Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The expression and function of glucocorticoid-induced leucine zipper (GILZ) in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease of unknown etiology. It is characterised by inflammation in the synovial joints and is known to affect other organs as well. For over 60 years, glucocorticoids have been used to treat RA, because of their powerful anti-inflammatory properties. However, the mechanism by which glucocorticoids modulate immune responses is not fully understood. Potently induced by glucocorticoids, glucocorticoid-induced leucine zipper (GILZ) is a protein with the unique ability to mimic the therapeutic effects of glucocorticoids. The immuno-suppressive effects of GILZ on key signalling pathways relevant to RA, represents a strategy to reduce pharmacological use of glucocorticoids in RA by harnessing the downstream effects of GILZ. Studies presented in this thesis examine the effect of GILZ in regulating inflammatory processes relevant to RA. The findings in Chapter 3 of this thesis show that endogenous GILZ acts as an anti-inflammatory mediator in collagen-induced arthritis (CIA). In this study, in vivo silencing of endogenous GILZ, using small-interfering RNA during the effector phase of CIA, resulted in the exacerbation of clinical and histological disease. The expression of GILZ in human rheumatoid synovial tissue, and the robust induction of GILZ by dexamethasone in RA fibroblast-like synoviocytes (FLS), suggests an important role for GILZ in human RA. Notably, inhibition of tumor necrosis factor (TNF)-induced cytokines was observed in GILZ overexpressing RA FLS. These findings suggest that modulation of GILZ expression may be therapeutically beneficial in RA. Chapter 4 demonstrates a therapeutic effect of exogenous GILZ in arthritis. In this study, overexpressing GILZ using a recombinant adeno-associated virus (rAAV) vector attenuated CIA severity, comparable to the effects of dexamethasone treatment. The inhibition of NF-κB luciferase activity in vitro by GILZ-rAAV in endothelial cells and macrophages demonstrates a mechanism by which GILZ suppress inflammation. In conjunction, the lack of effect on blood glucose concentrations in response to GILZ-rAAV, suggests anti-inflammatory and disease-modifying effects of GILZ may be achieved without the adverse effects that accompany glucocorticoid therapy. The findings in Chapter 5 report an unexpected role for GILZ in male fertility discovered during the generation of GILZ deficient (GILZ-/-) mice in the course of my PhD. GILZ-/- mice male mice exhibit a loss of germ cells in their testes, which resulted in the failure of spermatogenesis. Characterization of a normal blood-testis barrier and the absence of CD45 leukocytes in the testis demonstrate that this infertility phenotype was not of an autoimmune origin. These data suggest a role for GILZ in the process of spermatogenesis and development of the testes. In Chapter 6, data are presented which indicate that deficiency of physiological GILZ is without impact on the effector pathways of joint inflammation. GILZ-/- mice displayed increased delayed-type hypersensitivity (DTH) responses and increased accompanying ex vivo T cell responses to various antigens. This suggests a role for endogenous GILZ in mediating T cell activation. However, no enhancement of antigen-induced arthritis (AIA), CIA or K/BxN serum transfer arthritis susceptibility or severity was observed in GILZ-/- mice. A lack of effect of GILZ deletion on LPS-induced cytokinemia suggests that GILZ is not involved in mediating the myeloid effector responses of joint disease. Furthermore, contrary to what has been suggested by studies published without access to a GILZ-/- mouse, the results presented in Chapter 6 demonstrate that GILZ is not required for glucocorticoid effects in inflammation. This suggests a redundant effect of physiological GILZ in glucocorticoid suppression of inflammation, not withstanding the clear inhibitory effects of GILZ overexpression. This thesis extends the current understanding of the relationship between glucocorticoid function and GILZ and demonstrates the significant impacts that GILZ may have in the regulation of inflammation

