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Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

Cardiomyogenesis, the process by which the body generates cardiomyocytes, is poorly understood. We have recently shown that Sfrp2 promotes cardiomyogenesis in vitro. The objective of this study was to determine if Sfrp2 would similarly promote cardiomyogenesis in vivo. To test this hypothesis, we tracked multipotent cKit(+) cells in response to Sfrp2 treatment. In control adult mice, multipotent cKit(+) cells typically differentiated into endothelial cells but not cardiomyocytes. In contrast, Sfrp2 switched the fate of these cells. Following Sfrp2 injection, multipotent cKit(+) cells differentiated solely into cardiomyocytes. Sfrp2-derived cardiomyocytes integrated into the myocardium and exhibited identical physiological properties to preexisting native cardiomyocytes. The ability of Sfrp2 to promote cardiomyogenesis was further supported by tracking EdU-labeled cells. In addition, Sfrp2 did not promote the formation of new cardiomyocytes when the cKit(+) cell population was selectively ablated in vivo using a diphtheria toxin receptor-diphtheria toxin model. Notably, Sfrp2-induced cardiomyogenesis was associated with significant functional improvements in a cardiac injury model. In summary, our study further demonstrates the importance of Sfrp2 in cardiomyogenesis.

The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the development of new antitubercular agents, preferably working

Summary Background Benzodiazepine-refractory, or established, status epilepticus is thought to be of similar pathophysiology in children and adults, but differences in underlying aetiology and pharmacodynamics might differentially affect response to therapy. In the Established Status Epilepticus Treatment Trial (ESETT) we compared the efficacy and safety of levetiracetam, fosphenytoin, and valproate in established status epilepticus, and here we describe our results after extending enrolment in children to compare outcomes in three age groups. Methods In this multicentre, double-blind, response-adaptive, randomised controlled trial, we recruited patients from 58 hospital emergency departments across the USA. Patients were eligible for inclusion if they were aged 2 years or older, had been treated for a generalised convulsive seizure of longer than 5 min duration with adequate doses of benzodiazepines, and continued to have persistent or recurrent convulsions in the emergency department for at least 5 min and no more than 30 min after the last dose of benzodiazepine. Patients were randomly assigned in a response-adaptive manner, using Bayesian methods and stratified by age group ( 65 years), to levetiracetam, fosphenytoin, or valproate. All patients, investigators, study staff, and pharmacists were masked to treatment allocation. The primary outcome was absence of clinically apparent seizures with improved consciousness and without additional antiseizure medication at 1 h from start of drug infusion. The primary safety outcome was life-threatening hypotension or cardiac arrhythmia. The efficacy and safety outcomes were analysed by intention to treat. This study is registered in ClinicalTrials.gov , NCT01960075 . Findings Between Nov 3, 2015, and Dec 29, 2018, we enrolled 478 patients and 462 unique patients were included: 225 children (aged 65 years). 175 (38%) patients were randomly assigned to levetiracetam, 142 (31%) to fosphenyltoin, and 145 (31%) were to valproate. Baseline characteristics were balanced across treatments within age groups. The primary efficacy outcome was met in those treated with levetiracetam for 52% (95% credible interval 41–62) of children, 44% (33–55) of adults, and 37% (19–59) of older adults; with fosphenytoin in 49% (38–61) of children, 46% (34–59) of adults, and 35% (17–59) of older adults; and with valproate in 52% (41–63) of children, 46% (34–58) of adults, and 47% (25–70) of older adults. No differences were detected in efficacy or primary safety outcome by drug within each age group. With the exception of endotracheal intubation in children, secondary safety outcomes did not significantly differ by drug within each age group. Interpretation Children, adults, and older adults with established status epilepticus respond similarly to levetiracetam, fosphenytoin, and valproate, with treatment success in approximately half of patients. Any of the three drugs can be considered as a potential first-choice, second-line drug for benzodiazepine-refractory status epilepticus. Funding National Institute of Neurological Disorders and Stroke, National Institutes of Health.

