ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation

Inverted orbital polarization in strained correlated oxide films

Manipulating the orbital occupation of valence electrons via epitaxial strain in an effort to induce new functional properties requires considerations of how changes in the local bonding environment affect the band structure at the Fermi level. Using synchrotron radiation to measure the x-ray linear dichroism of epitaxially strained films of the correlated oxide CaFeO3, we demonstrate that the orbital polarization of the Fe valence electrons is opposite from conventional understanding. Although the energetic ordering of the Fe 3d orbitals is confirmed by multiplet ligand field theory analysis to be consistent with previously reported strain-induced behavior, we find that the nominally higher energy orbital is more populated than the lower. We ascribe this inverted orbital polarization to an anisotropic bandwidth response to strain in a compound with nearly filled bands. These findings provide an important counterexample to the traditional understanding of strain-induced orbital polarization and reveal a new method to engineer otherwise unachievable orbital occupations in correlated oxides

Conflicting evidence for the role of JNK as a target in breast cancer cell proliferation: comparisons between pharmacological inhibition and selective shRNA knockdown approaches

As a target, the JNK pathway has been implicated in roles including cell death, proliferation, and inflammation in variety of contexts which span cardiovascular disease, neurodegenerative pathologies, and cancer. JNK1 and JNK2 have recently been demonstrated to function independently, highlighting a new parameter in the study of the JNK pathway. In order for JNK1 and JNK2-specific roles to be defined, better tools need to be employed. Previous studies have relied upon the broad spectrum JNK inhibitor, SP600125, to characterize the role of JNK signaling in a number of cell lines, including the breast cancer cell line MCF-7. In line with previous literature, our study has demonstrated that SP600125 treatment inhibited c-Jun and JNK phosphorylation and MCF-7 proliferation. However, in addition to targeting JNK1, JNK2, and JNK3, SP600125 has been previously demonstrated to suppress the activity of a number of other serine/threonine kinases, making SP600125 an inadequate tool for JNK isoform-specific roles to be determined. In this study, lentiviral shRNA was employed to selectively knockdown JNK1, JNK2, and JNK1/2 in MCF-7 cells. Using this approach, JNK phosphorylation was fully inhibited following stable knockdown of respective JNK isoforms. Interestingly, despite suppression of JNK phosphorylation, MCF-7 cell proliferation, cell cycle progression, or cell death remained unaffected. These findings raise the question of whether JNK phosphorylation really is pivotal in MCF-7 cell growth and death or if suppression of these events is a result of one of the many off-targets cited for SP600125

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC-II and costimulatory molecules, which was associated with increased capacity of activating antigen-specific T cells, at first day but not three weeks after five daily treatment with anti-CD52-Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC-II and costimulatory molecules after antibody treatment at both time-points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti-CD52-Ab treatment was also observed in the lymphocyte-deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue-specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS-specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients

Chlorhexidine hexametaphosphate as a wound care material coating: antimicrobial efficacy, toxicity and effect on healing.

AIM: In this study, chlorhexidine hexametaphosphate (CHX-HMP) is investigated as a persistent antimicrobial coating for wound care materials. MATERIALS & METHODS: CHX-HMP was used as a wound care material coating and compared with chlorhexidine digluconate materials with respect to antimicrobial efficacy, toxicity and wound closure. RESULTS: Antimicrobial efficacy at day 1, 3 and 7 was observed with experimental and commercial materials. CHX-HMP coated materials had less toxic effect on human placental cells than commercial chlorhexidine dressings. CHX-HMP in pluronic gel did not delay healing but reduced wound colonization by E. faecalis. CONCLUSION: CHX-HMP could become a useful component of wound care materials with sustained antimicrobial efficacy, lower toxicity than chlorhexidine digluconate materials, and reduction in wound colonization without affecting closure

Baseline neutrophil-lymphocyte ratio holds no prognostic value for esophageal and junctional adenocarcinoma in patients treated with neoadjuvant chemotherapy

