MyD88 Is a Critical Regulator of Hematopoietic Cell-Mediated Neuroprotection Seen after Stroke

Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88^−/− animals or cells were compared with wild type (WT). We found that after stroke Myd88^−/− animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88^−/− and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88^−/− animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88^−/−. We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke.     

Neuronal brain-derived neurotrophic factor is synthesized in excess, with levels regulated by sortilin-mediated trafficking and lysosomal degradation

Brain-derived neurotrophic factor (BDNF) regulates neuronal differentiation, synaptic plasticity, and morphology, and modest changes in BDNF levels results in complex behavioral phenotypes. BDNF levels and intracellular localization in neurons are regulated by multiple mechanisms, including use of distinct promoters, mRNA and protein transport, and regulated cleavage of proBDNF to mature BDNF. Sortilin is an intracellular chaperone that binds to the prodomain of BDNF to traffic it to the regulated secretory pathway. However, sortilin binds to numerous ligands and plays a major role in mannose 6-phosphate receptor-independent transport of lysosomal hydrolases utilizing motifs in the intracellular domain that mediate trafficking from the Golgi and late endosomes. Sortilin is modified by ectodomain shedding, although the biological implications of this are not known. Here we demonstrate that ADAM10 is the preferred protease to cleave sortilin in the extracellular stalk region, to release the ligand binding sortilin ectodomain from the transmembrane and cytoplasmic domains. We identify sortilin shedding at the cell surface and in an intracellular compartment. Both sortilin and BDNF are trafficked to and degraded by the lysosome in neurons, and this is dependent upon the sortilin cytoplasmic tail. Indeed, expression of the sortilin ectodomain, which corresponds to the domain released after shedding, impairs lysosomal targeting and degradation of BDNF. These findings characterize the regulation of sortilin shedding and identify a novel mechanism by which sortilin ectodomain shedding acts as a regulatory switch for delivery of BDNF to the secretory pathway or to the lysosome, thus modulating the bioavailability of endogenous BDNF.     

Control of megakaryocyte expansion and bone marrow fibrosis by lysyl oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.     

Nanomaterials in biological environment: a review of computer modelling studies

Nanotechnology is set to impact a vast range of fields, including computer science, materials technology, engineering/manufacturing and medicine. As nanotechnology grows so does exposure to nanostructured materials, thus investigation of the effects of nanomaterials on biological systems is paramount. Computational techniques can allow investigation of these systems at the nanoscale, providing insight into otherwise unexaminable properties, related to both the intentional and unintentional effects of nanomaterials. Herein, we review the current literature involving computational modelling of nanoparticles and biological systems. This literature has highlighted the common modes in which nanostructured materials interact with biological molecules such as membranes, peptides/proteins and DNA. Hydrophobic interactions are the most favoured, with π-stacking of the aromatic side-chains common when binding to a carbonaceous nanoparticle or surface. van der Waals forces are found to dominate in the insertion process of DNA molecules into carbon nanotubes. Generally, nanoparticles have been observed to disrupt the tertiary structure of proteins due to the curvature and atomic arrangement of the particle surface. Many hydrophobic nanoparticles are found to be able to transverse a lipid membrane, with some nanoparticles even causing mechanical damage to the membrane, thus potentially leading to cytotoxic effects. Current computational techniques have revealed how some nanoparticles interact with biological systems. However, further research is required to determine both useful applications and possible cytotoxic effects that nanoparticles may have on DNA, protein and membrane structure and function within biosystems.     