The development of biased agonist drugs is widely recognized to be important for the treatment of many diseases, including cardiovascular disease. While GPCR biased agonism has been heavily characterized there is a distinct lack of information with respect to RTK biased agonism both in the identification of biased agonists as well as their attendant mechanisms. One such RTK, the Insulin-like Growth Factor 1 Receptor (IGF1R) plays an important role in a range of biological and disease processes. The micropeptide LL37 has been described as a biased agonist of the IGF1R. We were interested to further understand the mechanism by which LL37 promotes biased signaling through the IGF1R. We found that LL37 biased agonism is dependent on β-arrestin 2. Moreover, BRET assays indicated that LL37 biased agonism is explained by the inability of LL37 to promote the recruitment of IRS1 to the IGF1R compared to IGF1. LL37 promotes an altered association of IGF1R with GRK6, which could also serve as an explanation for bias. We also demonstrated a functional consequence of this bias by showing that while LL37 can promote cell proliferation, it does not induce protein synthesis, unlike IGF1, which does both. We have recently identified HASF, a natural protein released by mesenchymal stem cells, as a novel ligand of the IGF1R. HASF is a paracrine factor with potent cardioprotective and cardio-regenerative properties which also acts via IGF1R biased signaling, preferentially activated ERK over Akt.

We have recently shown that hypoxia and Akt-induced stem cell factor (HASF) protects the heart from ischemia-induced damage and promotes cardiomyocyte proliferation. While we have identified certain signaling pathways responsible for these protective effects, the receptor mediating these effects was unknown. Here, we undertook studies to identify the HASF receptor. A yeast two-hybrid screen identified a partial fragment of insulin-like growth factor 1 receptor (IGF1R) as a binding partner of HASF. Subsequent co-immunoprecipitation experiments showed that HASF bound to full-length IGF1R. Binding assays revealed a high affinity of HASF for IGF1R. The treatment of neonatal ventricular cardiomyocytes with HASF resulted in the phosphorylation of IGF1R and other proteins known to be involved in IGF1R-mediated signaling pathways. HASF-mediated ERK activation was abrogated by IGF1R pharmacological inhibitors and siRNAs that targeted IGF1R. However, siRNA-mediated knockdown of either IGF2R or the insulin receptor had no effect on HASF-induced cell signaling. Additionally, pharmacologic inhibition of IGF1R impeded HASF's ability to induce cardiomyocyte proliferation. Finally, we documented that in vivo deletion of the IGF1R completely abolished the ability of HASF to promote cardiomyocyte proliferation in an overexpression mouse model providing further evidence in vivo that the IGF1R is the functional receptor for HASF.

Background\ud The choice of drugs for patients with status epilepticus that is refractory to treatment with benzodiazepines has not been thoroughly studied.\ud \ud Methods\ud In a randomized, blinded, adaptive trial, we compared the efficacy and safety of three intravenous anticonvulsive agents — levetiracetam, fosphenytoin, and valproate — in children and adults with convulsive status epilepticus that was unresponsive to treatment with benzodiazepines. The primary outcome was absence of clinically evident seizures and improvement in the level of consciousness by 60 minutes after the start of drug infusion, without additional anticonvulsant medication. The posterior probabilities that each drug was the most or least effective were calculated. Safety outcomes included life-threatening hypotension or cardiac arrhythmia, endotracheal intubation, seizure recurrence, and death.\ud \ud Results\ud A total of 384 patients were enrolled and randomly assigned to receive levetiracetam (145 patients), fosphenytoin (118), or valproate (121). Reenrollment of patients with a second episode of status epilepticus accounted for 16 additional instances of randomization. In accordance with a prespecified stopping rule for futility of finding one drug to be superior or inferior, a planned interim analysis led to the trial being stopped. Of the enrolled patients, 10% were determined to have had psychogenic seizures. The primary outcome of cessation of status epilepticus and improvement in the level of consciousness at 60 minutes occurred in 68 patients assigned to levetiracetam (47%; 95% credible interval, 39 to 55), 53 patients assigned to fosphenytoin (45%; 95% credible interval, 36 to 54), and 56 patients assigned to valproate (46%; 95% credible interval, 38 to 55). The posterior probability that each drug was the most effective was 0.41, 0.24, and 0.35, respectively. Numerically more episodes of hypotension and intubation occurred in the fosphenytoin group and more deaths occurred in the levetiracetam group than in the other groups, but these differences were not significant.\ud \ud Conclusions\ud In the context of benzodiazepine-refractory convulsive status epilepticus, the anticonvulsant drugs levetiracetam, fosphenytoin, and valproate each led to seizure cessation and improved alertness by 60 minutes in approximately half the patients, and the three drugs were associated with similar incidences of adverse events. (Funded by the National Institute of Neurological Disorders and Stroke; ESETT ClinicalTrials.gov number, NCT01960075. opens in new tab.)