BACKGROUND: Several studies have reported that neutrophil-lymphocyte ratio (NLR) can predict survival in esophageal and gastroesophageal junction adenocarcinoma, as it reflects systemic inflammation. Hence, we aimed to determine whether baseline NLR holds prognostic value for esophageal adenocarcinoma patients treated with neoadjuvant chemotherapy (nCT) followed by surgery. METHODS: We studied the data of 139 patients that received nCT before undergoing esophagectomy with curative intent, all identified from a prospectively maintained database (1998-2016). Pretreatment hematology reports were used to calculate the baseline NLR. A receiver operating characteristic curve (ROC-curve) was plotted to determine an optimal cutoff value. NLR quartiles were used to display possible differences between groups in relation to overall survival (OS) and disease-free survival (DFS) using the method of Kaplan-Meier. Cox regression analysis was performed to assess the prognostic value of NLR. RESULTS: The median OS and DFS times were 46 months (interquartile range [IQR]: 19-166) and 30 months (IQR: 13-166], respectively, for the entire cohort. The ROC-curve showed that NLR has no discriminating power for survival status (area under the curve = 0.462) and therefore no optimal cutoff value could be determined. There were no statistically significant differences in median OS times for NLR quartiles: 65 (Q1), 32 (Q2), 45 (Q3), and 46 months (Q4) (P = 0.926). Similarly, DFS showed no difference between quartile groups, with median survival times of 27 (Q1), 19 (Q2), 36 (Q3), and 20 months (Q4) (P = 0.973). Age, pN, pM, and resection margin were independent prognostic factors for both OS and DFS. On the contrary, NLR was not associated with OS or DFS in univariable and multivariable analyses. CONCLUSION: Baseline NLR holds no prognostic value for esophageal and gastroesophageal junction adenocarcinoma patients treated with nCT in this study, in contrast to other recently published papers. This result questions the validity of NLR as a reliable prognostic indicator and its clinical usefulness in these patients

A multinuclear solid state NMR, density functional theory and X-Ray diffraction study of hydrogen bonding in Group I hydrogen dibenzoates

An NMR crystallographic approach incorporating multinuclear solid state NMR (SSNMR), X-ray structure determinations and density functional theory (DFT) are used to characterise the H bonding arrangements in benzoic acid (BZA) and the corresponding Group I alkali metal hydrogen dibenzoates (HD) systems. Since the XRD data often cannot precisely confirm the proton position within the hydrogen bond, the relationship between the experimental SSNMR parameters and the ability of gauge included plane augmented wave (GIPAW) DFT to predict them becomes a powerful constraint that can assist with further structure refinement. Both the 1H and 13C MAS NMR methods provide primary descriptions of the H bonding via accurate measurements of the 1H and 13C isotropic chemical shifts, and the individual 13C chemical shift tensor elements; these are unequivocally corroborated by DFT calculations, which together accurately describe the trend of the H bonding strength as the size of the monovalent cation changes. In addition, 17O MAS and DOR NMR form a powerful combination to characterise the O environments, with the DOR technique providing highly resolved 17O NMR data which helps verify unequivocally the number of inequivalent O positions for the conventional 17O MAS NMR to process. Further multinuclear MAS and static NMR studies involving the quadrupolar 7Li, 39K, 87Rb and 133Cs nuclei, and the associated DFT calculations, provide trends and a corroboration of the H bond geometry which assist in the understanding of these arrangements. Even though the crystallographic H positions in each H bonding arrangement reported from the single crystal X-ray studies are prone to uncertainty, the good corroboration between the measured and DFT calculated chemical shift and quadrupole tensor parameters for the Group I alkali species suggest that these reported H positions are reliable

Effect of sphingosine kinase modulators on interleukin-1β release, sphingosine 1-phosphate receptor 1 expression and experimental autoimmune encephalomyelitis : FTY720 analogues and inflammation

