Endoplasmic reticulum stress in wake-active neurons progresses with aging

Fragmentation of wakefulness and sleep are expected outcomes of advanced aging. We hypothesize that wake neurons develop endoplasmic reticulum dyshomeostasis with aging, in parallel with impaired wakefulness. In this series of experiments, we sought to more fully characterize age-related changes in wakefulness and then, in relevant wake neuronal populations, explore functionality and endoplasmic reticulum homeostasis. We report that old mice show greater sleep/wake transitions in the active period with markedly shortened wake periods, shortened latencies to sleep, and less wake time in the subjective day in response to a novel social encounter. Consistent with sleep/wake instability and reduced social encounter wakefulness, orexinergic and noradrenergic wake neurons in aged mice show reduced c-fos response to wakefulness and endoplasmic reticulum dyshomeostasis with increased nuclear translocation of CHOP and GADD34. We have identified an age-related unfolded protein response injury to and dysfunction of wake neurons. It is anticipated that these changes contribute to sleep/wake fragmentation and cognitive impairment in aging.     

Protective immunity to pre-erythrocytic stage malaria

The development of a vaccine against malaria is a major research priority given the burden of disease, death and economic loss inflicted upon the tropical world by this parasite. Despite decades of effort, however, a vaccine remains elusive. The best candidate is a subunit vaccine termed RTS,S but this provides only partial protection against clinical disease. This review examines what is known about protective immunity against pre-erythrocytic stage malaria by considering the humoral and T cell-mediated immune responses that are induced by attenuated sporozoites and by the RTS,S vaccine. On the basis of these observations a set of research priorities are defined that are crucial for the development of a vaccine capable of inducing long-lasting and high-grade protection against malaria.     

Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.     

Methane emissions from sheep pasture,measured with an open‐path eddy covariance system

Methane (CH₄) is an important greenhouse gas, contributing 0.4–0.5 W m^?2 to global warming. Methane emissions originate from several sources, including wetlands, rice paddies, termites and ruminating animals. Previous measurements of methane flux from farm animals have been carried out on animals in unnatural conditions, in laboratory chambers or fitted with cumbersome masks. This study introduces eddy covariance measurements of CH₄, using the newly developed LI‐COR LI‐7700 open‐path methane analyser, to measure field‐scale fluxes from sheep grazing freely on pasture. Under summer conditions, fluxes of methane in the morning averaged 30 nmol m^?2 s^?1, whereas those in the afternoon were above 100 nmol m^?2 s^?1, and were roughly two orders of magnitude larger than the small methane emissions from the soil. Methane emissions showed no clear relationship with air temperature or photosynthetically active radiation, but some diurnal pattern was apparent, probably linked to sheep grazing behaviour and metabolism. Over the measurement period (days 60–277, year 2010), cumulative methane fluxes were 0.34 mol CH₄ m^?2, equating to 134.3 g CO₂ equivalents m^?2. By comparison, a carbon dioxide (CO₂) sink of 819 g CO₂ equivalents m^?2 was measured over the same period, but it is likely that much of this would be released back to the atmosphere during the winter or as off‐site losses (through microbial and animal respiration). By dividing methane fluxes by the number of sheep in the field each day, we calculated CH₄ emissions per head of livestock as 7.4 kg CH₄ sheep^?1 yr^?1, close to the published IPCC emission factor of 8 kg CH₄ sheep^?1 yr^?1.     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.     

Neuropeptide profiling of the bovine hypothalamus: thermal stabilization is an effective tool in inhibiting post-mortem degradation

The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated samples contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized samples, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post-translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue samples, whereas we detected many trimmed or truncated peptides resulting from post-mortem degradation in the untreated tissue samples. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin-2 and proTRH.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4(+) T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM)) and/or central memory (T(CM)) CD4(+) T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM) and T(CM) cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+) T(E/EM) cells and of CD4(+) T(CM) cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+) T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+) T(E/EM) cells and of CD4(+) T(E/EM) cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM) and T(CM) cells are major producers of IL-2.