Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.Syt‐7 is a high‐affinity calcium sensor expressed on chromaffin cell dense core granules. The purpose of this study was to assess the role of Syt‐7 in regulating the secretory response to cholinergic stimulation. Acetylcholine elicits secretion by elevating cytosolic calcium. The calcium sensitivity of exocytosis in cells lacking Syt‐7 is impaired. Cells that lack Syt‐7 also release peptide hormones at faster rates, implicating a role for Syt‐7 in regulating the exocytotic fusion pore. These data demonstrate that Syt‐7 has an important role in triggering exocytosis in cells and is likely to play a role in controlling hormone output, in situ.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/3/jnc14986.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/2/jnc14986-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/1/jnc14986_am.pd

Context Dependent Neuroprotective Properties of Prion Protein (Prp)

Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure—independent of infectious prion conformation—to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-β, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer’s disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP’s neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson’s disease or tauopathy. Deletion of PrP in one of two Huntington’s disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington’s motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.Ellison Medical FoundationWhitaker Health Sciences Fund Fellowshi

Efficiency Theory: a Unifying Theory for Information, Computation and Intelligence

The paper serves as the first contribution towards the development of the theory of efficiency: a unifying framework for the currently disjoint theories of information, complexity, communication and computation. Realizing the defining nature of the brute force approach in the fundamental concepts in all of the above mentioned fields, the paper suggests using efficiency or improvement over the brute force algorithm as a common unifying factor necessary for the creation of a unified theory of information manipulation. By defining such diverse terms as randomness, knowledge, intelligence and computability in terms of a common denominator we are able to bring together contributions from Shannon, Levin, Kolmogorov, Solomonoff, Chaitin, Yao and many others under a common umbrella of the efficiency theory

