The role of metabotropic glutamate receptor 5 in the pathogenesis of mood disorders and addiction: combining preclinical evidence with human Positron Emission Tomography (PET) studies.

In the present review, we deliver an overview of the involvement of metabotropic glutamate receptor 5 (mGluR5) activity and density in pathological anxiety, mood disorders and addiction. Specifically, we will describe mGluR5 studies in humans that employed Positron Emission Tomography (PET) and combined the findings with preclinical animal research. This combined view of different methodological approaches-from basic neurobiological approaches to human studies-might give a more comprehensive and clinically relevant view of mGluR5 function in mental health than the view on preclinical data alone. We will also review the current research data on mGluR5 along the Research Domain Criteria (RDoC). Firstly, we found evidence of abnormal glutamate activity related to the positive and negative valence systems, which would suggest that antagonistic mGluR5 intervention has prominent anti-addictive, anti-depressive and anxiolytic effects. Secondly, there is evidence that mGluR5 plays an important role in systems for social functioning and the response to social stress. Finally, mGluR5's important role in sleep homeostasis suggests that this glutamate receptor may play an important role in RDoC's arousal and modulatory systems domain. Glutamate was previously mostly investigated in non-human studies, however initial human clinical PET research now also supports the hypothesis that, by mediating brain excitability, neuroplasticity and social cognition, abnormal metabotropic glutamate activity might predispose individuals to a broad range of psychiatric problems.

Written survey on recently deceased patients in germany and switzerland: how do general practitioners see their role?

BACKGROUND General practitioners (GPs) play an important role in end-of-life care due to their proximity to the patient's dwelling-place and their contact to relatives and other care providers. METHODS In order to get a better understanding of the role which the GP sees him- or herself as playing in end-of-life care and which care their dying patients get, we conducted this written survey. It asked questions about the most recently deceased patient of each physician. The questionnaire was sent to 1,201 GPs in southern North Rhine-Westphalia (Germany) and the Canton of Bern (Switzerland). RESULTS Response rate was 27.5 % (n = 330). The average age of responding physicians was 54.5 years (range: 34-76; standard derivation: 7.4), 68 % of them were male and 45 % worked alone in their practice. Primary outcome measures of this observational study are the characteristics of recently deceased patients as well as their care and the involvement of other professional caregivers. Almost half of the most recently deceased patients had cancer. Only 3 to 16 % of all deceased suffered from severe levels of pain, nausea, dyspnea or emesis. More than 80 % of the doctors considered themselves to be an indispensable part of their patient's end-of-life care. Almost 90 % of the doctors were in contact with the patient's family and 50 % with the responsible nursing service. The majority of the GPs had taken over the coordination of care and cooperation with other attending physicians. CONCLUSION The study confirms the relevance of caring for dying patients in GPs work and provides an important insight into their perception of their own role.

Serious Play as a Practice of Paradox

A recent stream of organizational research has used the term serious play to describe situations in which people engage in playful behaviors deliberately with the intention to achieve serious, work-related objectives. In this article, the authors reflect on the ambiguity of this term, and reframe serious play as a practice characterized by the paradox of intentionality (when actors engage deliberately in a fun, intrinsically motivating activity as a means to achieve a serious, extrinsically motivated work objective). This reframing not only extends the explanatory power of the concept of serious play but also helps bridge the concerns of scholars and practitioners: first, by enabling us to understand a variety of activities in organizations as serious play, which can help practitioners address specific organizational challenges; second, by recognizing the potential for emergent serious play, and the creation of the conditions to foster this emergence; third, by pointing toward specific, individual or group-level outcomes associated with the practice; and finally, by uncovering its ethical dimensions and encouraging the understanding of the role of serious play on ethical decision making.

Vegetation shift and laurophyllisation: the possible role of forest fires

Southern Switzerland is a fire prone area where fire has to be considered as a natural environmental factor. In the past decades, fire frequency has tended to increase due to changes in landscape management. The most common type of fire is surface fire which normally breaks out during the vegetation resting period. Usually this type of fire shows short residence time (rapid spread), low to medium fire intensity and limited size. South-facing slopes are particularly fire-prone, so that very high fire frequency is possible: under these conditions passive resistant species and postfire resprouting species are favoured, usually leading to a reduction in the number of surviving species to a few fire adapted sprouters. Evergreen broadleaves are extremely sensitive to repeated fires. A simulation of the potential vegetation of southern Switzerland under climatic changed conditions evidenced the coincidence of the potential area of spreading forests rich in evergreen broad-leaved species with the most fire-prone area of the region. Therefore, in future, wildfires could play an important regulating role: most probably they will not stop the large-scale laurophyllisation of the thermophilous forests of southern Switzerland, but at sites with high fire frequency the vegetation shift could be slowed or even prevented by fire-disturbances.

The Role of Platelets in Venous Thromboembolism

Multiple factors contribute to the risk of venous thromboembolism (VTE). Platelets have attracted much interest in arterial cardiovascular disease, whereas their role in VTE has received much less attention. Recent evidence suggests that platelets may play a more important role in VTE than previously anticipated. This review discusses the mechanisms that link platelets with venous thrombotic disease and their potential applications as novel risk factors for VTE. In addition, animal studies and randomized clinical trials that highlight the potential effect of antiplatelet therapy in venous thrombosis are evaluated to assess the role of platelets in VTE. The clinical significance of platelets for VTE risk assessment in specific patient cohorts and their role as a suitable therapeutic target for VTE prevention is acknowledged. The role of platelets in VTE is a promising field for future research.

The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

Cleavage and activation of calpain and its substrate caspase-7 in prostate cancer cells: Evidence for a role in apoptotic sensitization

Changes in the levels of intracellular calcium mediate multiple biological effects, including apoptosis, in some tumor cells. Early studies demonstrated that prostate cancer cells are highly sensitive to alterations in the levels of their intracellular calcium pools. Furthermore, it has been established that apoptosis in prostate cancer could be initiated through calcium-selective ionophores, or inhibitors of intracellular calcium pumps. High sensitivity to changes in intracellular calcium levels may therefore be exploited as a novel mechanism for controlling prostate cancer apoptotic thresholds; however, the mechanisms associated with this process are poorly understood. To investigate the role of calcium as a mediator of prostate cancer cell death and its effects on caspase activation, LNCaP and PC-3 cell response to the calcium ionophore A23187, were examined. LNCaP cells were highly sensitive to changes in intracellular calcium, and subtoxic concentrations of A23187 facilitated apoptosis initiated by cytokines (TNF or TRAIL). In contrast, PC-3 cell death was not affected by A23187 or cytokines. A23187 caused rapid and concentration-dependent activation of calpain in LNCaP (but not PC-3 cells) which correlated with cleavage of calpain substrates caspase-7 and PTP1B. Cleavage of PTP1B from a 50 kDa to 42 kDa protein correlated with its translocation from the endoplasmic reticulum to the cytosol and with inhibition of tyrosine phosphorylation. Caspase-7 was cleaved from a 35 kDa to 30 kDa protein in response to A23187 in LNCaP (but not PC-3) cells and correlated with activation of both upstream and downstream caspases. Extracts from A23187-treated LNCaP cells, or PC-3 cells transiently transfected with calpain, mediated similar processing of in vitro transcribed and translated (TNT) caspase-7. In vitro processing of caspase-7 correlated with its proteolytic activation, which was inhibited by calpain inhibitor (calpeptin) and to some degree, by caspase inhibitors (zVAD, DEVD). Together, these results suggest that calpain is directly involved in calcium-mediated apoptosis of prostate cancer cells through activation and cleavage of caspase-7 and other substrates. Loss of calpain activation may therefore play a critical role in apoptotic resistance of some prostate cancer cells. ^

