Extending Object-Oriented Network Protocols via alternative Transportation Bindings (Web Services over XMPP)

Distributed and/or composite web applications are driven by intercommunication via web services, which employ application-level protocols, such as SOAP. However, these protocols usually rely on the classic HTTP for transportation. HTTP is quite efficient for what it does — delivering web page content, but has never been intended to carry complex web service oriented communication. Today there exist modern protocols that are much better fit for the job. Such a candidate is XMPP. It is an XML-based, asynchronous, open protocol that has built-in security and authentication mechanisms and utilizes a network of federated servers. Sophisticated asynchronous multi-party communication patterns can be established, effectively aiding web service developers. This paper’s purpose is to prove by facts, comparisons, and practical examples that XMPP is not only better suited than HTTP to serve as middleware for web service protocols, but can also contribute to the overall development state of web services

Structural and Functional Aberrations in the Cerebral Cortex of Tenascin-C Deficient Mice

The extracellular matrix glycoprotein tenascin-C (TNC) has been implicated in neural development and plasticity but many of its functions in vivo remain obscure. Here we addressed the question as to whether the constitutive absence of TNC in mice affects cortical physiology and structure. Defined major cell populations (neurons and inhibitory neuronal subpopulations, astrocytes, oligodendrocytes and microglia) were quantified in the somatosensory and motor cortices of adult TNC deficient (TNC−/−) and wild-type (TNC+/+) mice by immunofluorescence labelling and stereology. In both areas studied we found abnormally high neuronal density, astrogliosis, low density of parvalbumin-positive interneurons and reduced ratios of oligodendrocytes to neurons and of inhibitory to excitatory neurons in the TNC deficient as opposed to the non-deficient animals. Analysis of Golgi-impregnated layer V pyramidal neurons in TNC−/− animals showed aberrant dendrite tortuosity and redistribution of stubby spines within first- to third-order dendritic arbors. Significantly enhanced responses upon whisker stimulation were recorded epicranially over the barrel and the motor cortices of TNC−/− as compared to TNC+/+ animals, and this effect might be associated with the diminished inhibitory circuitry. These results indicate that TNC is essential for normal cortical development and functio

Intraneural application of botulinum neurotoxin a improves motoneuron innervation and functional recovery after femoral nerve reconstruction in rats

