Fabrication of Custom Dynamic Pedorthoses for Clubfoot Correction via Additive-Based Technologies

Additive technologies have created many opportunities to improve the quality of life for patients in a range of medical applications. This paper provides an overview of how several of these technologies were leveraged to transform custom pedorthosis designs into physical, end-use, custom pedorthoses for clubfoot patients. The pedorthoses that were produced are currently being tested on the respective patients for their improvement in mobility and degree of clubfoot correction, and will continue through early 2010. The advantage of this approach is the reduction in labor and the increase in degrees of design freedom available, compared to conventional methods of fabricating orthotic devices. Several new approaches for fabricating custom orthotic devices that were explored, and the related results, are discussed.Mechanical Engineerin

β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated by islet amyloid polypeptide-induced UCH-L1 deficiency.

ObjectiveThe islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system.Research design and methodsWe used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated.ResultsWe report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis.ConclusionsOur results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability

Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas

Vesicular monoamine transporter 2 (VMAT2) is expressed in pancreatic beta cells and has recently been proposed as a target for measurement of beta cell mass in vivo. We questioned, (1) What proportion of beta cells express VMAT2? (2) Is VMAT2 expressed by other pancreatic endocrine or non-endocrine cells? (3) Is the relationship between VMAT2 and insulin expression disturbed in type 1 (T1DM) or type 2 diabetes (T2DM)? Human pancreas (7 non-diabetics, 5 T2DM, 10 T1DM) was immunostained for insulin, VMAT2 and other pancreatic hormones. Most beta cells expressed VMAT2. VMAT2 expression was not changed by the presence of diabetes. In tail of pancreas VMAT2 immunostaining closely correlated with insulin staining. However, VMAT2 was also expressed in some pancreatic polypeptide (PP) cells. Although VMAT2 was not excluded as a target for beta cell mass measurement, expression of VMAT2 in PP cells predicts residual VMAT2 expression in human pancreas even in the absence of beta cells

Insulin doseresponse curves for stimulation of splanchnic glucose uptake and suppression of endogenous glucose production differ in nondiabetic humans and are abnormal in people with type 2 diabetes.

To determine whether the insulin dose-response curves for suppression of endogenous glucose production (EGP) and stimulation of splanchnic glucose uptake (SGU) differ in nondiabetic humans and are abnormal in type 2 diabetes, 14 nondiabetic and 12 diabetic subjects were studied. Glucose was clamped at ϳ9.5 mmol/l and endogenous hormone secretion inhibited by somatostatin, while glucagon and growth hormone were replaced by an exogenous infusion. Insulin was progressively increased from ϳ150 to ϳ350 and ϳ700 pmol/l by means of an exogenous insulin infusion, while EGP, SGU, and leg glucose uptake (LGU) were measured using the splanchnic and leg catheterization methods, combined with a [3-3 H]glucose infusion. In nondiabetic subjects, an increase in insulin from ϳ150 to ϳ350 pmol/l resulted in maximal suppression of EGP, whereas SGU continued to increase (P < 0.001) when insulin was increased to ϳ700 pmol/l. In contrast, EGP progressively decreased (P < 0.001) and SGU progressively increased (P < 0.001) in the diabetic subjects as insulin increased from ϳ150 to ϳ700 pmol/l. Although EGP was higher (P < 0.01) in the diabetic than nondiabetic subjects only at the lowest insulin concentration, SGU was lower (P < 0.01) in the diabetic subjects at all insulin concentrations tested. On the other hand, in contrast to LGU and overall glucose disposal, the increment in SGU in response to both increments in insulin did not differ in the diabetic and nondiabetic subjects, implying a right shifted but parallel dose-response curve. These data indicate that the dose-response curves for suppression of glucose production and stimulation of glucose uptake differ in nondiabetic subjects and are abnormal in people with type 2 diabetes. Taken together, these data also suggest that agents that enhance SGU in diabetic patients (e.g. glucokinase activators) are likely to improve glucose tolerance. Diabete

Divergent mathematical treatments in utility theory

In this paper I study how divergent mathematical treatments affect mathematical modelling, with a special focus on utility theory. In particular I examine recent work on the ranking of information states and the discounting of future utilities, in order to show how, by replacing the standard analytical treatment of the models involved with one based on the framework of Nonstandard Analysis, diametrically opposite results are obtained. In both cases, the choice between the standard and nonstandard treatment amounts to a selection of set-theoretical parameters that cannot be made on purely empirical grounds. The analysis of this phenomenon gives rise to a simple logical account of the relativity of impossibility theorems in economic theory, which concludes the paper

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function

Loss of Ambra1 promotes melanoma growth and invasion

Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma. The absence of scaffold protein Ambra1 leads to hyperproliferation and growth in mouse models. Here the authors show that Ambra1 deficiency accelerates melanoma growth and increases metastasis in mouse models of melanoma through FAK1 hyperactivation

Characterization of Non-hormone Expressing Endocrine Cells in Fetal and Infant Human Pancreas

Context: Previously, we identified chromograninA positive hormone-negative (CPHN) cells in high frequency in human fetal and neonatal pancreas, likely representing nascent endocrine precursor cells. Here, we characterize the putative endocrine fate and replicative status of these newly formed cells.Objective: To establish the replicative frequency and transcriptional identity of CPHN cells, extending our observation on CPHN cell frequency to a larger cohort of fetal and infant pancreas.Design, Setting, and Participants: 8 fetal, 19 infant autopsy pancreata were evaluated for CPHN cell frequency; 12 fetal, 24 infant/child pancreata were evaluated for CPHN replication and identity.Results: CPHN cell frequency decreased 84% (islets) and 42% (clusters) from fetal to infant life. Unlike the beta-cells at this stage, CPHN cells were rarely observed to replicate (0.2 ± 0.1 vs. 4.7 ± 1.0%, CPHN vs. islet hormone positive cell replication, p < 0.001), indicated by the lack of Ki67 expression in CPHN cells whether located in the islets or in small clusters, and with no detectable difference between fetal and infant groups. While the majority of CPHN cells express (in overall compartments of pancreas) the pan-endocrine transcription factor NKX2.2 and beta-cell specific NKX6.1 in comparable frequency in fetal and infant/child cases (81.9 ± 6.3 vs. 82.8 ± 3.8% NKX6.1+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.9; 88.0 ± 4.7 vs. 82.1 ± 5.3% NKX2.2+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.4), the frequency of clustered CPHN cells expressing NKX6.1 or NKX2.2 is lower in infant/child vs. fetal cases (1.2 ± 0.3 vs. 16.7 ± 4.7 clustered NKX6.1+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01; 2.7 ± 1.0 vs. 16.0 ± 4.0 clustered NKX2.2+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01).Conclusions: The frequency of CPHN cells declines steeply from fetal to infant life, presumably as they differentiate to hormone-expressing cells. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.Precis: CPHN cell frequency declines steeply from fetal to infant life, as they mature to hormone expression. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis