Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.

Fixed-wing attitude estimation using temporal tracking of the horizon and optical flow

In this paper, a method has been developed for estimating pitch angle, roll angle and aircraft body rates based on horizon detection and temporal tracking using a forward-looking camera, without assistance from other sensors. Using an image processing front-end, we select several lines in an image that may or may not correspond to the true horizon. The optical flow at each candidate line is calculated, which may be used to measure the body rates of the aircraft. Using an Extended Kalman Filter (EKF), the aircraft state is propagated using a motion model and a candidate horizon line is associated using a statistical test based on the optical flow measurements and the location of the horizon. Once associated, the selected horizon line, along with the associated optical flow, is used as a measurement to the EKF. To test the accuracy of the algorithm, two flights were conducted, one using a highly dynamic Uninhabited Airborne Vehicle (UAV) in clear flight conditions and the other in a human-piloted Cessna 172 in conditions where the horizon was partially obscured by terrain, haze and smoke. The UAV flight resulted in pitch and roll error standard deviations of 0.42◦ and 0.71◦ respectively when compared with a truth attitude source. The Cessna flight resulted in pitch and roll error standard deviations of 1.79◦ and 1.75◦ respectively. The benefits of selecting and tracking the horizon using a motion model and optical flow rather than naively relying on the image processing front-end is also demonstrated.

A geometrical approach to the motion planning problem for a submerged rigid body

The main focus of this paper is the motion planning problem for a deeply submerged rigid body. The equations of motion are formulated and presented by use of the framework of differential geometry and these equations incorporate external dissipative and restoring forces. We consider a kinematic reduction of the affine connection control system for the rigid body submerged in an ideal fluid, and present an extension of this reduction to the forced affine connection control system for the rigid body submerged in a viscous fluid. The motion planning strategy is based on kinematic motions; the integral curves of rank one kinematic reductions. This method is of particular interest to autonomous underwater vehicles which can not directly control all six degrees of freedom (such as torpedo shaped AUVs) or in case of actuator failure (i.e., under-actuated scenario). A practical example is included to illustrate our technique.

Decoupled trajectory planning for a submerged rigid body subject to dissipative and potential forces

This paper studies the practical but challenging problem of motion planning for a deeply submerged rigid body. Here, we formulate the dynamic equations of motion of a submerged rigid body under the architecture of differential geometric mechanics and include external dissipative and potential forces. The mechanical system is represented as a forced affine-connection control system on the configuration space SE(3). Solutions to the motion planning problem are computed by concatenating and reparameterizing the integral curves of decoupling vector fields. We provide an extension to this inverse kinematic method to compensate for external potential forces caused by buoyancy and gravity. We present a mission scenario and implement the theoretically computed control strategy onto a test-bed autonomous underwater vehicle. This scenario emphasizes the use of this motion planning technique in the under-actuated situation; the vehicle loses direct control on one or more degrees of freedom. We include experimental results to illustrate our technique and validate our method.

Controlling a submerged rigid body : a geometric analysis

In this paper we analyze the equations of motion of a submerged rigid body. Our motivation is based on recent developments done in trajectory design for this problem. Our goal is to relate some properties of singular extremals to the existence of decoupling vector fields. The ideas displayed in this paper can be viewed as a starting point to a geometric formulation of the trajectory design problem for mechanical systems with potential and external forces.

Human resource flexibility and strong ties in entrepreneurial teams

Human resource flexibility is important in entrepreneurial ventures that need to respond to the changing challenges of growing the new business. This research investigates the impact of previously well-known people (strong ties) as entrepreneurial team members on the human resource flexibility of new ventures. Data collected from German founding entrepreneurs in technology-oriented, incubator-based firms shows that choosing a well known individual to join the entrepreneurial team increases the founder's ability to modify the team member's work role, but complicates asking the team member to leave the team if required. Hence, strong ties both increase and reduce human resource flexibility. However, the effect of strong ties on role modifiability is statistically significant only with novice entrepreneurs. These research findings counsel founders to discuss role modification and exit during partnership and entrepreneurial team membership negotiations.

Polybrominated diphenyl ether (PBDE) concentrations decrease with age : analysis of pooled human blood serum in the Australian population

Introduction Polybrominated diphenyl ethers (PBDEs) are considered to be a cost effective and efficient way to reduce the possibility of product ignition and inhibit the spread of fire, thereby limiting harm caused by fires. PBDEs are incorporated into a wide variety of manufactured products and are now considered an ubiquitous contaminant found worldwide in biological and environmental samples1 . In comparison to “traditional” persistent organic pollutants (POPs), the exposure modes of PBDEs in humans are less well defined, although dietary sources, inhalation (air/particulate matter) and dust ingestion have been reported 2-4. Limited investigations of population specific factors such as age or gender and PBDE concentrations report: no conclusive correlation by age in adults; higher concentrations in children ; similar concentrations in maternal and cord blood; and no gender differences. After preliminary findings of higher PBDE concentrations in children than in adults in Australia11 we sought to investigate at what age the PBDE concentrations peaked in an effort to focus exposure studies. This investigation involved the collection of blood samples from young age groups and the development of a simple model to predict PBDE concentrations by age in Australia.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Ethical fashion : the human stories driving a fashion movement

This paper examines and compares two stories, the novel Helen Fleetwood (Elizabeth, 1841) and the film China Blue (Teddy Bear Films, 2005), in relation to the Ethical Fashion movement. In 2005, more than 50 designers from around the world took part in The Ethical Fashion Show in Paris. This movement dictates that designers ensure that their garments are produced in an ethical manner, rather than support the ‘sweatshop’ environments of some industrialists determined to make a profit at the expense of workers rights. The momentum of the Ethical Fashion movement suggests that it is possible for fashion to be ethical, desirable and profitable in the 21st century. In 1841, after extensive research, Charlotte Elizabeth Tonna (using the pseudonym Charlotte Elizabeth) began to write about the atrocities of the factory system in industrialised England. Her novel, Helen Fleetwood, is one of the earliest examples of this kind of work, providing the reader with an extensive insight into the life of English factory workers in the mid-19th century. The story is about the Widow Green and her orphan dependents who are led, through circumstance, to leave their rural home and take up employment in the cotton mills of Manchester, with the hope of having an independent existence. Instead they discover the realities of factory life – extremely long hours, unsafe conditions, poor wages and a steady decline into extreme poverty. In his film China Blue (Teddy Bear Films, 2005), director Micha X. Peled tells an alarmingly similar tale set in 21st century China. This ‘docu-drama’ (a recreation from actual interviews and diary entries) tells the story of ‘Little Jasmine’ who leaves her family’s farm to pursue an independent life in Southern China’s manufacturing district. It is not long before the realities of modern factory life are revealed to the teenage ‘heroine’ – crowded dormitories, long working hours, arbitrary fines and wages that do not compare with those of workers in the Western world. While much of the human story remains unchanged, there have been significant improvements in technology and safety in the last 165 years that result in the reality that not all clothing manufacture is performed in ‘sweatshop’ conditions. After a recent visit to a manufacturing plant in China, consultation with peers in the industry and having worked in the Australian fashion industry for many years, the author compares these stories with her own experiences.