qRT-PCR of Small RNAs

Plant small RNAs are a class of 19- to 25-nucleotide (nt) RNA molecules that are essential for genome stability, development and differentiation, disease, cellular communication, signaling, and adaptive responses to biotic and abiotic stress. Small RNAs comprise two major RNA classes, short interfering RNAs (siRNAs) and microRNAs (miRNAs). Efficient and reliable detection and quantification of small RNA expression has become an essential step in understanding their roles in specific cells and tissues. Here we provide protocols for the detection of miRNAs by stem-loop RT-PCR. This method enables fast and reliable miRNA expression profiling from as little as 20 pg of total RNA extracted from plant tissue and is suitable for high-throughput miRNA expression analysis. In addition, this method can be used to detect other classes of small RNAs, provided the sequence is known and their GC contents are similar to those specific for miRNAs.

Design of a garment for data collection of toddler language and physical activity

Design process phases of development, evaluation and implementation were used to create a garment to simultaneously collect reliable data of speech production and intensity of movement of toddlers (18-36 months). A series of prototypes were developed and evaluated that housed accelerometer-based motion sensors and a digital transmitter with microphone. The approved test garment was a top constructed from loop-faced fabric with interior pockets to house devices. Extended side panels allowed for sizing. In total, 56 toddlers (28 male; 28 female; 16-36 months of age) participated in the study providing pilot and baseline data. The test garment was effective in collecting data as evaluated for accuracy and reliability using ANOVA for accelerometer data, transcription of video for type of movement, and number and length of utterances for speech production. The data collection garment has been implemented in various studies across disciplines.

Port-Hamiltonian theory of motion control for marine craft

Port-Hamiltonian Systems (PHS) have a particular form that incorporates explicitly a function of the total energy in the system (energy function) and also other functions that describe structure of the system in terms of energy distribution. For PHS, the product of the input and output variables gives the rate of energy change. This type of systems have the property that under certain conditions on the energy function, the system is passive; and thus, stable. Therefore, if one can design a controller such that the closed-loop system retains - or takes - a PHS form, such closed-loop system will inherit the properties of passivity and stability. In this paper, the classical model of marine craft is put into a PHS form. It is shown that models used for positioning control do not have a PHS form due to a kinematic transformation, but a control design can be done such that the closed-loop system takes a PHS form. It is further shown how integral action can be added and how the PHS-form can be exploited to provide a procedure for control design that ensures passivity and thus stability.

Control design for positioning of underwater vehicles with control allocation

In this paper, we address the control design problem of positioning of over-actuated underwater vehicles. The proposed design is based on a control architecture with combined position and velocity loops and a control tuning method based on the decoupled models. We derive analytical tuning rules based on requirements of closed-loop stability, positioning performance, and the vehicle velocity dynamic characteristics. The vehicle modelling is considered from force to motion with appropriate simplifications related to low-speed manoeuvring hydrodynamics and vehicle symmetry. The control design is considered together with a control allocation mapping. This approach makes the control tuning independent of the characteristics of the force actuators and provides the basis for control reconfiguration in the presence of actuator failure. We propose an anti-wind-up implementation of the controller, which ensures that the constraints related to actuation capacity are not violated. This approach simplifies the control allocation problem since the actuator constraints are mapped into generalised force constraints.

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Modeling and sensorless direct torque and flux control of a dual-airgap axial flux permanent-magnet machine with field-weakening operation

This paper presents the modeling and motion-sensorless direct torque and flux control of a novel dual-airgap axial-flux permanent-magnet machine optimized for use in flywheel energy storage system (FESS) applications. Independent closed-loop torque and stator flux regulation are performed in the stator flux ( x-y) reference frame via two PI controllers. This facilitates fast torque dynamics, which is critical as far as energy charging/discharging in the FESS is concerned. As FESS applications demand high-speed operation, a new field-weakening algorithm is proposed in this paper. Flux weakening is achieved autonomously once the y-axis voltage exceeds the available inverter voltage. An inherently speed sensorless stator flux observer immune to stator resistance variations and dc-offset effects is also proposed for accurate flux and speed estimation. The proposed observer eliminates the rotary encoder, which in turn reduces the overall weight and cost of the system while improving its reliability. The effectiveness of the proposed control scheme has been verified by simulations and experiments on a machine prototype.

