Sexual differentiation of the rodent brain is dependent upon the organizing actions of the steroid hormone, estradiol. In the preoptic area, a brain region critical for the expression of adult ...reproductive behavior, there are twice as many dendritic spine synapses per unit length on newborn male neurons compared to female neurons and this sex difference correlates with the expression of adult male copulatory behavior. The sex difference in the POA is achieved via estradiol's upregulation of the membrane-derived lipid signaling molecule prostaglandin E₂ (PGE₂); PGE₂ is necessary and sufficient to masculinize both dendritic spine density and adult sexual behavior in rats. We have previously shown that PGE₂ activates EP₂ and EP₄ receptors which increases protein kinase A (PKA) activity and that masculinized dendritic spine density and sex behavior are both dependent upon PKA as well as activation of AMPA type glutamate receptors. In the current experiments, we build upon this signaling cascade by determining that PGE₂ induces phosphorylation of the AMPA receptor subunit, GluR1, which leads to increased AMPA receptor insertion at the membrane. Treating female pups on the day of birth with PGE₂ induced the phosphorylation of GluR1 at the PKA-sensitive site within 2 hours of treatment, an effect that was blocked by co-administration of the PKA/AKAP inhibitor, HT31 with PGE₂. Brief treatment of mixed neuronal/glial POA cultures with PGE₂ or the cAMP/PKA stimulator, forskolin, increased membrane associated GluR1 in both neurons and glia. We speculate that PGE₂ induced increases in AMPA receptor associated with the membrane underlies our previously observed increase in dendritic spine density and is a critical component in the masculinization of rodent sex behavior.
Abstract
Objective. Determine outcomes of cooled radiofrequency ablation (C-RFA) of the genicular nerves for treatment of chronic knee pain due to osteoarthritis (OA).
Design. Cross-sectional survey.
...Setting. Academic pain medicine center.
Subjects. Consecutive patients with knee OA and 50% or greater pain relief following genicular nerve blocks who underwent genicular nerve C-RFA.
Methods. Survey administration six or more months after C-RFA. Pain numeric rating scale (NRS), Medication Quantification Scale III (MQSIII), Patient Global Impression of Change (PGIC), and total knee arthroplasty (TKA) data were collected. Logistic regression was used to identify factors that predicted treatment success.
Results. Thirty-three patients (52 discrete knees) met inclusion criteria. Thirty-five percent (95% confidence interval CI = 22–48) of procedures resulted in the combined outcome of 50% or greater reduction in NRS score, reduction of 3.4 or more points in MQSIII score, and PGIC score consistent with “very much improved/improved.” Nineteen percent (95% CI = 10–33) of procedures resulted in complete pain relief. Greater duration of pain and greater than 80% pain relief from diagnostic blocks were identified as predictors of treatment success. The accuracy of the model was 0.88 (95% CI = 0.78–0.97, P < 0.001).
Conclusions. Genicular C-RFA demonstrated a success rate of 35% based on a robust combination of outcome measures, and 19% of procedures resulted in complete relief of pain at a minimum of six months of follow-up. Report of 80% or greater relief from diagnostic blocks and duration of pain of less than five years are associated with high accuracy in predicting treatment success. Further prospective study is needed to optimize the patient selection protocol and success rate of this procedure.
Magnetic electrospun fibers are of interest for minimally invasive biomaterial applications that also strive to provide cell guidance. Magnetic electrospun fibers can be injected and then ...magnetically positioned in situ, and the aligned fiber scaffolds provide consistent topographical guidance to cells. In this study, magnetically responsive aligned poly-l-lactic acid electrospun fiber scaffolds were developed and tested for neural applications. Incorporating oleic acid-coated iron oxide nanoparticles significantly increased neurite outgrowth, reduced the fiber alignment, and increased the surface nanotopography of the electrospun fibers. After verifying neuron viability on two-dimensional scaffolds, the system was tested as an injectable three-dimensional scaffold. Small conduits of aligned magnetic fibers were easily injected in a collagen or fibrinogen hydrogel solution and repositioned using an external magnetic field. The aligned magnetic fibers provided internal directional guidance to neurites within a three-dimensional collagen or fibrin model hydrogel, supplemented with Matrigel. Neurites growing from dorsal root ganglion explants extended 1.4–3× farther on the aligned fibers compared with neurites extending in the hydrogel alone. Overall, these results show that magnetic electrospun fiber scaffolds can be injected and manipulated with a magnetic field in situ to provide directional guidance to neurons inside an injectable hydrogel. Most importantly, this injectable guidance system increased both neurite alignment and neurite length within the hydrogel scaffold.