The ability to detect latent fingermarks on porous surfaces, such as paper-based documents, is extremely important in resolving criminal cases. Detection methods that target amino acids present in latent fingermark deposits have achieved widespread use due to the binding of these acids to paper fibres resulting in a good representation of the fingermark. This communication is the first report of 6-(N,N-dimethylamino)fulvene (DMAF) as a novel reagent for the detection of latent fingermarks on porous surfaces. Through observation of undergraduate students showing poor laboratory hygiene, we noted that exposure to DMAF leads to brown staining on the students’ skin. We discovered that indeed it can develop latent fingermarks on paper surfaces as pale pink impressions, which are luminescent when illuminated at 505 nm and viewed through orange goggles. The procedure is simple, requiring treatment of the specimen with a solution of DMAF in hexane with subsequent heat treatment. Preliminary experiments indicate that DMAF is reacting with the amino acids present in the latent fingermark.

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells which give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation JG cells undergo expansion. Gene expression profiling comparing resident renal Mesenchymal Stromal Cells (MSCs) with JG cells indicate that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin producing cells. In vivo, Sox6 expression is up-regulated during JG cell expansion. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin expressing cells normally seen during JG cell expansion as well as the typical increase in renin. These results support a previously undefined role for Sox6 in renin expression during normal and pathophysiological conditions.

Wnt signaling has recently emerged as an important regulator of cardiac progenitor cell proliferation and differentiation, but the exact mechanisms by which Wnt signaling modulates these effects are not known. Understanding these mechanisms is essential for advancing our knowledge of cardiac progenitor cell biology and applying this knowledge to enhance cardiac therapy. Here, we explored the effects of Sfrp2, a canonical Wnt inhibitor, in adult cardiac progenitor cell (CPC) differentiation and investigated the molecular mechanisms involved. Our data show that Sfrp2 treatment can promote differentiation of CPCs after ischemia-reperfusion injury. Treatment of CPCs with Sfrp2 inhibited CPC proliferation and primed them for cardiac differentiation. Sfrp2 binding to Wnt6 and inhibition of Wnt6 canonical pathway was essential for the inhibition of CPC proliferation. This inhibition of Wnt6 canonical signaling by Sfrp2 was important for activation of the non-canonical Wnt/Planar Cell Polarity (PCP) pathway through JNK, which in turn induced expression of cardiac transcription factors and CPC differentiation. Taken together, these results demonstrate a novel role of Sfrp2 and Wnt6 in regulating the dynamic process of CPC proliferation and differentiation, as well as providing new insights into the mechanisms of Wnt signaling in cardiac differentiation.

Background Little is known about hemodynamics in adult, out-of-hospital (OHCA) patients following return of spontaneous circulation (ROSC). A 1994 study when “high-dose epinephrine” use was common showed consistently elevated systemic vascular resistance (SVR) lasting ≥6 h in 49 adult patients after return of spontaneous circulation (ROSC). Study aim To characterize hemodynamic abnormalities in adult OHCA patients soon after ROSC. Our hypothesis was that, unlike the consistently high SVR values reported when “high-dose” epinephrine was in common use, there would be a more heterogenous distribution of SVR values using current adrenergic therapy. Methods We included adult, OHCA patients transported by paramedics to the Emergency Department (ED) post-ROSC. Children, prisoners, pregnant women, and those with ongoing CPR or arrest due to traumatic injury were excluded. Hemodynamics were recorded non-invasively as soon as feasible after ED arrival but were not used to influence therapy, which was guided by clinical judgment of treating ED physicians. Results Hemodynamics were recorded on 30 patients 20 [16,25] minutes after ED arrival: 50% had a normal SVR, 30% had a high SVR, and 20% had a low SVR. There was no difference in survival to admission among groups, although there was a difference among groups in survival to discharge. Comparing the low SVR group vs the combined normal and high group revealed a trend for fewer 0/6 (0%) low vs. 10/24 (42%) normal or high SVR patients surviving to hospital discharge (p = .053). Conclusion A heterogeneous range of hemodynamic states exist post-ROSC rather than consistent vasoconstriction. Adequately powered, randomized clinical trials will be needed to determine whether noninvasively-derived, hemodynamic-directed therapy can play a role in improving neurologically-intact survival following OHCA in adults.

Rationale : Cardiac progenitor cells (CPCs) are thought to differentiate into the major cell types of the heart: cardiomyocytes, smooth muscle cells, and endothelial cells. We have recently identified ABI family, member 3 (NESH) binding protein (Abi3bp) as a protein important for mesenchymal stem cell biology. Because CPCs share several characteristics with mesenchymal stem cells, we hypothesized that Abi3bp would similarly affect CPC differentiation and proliferation. Objective : To determine whether Abi3bp regulates CPC proliferation and differentiation. Methods and Results : In vivo, genetic ablation of the Abi3bp gene inhibited CPC differentiation, whereas CPC number and proliferative capacity were increased. This correlated with adverse recovery after myocardial infarction. In vitro, CPCs, either isolated from Abi3bp knockout mice or expressing an Abi3bp shRNA construct, displayed a higher proliferative capacity and, under differentiating conditions, reduced expression of both early and late cardiomyocyte markers. Abi3bp controlled CPC differentiation via integrin-β1, protein kinase C-ζ, and v-akt murine thymoma viral oncogene homolog. Conclusions : We have identified Abi3bp as a protein important for CPC differentiation and proliferation.

Rationale: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. Objective: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. Methods and Results: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway–specific inhibitors and silencing of the Cdk7 gene. Conclusions: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Hypertension afflicts 33% of the U.S. adult population. Despite current treatments, approximately 50% of people are unresponsive to treatment. There is a critical need to develop new therapies to treat this disease and its complications. The Renin Angiotensin Aldosterone System plays a key role in regulating blood pressure in humans. Renin controls the rate-limiting step in the conversion of angiotensinogen to angiotensin I. In adults, renin is produced and stored by Juxtaglomerular (JG) cells in the kidney. However, the transcriptional mechanisms that govern formation of renin expressing cells under normal or pathophysiological conditions remain poorly understood. During blood pressure changes the number of adult renal cells expressing renin increase through a process known as JG cell expansion. We found that this process involves differentiation of mesenchymal stromal-like cells (MSC) to renin expressing cells among others. We aim to determine new regulators of renin expression and blood pressure control. Renin expression in vitro was induced by treatment of MSCs with 3-isobutyl-1-methylxanthine (IBMX) and Forskolin. MSCs were transduced with lentivirus carrying vectors Sox6 shRNA or control shRNA. A new transgenic mouse, in which Sox6 is deleted specifically in renin expressing cells (Ren1dCre/Sox6 fl/fl ), was used to study the impact of Sox6 in renin expression in vivo . Gene array comparing renal MSC and JG cells identified potential genes that control MSC differentiation, including Sox6. In vitro silencing of Sox6 by shRNA decreased differentiation of renal MSCs into renin producing cells (3.5 fold, n=4, P= 0.01). Preliminary IHC data showed that the transcription factor Sox6 is expressed by renin producing cells in the adult kidney during JG cell expansion. Plasma renin concentration (PRC) increased during JG cell expansion (induced with low sodium diet and furosemide) in wild type mice (27-fold), whereas in mice lacking Sox6 in JG cells PRC was as low as non-treated mice. We conclude that Sox6 has a novel role in modulating renin expression and thereby can contribute to renal physiology, opening new possibilities of addressing questions regarding physiological regulation of renin and hypertension.