Background and Purpose: The sphingosine analogue, FTY720 (Gilenya®) alleviates clinical disease progression in multiple sclerosis. Here we variously assessed the effects of an azide analogue of (S)-FTY720 vinylphosphonate (compound 5; a sphingosine kinase 1 activator), (R)-FTY720 methyl ether (ROMe, a sphingosine kinase 2 inhibitor) and RB-020 (a sphingosine kinase 1 inhibitor and sphingosine kinase 2 substrate) on IL-1beta formation, sphingosine 1-phosphate levels and S1P1 expression. We also assessed the effect of compound 5 and ROMe in an experimental autoimmune encephalomyelitis (EAE) model. Experimental Approach: We measured IL-1beta formation by macrophages, sphingosine 1-phosphate levels and S1P1 expression levels in vitro and clinical score and inflammatory cell infiltration into the spinal cord in vivo. Key Results: Treatment of differentiated U937 macrophages with compound 5, RB-020 or sphingosine (but not ROMe) enhanced IL-1beta release. This data suggests these compounds might be pro-inflammatory in vitro. However, compound 5 or ROMe reduced disease progression and infiltration of inflammatory cells into the spinal cord in EAE and ROMe induced a reduction in CD4+ and CD8+ T-cell levels in the blood (lymphopenia). Indeed, ROMe induced a marked decrease in cell surface S1P1 expression in vitro. Conclusion and Implications: This is the first demonstration that an activator of SK1 (compound 5) and an inhibitor of SK2 (ROMe, which also reduces cell surface S1P1 expression) have an anti-inflammatory action in EAE

Increased levels of IL-16 in the central nervous system during neuroinflammation are associated with infiltrating immune cells and resident glial cells

Interleukin (IL)-16, a CD4 + immune cell specific chemoattractant cytokine, has been shown to be involved in the development of multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). While immune cells such as T cells and macrophages are reported to be the producers of IL-16, the cellular source of IL-16 in the CNS is less clear. This study investigates the correlation of IL-16 expression levels in the CNS with the severity of neuroinflammation and determines the phenotype of cells which produce IL-16 in the CNS of experimental autoimmune encephalomyelitis (EAE) mice. Our data show that IL-16 expression is significantly increased in the brain and spinal cord tissues of EAE mice compared to phosphate buffered saline (PBS) immunised controls. Dual immunofluorescence staining reveals that the significantly increased IL-16 + cells in the CNS lesions of EAE mice are likely to be the CD45 + infiltrating immune cells such as CD4 + or F4/80 + cells and the CNS resident CD11b + microglia and GFAP + astrocytes, but not NeuN+ neurons. Our data suggest cytokine IL-16 is closely involved in EAE pathology as evidenced by its increased expression in the glial and infiltrating immune cells, which impacts the recruitment and activation of CD4 + immune cells in the neuroinflammation

The Internet and HIV study: design and methods

BACKGROUND: The Internet provides a new meeting ground, especially for gay men, that did not exist in the early 1990s. Several studies have found increased levels of high risk sexual behaviour and sexually transmissible infections (STI) among gay men who seek sex on the Internet, although the underlying processes are not fully understood. Research funded by the UK Medical Research Council (2002–2004) provided the opportunity to consider whether the Internet represents a new sexual risk environment for gay and bisexual men living in London. METHODS: The objectives of the Internet and HIV study are to: (i) measure the extent to which gay men living in London seek sexual partners on the Internet; (ii) compare the characteristics of London gay men who do and do not seek sex on the Internet; (iii) examine whether sex with Internet-partners is less safe than with other sexual partners; (iv) compare use of the Internet with other venues where men meet sexual partners; (v) establish whether gay men use the Internet to actively seek partners for unprotected anal intercourse; (vi) determine the potential for using the Internet for HIV prevention. These objectives have been explored using quantitative and qualitative research methods in four samples of London gay men recruited and interviewed both online and offline. The four samples were: (i) gay men recruited through Internet chat rooms and profiles; (ii) HIV positive gay men attending an NHS hospital outpatients clinic; (iii) gay men seeking an HIV test in an NHS HIV testing or sexual health clinic; (iv) gay men recruited in the community. RESULTS: Quantitative data were collected by means of confidential, anonymous self-administered questionnaires (n>4000) completed on-line by the Internet sample. Qualitative data were collected by means of one-to-one interviews (n = 128) conducted either face-to-face or on-line. CONCLUSION: The strength of the Internet and HIV study is its methodological plurality, drawing on both qualitative and quantitative research among online and offline samples, as well as taking advantage of recent advances in web survey design. The study's findings will help us better understand the role of the Internet in relation to gay men's sexual practic