Lymphocytes transfer [14C]‐labeled fatty acids to skeletal muscle in culture; modulation by exercise

Previous studies have shown that lipids are transferred from lymphocytes (Ly) to different cell types including macrophages, enterocytes, and pancreatic β cells in co‐culture. This study investigated whether [¹⁴C]‐labeled fatty acids (FA) can be transferred from Ly to skeletal muscle (SM), and the effects of exercise on such phenomenon. Ly obtained from exercised (EX) and control (C) male Wistar rats were preloaded with the [¹⁴C]‐labeled free FA palmitic (PA), oleic (OA), linoleic (LA), or arachidonic (AA). Radioactively loaded Ly were then co‐cultured with SM from the same Ly donor animals. Substantial amounts of FA were transferred to SM being the profile PA = OA > AA > LA to the C group, and PA > OA > LA > AA to the EX group. These FA were incorporated predominantly as phospholipids (PA = 66.75%; OA = 63.09%; LA = 43.86%; AA = 47.40%) in the C group and (PA = 63.99% OA = 52.72%; LA = 55.99%; AA = 63.40%) in the EX group. Also in this group, the remaining radioactivity from AA, LA, and OA acids was mainly incorporated in structural and energetic lipids. These results support the hypothesis that Ly are able to export lipids to SM in co‐culture. Furthermore, exercise modulates the lipid transference profile, and its incorporation on SM. The overall significance of this phenomenon in vivo remains to be elucidated. Copyright © 2010 John Wiley & Sons, Ltd.     

Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

Background

Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.

Methods

FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis.

Results

PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.

Conclusion

Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.     

Functional Changes in Littoral Macroinvertebrate Communities in Response to Watershed-Level Anthropogenic Stress

Watershed-scale anthropogenic stressors have profound effects on aquatic communities. Although several functional traits of stream macroinvertebrates change predictably in response to land development and urbanization, little is known about macroinvertebrate functional responses in lakes. We assessed functional community structure, functional diversity (Rao’s quadratic entropy) and voltinism in macroinvertebrate communities sampled across the full gradient of anthropogenic stress in Laurentian Great Lakes coastal wetlands. Functional diversity and voltinism significantly decreased with increasing development, whereas agriculture had smaller or non-significant effects. Functional community structure was affected by watershed-scale development, as demonstrated by an ordination analysis followed by regression. Because functional community structure affects energy flow and ecosystem function, and functional diversity is known to have important implications for ecosystem resilience to further environmental change, these results highlight the necessity of finding ways to remediate or at least ameliorate these effects.     

Effect of Cholecalciferol Supplementation on Inflammation and Cellular Alloimmunity in Hemodialysis Patients: Data from a Randomized Controlled Pilot Trial

Background

Memory T-cells are mediators of transplant injury, and no therapy is known to prevent the development of cross-reactive memory alloimmunity. Activated vitamin D is immunomodulatory, and vitamin D deficiency, common in hemodialysis patients awaiting transplantation, is associated with a heightened alloimmune response. Thus, we tested the hypothesis that vitamin D₃ supplementation would prevent alloreactive T-cell memory formation in vitamin D-deficient hemodialysis patients.

Methods and Findings

We performed a 12-month single-center pilot randomized, controlled trial of 50,000 IU/week of cholecalciferol (D₃) versus no supplementation in 96 hemodialysis patients with serum 25(OH)D<25 ng/mL, measuring effects on serum 25(OH)D and phenotypic and functional properties of T-cells. Participants were randomized 2∶1 to active treatment versus control. D₃ supplementation increased serum 25(OH)D at 6 weeks (13.5 [11.2] ng/mL to 42.5 [18.5] ng/mL, p<0.001) and for the duration of the study. No episodes of sustained hypercalcemia occurred in either group. Results of IFNγ ELISPOT-based panel of reactive T-cell assays (PRT), quantifying alloreactive memory, demonstrated greater increases in the controls over 1 year compared to the treatment group (delta PRT in treatment 104.8+/−330.8 vs 252.9+/−431.3 in control), but these changes in PRT between groups did not reach statistical significance (p = 0.25).

Conclusions

D₃ supplements are safe, effective at treating vitamin D deficiency, and may prevent time-dependent increases in T-cell alloimmunity in hemodialysis patients, but their effects on alloimmunity need to be confirmed in larger studies. These findings support the routine supplementation of vitamin D-deficient transplant candidates on hemodialysis and highlight the need for large-scale prospective studies of vitamin D supplementation in transplant candidates and recipients.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01175798","term_id":"NCT01175798"}}NCT01175798