this approach could be harnessed to improve control over MSC secretome post-transplantation, especially to prevent adverse remodeling post-myocardial infarction.United States. National Institutes of Health (HL097172)United States. National Institutes of Health (HL095722

A cell rolling cytometer reveals the correlation between mesenchymal stem cell dynamic adhesion and differentiation state

This communication presents quantitative studies of the dynamic adhesion behavior of mesenchymal stem cells (MSCs) enabled by the combination of cell-surface receptor–ligand interactions and three-dimensional hydrodynamic control by microtopography.National Institutes of Health (U.S.) (Grant HL-095722)National Institutes of Health (U.S.) (Grant HL-097172)National Science Foundation (U.S.) (CAREER Award 0952493)Korea (South). Ministry of Science, ICT and Future Planning (National Research Foundation of Korea. Pioneer Research Center Program 2013M3C1A3064777)National Research Foundation of Korea (Framework of International Cooperation Program 2013K2A1A2053078

Climatic Drivers for Multi-Decadal Shifts in Solute Transport and Methane Production Zones within a Large Peat Basin

Northern peatlands are an important source for greenhouse gases but their capacity to produce methane remains uncertain under changing climatic conditions. We therefore analyzed a 43-year time series of pore-water chemistry to determine if long-term shifts in precipitation altered the vertical transport of solutes within a large peat basin in northern Minnesota. These data suggest that rates of methane production can be finely tuned to multi-decadal shifts in precipitation that drive the vertical penetration of labile carbon substrates within the Glacial Lake Agassiz Peatlands. Tritium and cation profiles demonstrate that only the upper meter of these peat deposits was flushed by downwardly moving recharge from 1965 through 1983 during a Transitional Dry-to-Moist Period. However, a shift to a moister climate after 1984 drove surface waters much deeper, largely flushing the pore waters of all bogs and fens to depths of 2 m. Labile carbon compounds were transported downward from the rhizosphere to the basal peat at this time producing a substantial enrichment of methane in Delta C-14 with respect to the solid-phase peat from 1991 to 2008. These data indicate that labile carbon substrates can fuel deep production zones of methanogenesis that more than doubled in thickness across this large peat basin after 1984. Moreover, the entire peat profile apparently has the capacity to produce methane from labile carbon substrates depending on climate-driven modes of solute transport. Future changes in precipitation may therefore play a central role in determining the source strength of peatlands in the global methane cycle

Sulfa or Sulfone Prophylaxis and Geographic Region Predict Mutations in the Pneumocystis carinii Dihydropteroate Synthase Gene

To determine factors associated with mutations in the Pneumocystis carinii dihydropteroate synthase (DHPS) gene, a prospective study of human immunodeficiency virus (HIV)-infected patients with confirmed P. carinii pneumonia was conducted in Atlanta, Seattle, and San Francisco. Clinical information was obtained from patient interview and chart abstraction. DHPS genotype was determined from DNA sequencing. Overall, 76 (68.5%) of 111 patients had a mutant DHPS genotype, including 22 (81.5%) of 27 patients from San Francisco. In multivariate analysis, sulfa or sulfone prophylaxis and study site were independent predictors of a mutant genotype. Fourteen (53.8%) of 26 patients who were newly diagnosed with HIV infection and had never taken prophylaxis had a mutant genotype. The significance of geographic location as a risk factor for mutant genotype and the high proportion of mutant genotypes among persons never prescribed prophylaxis, including those newly diagnosed with HIV infection, provide indirect evidence that these mutations are transmitted from person to person either directly or through a common environmental source

Confounding factors in diagnosing brain death: a case report

BACKGROUND: Brain death is strictly defined medically and legally. This diagnosis depends on three cardinal neurological features: coma, absent brainstem reflexes, and apnea. The diagnosis can only be made, however, in the absence of intoxication, hypothermia, or certain medical illnesses. CASE PRESENTATION: A patient with severe hypoxic-ischemic brain injury met the three cardinal neurological features of brain death but concurrent profound hypothyroidism precluded the diagnosis. Our clinical and ethical decisions were further challenged by another facet of this complex case. Although her brain damage indicated a hopeless prognosis, we could not discontinue care based on futility because the only known surrogate was mentally retarded and unable to participate in medical planning. CONCLUSION: The presence of certain medical conditions prohibits a diagnosis of brain death, which is a medicolegal diagnosis of death, not a prediction or forecast of future outcome. While prognostication is important in deciding to withdraw care, it is not a component in diagnosing brain death

Costs of Illness in the 1993 Waterborne Cryptosporidium Outbreak, Milwaukee, Wisconsin

To assess the total medical costs and productivity losses associated with the 1993 waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, including the average cost per person with mild, moderate, and severe illness, we conducted a retrospective cost-of-illness analysis using data from 11 hospitals in the greater Milwaukee area and epidemiologic data collected during the outbreak. The total cost of outbreak-associated illness was $96.2 million:$31.7 million in medical costs and $64.6 million in productivity losses. The average total costs for persons with mild, moderate, and severe illness were$116, $475, and$7,808, respectively. The potentially high cost of waterborne disease outbreaks should be considered in economic decisions regarding the safety of public drinking water supplies

Demonstrating the effectiveness of the fundamentals of robotic surgery (FRS) curriculum on the RobotiX Mentor Virtual Reality Simulation Platform

Fundamentals of robotic surgery (FRS) is a proficiency-based progression curriculum developed by robotic surgery experts from multiple specialty areas to address gaps in existing robotic surgery training curricula. The RobotiX Mentor is a virtual reality training platform for robotic surgery. Our aims were to determine if robotic surgery novices would demonstrate improved technical skills after completing FRS training on the RobotiX Mentor, and to compare the effectiveness of FRS across training platforms. An observational, pre-post design, multi-institutional rater-blinded trial was conducted at two American College of Surgeons Accredited Education Institutes-certified simulation centers. Robotic surgery novices (n = 20) were enrolled and trained to expert-derived benchmarks using FRS on the RobotiX Mentor. Participants’ baseline skill was assessed before (pre-test) and after (post-test) training on an avian tissue model. Tests were video recorded and graded by blinded raters using the Global Evaluative Assessment of Robotic Skills (GEARS) and a 32-criteria psychomotor checklist. Post hoc comparisons were conducted against previously published comparator groups. On paired-samples T tests, participants demonstrated improved performance across all GEARS domains (p < 0.001 to p = 0.01) and for time (p < 0.001) and errors (p = 0.003) as measured by psychometric checklist. By ANOVA, improvement in novices’ skill after FRS training on the RobotiX Mentor was not inferior to improvement reported after FRS training on previously published platforms. Completion of FRS on the RobotiX Mentor resulted in improved robotic surgery skills among novices, proving effectiveness of training. These data provide additional validity evidence for FRS and support use of the RobotiX Mentor for robotic surgery skill acquisition