Bluetooth vehicle trajectory by fusing Bluetooth and loops : motorway travel time statistics

Loop detectors are widely used on the motorway networks where they provide point speed and traffic volumes. Models have been proposed for temporal and spatial generalization of speed for average travel time estimation. Advancement in technology provides complementary data sources such as Bluetooth MAC Scanner (BMS), detecting the MAC ID of the Bluetooth devices transported by the traveller. Matching the data from two BMS stations provides individual vehicle travel time. Generally, on the motorways loops are closely spaced, whereas BMS are placed few kilometres apart. In this research, we fuse BMSs and loops data to define the trajectories of the Bluetooth vehicles. The trajectories are utilised to estimate the travel time statistics between any two points along the motorway. The proposed model is tested using simulation and validated with real data from Pacific motorway, Brisbane. Comparing the model with the linear interpolation based trajectory provides significant improvements.

Beyond tasks and gateways: discovering BPMN models with subprocesses, boundary events and activity markers

Existing techniques for automated discovery of process models from event logs largely focus on extracting flat process models. In other words, they fail to exploit the notion of subprocess, as well as structured error handling and repetition constructs provided by contemporary process modeling notations, such as the Business Process Model and Notation (BPMN). This paper presents a technique for automated discovery of BPMN models containing subprocesses, interrupting and non-interrupting boundary events, and loop and multi-instance markers. The technique analyzes dependencies between data attributes associated with events, in order to identify subprocesses and to extract their associated logs. Parent process and subprocess models are then discovered separately using existing techniques for flat process model discovery. Finally, the resulting models and logs are heuristically analyzed in order to identify boundary events and markers. A validation with one synthetic and two real-life logs shows that process models derived using the proposed technique are more accurate and less complex than those derived with flat process model discovery techniques.

Pragmatic two-way communication : a practitioner perspective on dialogue in public relations

Public relations has traditionally claimed a close association with dialogue, but this research demonstrates that formal notions of dialogue have little relevance to the real world of public relations practice. Instead, practitioners undertake pragmatic forms of two-way communication, because the constraints within which they work mean dialogue is difficult if not impossible to carry out. This qualitative research project shows that although the label of 'dialogue' has been co-opted in both the theory and practice of public relations, this claimed connection is not supported by empirical evidence.

Molecular aspects of tissue engineering in the dental field

Tissue engineering is a multidisciplinary field with the potential to replace tissues lost as a result of trauma, cancer surgery, or organ dysfunction. The successful production, integration, and maintenance of any tissue-engineered product are a result of numerous molecular interactions inside and outside the cell. We consider the essential elements for successful tissue engineering to be a matrix scaffold, space, cells, and vasculature, each of which has a significant and distinct molecular underpinning (Fig. 1). Our approach capitalizes on these elements. Originally developed in the rat, our chamber model (Fig. 2) involves the placement of an arteriovenous loop (the vascular supply) in a polycarbonate chamber (protected space) with the addition of cells and an extracellular matrix such as Matrigel or endogenous fibrin (34, 153, 246, 247). This model has also been extended to the rabbit and pig (J. Dolderer, M. Findlay, W. Morrison, manuscript in preparation), and has been modified for the mouse to grow adipose tissue and islet cells (33, 114, 122) (Fig. 3)...

Monocyte chemoattractant protein-1 and nitric oxide promote adipogenesis in a model that mimics obesity

Objective: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low-level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute-phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)-I and zymosan-A (Zy) in a murine tissue engineering model. Research Methods and Procedures: MCP-I and Zy were added to chambers filled with Matrigel and fibroblastgrowth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. Results: Our results show that MCP-I generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. Discussion: Our findings demonstrate that low-grade inflammation and iNOS expression are important factors in adipogenesis, Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage-derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.

The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Enhancement of RAD51 recombinase activity by the tumor suppressor PALB2

Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.