Synthesis and biological evaluation of [6]-gingerol analogues as transient receptor potential channel TRPV1 and TRPA1 modulators

In order to explore the structural determinants for the TRPV1 and TRPA1 agonist properties of gingerols, a series of nineteen analogues (1b-5) of racemic [6]-gingerol (1a) was synthesized and tested on TRPV1 and TRPA1 channels. The exploration of the structure-activity relationships, by modulating the three pharmacophoric regions of [6]-gingerol, led to the identification of some selective TRPV1 agonists/desensitizers of TRPV1 channels (3a, 3f, and 4) and of some full TRPA1 antagonists (2c, 2d, 3b, and 3d).     

Overtime work as a predictor of major depressive episode: a 5-year follow-up of the Whitehall II study

Background

The association between overtime work and depression is still unclear. This study examined the association between overtime work and the onset of a major depressive episode (MDE).

Methodology/Principal Findings

Prospective cohort study with a baseline examination of working hours, psychological morbidity (an indicator of baseline depression) and depression risk factors in 1991–1993 and a follow-up of major depressive episode in 1997–1999 (mean follow-up 5.8 years) among British civil servants (the Whitehall II study; 1626 men, 497 women, mean age 47 years at baseline). Onset of 12-month MDE was assessed by the Composite International Diagnostic Interview (CIDI) at follow-up. In prospective analysis of participants with no psychological morbidity at baseline, the odds ratio for a subsequent major depressive episode was 2.43 (95% confidence interval 1.11 to 5.30) times higher for those working 11+ hours a day compared to employees working 7–8 hours a day, when adjusted for socio-demographic factors at baseline. Further adjustment for chronic physical disease, smoking, alcohol use, job strain and work-related social support had little effect on this association (odds ratio 2.52; 95% confidence interval 1.12 to 5.65).

Conclusions/Significance

Data from middle-aged civil servants suggest that working long hours of overtime may predispose to major depressive episodes.     

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.     

Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal

We identified a new calmodulin kinase I (CaMKI) substrate, cytidyltransferase (CCTα), a crucial enzyme required for maintenance of cell membranes. CCTα becomes activated with translocation from the cytoplasm to the nuclear membrane, resulting in increased membrane phospholipids. Calcium-activated CCTα nuclear import is mediated by binding of its C-terminus to 14-3-3 ζ, a regulator of nuclear trafficking. Here CaMK1 phosphorylates residues within this C-terminus that signals association of CCTα with 14-3-3 ζ to initiate calcium-induced nuclear entry. CaMKI docks within the CCTα membrane-binding domain (residues 290-299), a sequence that displays similarities to a canonical nuclear export signal (NES) that also binds CRM1/exportin 1. Expression of a CFP-CCTα mutant lacking residues 290-299 in cells results in cytosolically retained enzyme. CRM1/exportin 1 was required for CCTα nuclear export, and its overexpression in cells was partially sufficient to trigger CCTα nuclear export despite calcium stimulation. An isolated CFP-290-299 peptide remained in the nucleus in the presence of leptomycin B but was able to target to the cytoplasm with farnesol. Thus CaMKI vies with CRM1/exportin 1 for access to a NES, and assembly of a CaMKI-14-3-3 ζ-CCTα complex is a key effector mechanism that drives nuclear CCTα translocation.     

Suppression of FOXM1 sensitizes human cancer cells to cell death induced by DNA-damage

Irradiation and DNA-damaging chemotherapeutic agents are commonly used in anticancer treatments. Following DNA damage FOXM1 protein levels are often elevated. In this study, we sought to investigate the potential role of FOXM1 in programmed cell death induced by DNA-damage. Human cancer cells after FOXM1 suppression were subjected to doxorubicin or γ-irradiation treatment. Our findings indicate that FOXM1 downregulation by stable or transient knockdown using RNAi or by treatment with proteasome inhibitors that target FOXM1 strongly sensitized human cancer cells of different origin to DNA-damage-induced apoptosis. We showed that FOXM1 suppresses the activation of pro-apoptotic JNK and positively regulates anti-apoptotic Bcl-2, suggesting that JNK activation and Bcl-2 down-regulation could mediate sensitivity to DNA-damaging agent-induced apoptosis after targeting FOXM1. Since FOXM1 is widely expressed in human cancers, our data further support the fact that it is a valid target for combinatorial anticancer therapy.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.