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Castrate resistant prostate cancer (CRPC) remains a major challenge for healthcare professionals. Immunotherapeutic approaches, including DNA vaccination, hold the potential to ...harness the host’s own immune system to mount a cell-mediated, anti-tumour response, capable of clearing disseminated tumour deposits. These anti-cancer vaccines represent a promising strategy for patients with advanced disease, however, to date DNA vaccines have demonstrated limited efficacy in clinical trials, owing to the lack of a suitable DNA delivery system. This study was designed to evaluate the efficacy of a two-tier delivery system incorporating cationic RALA/pDNA nanoparticles (NPs) into a dissolvable microneedle (MN) patch for the purposes of DNA vaccination against prostate cancer. Application of NP-loaded MN patches successfully resulted in endogenous production of the encoded Prostate Stem Cell Antigen (PSCA). Furthermore, immunisation with RALA/pPSCA loaded MNs elicited a tumour-specific immune response against TRAMP-C1 tumours ex vivo. Finally, vaccination with RALA/pPSCA loaded MNs demonstrated anti-tumour activity in both prophylactic and therapeutic prostate cancer models in vivo. This is further evidence that this two-tier MN delivery system is a robust platform for prostate cancer DNA vaccination.
This research describes the development and utilisation of our unique microneedle (MN) DNA delivery system, which enables penetration through the stratum corneum and deposition of the DNA within the highly immunogenic skin layers via a dissolvable MN matrix, and facilitates cellular uptake via complexation of pDNA cargo into nanoparticles (NPs) with the RALA delivery peptide. We report for the first time on using the NP-MN platform to immunise mice with encoded Prostate Stem Cell Antigen (mPSCA) for prostate cancer DNA vaccination. Application of the NP-MN system resulted in local mPSCA expression in vivo. Furthermore, immunisation with the NP-MN system induced a tumour-specific cellular immune response, and inhibited the growth of TRAMP-C1 prostate tumours in both prophylactic and therapeutic challenge models in vivo.
Context: Viscoelastic hemostatic assays (VHA) are commonly used to identify specific cellular and humoral causes for bleeding in cardiac surgery patients. Cardiopulmonary bypass (CPB) alterations to ...coagulation are observable on VHA. Citrated VHA can approximate fresh whole blood VHA when kaolin is used as the activator in healthy volunteers. Some have suggested that noncitrated blood is more optimal than citrated blood for point-of-care analysis in some populations.
Aims: To determine if storage of blood samples in citrate after CPB alters kaolin activated VHA results.
Settings and Design: This was a prospective observational cohort study at a single tertiary care teaching hospital.
Methods and Material: Blood samples were subjected to VHA immediately after collection and compared to samples drawn at the same time and stored in citrate for 30, 90, and 150 min prior to kaolin activated VHA both before and after CPB.
Statistical Analysis Used: VHA results were compared using paired T-tests and Bland-Altman analysis.
Results: Maximum clot strength and time to clot initiation were not considerably different before or after CPB using paired T-tests or Bland-Altman Analysis.
Conclusions: Citrated samples appear to be a clinically reliable substitute for fresh samples for maximum clot strength and time to VHA clot initiation after CPB. Concerns about the role of citrate in altering the validity of the VHA samples in the cardiac surgery population seem unfounded.
Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and ...virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.
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Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy ...of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer.
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