A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.     

Somatosensory Abnormalities for Painful and Innocuous Stimuli at the Back and at a Site Distinct from the Region of Pain in Chronic Back Pain Patients

Chronic low back pain (CLBP) was shown to be associated with pathophysiological changes at several levels of the sensorimotor system. Changes in sensory thresholds have been reported but complete profiles of Quantitative Sensory Testing (QST) were only rarely obtained in CLBP patients. The aim of the present study was to investigate comprehensive QST profiles in CLBP at the painful site (back) and at a site distinct from their painful region (hand) and to compare these data with similar data in healthy controls. We found increased detection thresholds in CLBP patients compared to healthy controls for all innocuous stimuli at the back and extraterritorial to the painful region at the hand. Additionally, CLBP patients showed decreased pain thresholds at both sites. Importantly, there was no interaction between the investigated site and group, i.e. thresholds were changed both at the affected body site and for the site distinct from the painful region (hand). Our results demonstrate severe, widespread changes in somatosensory sensitivity in CLBP patients. These widespread changes point to alterations at higher levels of the neuraxis or/and to a vulnerability to nociceptive plasticity in CLBP patients.     

TNF-α induced secretion of HMGB1 from non-immune canine mammary epithelial cells (MTH53A)

BackgroundMammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas.Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue.MethodsThe canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1–GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging.ResultsThe observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24 h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1–GFP after TNF-α stimulation was visible.ConclusionOur results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.     

Prevalence and trends of common mental disorders from 2007-2009 to 2019-2022: results from the Netherlands Mental Health Survey and Incidence Studies (NEMESIS), including comparison of prevalence rates before vs. during the COVID-19 pandemic

Up-to-date information on the prevalence and trends of common mental disorders is relevant to health care policy and planning, owing to the high burden associated with these disorders. In the first wave of the third Netherlands Mental Health Survey and Incidence Study (NEMESIS-3), a nationally representative sample was interviewed face-to-face from November 2019 to March 2022 (6,194 subjects; 1,576 interviewed before and 4,618 during the COVID-19 pandemic; age range: 18-75 years). A slightly modified version of the Composite International Diagnostic Interview 3.0 was used to assess DSM-IV and DSM-5 diagnoses. Trends in 12-month prevalence rates of DSM-IV mental disorders were examined by comparing these rates between NEMESIS-3 and NEMESIS-2 (6,646 subjects; age range: 18-64 years; interviewed from November 2007 to July 2009). Lifetime DSM-5 prevalence estimates in NEMESIS-3 were 28.6% for anxiety disorders, 27.6% for mood disorders, 16.7% for substance use disorders, and 3.6% for attention-deficit/hyperactivity disorder. Over the last 12 months, prevalence rates were 15.2%, 9.8%, 7.1%, and 3.2%, respectively. No differences in 12-month prevalence rates before vs. during the COVID-19 pandemic were found (26.7% pre-pandemic vs. 25.7% during the pandemic), even after controlling for differences in socio-demographic characteristics of the respondents interviewed in these two periods. This was the case for all four disorder categories. From 2007-2009 to 2019-2022, the 12-month prevalence rate of any DSM-IV disorder significantly increased from 17.4% to 26.1%. A stronger increase in prevalence was found for students, younger adults (18-34 years) and city dwellers. These data suggest that the prevalence of mental disorders has increased in the past decade, but this is not explained by the COVID-19 pandemic. The already high mental disorder risk of young adults has particularly further increased in recent years.     

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.