Search

Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott–Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.

Background Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. Objectives To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). Methods To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Results Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. Conclusion We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

Inherited or acquired disorders of platelet production and function can result in thrombocytopenia and bleeding. Mouse models have proven useful for investigating the mechanisms that underlie these defects in humans. Precise methods for blood withdrawal, platelet isolation and measurement of platelet parameters are key for the generation of reproducible and conclusive data. Here, we provide three different protocols for mouse platelet isolation to encourage research knowledge transfer between experienced laboratories, while at the same time enabling less experienced researchers to implement a protocol that best suits their local expertise and equipment. We also address the issue that reported mouse platelet count and size vary considerably in the literature by investigating different factors that influence these important platelet parameters, namely: 1) genetic background and gender, 2) choice of analysis method (hematological analyzer or flow cytometry), 3) dilution of the blood sample and 4) choice of anticoagulant. The herein presented results and considerations may serve as a practical guide for both experienced and new researchers in the platelet field.

Bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal-recessive small-platelet thrombocytopenia (CARST) was described; it is caused by mutations in the adhesion and degranulation-promoting adaptor protein (ADAP; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice (Adap-/- ) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap-/- mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap-/- platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole-sternum 3-dimensional confocal imaging and intravital 2-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the BM compartment. In addition, cultured BM-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP-deficient mice (PF4-cre) also produced fewer and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is an MK-intrinsic defect. Taken together, these results point to an as-yet-unidentified role of ADAP in the process of MK polarization and platelet biogenesis.

Automatisierte Materialflusssysteme sind aufgrund ihrer hohen Durchsatze und kurzen Durchlaufzeiten in der Industrie weit verbreitet. Um ihr volles Potential entfalten zu konnen, werden diese Systeme kundenspezifisch projektiert, weshalb eine individuelle Steuerungssoftware notwendig ist. Das Erstellen dieser Steuerungssoftware erfordert umfangreiches manuelles Programmieren, da haufig eine Steuerungssoftware mit vordefinierten Modulen fehlt. In dieser Arbeit wird diese Lucke geschlossen, indem ein modulares Konzept zur Erstellung der Steuerungssoftware fur hierarchisch gesteuerte Materialflusssysteme vorgestellt wird. Dieses Konzept basiert auf einer Zwei-Schichten-Architektur, in welcher die Ansteuerung der Hardware von den Entscheidungen des Materialflusses getrennt wird. Die Steuerung erfolgt anhand eines Steuerungs-Zyklus, der in funf Zyklus-Phasen unterteilt ist. Dieser funf Phasen umfassende Steuerungs-Zyklus wird in einer generischen Form vorgestellt, welcher einer Vorlage entspricht, anhand derer die Module fur spezifische Fordermittel und logistische Funktionen ausgearbeitet werden konnen. Diese Vorlage wird daraufhin fur Module eines Beispiellayouts ausgearbeitet und die korrekte Funktion in einer Emulation aufgezeigt. Somit konnen anhand des Konzeptes Module fur die Hardwareschicht und die Materialflussschicht erstellt werden, die dann zur individuellen Steuerungssoftware eines Materialflusssystems zusammengefugt werden, wodurch die manuelle Programmierung bei der Steuerungssoftwareerstellung entfallt.

The (re)development of industrial production systems have to deal with high flexibility due to customers' demands as well as constraints such as the dimensions of the manufactory. Therefore, also the Intralogistics system, which transport the goods or products between the machine tools in the production system must be flexibly assembled to fit into the given space. This presuppose that parts of the Intralogistics system, such as a belt conveyor, may be modified or exchanged by different ones, with less effort. Additionally, due to the different lifespan of mechanical, electrical and software components often electrical devices need to be replaced by other ones during the operation, due to lacking availability because of shorter life cycles. Nowadays in industry, a manual adaptation of the control software is necessary after exchanging an electrical/mechanical device with a newer one. In order to ease the software adaptation, in this paper an automatic analysis of differences between incompatible software and an adaptation approach focusing on the functional behavior of the software is introduced. After identifying seven interaction points of control software which need to be adapted the approach is evaluated with an industrial case study and feedback from industrial experts, to proof industrial scalability.

Today’s industrial automation production systems (aPS) have to deal with the requirement to produce many different products. Hence, not only a flexible modularized aPS, but also a flexible modularized material flow system (MFS) is required in order to improve modifications to the system, i.e. extending, reducing or modifying parts of the system. To reduce the engineering effort involved in setting up modularized MFS and to deal with the complexity of the system, a definition of the architecture, behavior and (pre)defined interfaces of such modules is provided by a model-based engineering approach, which is based on an appropriate meta model. In addition, the modules can interact with neighboring modules to execute specific logistic functionalities by using (pre)defined interfaces. Since the development of automated material flow modules (aMFM) can be considered as an evolutional process, the aMFM are frequently redeveloped using current-state-of-the-art technologies, which, in some circumstances, also require newly-developed interfaces. Thus, an inconsistency between older and newer modules containing different version numbers occurs when different (pre)defined interfaces are used, leading to a reduction in system flexibility. To implement the backward compatibility of newer modules, the (pre)defined software interfaces of the newer modules, or even of its neighboring modules, usually have to be adapted manually, which increases the engineering effort and propensity for errors. This paper introduces a model-based approach to analyzing and adapting the interface of aMFM automatically in order to enable interaction with neighboring modules across different version numbers, i.e. modules containing a different version number. Hence, the engineering effort and error-proneness for the adaption can be reduced. For instance, the automatic analysis and adaption of the interface of a newly-developed QR Code Scanner is considered here.

