Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering

Regulation of type 1 diabetes development and B-cell activation in nonobese diabetic mice by early life exposure to a diabetogenic environment

Microbes, including viruses, influence type 1 diabetes (T1D) development, but many such influences remain undefined. Previous work on underlying immune mechanisms has focussed on cytokines and T cells. Here, we compared two nonobese diabetic (NOD) mouse colonies, NODlow and NODhigh, differing markedly in their cumulative T1D incidence (22% vs. 90% by 30 weeks in females). NODhigh mice harbored more complex intestinal microbiota, including several pathobionts; both colonies harbored segmented filamentous bacteria (SFB), thought to suppress T1D. Young NODhigh females had increased B-cell activation in their mesenteric lymph nodes. These phenotypes were transmissible. Co-housing of NODlow with NODhigh mice after weaning did not change T1D development, but T1D incidence was increased in female offspring of co-housed NODlow mice, which were exposed to the NODhigh environment both before and after weaning. These offspring also acquired microbiota and B-cell activation approaching those of NODhigh mice. In NODlow females, the low rate of T1D was unaffected by cyclophosphamide but increased by PD-L1 blockade. Thus, environmental exposures that are innocuous later in life may promote T1D progression if acquired early during immune development, possibly by altering B-cell activation and/or PD-L1 function. Moreover, T1D suppression in NOD mice by SFB may depend on the presence of other microbial influences. The complexity of microbial immune regulation revealed in this murine model may also be relevant to the environmental regulation of human T1D

How to Reduce Fluid-Injection-Induced Seismicity

The recent growth in energy technologies and the management of subsurface reservoirs has led to increased human interaction with the Earth's crust. One consequence of this is the overall increase of anthropogenic earthquakes. To manage fluid-injection-induced seismicity, in this study, we propose to use an advanced fluid-injection scheme. First, long-term fluid-injection experiments are separated from short-term fluid-injection experiments. Of the short-term experiments, enhanced geothermal systems stimulations have shown a higher propensity to produce larger seismic events compared to hydraulic fracturing in oil and gas. Among the factors discussed for influencing the likelihood of an induced seismic event to occur are injection rate, cumulative injected volume, wellhead pressure, injection depth, stress state, rock type, and proximity to faults. We present and discuss the concept of fatigue hydraulic fracturing at different scales in geothermal applications. In contrast to the conventional hydraulic fracturing with monotonic injection of high-pressure fluids, in fatigue hydraulic fracturing, the fluid is injected in pressure cycles with increasing target pressure, separated by depressurization phases for relaxing the crack tip stresses. During pressurization phases, the target pressure level is modified by pulse hydraulic fracturing generated with a second pump system. This combination of two pumps with multiple-flow rates may allow a more complex fracture pattern to be designed, with arresting and branching fractures, forming a broader fracture process zone. Small-scale laboratory fluid-injection tests on granite cores and intermediate-scale fluid-injection experiments in a hard rock underground test siteare described. At laboratory scale, cyclic fluid-injection tests with acoustic emission analysis arereported with subsequent X-ray CT fracture pattern analysis. At intermediate scale, in a controlled underground experiment at constant depth with well-known stress state in granitic rock, we test advanced fluid-injection schemes. The goal is to optimize the fracture network and mitigate larger seismic events. General findings in granitic rock, independent of scale, are summarized. First, the fracture breakdown pressure in fatigue hydraulic testing is lower than that in the conventional hydraulic fracturing. Second, compared to continuous injection, the magnitude of the largest induced seismic event seems to be systematically reduced by cyclic injection. Third, the fracture pattern in fatigue testing is different from that in the conventional injection tests at high pressures. Cyclic fracture patterns seem to result from chiefly generated low energy grain boundary cracks forming a wider process zone. Fourth, cyclic injection increases the permeability of the system. A combination of cyclic progressive and pulse pressurization leads to the best hydraulic performance of all schemes tested. One advantage of fatigue testing is the fact that this soft stimulation method can be applied in circumstances where the conventional stimulation might otherwise be abandoned based on site-specific seismic hazard estimates.Y