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Medical applications of nanotechnology typically focus on drug delivery and biosensors. Here, we combine nanotechnology and bioengineering to demonstrate that nanoparticles can be used to remotely regulate protein production in vivo. We decorated a modified temperature-sensitive channel, TRPV1, with antibody-coated iron oxide nanoparticles that are heated in a low-frequency magnetic field. When local temperature rises, TRPV1 gates calcium to stimulate synthesis and release of bioengineered insulin driven by a Ca2+-sensitive promoter. Studying tumor xenografts expressing the bioengineered insulin gene, we show that exposure to radio waves stimulates insulin release from the tumors and lowers blood glucose in mice. We further show that cells can be engineered to synthesize genetically encoded ferritin nanoparticles and inducibly release insulin. These approaches provide a platform for using nanotechnology to activate cells.

Targeted, temporally regulated neural modulation is invaluable in determining the physiological roles of specific neural populations or circuits. Here we describe a system for non-invasive, temporal activation or inhibition of neuronal activity in vivo and its use to study central nervous system control of glucose homeostasis and feeding in mice. We are able to induce neuronal activation remotely using radio waves or magnetic fields via Cre-dependent expression of a GFP-tagged ferritin fusion protein tethered to the cation-conducting transient receptor potential vanilloid 1 (TRPV1) by a camelid anti-GFP antibody (anti-GFP–TRPV1)1. Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase–Cre mice, which express Cre in glucose-sensing neurons2. Acute activation of glucose-sensing neurons in this region increases plasma glucose and glucagon, lowers insulin levels and stimulates feeding, while inhibition reduces blood glucose, raises insulin levels and suppresses feeding. These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behaviour. The method we employ obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

The autonomic nervous system regulates fuel availability and energy storage in the liver, adipose tissue, and other organs; however, the molecular components of this neural circuit are poorly understood. We sought to identify neural populations that project from the CNS indirectly through multisynaptic pathways to liver and epididymal white fat in mice using pseudorabies virus strains expressing different reporters together with BAC transgenesis and immunohistochemistry. Neurons common to both circuits were identified in subpopulations of the paraventricular nucleus of the hypothalamus (PVH) by double labeling with markers expressed in viruses injected in both sites. The lateral hypothalamus and arcuate nucleus of the hypothalamus and brainstem regions (nucleus of the solitary tract and A5 region) also project to both tissues but are labeled at later times. Connections from these same sites to the PVH were evident after direct injection of virus into the PVH, suggesting that these regions lie upstream of the PVH in a common pathway to liver and adipose tissue (two metabolically active organs). These common populations of brainstem and hypothalamic neurons express neuropeptide Y and proopiomelanocortin in the arcuate nucleus, melanin-concentrating hormone, and orexin in the lateral hypothalamus and in the corticotrophin-releasing hormone and oxytocin in the PVH. The delineation of this circuitry will facilitate a functional analysis of the possible role of these potential commandlike neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.autonomic nervous system | neuronal tracing | pseudorabies virus T he autonomic nervous system plays a prominent role in modulating carbohydrate and lipid metabolism. The original theories of sympathetic activation (1) proposed a body-wide increase in sympathetic outflow. However, later studies suggested differential sympathetic activation on specific organs through distinct autonomic projections, which permits more finely tuned control of metabolism (2, 3).The liver and adipose tissue play important roles in fuel storage and release. These organs respond to altered energy availability with a set of homeostatic responses mediated by humoral factors and autonomic outflow. For example, activation of hepatic sympathetic aminergic and peptidergic innervation increases glucose output and modulates fatty acid transport. Conversely, parasympathetic activity decreases hepatic glucose output and increases carbohydrate storage (4). Likewise, the sympathetic innervation of white adipose tissue induces lipolysis (5) and alters glucose uptake. Thus, in general, parasympathetic activity favors fuel storage, whereas sympathetic activity increases the fuel available for immediate use.Many CNS regions regulate autonomic outflow to hepatic or adipose tissue to influence peripheral energy homeostasis. In particular, the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), and nucleus of the solitary tract (NTS) (6)...

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We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

The central nervous system relies on a continual supply of glucose, and must be able to detect glucose levels and regulate peripheral organ functions to ensure that its energy requirements are met. Specialized glucose-sensing neurons, first described half a century ago, use glucose as a signal and modulate their firing rates as glucose levels change. Glucose-excited neurons are activated by increasing glucose concentrations, while glucose-inhibited neurons increase their firing rate as glucose concentrations fall and decrease their firing rate as glucose concentrations rise. Glucose-sensing neurons are present in multiple brain regions and are highly expressed in hypothalamic regions, where they are involved in functions related to glucose homeostasis. However, the roles of glucose-sensing neurons in healthy and disease states remain poorly understood. Technologies that can rapidly and reversibly activate or inhibit defined neural populations provide invaluable tools to investigate how specific neural populations regulate metabolism and other physiological roles. Optogenetics has high temporal and spatial resolutions, requires implants for neural stimulation, and is suitable for modulating local neural populations. Chemogenetics, which requires injection of a synthetic ligand, can target both local and widespread populations. Radio- and magnetogenetics offer rapid neural activation in localized or widespread neural populations without the need for implants or injections. These tools will allow us to better understand glucose-sensing neurons and their metabolism-regulating circuits.

Stabbing with a kitchen knife is a common method of homicide in Europe. Serrated knives may leave tool markings (striations) in tissues. Documentation of striations is necessary for their use as forensic evidence. Traditional methods (physical casting and photography) have significant limitations, and micro-computed tomography (micro-CT) has been trialled in cartilage to Bvirtually cast^wounds. Previous research has shown the proportion of striations in cartilage falls following decomposition. This project has investigated the effects of taphonomic alteration and documentation methods of striations in porcine skin. Fresh, decomposed, mummified, burnt and waterlogged stab wounds in a porcine analogue were excised and imaged using photography, stereo-optical microscopy and micro-CT. The proportion of striations in each taphonomic group was determined from the images by independent analysts. Striations were observed more frequently in serrated blade wounds, although they were also identified in non-serrated blade wounds. The proportion of wounds showing striations declined following decomposition. An inversely proportional linear correlation between advancing decomposition and proportion of striations existed. Dehydration (mummification and burning) rendered serrated and nonserrated blade wounds indistinguishable. Water composition affected the preservation of striations. Identification of striations gradually declined after decomposition in tap water, but persisted to a point when left in brackish water. All three techniques imaged striations; however, the optimum technique was stereo-optical microscopy due to practical advantages and specific limitations affecting photography and micro-CT. This study demonstrates the effects of taphonomic alteration on striations and suggests stereo-optical microscopy is the optimum method for their documentation.