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Repeated administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPARγ is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal (i.t.) injection of pioglitazone, a PPARγ agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid antihyperalgesic actions of PPARγ activation, we administered pioglitazone to rats with spared nerve injury and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 minutes of injection, consistent with a nongenomic mechanism. Systemic or i.t. administration of GW9662, a PPARγ antagonist, inhibited the antihyperalgesic actions of intraperitoneal or i.t. pioglitazone, suggesting a spinal PPARγ-dependent mechanism. To further address the contribution of nongenomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When coadministered intrathecally, anisomycin did not change pioglitazone antihyperalgesia at an early 7.5-minute time point, further supporting a rapid nongenomic mechanism. At later time points, anisomycin reduced pioglitazone antihyperalgesia, suggesting delayed recruitment of genomic mechanisms. Pioglitazone reduction of spared nerve injury-induced increases in GFAP expression occurred more rapidly than expected, within 60 minutes. We are the first to show that activation of spinal PPARγ rapidly reduces neuropathic pain independent of canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation and through both genomic and nongenomic PPARγ mechanisms.

Conceived in terms of diaspora, the impact of CI enterprises can be thought of in terms of workforce development, strengthening leading edge industrial knowledge, and building national capabilities. To investigate how we might think through the diaspora of CI enterprises, we conducted a pilot study of one major CI enterprise: the National Center for Supercomputing Applications (NCSA), housed at the University of Illinois at Urbana-Champaign. We compiled and analyzed data from 425 key employees who departed NCSA in the decade from 2003-2013. Often the best way to make sense of complex data involves visualization, and the purpose of this effort was to experiment with visualizations of this diaspora data in an effort to better understand the impact of NCSA’s diaspora.

Indomethacin is a non-narcotic and non-steroidal anti-inflammatory drug used in the treatment of various inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In neonates, it is also used for induction of closure of patent ductus arteriosus (PDA). Its mechanism of action is believed to be through the inhibition of cyclooxygenase. Due to narrow therapeutic window and number of side effects, it's monitoring, particularly in neonates, is recommended. In the gas chromatography method described here, the drug is extracted from serum or plasma using methylene chloride and phosphate buffer (pH 6). The methylene chloride phase containing drug is separated and dried under stream of nitrogen. The drug is derivatized using Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS (trimethylchlorosilane). The derivatized drug is analyzed using gas chromatography-mass spectrometry. Quantitation of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring. Meclofenamic acid is used as an internal standard.

Indomethacin is a non-narcotic and non-steroidal anti-inflammatory drug used in the treatment of various inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In neonates, it is also used for induction of closure of patent ductus arteriosus (PDA). Its mechanism of action is believed to be through the inhibition of cyclooxygenase. Due to narrow therapeutic window and number of side effects, it's monitoring, particularly in neonates, is recommended. In the gas chromatography method described here, the drug is extracted from serum or plasma using methylene chloride and phosphate buffer (pH 6). The methylene chloride phase containing drug is separated and dried under stream of nitrogen. The drug is derivatized using Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS (trimethylchlorosilane). The derivatized drug is analyzed using gas chromatography-mass spectrometry. Quantitation of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring. Meclofenamic acid is used as an internal standard.

Process-mapping of the patient pathway at the Birmingham Women's Hospital revealed that, after an abnormal smear was identified, women could experience a significant delay before referral for treatment. The direct referral policy was introduced in 2007 as part of a wider pan-Birmingham project to address this.To investigate the impact of the direct referral policy on patient experience.An integrated survey was conducted (n=200) and collected both quantitative and qualitative data. Following the introduction of a new information booklet, the same survey was repeated (n=200).The findings showed that, although there was improvement in the information provided, some women stated that no information was received.This highlights the fact that, while there is a need to repeat annual patient surveys to ensure the patient pathway is as robust as possible, surveys may not capture the quality of patients' clinical experience. Suggestions are made to address this (see Implications for practice).This study shows the importance of evaluating any changes in care delivery. The direct referral policy has reduced the time it takes for women to be seen in the colposcopy department following an abnormal smear.