Today's industry increasingly requires flexibility and adaptability in the development of automation control software, especially for automated production systems (aPS) including an automated material flow system (aMFS). To meet these demands, the development of the automation control software and the modification during the operation of the aMFS has to become as easy as possible without taking a lot of time. Using a proven approach from computer science that describes the separation of the control software in different layers in order to meet high-level and low-level requirements individually, the results of a feasibility study regarding the implementation of this approach for aMFS are shown. Moreover, to deal with the complexity of developing the control software, a model-based development approach is applied. In order to model aMFS, the modeling language has to cover different aspects such as logistics functions, different interfaces and abstractions of control levels compliant to international standards. Furthermore, a feasibility study how to implement such model-based two-layer architectures considering the commissioning of an industrial aMFS is shown.

At sites of vascular injury, exposed subendothelial collagens trigger platelet activation and thrombus formation by interacting with the immunoreceptor tyrosine-based activation motif (ITAM)-coupled glycoprotein VI (GPVI) on the platelet surface. Platelets are derived from the cytoplasm of megakaryocytes (MKs), which extend large proplatelets into bone marrow (BM) sinusoids that are then released into the bloodstream, where final platelet sizing and maturation occurs. The mechanisms that prevent activation of MKs and forming proplatelets in the collagen-rich BM environment remain largely elusive. Here, we demonstrate that newly formed young platelets (NFYPs) released after antibody-mediated thrombocytopenia in mice display a severe and highly selective signaling defect downstream of GPVI resulting in impaired collagen-dependent activation and thrombus formation in vitro and in vivo. The diminished GPVI signaling in NFYPs is linked to reduced phosphorylation of key downstream signaling proteins, including Syk, LAT, and phospholipase Cγ2, whereas the G protein-coupled receptor and C-type lectin-like receptor 2 signaling pathways remained unaffected. This GPVI signaling defect was overcome once the platelet counts were restored to normal in the circulation. Overall, these results indicate that the GPVI-ITAM signaling machinery in NFYPs after antibody-mediated thrombocytopenia only becomes fully functional in the blood circulation.

This paper proposes surveillance trajectories for a network of autonomous cameras to detect intruders. We consider smart intruders, which appear at arbitrary times and locations, are aware of the cameras configuration, and move to avoid detection for as long as possible. As performance criteria, we consider the worst case detection time (WDT) and the average detection time (ADT). We focus on the case of a chain of cameras, and we obtain the following results. First, we characterize a lower bound on the WDT and on the ADT of smart intruders. Second, we propose a team trajectory for the cameras, namely equal- waiting trajectory, with minimum WDT and with guarantees on the ADT. Third, we design a distributed algorithm to coordinate the cameras along an equal-waiting trajectory. Fourth, we design a distributed algorithm for cameras reconfiguration in the case of failure or network change. Finally, we illustrate the effectiveness and robustness of our algorithms via numerical studies and experiments. development of distributed and autonomous surveillance strategies for the detection of moving intruders. We make combined assumptions on the environment to be monitored, the cameras, and the intruders. We assume the environment to be 1-D, in the sense that it can be completely observed by a chain of cameras using linear motion only (the perimeter surveillance problem is a special case of this framework). We assume the cameras to be subjected to physical constraints, such as limited field of view (f.o.v.) and speed, and to be equipped with a low-level routine to detect intruders that fall within their f.o.v. We assume intruders to be smart, in the sense that they have access to the cameras configuration at every time instant, and schedule their trajectory to avoid detection for as long as possible. Since the probability of success of an intrusion increases with the time an intruder remains unde- tected in the environment (5), we propose cameras trajectories and control algorithms to minimize the worst case detection time (WDT) and the average detection time (ADT) of smart intruders.

Automated material handling systems are widely used in industry due to high throughputs and good process quality. In order to fully utilize their potential, these systems have to be custom-built, requiring individually created control software. The engineering process of the control software includes extensive manual programming, because a modular structure of the control algorithm with predefined modules is often not available. We describe one such modular control algorithm, based upon a two-layer architecture that separates the hardware control from the material flow control. Our modular control algorithm allows predefined modules for both the conveyor hardware and material flow elements to be created and then assembled in the needed configuration. As a result, the control software for a custom-built material handling system can be created by arranging predefined modules and setting their parameters, thus without manual programming. The proper functionality of our control algorithm is demonstrated by a simulation example using emulated hardware.

Body weight supported treadmill training is an approach to gait rehabilitation following a stroke or spinal cord injury. Although lateral control of balance is an important aspect of walking, many of the currently available body weight support systems have a fixed pulley configuration which can lead to lateral forces being developed in the supporting cables, interfering with the lateral balance task. In this paper, a novel extension for body weight support systems, used for treadmill walking, is presented which features a system of pulleys and trolleys. A model is developed for the device along with a basic feedback controller in order to enable simulation of the concept. The lateral forces induced by the novel system are greatly reduced in comparison to a fixed pulley system. This device has applications in balance training within gait rehabilitation programs.

This work proposes algorithms to control the trajectory of a team of cameras for video surveillance. We consider a chain of cameras installed in an environment. These cameras are used to detect smart intruders, who are aware of the cameras' configuration at each time instant, and who schedule their motion to avoid detection, if possible. For this problem setup, we first obtain a lower bound on the worst-case and on the average detection time of a smart intruder. We then propose a team trajectory for the cameras, called the equal-waiting trajectory, with minimum worst-case detection time and constant-factor optimal average detection time. Additionally, we design and analyse a distributed algorithm to steer the cameras in finite time towards the equal-waiting trajectory. Finally, the performance and robustness of our distributed algorithm is further analyzed through a simulation study.