Species‐specific differences in the medial prefrontal projections to the pons between rat and rabbit

The medial prefrontal cortex (mPFC) of both rats and rabbits has been shown to support trace eyeblink conditioning, presumably by providing an input to the cerebellum via the pons that bridges the temporal gap between conditioning stimuli. The pons of rats and rabbits, however, shows divergence in gross anatomical organization, leaving open the question of whether the topography of prefrontal inputs to the pons is similar in rats and rabbits. To investigate this question, we injected anterograde tracer into the mPFC of rats and rabbits to visualize and map in 3D the distribution of labeled terminals in the pons. Effective mPFC injections showed labeled axons in the ipsilateral descending pyramidal tract in both species. In rats, discrete clusters of densely labeled terminals were observed primarily in the rostromedial pons. Clusters of labeled terminals were also observed contralateral to mPFC injection sites in rats, appearing as a less dense "mirror‐image" of ipsilateral labeling. In rabbits, mPFC labeled corticopontine terminals were absent in the rostral pons, and instead were restricted to the intermediate pons. The densest terminal fields were typically observed in association with the ipsilateral pyramidal tract as it descended ventromedially through the rabbit pons. No contralateral terminal labeling was observed for any injections made in the rabbit mPFC. The results suggest the possibility that mPFC inputs to the pons may be integrated with different sources of cortical inputs between rats and rabbits. The resulting implications for mPFC or pons manipulations for studies of trace eyeblink in each species are discussed. J. Comp. Neurol. 522:3052–3074, 2014. © 2014 Wiley Periodicals, Inc.     

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram^®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.     

Anti‐gravity treadmills are effective in reducing knee forces

Lower body positive pressure (LBPP) treadmills permit significant unweighting of patients and have the potential to enhance recovery following lower limb surgery. We determined the efficacy of an LBPP treadmill in reducing knee forces in vivo. Subjects, implanted with custom electronic tibial prostheses to measure forces in the knee, were tested on a treadmill housed within a LBPP chamber. Tibiofemoral forces were monitored at treadmill speeds from 1.5 mph (0.67 m/s) to 4.5 mph (2.01 m/s), treadmill incline from ?10° to +10°, and four treadmill chamber pressure settings adjusted to decrease net treadmill reaction force from 100% to 25% of the subject's body weight (BW). The peak axial tibiofemoral force ranged from 5.1 times BW at a treadmill speed of 4.5 mph (2.01 m/s) and a pressure setting of 100% BW to 0.8 times BW at 1.5 mph (0.67 m/s) and a pressure setting of 25% BW. Peak knee forces were significantly correlated with walking speed and treadmill reaction force (R² = 0.77, p = 0.04). The LBPP treadmill might be an effective tool in the rehabilitation of patients following lower‐extremity surgery. The strong correlation between tibiofemoral force and walking speed and treadmill reaction forces allows for more precisely achieving the target knee forces desired during early rehabilitation. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 672–679, 2013     

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.     

The anatomy and pathophysiology of neck pain

This article carefully itemizes the various anatomic structures that can evoke neck pain, putting in perspective what clinicians know, what they assume, and what they need to understand better about neck pain and pain referred from the neck. The critique of many of the accepted entities in the differential diagnosis of neck pain is crucial to an understanding of the causes of neck pain and an ability to implement appropriate therapies.     

Genetically encoded Ca2+-sensor reveals details of porcine endothelial cell activation upon contact with human serum

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca²⁺-messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca²⁺-sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca²⁺-sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca²⁺ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca²⁺-sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.     

Altitude training causes haematological fluctuations with relevance for the Athlete Biological Passport

The impact of altitude training on haematological parameters and the Athlete Biological Passport (ABP) was evaluated in international‐level elite athletes. One group of swimmers lived high and trained high (LHTH, n = 10) for three to four weeks at 2130 m or higher whereas a control group (n = 10) completed a three‐week training camp at sea‐level. Haematological parameters were determined weekly three times before and four times after the training camps. ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (RET%), OFF score and the abnormal blood profile score (ABPS) were calculated using the Bayesian model. After altitude training, six swimmers exceeded the 99% ABP thresholds: two swimmers exceeded the OFF score thresholds at day +7; one swimmer exceeded the OFF score threshold at day +28; one swimmer exceeded the threshold for RET% at day +14; and one swimmer surpassed the ABPS threshold at day +14. In the control group, no values exceeded the individual ABP reference range. In conclusion, LHTH induces haematological changes in Olympic‐level elite athletes which can exceed the individually generated references in the ABP. Training at altitude should be considered a confounding factor for ABP interpretation for up to four weeks after altitude exposure but does not consistently cause abnormal values in the ABP. Copyright © 2014 John Wiley & Sons, Ltd.