Ca2+ is an obligatory factor for both the extrinsic and intrinsic pathways of coagulation. In majority of in vitro studies, investigators use saturating concentrations of Ca2+ (5 to 10 mM) for FVIIa/tissue factor (TF) activation of factor IX (FIX), and factor X (FX) (extrinsic coagulation), as well as for the activation of FIX by FXIa, FX by FIXa/FVIIIa, and prothrombin by FXa/FVa (intrinsic coagulation). However, the concentration of Ca2+ in plasma is only 1.1 mM, which is considerably below the saturating concentration needed for optimal coagulation. Importantly, plasma also contains 0.6 mM Mg2+ that could compensate for subsaturating concentrations of Ca2+ in promoting coagulation. Previous studies have attempted to clarify this concept in FIX, FX and prothrombin activation. However, these studies are sparse and in virtually all cases not detailed. We have systematically examined the role of plasma concentration of Mg2+ (in addition to the plasma concentration of Ca2+) in promoting all Ca2+ dependent steps of extrinsic and intrinsic coagulation and compared it with the saturating concentration of Ca2+. The Km (~ 100 nM) for activation of FIX by FXIa was similar in the presence of plasma concentrations of Ca2+/Mg2+ or 5 mM Ca2+. Furthermore, the Km and Vmax for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The Km and Vmax for the activation of FX by FVIIIa/FIXa ± phospholipid, and prothrombin by FXa/FVa ± phospholipid were also indistinguishable in these two different metal ion conditions. Notably, when only plasma concentrations of Ca2+ (1.1mM or 1.7mM) were used in all reactions mentioned above, coagulation proceeded at suboptimal rates. In further studies, we used Biacore to investigate the binding of FXIa and FVIII to FIXa, soluble TF to FVIIa, and FVa to FXa. Soluble TF, dansyl-Glu-Gly-Arg (dEGR)-IXa and dEGR-Xa were coupled to CM5 chips in the presence of 10 mM Ca2+. At 5 mM Ca2+, the binding of FXIa to dEGR-IXa was characterized by a Kd of ~40 nM, binding of FVIII to dEGR-IXa by a Kd of ~100 nM, and FVa to dEGR-Xa by a Kd of ~120 nM. In the presence of plasma concentrations of Ca2+ and Mg2+, binding constants were similar to those obtained in the presence of 5 mM saturating Ca2+ concentration. Additional 45Ca2+ binding studies using equilibrium dialysis and prothrombin fragment 1, dEGR-VIIa and decarboxylated dEGR-VIIa, FIX and decarboxylated FIX, and FX and decarboxylated FX, indicated that in the g-carboxyglutamic acid (Gla) domain, 2-3 Ca2+ binding sites (Shikimoto, et al., J. Biol. Chem. 278, 24090-24094, 2003; Wang, et al., Biochemistry42, 7959-7966, 2003; Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006) out of seven core divalent ion binding sites (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992) could be replaced by Mg2+. Conversely, Mg2+ could not displace the Ca2+ binding sites in the epidermal growth factor-like domain 1 (EGF1) and protease domains of FIX or FX. Overall these studies indicate that (1) saturating concentrations of Ca2+ used in in vitro investigations are valid representations of coagulation studies, except for that Mg2+ compensates for suboptimal concentrations of Ca2+ under physiological conditions; (2) two of the Ca2+-binding sites in the Gla domain (numbers 1 and 7, per Tulinsky numbering (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992)), and possibly a third site (number 4) are specific for Mg2+ under physiologic conditions; and (3) the Ca2+-binding sites in the EGF1 and protease domains are specific for Ca2+ and can not be occupied by Mg2+ under physiologic conditions. In conclusion, Ca2+ and Mg2+ act in concert to promote optimal coagulation under physiologic conditions. Mg2+ alone does not promote coagulation since it cannot bind to the Ca2+ specific sites in the Gla domain necessary for folding of the Gla domain omega loop.