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

The Role of EphA4 in Glial Scar Formation Following Injury

Although many areas of the brain lose their regenerative capacity with age, stem cell niches have been identified in both the subventricular zone (SVZ) along the lateral walls of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (Gage, 2000; Alvarez-Buylla et al., 2001; Alvarez-Buylla and Lim, 2004). The SVZ niche utilizes many mechanisms to determine the migration patterns of neuroblasts along the RMS into the olfactory bulb, one being Eph/ephrin signaling (Conover et al., 2000; Holmberg et al., 2005). EphA4-mediated signaling is necessary for axon guidance during development, and its continued expression in the SVZ niche suggests a regulatory role throughout adulthood. Previous studies have suggested that EphA4 plays a role in the regulation of astrocytic gliosis and glial scar formation, which inhibits axonal regeneration in these areas following spinal cord injury (Goldshmit et al., 2004). Blood vessels may also play an important role in SVZ cell proliferation and neuroblast migration following injury (Tavazoie et al., 2008; Yamashita et al., 2006). The goal of this project is to examine glial scar formation as well as the relationship between SVZ vasculature, neuroblasts, and neural stem cells in EphA4 +/+, EphA4 +/-, and EphA4 -/- mice following a needle stick injury in the cortex or striatum. The outcome of these experiments will determine whether invasive procedures such as injections will affect neuroblast migration and/or the organization of the SVZ.

Investigating the Role of Small, Acid-Soluble Spore Proteins (SASPs) in the Resistance of Clostridium Perfringens Spores to Heat.

BACKGROUND: Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates. RESULTS: Our current study demonstrated the presence of all three SASP-encoding genes (ssp1, 2 and 3) in five surveyed C. perfringens clinical food poisoning isolates. beta-Glucuronidase assay showed that these ssp genes are expressed specifically during sporulation. Consistent with these expression results, our study also demonstrated the production of SASPs by C. perfringens food poisoning isolates. When the heat sensitivities of spores produced by a ssp3 knock-out mutant of a C. perfringens food poisoning isolate was compared with that of spores of the wild-type strain, spores of the ssp3 mutant were found to exhibit a lower decimal reduction value (D value) at 100 degrees C than exhibited by the spores of wild-type strain. This effect was restored by complementing the ssp3 mutant with a recombinant plasmid carrying wild-type ssp3, suggesting that the observed differences in D values between spores of wild-type versus ssp3 mutant was due to the specific inactivation of ssp3. Furthermore, our DNA protection assay demonstrated that C. perfringens SASPs can protect DNA from DNase I digestion. CONCLUSION: The results from our current study provide evidences that SASPs produced by C. perfringens food poisoning isolates play a role in protecting their spores from heat-damage, which is highly significant and relevant from a food safety perspective. Further detailed studies on mechanism of action of SASPs from C. perfringens should help in understanding the mechanism of protection of C. perfringens spores from heat-damage.

Intrinsic pH-Sensitivity of Cells in the Retrotrapezoid Nucleus: Possible Role of Glia in Respiratory Drive

An increase in carbon dioxide (CO2) and protons (H+) are the primary signals for breathing. Cells that sense changes in CO2/H+ levels and increase breathing accordingly are located in a region of the caudal medulla oblongata called the retrotrapezoid nucleus (RTN). Specifically, select RTN neurons are intrinsically pH sensitive and send excitatory projections to the respiratory rhythm generator to drive breathing. Glial cells in the RTN are thought to contribute to this respiratory drive, possibly by releasing ATP in response to increases in CO2/H+ levels. However, pH sensitivity of RTN glial cells has yet to be determined. Therefore, the goal of my thesis is to determine if acutely dissociated RTN cells can respond to changes in pH in isolation. To make this determination I used ratiometric fluorescent microscopy to measure intracellular calcium in dissociated RTN cells during changes in bath pH. I found that a small percentage of RTN cells (16%) respond to bath acidification from pH 7.3 to pH 6.9 with an increase in fluorescence indicating an increase in intracellular calcium. Preliminary electrophysiological findings suggest that responsive cells are unable to make action potentials, thus suggesting their identity to be glia. These results indicate that a subset of pH sensitive cells in the RTN are intrinsically pH sensitive and that glia cells may possibly play a role in central chemoreception.

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^