On the nature and impact of self-similarity in real-time systems

In real-time systems with highly variable task execution times simplistic task models are insufficient to accurately model and to analyze the system. Variability can be tackled using distributions rather than a single value, but the proper charac- terization depends on the degree of variability. Self-similarity is one of the deep- est kinds of variability. It characterizes the fact that a workload is not only highly variable, but it is also bursty on many time-scales. This paper identifies in which situations this source of indeterminism can appear in a real-time system: the com- bination of variability in task inter-arrival times and execution times. Although self- similarity is not a claim for all systems with variable execution times, it is not unusual in some applications with real-time requirements, like video processing, networking and gaming. The paper shows how to properly model and to analyze self-similar task sets and how improper modeling can mask deadline misses. The paper derives an analyti- cal expression for the dependence of the deadline miss ratio on the degree of self- similarity and proofs its negative impact on real-time systems performance through system¿s modeling and simulation. This study about the nature and impact of self- similarity on soft real-time systems can help to reduce its effects, to choose the proper scheduling policies, and to avoid its causes at system design time.This work was developed under a grant from the European Union (FRESCOR-FP6/2005/IST/5-03402).Enrique Hernández-Orallo; Vila Carbó, JA. (2012). On the nature and impact of self-similarity in real-time systems. Real-Time Systems. 48(3):294-319. doi:10.1007/s11241-012-9146-0S294319483Abdelzaher TF, Sharma V, Lu C (2004) A utilization bound for aperiodic tasks and priority driven scheduling. IEEE Trans Comput 53(3):334–350Abeni L, Buttazzo G (1999) QoS guarantee using probabilistic deadlines. In: Proc of the Euromicro confererence on real-time systemsAbeni L, Buttazzo G (2004) Resource reservation in dynamic real-time systems. Real-Time Syst 37(2):123–167Anantharam V (1999) Scheduling strategies and long-range dependence. Queueing Syst 33(1–3):73–89Beran J (1994) Statistics for long-memory processes. Chapman and Hall, LondonBeran J, Sherman R, Taqqu M, Willinger W (1995) Long-range dependence in variable-bit-rate video traffic. IEEE Trans Commun 43(2):1566–1579Boxma O, Zwart B (2007) Tails in scheduling. SIGMETRICS Perform Eval Rev 34(4):13–20Brichet F, Roberts J, Simonian A, Veitch D (1996) Heavy traffic analysis of a storage model with long range dependent on/off sources. Queueing Syst 23(1):197–215Crovella M, Bestavros A (1997) Self-similarity in world wide web traffic: evidence and possible causes. IEEE/ACM Trans Netw 5(6):835–846Dìaz J, Garcìa D, Kim K, Lee C, Bello LL, López J, Min LS, Mirabella O (2002) Stochastic analysis of periodic real-time systems. In: Proc of the 23rd IEEE real-time systems symposium, pp 289–300Erramilli A, Narayan O, Willinger W (1996) Experimental queueing analysis with long-range dependent packet traffic. IEEE/ACM Trans Netw 4(2):209–223Erramilli A, Roughan M, Veitch D, Willinger W (2002) Self-similar traffic and network dynamics. Proc IEEE 90(5):800–819Gardner M (1999) Probabilistic analysis and scheduling of critical soft real-time systems. Phd thesis, University of Illinois, Urbana-ChampaignGarrett MW, Willinger W (1994) Analysis, modeling and generation of self-similar vbr video traffic. In: ACM SIGCOMMHarchol-Balter M (2002) Task assignment with unknown duration. J ACM 49(2):260–288Harchol-Balter M (2007) Foreword: Special issue on new perspective in scheduling. SIGMETRICS Perform Eval Rev 34(4):2–3Harchol-Balter M, Downey AB (1997) Exploiting process lifetime distributions for dynamic load balancing. ACM Trans Comput Syst 15(3):253–285Hernandez-Orallo E, Vila-Carbo J (2007) Network performance analysis based on histogram workload models. In: Proceedings of the 15th international symposium on modeling, analysis, and simulation of computer and telecommunication systems (MASCOTS), pp 331–336Hernandez-Orallo E, Vila-Carbo J (2010) Analysis of self-similar workload on real-time systems. In: IEEE real-time and embedded technology and applications symposium (RTAS). IEEE Computer Society, Washington, pp 343–352Hernández-Orallo E, Vila-Carbó J (2010) Network queue and loss analysis using histogram-based traffic models. Comput Commun 33(2):190–201Hughes CJ, Kaul P, Adve SV, Jain R, Park C, Srinivasan J (2001) Variability in the execution of multimedia applications and implications for architecture. SIGARCH Comput Archit News 29(2):254–265Leland W, Ott TJ (1986) Load-balancing heuristics and process behavior. SIGMETRICS Perform Eval Rev 14(1):54–69Leland WE, Taqqu MS, Willinger W, Wilson DV (1994) On the self-similar nature of ethernet traffic (extended version). IEEE/ACM Trans Netw 2(1):1–15Liu CL, Layland JW (1973) Scheduling algorithms for multiprogramming in a hard-real-time environment. J ACM 20(1):46–61Mandelbrot B (1965) Self-similar error clusters in communication systems and the concept of conditional stationarity. IEEE Trans Commun 13(1):71–90Mandelbrot BB (1969) Long run linearity, locally Gaussian processes, h-spectra and infinite variances. Int Econ Rev 10:82–113Norros I (1994) A storage model with self-similar input. Queueing Syst 16(3):387–396Norros I (2000) Queueing behavior under fractional Brownian traffic. In: Park K, Willinger W (eds) Self-similar network traffic and performance evaluation. Willey, New York, Chap 4Park K, Willinger W (2000) Self-similar network traffic: An overview. In: Park K, Willinger W (eds) Self-similar network traffic and performance evaluation. Willey, New York, Chap 1Paxson V, Floyd S (1995) Wide area traffic: the failure of Poisson modeling. IEEE/ACM Trans Netw 3(3):226–244Rolls DA, Michailidis G, Hernández-Campos F (2005) Queueing analysis of network traffic: methodology and visualization tools. Comput Netw 48(3):447–473Rose O (1995) Statistical properties of mpeg video traffic and their impact on traffic modeling in atm systems. In: Conference on local computer networksRoy N, Hamm N, Madhukar M, Schmidt DC, Dowdy L (2009) The impact of variability on soft real-time system scheduling. In: RTCSA ’09: Proceedings of the 2009 15th IEEE international conference on embedded and real-time computing systems and applications. IEEE Computer Society, Washington, pp 527–532Sha L, Abdelzaher T, Årzén KE, Cervin A, Baker T, Burns A, Buttazzo G, Caccamo M, Lehoczky J, Mok AK (2004) Real time scheduling theory: A historical perspective. Real-Time Syst 28(2):101–155Taqqu MS, Willinger W, Sherman R (1997) Proof of a fundamental result in self-similar traffic modeling. SIGCOMM Comput Commun Rev 27(2):5–23Tia T, Deng Z, Shankar M, Storch M, Sun J, Wu L, Liu J (1995) Probabilistic performance guarantee for real-time tasks with varying computation times. In: Proc of the real-time technology and applications symposium, pp 164–173Vila-Carbó J, Hernández-Orallo E (2008) An analysis method for variable execution time tasks based on histograms. Real-Time Syst 38(1):1–37Willinger W, Taqqu M, Erramilli A (1996) A bibliographical guide to self-similar traffic and performance modeling for modern high-speed networks. In: Stochastic networks: Theory and applications, pp 339–366Willinger W, Taqqu MS, Sherman R, Wilson DV (1997) Self-similarity through high-variability: statistical analysis of ethernet lan traffic at the source level. IEEE/ACM Trans Netw 5(1):71–8

Very early invasive strategy in higher risk non-ST-elevation acute coronary syndrome: the RAPID NSTEMI trial

