A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport

In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.     

Cumulative life stress in chronic fatigue syndrome

We studied the impact of cumulative life stress on CFS in a population-based study. We found that exposure to stressors was significantly more common in persons with CFS compared to NF controls; those with CFS reported experiencing significantly higher levels of psychological distress. Also, post-traumatic stress disorder was significantly more common in people with CFS. These results not only corroborate findings from other studies but, importantly, extend those by: a) measuring a comprehensive spectrum of stress variables, b) for the first time presenting data on stress in a population-based study, thus minimizing the effects of recruitment bias, and c) diagnosing CFS by means of standardized, validated scales, thus allowing replication and extension of our findings. Stress may be an important factor in the pathophysiology of CFS. Consequently, future studies should provide a more detailed understanding of the processes that lead from stress to CFS using longitudinal designs.     

Effects of stressors and immune activating agents on peripheral and central cytokines in mouse strains that differ in stressor responsivity

The impact of inflammatory immune activation on behavioral and physiological processes varies with antecedent stressor experiences. We assessed whether immune activation would differentially influence such outcomes as a function of stressor reactivity related to genetic differences. To this end, we assessed the influence of a social stressor (exposure to a dominant mouse) in combination with an acute immune challenge on behavior and on peripheral and central cytokines in stressor-reactive BALB/cByJ mice and the less reactive C57BL/6ByJ strain. As C57BL/6ByJ and BALB/cByJ mice are highly T helper type-1 (Th1) and Th2 responsive, respectively, the stressor effects were assessed in response to different challenges, namely the viral analogue poly I:C and the bacterial endotoxin lipopolysaccharide (LPS). The stressor enhanced the effects of LPS on sickness behaviors and plasma corticosterone particularly in BALB/cByJ mice, whereas the effects of poly I:C, which primarily affects Th1 processes, were not augmented by the stressor. As well, the stressor increased circulating cytokines in LPS treated C57BL/6ByJ mice, whereas the effects of poly I:C were diminished. Finally, like circulating cytokines, mRNA expression of pro-inflammatory cytokines within the prefrontal cortex and hippocampus varied with the mouse strain and with the stressor experience, and with the specific cytokine considered. Together, the experiments indicated that the impact of stressors vary with the nature of the immune challenge to which animals had been exposed. Moreover, given the diversity of the stressor effects on central and peripheral processes, it seems likely that the cytokine changes, HPA activity and sickness operate through independent mechanisms.     

A neural region of abstract working memory

Over 350 years ago, Descartes proposed that the neural basis of consciousness must be a brain region in which sensory inputs are combined. Using fMRI, we identified at least one such area for working memory, the limited information held in mind, described by William James as the trailing edge of consciousness. Specifically, a region in the left intraparietal sulcus was found to demonstrate load-dependent activity for either visual stimuli (colored squares) or a combination of visual and auditory stimuli (spoken letters). This result was replicated across two experiments with different participants and methods. The results suggest that this brain region, previously well known for working memory of visually presented materials, actually holds or refers to information from more than one modality.     

Aggressive Care After a Massive Stroke in Young Patients: Is That What They Want?

Background  

The assumption is often made that aggressive care in the form of early decompressive hemicraniectomy is appropriate for young patients who suffer a massive stroke. However, neither their attitude toward aggressive treatment, nor their perception of acceptable quality of life after a stroke, has been adequately studied.