Axonal injuries to motoneurons of adult mammals cause, among other responses, loss of synaptic terminals from their cell bodies and dendrites. This “synaptic stripping” is largely, but not always completely reversed after successful axonal regeneration and muscle reinnervation. Long-lasting deficits in, e.g., cholinergic and glutamatergic afferent terminals, correlate negatively with degree of functional recovery in rats suggesting that persistent partial deafferentation of motoneurons may be a factor limiting functional recovery after peripheral nerve injury. AimsTo further explore the idea that functional recovery is partially linked to restoration of synaptic inputs to regenerated motoneurons, we pursued to modulate the deafferentation of motoneurons following nerve section/suture and monitor the effects of this manipulation on the outcome of peripheral nerve regeneration. Two neuroactive molecules, botulinum neurotoxin A (BoNT) and brain-derived neurotrophic factor (BDNF), known for their ability to influence synaptic inputs to neurons, were selected as manipulation tools. Drug solutions were applied to the proximal stump of the freshly cut femoral nerve of rats to achieve drug uptake and transport to the neuronal perikarya and possibly transcytosis to afferent synapses. The experiments were performed in adult (10-week-old) female Wistar rats which received either BoNT (N = 17), BDNF (N = 15) or bovine serum albumin treatment (BSA, control, N = 13). After drug application, the femoral nerve was surgically reconstructed and functional recovery was monitored over a 5-month period using an established gait analysis protocol. Other outcome measures were quality of endplate reinnervation (occurrence of abnormal polyinnervation assessed morphologically) and numbers of cholinergic, GABA/glycinergic and glutamatergic synaptic terminals in the femoral motor nucleus in the spinal cord (assessed using stereological approaches). Application of BoNT, but not BDNF, resulted in a marked, as compared with BSA, improvement of motor recovery at 2 to 20 weeks after injury. At two months, BoNT-treated rats had an attenuated loss of perisomatic cholinergic terminals compared with the other two treatments. Analysis of individual animal data revealed significant linear correlations between functional parameters and numbers of cholinergic terminals. Quality of endplate reinnervation was not affected by treatment with BoNT or BDNF. The effect of BoNT on synaptic terminals is possibly related to transcytosis of BoNT into perisomatic nerve terminals as suggested by immunohistochemical analysis of cleaved SNAP-25. In turn, better preservation of modulatory cholinergic terminals, which are crucial for normal motoneuron excitability, might underlie enhanced recovery of function in BoNT-treated rats. The findings support the idea that persistent partial deafferentation of axotomized motoneurons is a factor contributing to deficient functional recovery after nerve injury. Intraneural application of drugs appears to be a promising way to analyze causal relationships between synaptic plasticity and restoration of function. In addition, it is thinkable that the described drug application approach may evolve into a clinically feasible therapy if further controlled animal experiments provide convincing evidence for its safety and efficacy.Läsionen peripherer Nerven von erwachsenen Säugetieren verursachen Verlust von Synapsen an Zellkörpern und Dendriten von Motoneuronen. Diese Deafferenzierung (“synaptic stripping“) ist, selbst nach erfolgreicher Nervenregeneration und Reinnervation der Muskulatur, nicht immer vollkommen reversibel. Langfristige Defizite sind für cholinerge und glutamaterge Afferenzen nachgewiesen und diese korrelieren negativ mit dem Grad der funktionellen Erholung bei Ratten. Es kann angenommen werden, dass eine persistierende partielle Deafferenzierung von Motoneuronen das funktionelle Outcome einer peripheren Nervenverletzung beeinträchtigen kann. Um die o. g. Hypothese zu überprüfen, wurde hier versucht die Deafferenzierung der Motoneurone nach Durchtrennung und Naht des Nervus femoralis der Ratte zu manipulieren. Zwei neuroaktive Moleküle, Botulinum-Neurotoxin A (BoNT) und Brain-derived neurotrophic factor (BDNF), die bekanntlich synaptische Eingänge an Neuronen beeinflussen, wurden als „Manipulationswerkzeuge“ ausgewählt. Die Effekte dieser Behandlungen auf das Outcome der Nervenregeneration wurden funktionell und strukturell untersucht. BoNT oder BDNF wurden an den proximalen Stumpf des durchtrennten Nervus femoralis appliziert, um ihre Aufnahme und den Transport zu den Perikarya sowie eventuell die Transzytose zu afferenten Synapsen zu erreichen. Als Kontrolle diente bovines Serumalbumin (BSA). Die Experimente wurden an erwachsenen, zehn Wochen alten weiblichen Wistar Ratten durchgeführt, die entweder eine BoNT (N = 17), BDNF (N = 15) oder BSA (N = 13) Behandlung erhielten. Nach der Applikation wurde der Nerv chirurgisch rekonstruiert und die funktionelle Erholung mithilfe eines etablierten Ganganalyseverfahrens in einem Zeitraum von fünf Monaten dokumentiert. Weitere Outcome-Parameter waren die Qualität der Endplattenreinnervation (Auftreten anormaler Polyinnervation) und die Anzahl der 8 cholinergen, GABA/glycinergen und glutamatergen synaptischen Terminale im motorischen Kern des N. femoralis im Rückenmark. Zwei bis 20 Wochen nach der Läsion war die funktionelle Erholung nach BoNT Applikation signifikant besser im Vergleich zu den anderen beiden Behandlungen. Der posttraumatische Verlust an perisomatischen cholinergen Synapsen zwei Monate nach der Operation war ebenso reduziert in BoNT-behandelten Tieren. Regressionsanalysen zeigten signifikante lineare Korrelationen zwischen Funktionsparametern und der Anzahl cholinerger Terminale. Die Qualität der Endplattenreinnervation wurde nicht durch die Behandlung mit BoNT oder BDNF beeinflusst. Die Effekte von BoNT beruhen, wie durch Nachweis von gespaltetem SNAP-25 in perisomatischen Nerventerminale angedeutet, möglicherweise auf BoNT Transzytose. Eine bessere Erhaltung der modulatorischen cholinergen Terminale, die für die normale Erregbarkeit von Motoneuronen von entscheidender Bedeutung sind, könnte wiederum einer verbesserten Erholung der Funktion bei mit BoNT behandelten Ratten zugrunde liegen Die Ergebnisse stützen die Hypothese, dass langanhaltende partielle Deafferenzierungen von Motoneuronen negative Auswirkungen auf die funktionelle Erholung nach Nervenläsionen haben können. Die intraneurale Anwendung von neuroaktiven Substanzen scheint ein vielversprechender Weg zu sein, um kausale Zusammenhänge zwischen synaptischer Plastizität und Wiederherstellung der Funktion zu analysieren. Es ist außerdem denkbar, dass sich der beschriebene Ansatz der Arzneimittelapplikation zu einer klinisch durchführbaren Therapie entwickeln kann, wenn weitere kontrollierte Tierversuche überzeugende Beweise für seine Sicherheit und Wirksamkeit liefern

