The Prying Nose: Florida v. Jardines and Warrantless Dog-Sniff Tests on Private Property

This commentary previews an upcoming Supreme Court case, Florida v. Jardines, in which the Court will decide whether a dog-sniff test at the front door of a home constitutes a Fourth Amendment search. The case asks the Court to resolve its prior decisions holding that dog-sniff tests are minimally intrusive when conducted in public with its decisions affording higher protections for searches of private residences

Mega, Digital Storage Lockers, and the DMCA: Will Innovation Be Stifled by Fears of Piracy?

Kim Dotcom, founder of Megaupload Limited, has been in many news headlines over the past year. Megaupload—one of Dotcom’s many peer-to-peer sharing sites—was the center of controversy, as it allowed users to upload and share all sorts of files, including copyrighted material. After an organized effort by the Department of Justice and several foreign governments, Dotcom was arrested for (secondary) copyright infringement and his site was ultimately shut down. Dotcom has recently launched a new service, MEGA, which he claims will evade copyright laws entirely. Like other well-known cloud-sharing services such as Dropbox and Google Drive, MEGA allows users to upload files and to share them with select users. In an attempt to avoid liability, MEGA locally encrypts all files on the user’s computer before they are uploaded to the site. The private key and public key used to encrypt and decrypt the file are retained solely by the user; MEGA gets no part of that information. This, Dotcom argues, will shift the entirety of the copyright onus to the user. This Issue Brief analyzes the protections afforded cyberlocker services like MEGA by the DMCA, including tensions raised in actual litigation. This Issue Brief argues that, while an ex ante secondary-liability analysis is difficult due to its contextual nature, MEGA’s use of user-controlled encryption (UCE), deduplication, and distributed host servers may lend to an affirmative finding of liability

Therapeutic potential of adipose-derived stromal cells for the treatment of senile osteoporosis

Summary Impaired osteogenic differentiation of resident bone marrow stromal cells (BMSCs) of aged patients with low turnover osteoporosis is considered to be one of the major contributing factors in the development of structural and mechanical deficiencies associated with osteoporotic bone. As such, therapies designed to counteract these effects are currently being pursued with an aim to restoring the stem cell niche and enhancing bone quality. In the current thesis, evidence is provided which supports the use of autologous adipose-derived stromal cells (ASCs) as a means by which to combat age-related bone loss. Initial studies were undertaken to compare the differentiation potential of ASCs from osteoporotic SAMP6 and non-osteoporotic SAMR1 mice in 2D in vitro culture systems. Their ability to differentiate towards osteoblast and adipocyte cell lineages was evaluated by histological, biochemical and molecular techniques. In contrast to BMSCs, the differentiation potential of ASCs was found to be comparable between both strains. Further in vitro studies were performed to assess the osteogenic potential of SAMP6-derived ASCs cultured under 3D conditions using long-term silk fibroin scaffold cultures and short-term scaffold free microtissue spheroid (ASC-MT) cultures. In both cases, ASCs underwent efficient osteogenesis resulting in mineralized tissue formation. In the final study, ASCs and ASC-MT were evaluated for their ability to enhance trabecular bone quality following a single intratibial injection in SAMP6 mice using micro-CT, histological and molecular based analyses. Both ASCs and ASC-MT could be identified within treated bones after 42 days and moreover, resulted in significant improvements in several parameters of trabecular bone quality. Furthermore, human ASCs harvested from osteoporotic patients were also demonstrated as having a positive influence on the osteogenic differentiation potential of dysfunctional osteoporotic patient-derived BMSCs in vitro. In conclusion, these studies have demonstrated that adipose tissue may represent a promising autologous cell source for the development of novel bone regenerative therapeutic strategies for the treatment of age-related bone loss. Zusammenfassung Als einer der Hauptgründe für die strukturellen und mechanischen Defizite des Knochengewebes von Patienten mit altersbedingter Osteoporose wird eine beeinträchtigte Differenzierung von adulten Stammzellen aus dem Knochenmark (bone marrow stromal cells, BMSCs) zu knochenbildenden Osteoblasten angenommen. Dementsprechend werden gegenwärtig Therapieansätze verfolgt, die diese verminderte Differenzierungskapazität von Stammzellen kompensieren und dadurch die Knochenqualität verbessern sollen. Im Rahmen dieser Promotion konnte gezeigt werden, dass autologe adulte Stammzellen aus dem Fettgewebe (adipose-derived stromal cells, ASCs) ein geeignetes Mittel zur Behandlung von altersbedingter Osteoporose darstellen könnten. Zunächst wurde das Differenzierungspotential von ASCs aus osteoporotischen SAMP6- und nicht osteoporotischen SAMR1-Mäusen zu Osteoblasten und Adipozyten in vitro in 2D- Zellkulturen mit Hilfe von histologischen, biochemischen und molekularbiologischen Methoden untersucht. Im Gegensatz zu BMSCs konnte dabei kein Unterschied in der Differenzierungsfähigkeit von ASCs aus osteoporotischen und nicht-osteoporotischen Mäusen festgestellt werden. Daraufhin wurde das osteogene Potential von ASCs aus osteoporotischen SAMP6-Mäusen in 3D-Zellkulturen untersucht. Zum einen wurden die Zellen auf einer Seidenfibroin-Matrix kultiviert, zum anderen Mikrogewebe ohne Matrix (ASC microtissues, ASC-MTs) hergestellt. ASCs zeigten unter beiden Kulturbedingungen eine effiziente Osteogenese mit einhergehender Mineralisierung. Im abschliessenden Teil wurden ASCs und ASC-MT einmalig direkt in die Tibia von SAMP6-Mäusen injiziert und anschliessend die trabekuläre Knochenqualität mittels Mikro- CT sowie histologischen und molekularbiologischen Methoden bestimmt. Sowohl ASCs als auch ASC-MTs konnten noch 42 Tage nach der Injektion in den behandelten Knochen nachgewiesen werden, und ihre Applikation resultierte in einer signifikanten Verbesserung verschiedener Parameter der trabekulären Knochenqualität. Des Weiteren konnte gezeigt werden, dass ASCs osteoporotischer Patienten in vitro einen positiven Einfluss auf die osteogene Differenzierung von dysfunktionellen BMSCs von Osteoporose-Patienten haben. Zusammenfassend kann gesagt werden, dass das Fettgewebe eine vielversprechende, autologe Quelle für adulte Stammzellen zur Entwicklung von neuen Therapieansätzen zur Behandlung von altersbedingter Osteoporose darstellen könnte

Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation

Abstract Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions.</jats:p

ARTD1 regulates osteoclastogenesis and bone homeostasis by dampening NF-κB-dependent transcription of IL-1β

While ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) and its enzymatic activity have been shown to be important for reprogramming and differentiation of cells, such as during adipogenesis, their role and mechanism in regulating osteoclastogenesis and bone homeostasis are largely unknown. Here, in cell culture-based RANKL-induced osteoclastogenesis models, we show that silencing of ARTD1 or inhibition of its enzymatic activity enhances osteoclast differentiation and function. As a consequence of ARTD1 silencing or inhibition, the recruitment of p65/RelA to the IL-1β promoter, which is associated with transcriptionally active histone marks, IL-1β expression and inflammasome-dependent secretion of IL-1β are enhanced. This subsequently promotes sustained induction of the transcription factor Nfatc1/A and osteoclastogenesis in an autocrine manner via the IL-1 receptor. In vivo, Artd1-deficient mice display significantly decreased bone mass as a consequence of increased osteoclast differentiation. Accordingly, the expression of osteoclast markers is enhanced in mutant compared to wild-type mice. Together, these results indicate that ARTD1 controls osteoclast development and bone remodelling via its enzymatic activity by modulating the epigenetic marks surrounding the IL-1β promoter and expression of IL-1β and subsequently also Nfatc1/A

ARTD1-induced poly-ADP-ribose formation enhances PPARγ ligand binding and co-factor exchange

PPARγ-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPARγ target genes by ligand-dependent co-factor exchange. However, how endogenous PPARγ ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown. Moreover, the significance of PAR formation in PPARγ-regulated adipose tissue function is also unknown. Here, we show that inhibition of PAR formation in mice on a high-fat diet reduces weight gain and cell size of adipocytes, as well as PPARγ target gene expression in white adipose tissue. Mechanistically, topoisomerase II activity induces ARTD1 recruitment to PPARγ target genes, and ARTD1 automodification enhances ligand binding to PPARγ, thus promoting sufficient transcriptional co-factor exchange in adipocytes. Thus, ARTD1-mediated PAR formation during adipogenesis is necessary to adequately convey the low signal of endogenous PPARγ ligand to effective gene expression. These results uncover a new regulatory mechanism of ARTD1-induced ADP-ribosylation and highlight its importance for nuclear factor-regulated gene expression

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix

ARTD1 deletion causes increased hepatic lipid accumulation in mice fed a high-fat diet and impairs adipocyte function and differentiation

ADP-ribosyltransferase Diphtheria toxin-like 1 [ARTD1; formerly called poly-ADP-ribose polymerase 1 (PARP1)] is a chromatin-associated enzyme involved in regulating metabolic homeostasis. The liver is at the core of glucose and lipid metabolism and is significantly affected by obesity and the metabolic syndrome. Here, we show that when fed a high-fat diet (HFD), mice lacking ARTD1 developed exacerbated hepatic steatosis. ARTD1(-/-) mice had a 19% higher liver weight than wild-type (WT) animals and exhibited a significantly increased serum concentration of cholesterol (38%) and impaired glucose tolerance. In addition, adipocyte function and size were significantly reduced in ARTD1(-/-) mice fed an HFD (7794 μm(2) for WT and 5579 μm(2) for ARTD1(-/-) mice). The significantly reduced adipogenic differentiation of adipose-derived stromal cells (ASCs) isolated from ARTD1(-/-) mice (28 vs. 11% Oil red O-positive cells in WT and ARTD1(-/-) ASCs, respectively) suggested that impaired adipogenesis as the underlying cause for this adipose tissue malfunction. This function of ARTD1 was specific for adipogenesis, since osteogenic differentiation was not affected by the ARTD1 deletion. In summary, we show that ARTD1(-/-) mice fed an HFD display impaired adipogenesis and show exacerbated hepatic steatosis, which can have important implications for nonalcoholic fatty liver disease.-Erener, S., Mirsaidi, A., Hesse, M., Tiaden, A. N., Ellingsgaard, H., Kostadinova, R., Donath, M. Y., Richards, P. J., Hottiger, M. O. ARTD1 deletion causes increased hepatic lipid accumulation in mice fed a high-fat diet and impairs adipocyte function and differentiation