The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn’t require expert microsurgical skills.The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.     

Elevated alkalinity and sulfate adversely affect the aquatic macrophyte Lobelia dortmanna

The increase in alkalinity and SO₄ ^2? in softwater lakes can negatively affect pristine isoetid population because the increase in alkalinity and SO₄ ^2? can stimulate sediment mineralization and consequently cause anoxia. The consequences of increased sediment mineralization depend on the ability of isoetids such as Lobelia dortmanna to oxidize the rhizosphere via radial O₂ loss. To study how alkalinity and SO₄ ^2? affect the isoetid L. dortmanna, and if negative effects could be alleviated by neighboring plants, three densities of L. dortmanna (“Low”?=?64 plants m^?2, “Medium”?=?256 plants m^?2 and “High”?=?1,024 plants m^?2) were exposed to elevated alkalinity in the water column, or a combination of both elevated alkalinity and SO₄ ^2?, and compared to a control situation. The combination of SO₄ ^2? and alkalinity significantly increased mortality, lowered areal biomass and reduced actual photosynthetic efficiency. Plant density did not significantly alleviate the negative effects caused by SO₄ ^2? and alkalinity. However, actual photosynthetic efficiency was significantly positively correlated to redox potential in the sediment, indicating a positive relationship between plant performance and sediment oxidation. The negative effects on L. dortmanna were probably caused by long periods of tissue anoxia by itself or in combination with H₂S intrusion. Therefore, increase in both SO₄ ^2? and alkalinity surface water can dramatically affect L. dortmanna populations, causing reduction or even disappearance of this icon species.     

Surfactant protein d deficiency in mice is associated with hyperphagia, altered fat deposition, insulin resistance, and increased basal endotoxemia

Pulmonary surfactant protein D (SP-D) is a host defence lectin of the innate immune system that enhances clearance of pathogens and modulates inflammatory responses. Recently it has been found that systemic SP-D is associated with metabolic disturbances and that SP-D deficient mice are mildly obese. However, the mechanism behind SP-D's role in energy metabolism is not known.Here we report that SP-D deficient mice had significantly higher ad libitum energy intake compared to wild-type mice and unchanged energy expenditure. This resulted in accumulation but also redistribution of fat tissue. Blood pressure was unchanged. The change in energy intake was unrelated to the basal levels of hypothalamic Pro-opiomelanocortin (POMC) and Agouti-related peptide (AgRP) gene expression. Neither short time systemic, nor intracereberoventricular SP-D treatment altered the hypothalamic signalling or body weight accumulation.In ad libitum fed animals, serum leptin, insulin, and glucose were significantly increased in mice deficient in SP-D, and indicative of insulin resistance. However, restricted diets eliminated all metabolic differences except the distribution of body fat. SP-D deficiency was further associated with elevated levels of systemic bacterial lipopolysaccharide.In conclusion, our findings suggest that lack of SP-D mediates modulation of food intake not directly involving hypothalamic regulatory pathways. The resulting accumulation of adipose tissue was associated with insulin resistance. The data suggest SP-D as a regulator of energy intake and body composition and an inhibitor of metabolic endotoxemia. SP-D may play a causal role at the crossroads of inflammation, obesity, and insulin resistance.     

Strategy tool trial for office furniture

Purpose

A strategic product development tool combining REACH and environmental and financial factors was previously developed for a coatings company. This paper presents results from refining this tool for an office furniture company, using life cycle assessment (LCA)-based environmental information, addressing the research questions: ? Is it possible to combine information from REACH with the LCA approach to provide useful information for a furniture producer in their environmental product development process? ? Does the approach developed for substances in mixtures need to be adapted for articles? ? Is there a correlation between energy consumption and the environmental impacts analysed? ? Will product designers get the same information independent of the environmental impact category used? ?C Will the strategy tool indicate the same ranking of products for all environmental impacts? ?C Does REACH information indicate the same set of priorities as those arising from LCA environmental data alone? (Do they agree, or is there a conflict?) ? Will strategic decisions differ if different environmental indicators are in focus? The strategy tool??s purpose is to analyse company product portfolios, identifying products that need redevelopment or redesign because of issues concerning hazardous substances, or environmental performance.

Methods

The LCA data used is cradle-to-gate data from type III environmental declarations for 11 seating solutions. REACH Complexity, health hazard and environmental class indicators (based on risk phrases) are combined with financial data and LCA-based indicators. Correlations between energy consumption and environmental impact factors for these specific furniture products are investigated. Establishing any such correlations serves to simplify subsequent analysis in the product development process, by effectively reducing the number of indicators that need to be taken into consideration.