Objective To investigate whether a very early invasive strategy (IS)±revascularisation improves clinical outcomes compared with standard care IS in higher risk patients with non-ST-elevation acute coronary syndrome (NSTE-ACS). Methods Multicentre, randomised, controlled, pragmatic strategy trial of higher risk patients with NSTE-ACS, defined by Global Registry of Acute Coronary Events 2.0 score of ≥118, or ≥90 with at least one additional high-risk feature. Participants were randomly assigned to very early IS±revascularisation (<90 min from randomisation) or standard care IS±revascularisation (<72 hours). The primary outcome was a composite of all-cause mortality, new myocardial infarction or hospitalisation for heart failure at 12 months. Results The trial was discontinued early by the funder due to slow recruitment during the COVID-19 pandemic. 425 patients were randomised, of whom 413 underwent an IS: 204 to very early IS (median time from randomisation: 1.5 hours (IQR: 0.9–2.0)) and 209 to standard care IS (median: 44.0 hours (IQR: 22.9–72.6)). At 12 months, there was no significant difference in the primary outcome between the early IS (5.9%) and standard IS (6.7%) groups (OR 0.93, 95% CI 0.42 to 2.09; p=0.86). The incidence of stroke and major bleeding was similar. The length of hospital stay was reduced with a very early IS (3.9 days (SD 6.5) vs 6.3 days (SD 7.6), p<0.01). Conclusions A strategy of very early IS did not improve clinical outcomes compared with a standard care IS in higher risk patients with NSTE-ACS. However, the primary outcome rate was low and the trial was underpowered to detect such a difference

PKCδ Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and DNA Damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches

Adult Neurogenesis Transiently Generates Oxidative Stress

An increasing body of evidence suggests that alterations in neurogenesis and oxidative stress are associated with a wide variety of CNS diseases, including Alzheimer’s disease, schizophrenia and Parkinson’s disease, as well as routine loss of function accompanying aging. Interestingly, the association between neurogenesis and the production of reactive oxidative species (ROS) remains largely unexamined. The adult CNS harbors two regions of persistent lifelong neurogenesis: the subventricular zone and the dentate gyrus (DG). These regions contain populations of quiescent neural stem cells (NSCs) that generate mature progeny via rapidly-dividing progenitor cells. We hypothesized that the energetic demands of highly proliferative progenitors generates localized oxidative stress that contributes to ROS-mediated damage within the neuropoietic microenvironment. In vivo examination of germinal niches in adult rodents revealed increases in oxidized DNA and lipid markers, particularly in the subgranular zone (SGZ) of the dentate gyrus. To further pinpoint the cell types responsible for oxidative stress, we employed an in vitro cell culture model allowing for the synchronous terminal differentiation of primary hippocampal NSCs. Inducing differentiation in primary NSCs resulted in an immediate increase in total mitochondria number and overall ROS production, suggesting oxidative stress is generated during a transient window of elevated neurogenesis accompanying normal neurogenesis. To confirm these findings in vivo, we identified a set of oxidation-responsive genes, which respond to antioxidant administration and are significantly elevated in genetic- and exercise-induced model of hyperactive hippocampal neurogenesis. While no direct evidence exists coupling neurogenesis-associated stress to CNS disease, our data suggest that oxidative stress is produced as a result of routine adult neurogenesis

Hox10 Genes Function in Kidney Development in the Differentiation and Integration of the Cortical Stroma

Organogenesis requires the differentiation and integration of distinct populations of cells to form a functional organ. In the kidney, reciprocal interactions between the ureter and the nephrogenic mesenchyme are required for organ formation. Additionally, the differentiation and integration of stromal cells are also necessary for the proper development of this organ. Much remains to be understood regarding the origin of cortical stromal cells and the pathways involved in their formation and function. By generating triple mutants in the Hox10 paralogous group genes, we demonstrate that Hox10 genes play a critical role in the developing kidney. Careful examination of control kidneys show that Foxd1-expressing stromal precursor cells are first observed in a cap-like pattern anterior to the metanephric mesenchyme and these cells subsequently integrate posteriorly into the kidney periphery as development proceeds. While the initial cap-like pattern of Foxd1-expressing cortical stromal cells is unaffected in Hox10 mutants, these cells fail to become properly integrated into the kidney, and do not differentiate to form the kidney capsule. Consistent with loss of cortical stromal cell function, Hox10 mutant kidneys display reduced and aberrant ureter branching, decreased nephrogenesis. These data therefore provide critical novel insights into the cellular and genetic mechanisms governing cortical cell development during kidney organogenesis. These results, combined with previous evidence demonstrating that Hox11 genes are necessary for patterning the metanephric mesenchyme, support a model whereby distinct populations in the nephrogenic cord are regulated by unique Hox codes, and that differential Hox function along the AP axis of the nephrogenic cord is critical for the differentiation and integration of these cell types during kidney organogenesis