We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. However, direct reprogramming strategies are inefficient and slow. Moving towards the eventual goal of clinical application it is necessary to develop new methodologies to overcome these limitations. Here, we report the identification of a specific media composition, reprogramming media (RM), which augmented the effect of miR combo by 5–15-fold depending upon the cardiac marker tested. RM alone was sufficient to strongly induce cardiac gene and protein expression in neonatal tail-tip as well as cardiac fibroblasts. Expression of pluripotency markers Nanog, Oct4, Sox2 and Klf4 was significantly enhanced by RM, with miR combo augmenting the effect further. Knockdown of Nanog by siRNA inhibited the effect of RM on cardiac gene expression. Removal of insulin-transferrin-selenium completely inhibited the effect of reprogramming media upon cardiac gene expression and the addition of selenium to standard culture media recapitulated the effects of RM. Moreover, selenium enhanced the reprogramming efficiency of miR combo.

Introduction: Resident c-Kit + cardiac progenitor cells (CPCs) differentiation into cardiomyocytes in vivo is controversial. Using lineage tracing and gene ablation techniques we documented that the Wnt signaling modulator Sfrp2 substantially increased differentiation of c-Kit + CPCs into cardiomyocytes in post-MI heart and restored cardiac function. Methods: c-Kit-CreERT2/mTeG mice were used where the c-Kit promoter drives expression of a tamoxifen (TX)-inducible Cre. Upon TX treatment Cre-mediated recombination drives expression of eGFP exclusively in c-Kit cells. Mice were crossed with a Diphtheria toxin receptor (DT) strain to selectively ablate heart c-Kit cells. Mice were subjected to myocardial infarction. 2 days later, mice were injected with Sfrp2 (0.5 μg) or saline at the infarct border zone. Heart tissue was analyzed 2 months post-MI for eGFP expression and various markers by microscopy. Results: c-Kit-CreERT2/mTeG model was tested. eGFP expression had no effect on in vitro c-Kit CPC differentiation or proliferation. In TX treated mice, >90% of c-Kit+ cells were eGFP+. No eGFP+ c-Kit+ cells were seen in vehicle-only mice (N=6, p95% N=4, P=0.01). Sfrp2 induction of c-Kit differentiation to cardiomyocytes and functional improvements were completely ablated by DT. Conclusion: Using c-Kit lineage-tracing and genetic ablation we have identified that these cells are important in cardiomyocyte generation in vivo in response to Sfrp2 and improves cardiac function following MI.

Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell–like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate bone resorption in the rat calvarial model.

A novel series of ketoamide-based inhibitors of cathepsin K has been identified. Modifications to P(2) and P(3) elements were crucial to enhancing inhibitory activity. Although not optimized, a selected inhibitor was effective in attenuating type I collagen hydrolysis in a surrogate assay of bone resorption.

Brooks and Orr [R.R. Brooks and N. Orr, A model for mobile code using interacting automata. IEEE Trans Mobile Computing 2002, 1(4)] present a model for analysis and simulation of mobile code systems based on cellular automata (CA) abstractions. One flaw with that article was a lack of experimental support showing that CA can model IP networks. This article presents CA models, consistent with those in the work of Brooks and Orr, that model the transport layer of IP networks. We show how these models may be generalized for more complicated network topologies. We provide quantitative results comparing the quality of our CA implementation versus the standard network modeling tool ns-2. The results from the CA model are qualitatively similar to ns-2, but the CA simulation runs significantly faster and scales better. © 2004 Wiley Periodicals, Inc. Complexity 9:32–40, 2004