The extracellular-matrix protein matrilin 2 participates in peripheral nerve regeneration

Matrilins are adaptor proteins of the extracellular matrix involved in the formation of both collagen-dependent and collagen-independent filamentous networks. Although their molecular structure and binding partners have been characterized, the functional roles of the four matrilin family members in vivo are still largely unknown. Here, we show that matrilin 2, expressed in pre-myelinating Schwann cells during normal development, profoundly influences the behaviour of glial cells and neurons in vitro. When offered as a uniform substrate, matrilin 2 increased neurite outgrowth of dorsal root ganglia (DRG) neurons and enhanced the migration of both cell line- and embryonic DRG-derived Schwann cells. Vice versa, axonal outgrowth and cell migration were decreased in DRG cultures prepared from matrilin-2-deficient mice compared with wild-type (wt) cultures. In stripe assays, matrilin 2 alone was sufficient to guide axonal growth and, interestingly, axons favoured the combination of matrilin 2 and laminin over laminin alone. In vivo, matrilin 2 was strongly upregulated in injured peripheral nerves of adult wild-type mice and failure of protein upregulation in knockout mice resulted in delayed regrowth of regenerating axons and delayed time-course of functional recovery. Strikingly, the functional recovery 2 months after nerve injury was inferior in matrilin-2-deficient mice compared with wild-type littermates, although motoneuron survival, quality of axonal regeneration, estimated by analyses of axonal diameters and degrees of myelination, and Schwann cell proliferation were not influenced by the mutation. These results show that matrilin 2 is a permissive substrate for axonal growth and cell migration, and that it is required for successful nerve regeneratio

Tumor prevention facilitates delayed transplant of stem cell‐derived motoneurons

Objective Nerve injuries resulting in prolonged periods of denervation result in poor recovery of motor function. We have previously shown that embryonic stem cell-derived motoneurons transplanted at the time of transection into a peripheral nerve can functionally reinnervate muscle. For clinical relevance, we now focused on delaying transplantation to assess reinnervation after prolonged denervation. Methods Embryonic stem cell-derived motoneurons were transplanted into the distal segments of transected tibial nerves in adult mice after prolonged denervation of 1–8 weeks. Twitch and tetanic forces were measured ex vivo 3 months posttransplantation. Tissue was harvested from the transplants for culture and immunohistochemical analysis. Results In this delayed reinnervation model, teratocarcinomas developed in about one half of transplants. A residual multipotent cell population (~ 6% of cells) was found despite neural differentiation. Exposure to the alkylating drug mitomycin C eliminated this multipotent population in vitro while preserving motoneurons. Treating neural differentiated stem cells prior to delayed transplantation prevented tumor formation and resulted in twitch and tetanic forces similar to those in animals transplanted acutely after denervation. Interpretation Despite a neural differentiation protocol, embryonic stem cell-derived motoneurons still carry a risk of tumorigenicity. Pretreating with an antimitotic agent leads to survival and functional muscle reinnervation if performed within 4 weeks of denervation in the mouse

Differential expression of caveolins and myosin heavy chains in response to forced exercise in rats

Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFβ-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFβ-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke

Expression of Cd34 and Myf5 Defines the Majority of Quiescent Adult Skeletal Muscle Satellite Cells

Skeletal muscle is one of a several adult postmitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express p-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majorit

Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions

Embryonic Stem Cell-Derived L1 Overexpressing Neural Aggregates Enhance Recovery after Spinal Cord Injury in Mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system