Factor VII (FVII) consists of an N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. The complex of tissue factor (TF) and FVIIa activates FIX and FX during coagulation. FVIIa on its own is structurally more “zymogen-like” and when bound to TF it is more “active enzyme-like.” We obtained crystal structures of EGR-VIIa/soluble (s) TF (2.9 Å resolution), dansyl-EGR-VIIa/sTF (1.9 Å resolution) and benzamidine-VIIa/sTF (1.6 Å resolution). We also investigated the effect of TF binding on the S1, S2, and S3/S4 subsites (Schechter and Berger, BBRC, 27:157-162, 1967) in FVIIa. The affinity of variously inhibited FVIIa to sTF was also measured using Biacore technology. For obtaining second order inhibition rate constants, FVIIa ± soluble (s) TF was incubated with each inhibitor for various times, diluted several fold and assayed for the residual FVIIa activity. The second order rate constants were obtained by plotting the first order rate constants versus the inhibitor concentrations. These data are summarized in the table below. From these data it appears that all subsites are affected upon FVIIa binding to sTF. Since in the crystal structure of EGR-VIIa/sTF the P1 Arg residue is the only residue that makes contact with FVIIa, it follows that the S1 site is affected ~10-fold upon binding to sTF. Adding a dansyl group that partially occupies the S3/S4 position (1.9 Å structure) increases the second order rate constant 7-fold (2.41 versus 0.35) over that of EGR-ck. Moreover the addition of Pro (DFPR-ck) or Phe (DFFR-ck) residue occupying the S2 position increases the second order rate constant 357-fold and 1500- fold, respectively (125 and 525 versus 0.35). Thus, comparison of dEGR, DFPR, DFFR inhibition suggests that FVIIa prefers Phe at S2 and at S3/S4 positions, and that TF opens up the S1/S2/S3/S4 sites for substrate or inhibitor occupancy. These data are consistent with LTR (P3/P2/P1) residues in FX at its activation cleavage site as well as with LTR (P3/P2/P1) residues and FTR (P3/P2/P1) residues at the 145-146 and 180-181 FIX activation cleavage sites, respectively. Thus, these studies with chloromethylketone inhibitors have biologic relevance. For Biacore studies, sTF was amine coupled to a CM5 chip. The binding of unoccupied active site FVIIa in 5 mM calcium to sTF was characterized by a KD of 7 nM. Benzamidine (10 mM)-VIIa, p-aminobenzamidine (pAB, 1 mM)-VIIa, EGR-VIIa, dEGR-VIIa, DFPR-VIIa and DFFR-VIIa each bound to sTF with KD values ranging from 1- 2 nM. These affinity measurements indicate that the S1 site occupied FVIIa molecule (benzamidine-FVIIa, pAB-VIIa) has essentially the same conformation as the S1/S2/S3/S4 occupied FVIIa. This conclusion is consistent with similar crystal structures of variously inhibited FVIIa molecules complexed with sTF. The differential rates of incorporation of various chloromethylketone inhibitors could be due to the interaction of various residues (P1, P2, P3, P4) with the corresponding active subsites (S1/S2/S3/S4) of FVIIa. Additionally, the rate of incorporation of chloromethylketone inhibitors into FVIIa also involves the irreversible alkylation step, which could be faster for DFFR-ck and DFPR-ck. Once these inhibitors are incorporated, it appears that they induce the same conformation in FVIIa as achieved by S1 site occupancy alone. Thus S1 site occupancy in FVIIa induces the required conformation to modestly increase the affinity for TF. Second Order Rate Constants for Inhibition of FVIIa ± sTF with Various Chloromethylketone (ck) Inhibitors Inhibitor Minus sTF k (min−1 mM−1) Plus sTF k (min−1 mM−1) Fold Difference EGR-ck 0.04 0.35 8.8 dansyl EGR-ck 0.07 2.41 34.4 (D)FPR-ck 2.3 125 54.3 D)FFR-ck 5.6 525 93.8