Background: We have previously shown that CD44+ MSCs exist in the adult kidney and that under chronic sodium deprivation these cells migrate to the juxtaglomerular (JG) area and differentiate to new renin expressing cells. The mechanisms mediating these biological processes remain elusive. Renin expression and release by JG cells is initiated at the Macula densa (MD) primarily by the release of PGE2. Interestingly, renal MSC expresses the PGE2 receptor EP4. Here, we hypothesized that MD-derived PGE2 acting via the EP4 is responsible for the migration of renal MSCs and their differentiation to renin expressing cells. Methods: MSCs were isolated by FACS from the kidney of C57BL/6 adult mice and co-cultured in a transwell system with the MD cell line MMDD1 at normal or low NaCl medium. For the in vivo study, wild type and EP4 knockout mice were fed a low salt diet and given furosemide to induce JG hyperplasia. Renin, CD44 and B-catenin stabilization were detected by immunochemistry staining and Western blot analysis. Results: Low salt stimulation of MMDD1 cells significantly increased MSC migration in transwell assays (P Conclusion: The MD/ PGE2/ EP4 axis plays a key role in the activation of MSCs and their differentiation to renin producing cells. This process could potentially be an important physiologic mechanism of maintaining blood pressure under conditions of sodium deprivation.

Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

Museums and galleries have in recent years spent considerable time and effort in digitisation projects, yet the software resources available to fully explore their collections are still largely unsatisfactory. In a joint project between the new media company Deep Visuals, the Scott Polar Research Institute, and Anglia Ruskin University we are developing a new browsing system aimed at enabling museum visitors to experience more fully the museum's digital assets beyond the exhibited artefacts.

The U.S. Department of Homeland Security has targeted bulk food contamination as a focus for attention because it poses a high consequence health threat. The current manual methods used for securing milk during transport are paper intensive and prone to errors. The bulk milk transportation sector needs a security enhancement that will reduce recording errors and enable normal transport activities while providing security. The goal was to develop a milk transport security system to meet these demands; demonstrate it; and transfer the technology to the public.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinasein vitroat 0.06 μM and was inactive against 26 other kinases. GW2580 at 1 μM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophagesin vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cellsin vivomakes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Starting from potent aldehyde inhibitors with poor drug properties, derivatization to semicarbazones led to the identification of a series of semicarbazone-based cathepsin K inhibitors with greater solubility and better pharmacokinetic profiles than their parent aldehydes. Furthermore, a representative semicarbazone inhibitor attenuated bone resorption in an ex vivo rat calvarial bone resorption model. However, based on enzyme inhibition comparisons at neutral pH, semicarbazone hydrolysis rates, and 13 C NMR experiments, these semicarbazones probably function as prodrugs of aldehydes.

An orally bioavailable series of ketoamide-based cathepsin K inhibitors with good pharmacokinetic properties has been identified. Starting from a potent inhibitor endowed with poor drug properties, conformational constraint of the P2-P3 linker and modifications to P 1 ′ elements led to an enhancement in potency, solubility, clearance, and bioavailability. These optimized inhibitors attenuated bone resorption in a rat TPTX hypocalcemic bone resorption model.

Using differential display polymerase chain reaction, we cloned a novel cDNA named RoBo-1 from rat tibia. RoBo-1 is abundantly expressed in bone, including the hypertrophic chondrocytes of the growth plate where cartilage is remodeled into bone. RoBo-1 mRNA expression increased in response to two modulators of bone metabolism, estradiol and intermittent mechanical loading, suggesting a role in bone homeostasis. The 1.6-kilobase cDNA encodes a 240-amino acid protein with a cysteine spacing pattern, suggesting that RoBo-1 is a novel member of the urokinase plasminogen activator receptor/CD59/Ly-6/snake toxin family. Furthermore, the C-terminal contains a glycosyl-phosphatidylinositol attachment site, suggesting that it is a cell surface protein similar to other mammalian members of this family. The strongest homology of RoBo-1 is to the snake serum-derived phospholipase A2 inhibitors, which uniquely contain two of the cysteine domains but are secreted proteins. Interestingly, RoBo-1 is likely the first membrane-anchored member of this family containing two cysteine domains. Thus, the tissue specificity, responsiveness to bone protective mediators, along with its relationship to the multifunctional urokinase plasminogen activator receptor/CD59/Ly-6/snake toxin family suggests that RoBo-1 may play a novel role in the growth or remodeling of bone.