Results

Correlations between energy consumption and the environmental impacts global warming, acidification, eutrophication and heavy metals are presented. Strategy tool figures are shown for energy consumption, ozone depletion potential and photochemical oxidation potential. The results for office chairs and conference/visitor chairs are presented separately, as the two types of chairs fulfil different functions.

Conclusions

The correlation between energy consumption and certain environmental impact indicators affords a simplification of the product development process, since energy consumption can be used as a reasonable proxy for these indicators in this specific case. The results support acknowledged principles of Ecodesign. Energy and materials minimization improves environmental performance??higher recycled material content and proportion of renewable energy resources are also beneficial. Designers have to consider multiple aspects in parallel and the strategy tool is useful for this purpose; the furniture producer has gained useful product development insight. The tool is applicable for strategic choice of products for development or redesign that can be useful across many business sectors.     

A novel role for Bcl-2 in regulation of cellular calcium extrusion

The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.     

Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma

Background

Prostaglandin E₂ (PGE₂) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE₂ production, the expression pattern and localization of PGE₂ receptors and intracellular signal transduction pathways activated by PGE₂.

Principal Findings

A high expression of the PGE₂ receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE₂ and stimulation of serum-starved neuroblastoma cells with PGE₂ increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE₂ (dmPGE₂) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE₂. Similarly, PGE₂ receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner.

Conclusions

These findings demonstrate that PGE₂ acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE₂ signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.     

Silencing of renal DNaseI in murine lupus nephritis imposes exposure of large chromatin fragments and activation of Toll like receptors and the Clec4e

Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression levels of DNaseI, Toll like receptors 7-9, Clec4e, pro-inflammatory cytokines and MMP2/MMP9 were determined and compared with in situ protein expression profiles and clinical data. We demonstrate that exposure of chromatin significantly up-regulate Toll like receptors and Clec4e in mice, and also but less pronounced in patients with lupus nephritis treated with immunosuppresants. In conclusion, silencing of renal DNaseI gene expression initiates a cascade of inflammatory signals leading to progression of both murine and human lupus nephritis. Principal component analyses biplot of data from murine and human lupus nephrits demonstrate the importance of DNaseI gene shut down for progression of the organ disease.     

Characterization of HIV-specific CD4+ T cell responses against peptides selected with broad population and pathogen coverage

CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73% of the predicted peptides were found to induce HIV-specific CD4+ T cell responses. The Gag and Nef peptides induced most responses. The vast majority of the peptides (93%) had predicted restriction to the patient's HLA alleles. Interestingly, the viral load in viremic patients was inversely correlated to the number of targeted Gag peptides. In addition, the predicted Gag peptides were found to induce broader polyfunctional CD4+ T cell responses compared to the commonly used Gag-p55 peptide pool. These results demonstrate the power of the PopCover method for the identification of broadly recognized HLA class II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines.     

No effect of NGAL/lipocalin-2 on aggressiveness of cancer in the MMTV-PyMT/FVB/N mouse model for breast cancer

NGAL/lipocalin-2 is a siderophore-binding protein that is highly expressed in several cancers. It is suggested to confer a proliferative advantage to cancer cells. Its expression has been correlated with aggressiveness of breast cancer as determined both in patients and in mouse breast cancer models. This was recently confirmed in two mouse models of spontaneous breast cancer in wild-type and lipocalin-2-deficient mice. We used a similar strategy using a different mouse strain. Lipocalin-2-deficient mice and mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) mice were crossed into the same FVB/N background. All mice developed tumors by week 8. The mice were sacrificed on week 13 and tissue was processed for biochemical and histological analysis. The total tumor volume and number of metastases were quantitated in 26 lipocalin-2-deficient mice and 34 wild-type controls. Lipocalin-2 expression in tumors of MMTV-PyMT-positive and wild-type mice was assessed by quantitative real-time PCR and by immunohistochemistry. The expression of the lipocalin-2 receptors 24p3R and megalin and of Mmp-9, transferrin receptor, and Bdh2 (a producer of a mammalian siderophore) were quantitated by real-time PCR. No significant difference was observed between wild-type and lipocalin-2-deficient mice. Lipocalin-2 was highly expressed in tumors from wild-type mice, but the expression did not correlate with tumor size. No effect of lipocalin-2 was observed with respect to time to tumor appearance, total tumor volume, or to the number of metastases. Histology and gelatinolytic activity of the mammary tumors did not differ between wild-type and lipocalin-2-deficient mice. We conclude that NGAL/lipocalin-2 does not invariably affect the aggressiveness of breast cancers as assessed in mouse models, thus questioning the role of lipocalin-2 in cancer development.     

Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghum variety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.