The expression and function of glucocorticoid-induced leucine zipper (GILZ) in models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease of unknown etiology. It is characterised by inflammation in the synovial joints and is known to affect other organs as well. For over 60 years, glucocorticoids have been used to treat RA, because of their powerful anti-inflammatory properties. However, the mechanism by which glucocorticoids modulate immune responses is not fully understood. Potently induced by glucocorticoids, glucocorticoid-induced leucine zipper (GILZ) is a protein with the unique ability to mimic the therapeutic effects of glucocorticoids. The immuno-suppressive effects of GILZ on key signalling pathways relevant to RA, represents a strategy to reduce pharmacological use of glucocorticoids in RA by harnessing the downstream effects of GILZ. Studies presented in this thesis examine the effect of GILZ in regulating inflammatory processes relevant to RA. The findings in Chapter 3 of this thesis show that endogenous GILZ acts as an anti-inflammatory mediator in collagen-induced arthritis (CIA). In this study, in vivo silencing of endogenous GILZ, using small-interfering RNA during the effector phase of CIA, resulted in the exacerbation of clinical and histological disease. The expression of GILZ in human rheumatoid synovial tissue, and the robust induction of GILZ by dexamethasone in RA fibroblast-like synoviocytes (FLS), suggests an important role for GILZ in human RA. Notably, inhibition of tumor necrosis factor (TNF)-induced cytokines was observed in GILZ overexpressing RA FLS. These findings suggest that modulation of GILZ expression may be therapeutically beneficial in RA. Chapter 4 demonstrates a therapeutic effect of exogenous GILZ in arthritis. In this study, overexpressing GILZ using a recombinant adeno-associated virus (rAAV) vector attenuated CIA severity, comparable to the effects of dexamethasone treatment. The inhibition of NF-κB luciferase activity in vitro by GILZ-rAAV in endothelial cells and macrophages demonstrates a mechanism by which GILZ suppress inflammation. In conjunction, the lack of effect on blood glucose concentrations in response to GILZ-rAAV, suggests anti-inflammatory and disease-modifying effects of GILZ may be achieved without the adverse effects that accompany glucocorticoid therapy. The findings in Chapter 5 report an unexpected role for GILZ in male fertility discovered during the generation of GILZ deficient (GILZ-/-) mice in the course of my PhD. GILZ-/- mice male mice exhibit a loss of germ cells in their testes, which resulted in the failure of spermatogenesis. Characterization of a normal blood-testis barrier and the absence of CD45 leukocytes in the testis demonstrate that this infertility phenotype was not of an autoimmune origin. These data suggest a role for GILZ in the process of spermatogenesis and development of the testes. In Chapter 6, data are presented which indicate that deficiency of physiological GILZ is without impact on the effector pathways of joint inflammation. GILZ-/- mice displayed increased delayed-type hypersensitivity (DTH) responses and increased accompanying ex vivo T cell responses to various antigens. This suggests a role for endogenous GILZ in mediating T cell activation. However, no enhancement of antigen-induced arthritis (AIA), CIA or K/BxN serum transfer arthritis susceptibility or severity was observed in GILZ-/- mice. A lack of effect of GILZ deletion on LPS-induced cytokinemia suggests that GILZ is not involved in mediating the myeloid effector responses of joint disease. Furthermore, contrary to what has been suggested by studies published without access to a GILZ-/- mouse, the results presented in Chapter 6 demonstrate that GILZ is not required for glucocorticoid effects in inflammation. This suggests a redundant effect of physiological GILZ in glucocorticoid suppression of inflammation, not withstanding the clear inhibitory effects of GILZ overexpression. This thesis extends the current understanding of the relationship between glucocorticoid function and GILZ and demonstrates the significant impacts that GILZ may have in the regulation of inflammation

Kemampuan Dividen Payout Ratio dan Investment Opportunity Set untuk Mempengaruhi Future Earning Growth pada Badan Usaha Go Public yang Terdaftar di BEI Periode 2007-2009

Penelitian ini membahas mengenai pengaruh dividend payout ratio dan investment opportunity set terhadap future earning growth. Dimana sektor penelitian yang diambil adalah seluruh badan usaha go public yang terdaftar di Bursa Efek Indonesia pada 2007 -2009. Penelitian dividend payout ratio terhadap future earning growth ditujukan untuk melihat implikasi dari dividend signaling theory, yang mengatakan bahwa dividen dapat dijadikan sebagai salah satu alat yang dapat digunakan investor untuk memprediksi future earning growth perusahaan di masa yang akan datang. Dan ditemukan tidak adanya pengaruh antaradividend payout ratio terhadap future earning growth pada badan usaha go public di Indonesia. Sedangkan penelitian investment opportunity set terhadap future earning growth ditujukan untuk melihat apakah peluang investasi yang dimiliki oleh perusahaan di masa yang akan datang dapat berpengaruh terhadap future earning growth.Dan dari penelitian ditemukan tidak adanya pengaruh antara investment opportunity set terhadap future earning growth

Reflection spectroscopy study of the 16O12C16O ν

International audienceReflection spectra of pure CO2 gas have been recorded in the 4.3 μ m region by using a high resolution Fourier transform spectrometer, providing measured values of the refractive index. Comparisons with the results of calculations using available spectroscopic data, and free of any adjusted line parameter, show a very good agreement. This confirms that this experimental technique provides an alternative approach for the test or determination of spectroscopic parameters. Its advantages for studies of extremely strongly absorbing species and investigations of spatially (tightly) confined molecular gases are discussed

Glucocorticoid-induced leucine zipper (GILZ) regulates testicular FOXO1 activity and spermatogonial stem cell (SSC) function

Spermatogonia stem cell (SSC) self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ) in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF(+) cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF(+) cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility

Macrophage Migration Inhibitory Factor Inhibits the Antiinflammatory Effects of Glucocorticoids via Glucocorticoid-Induced Leucine Zipper

Objective. Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. Methods. GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. Results. GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. Conclusion. MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity