Differential influence of basic psychological needs on burnout and academic achievement in three southeast European countries.

Several studies show the importance of basic psychological needs (BPN) for decreasing burnout and increasing grade point average (GPA), but, to our knowledge, no prior study has explored the potential contextual differences in Southeastern European countries. Moreover, even less is known about how this relationship may differ during stressful (exam) and less stressful (beginning of the semester) periods. Measures of the Maslach Burnout Inventory-Student Survey and BPN Satisfaction and Frustration Scale were translated and adapted. The study included a cross-sectional sample of students from Croatia, Serbia and Slovenia during beginning of the semester and exam period. Across all countries and both contexts, students with high autonomy need satisfaction showed the strongest decrease in burnout, followed by those with high competence need satisfaction. Students with high academic achievement showed an increase due to competence need satisfaction. Competence and autonomy need satisfaction were higher beginning of a semester, while burnout was higher during the exam period. BPN play an important role in educational settings-satisfaction of the need for autonomy and competence protects students from burnout, and the need for competence predicts greater academic achievement regardless of culture or time of the semester.

Early Cell Cultures from Prostate Cancer Tissue Express Tissue Specific Epithelial and Cancer Markers.

Prostate cancer (PCa) is a widespread oncological disease that proceeds in the indolent form in most patients. However, in some cases, the indolent form can transform into aggressive metastatic incurable cancer. The most important task of PCa diagnostics is to search for early markers that can be used for predicting the transition of indolent cancer into its aggressive form. Currently, there are two effective preclinical models to study PCa pathogenesis: patients derived xenografts (PDXs) and patients derived organoids (PDOs). Both models have limitations that restrict their use in research. In this work, we investigated the ability of the primary 2D prostate cell cultures (PCCs) from PCa patients to express epithelial and cancer markers. Early PCCs were formed by epithelial cells that were progressively replaced with the fibroblast-like cells. Early PCCs contained tissue-specific stem cells that could grow in a 3D culture and form PDOs similar to those produced from the prostate tissue. Early PCCs and PDOs derived from the tissues of PCa patients expressed prostate basal and luminal epithelial markers, as well as cancer markers AMACR, TMPRSS2-ERG, and EZH2, the latter being a promising candidate to mark the transition from the indolent to aggressive PCa. We also identified various TMPRSS2-ERG fusion transcripts in PCCs and PDOs, including new chimeric variants resulting from the intra- and interchromosomal translocations. The results suggest that early PCCs derived from cancerous and normal prostate tissues sustain the phenotype of prostate cells and can be used as a preclinical model to study the pathogenesis of PCa.

RETRO-POPE: A Retrospective, Multicenter, Real-World Study of All-Cause Mortality in COPD.

Purpose: The Phenotypes of COPD in Central and Eastern Europe (POPE) study assessed the prevalence and clinical characteristics of four clinical COPD phenotypes, but not mortality. This retrospective analysis of the POPE study (RETRO-POPE) investigated the relationship between all-cause mortality and patient characteristics using two grouping methods: clinical phenotyping (as in POPE) and Burgel clustering, to better identify high-risk patients. Patients and Methods: The two largest POPE study patient cohorts (Czech Republic and Serbia) were categorized into one of four clinical phenotypes (acute exacerbators [with/without chronic bronchitis], non-exacerbators, asthma-COPD overlap), and one of five Burgel clusters based on comorbidities, lung function, age, body mass index (BMI) and dyspnea (very severe comorbid, very severe respiratory, moderate-to-severe respiratory, moderate-to-severe comorbid/obese, and mild respiratory). Patients were followed-up for approximately 7 years for survival status. Results: Overall, 801 of 1,003 screened patients had sufficient data for analysis. Of these, 440 patients (54.9%) were alive and 361 (45.1%) had died at the end of follow-up. Analysis of survival by clinical phenotype showed no significant differences between the phenotypes (P=0.211). However, Burgel clustering demonstrated significant differences in survival between clusters (P<0.001), with patients in the "very severe comorbid" and "very severe respiratory" clusters most likely to die. Overall survival was not significantly different between Serbia and the Czech Republic after adjustment for age, BMI, comorbidities and forced expiratory volume in 1 second (hazard ratio [HR] 0.80, 95% confidence interval [CI] 0.65-0.99; P=0.036 [unadjusted]; HR 0.88, 95% CI 0.7-1.1; P=0.257 [adjusted]). The most common causes of death were respiratory-related (36.8%), followed by cardiovascular (25.2%) then neoplasm (15.2%). Conclusion: Patient clusters based on comorbidities, lung function, age, BMI and dyspnea were more likely to show differences in COPD mortality risk than phenotypes defined by exacerbation history and presence/absence of chronic bronchitis and/or asthmatic features.

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter.

The retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB, despite being G-418 resistant. However, the consequences of the transient production of exogenous RB had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except -60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.

p130 And pRb in the Maintenance of Transient Quiescence of Mesenchymal Stem Cells.

An effective regulation of quiescence plays a key role in the differentiation, plasticity, and prevention of stem cells from becoming malignant. The state of quiescence is being controlled by the pRb family proteins which show overlapping functions in cell cycle regulation; however, their roles in controlling the proliferation of mesenchymal stem cells (MSCs) remain to be understood. This study investigated the regulation of transient quiescence using growth curves, proliferation assay, the cytometric evaluation of cell cycle, Western blotting, and the electromobility gel shift assay (EMSA) on synchronized MSCs of the C3H10Т1/2 and control cells with different statuses of pRb proteins. It has been found that functional steady-state level of p130 but not pRb plays a critical role for entering, exiting, and maintenance of transient quiescence in multipotent mesenchymal stem cells.

Preparation and characterization of the antibody recognizing AMACR inside its catalytic center.

Alpha-methylacyl-CoA racemase (AMACR) catalyzes the ß-oxidation of fatty acids and is overexpressed in carcinomas in various organs, while its inactivation results in the inhibition of cancer growth. In the present study, we prepared and characterized 20 different mouse monoclonal antibodies against human AMACR. In the course of biopanning of a phage peptide commercial library against in-house prepared 6H9 and 2A5, and commercial 13H4 antibodies, 10 phage mimotopes recognized by each type of the antibody were selected. Using the program Pepitope and the crystal structure of AMACR from Mycobacterium tuberculosis, we reveal for the first time, at least to the best of our knowledge, that the epitopes recognizing the antibody against AMACR are composed of conformation sequences localized inside the AMACR catalytic center. When delivered into live HeLa cells using cationic lipid-based PULSin reagent, the specific antibodies against AMACR were co-localized with peroxisomes. The in-house made 6H9 antibody exhibited a low level of this co-localization compared to the commercially available 63340 antibody, and did not inhibit the growth rate of HeLa and T98G cells. The results obtained suggest that antibody against AMACR may possess anti-AMACR catalytic activity and needs to be further investigated as a potential drug for use in anticancer therapy. On the whole, in this study, we generated several clones of AMACR antibodies and demonstrated that these antibodies can be colonized into live cells. Currently, we are testing the growth inhibitory properties of these antibodies against AMACR.

Lung epithelial cells induce endodermal differentiation in mouse mesenchymal bone marrow stem cells by paracrine mechanism.

Mesenchymal stem cells (MSCs) from bone marrow are a potential source for reconstructive therapy. In vitro, MSCs differentiate into cells of mesodermal and ectodermal lineages but rarely into cells of endodermal lineage. We developed an in vitro model to study the endodermal differentiation of MSCs using co-culture of MSCs and transformed lung epithelial (A-549) cells. The cells were separated using a cell-impermeable membrane to eliminate the possibility of cell fusion. Under these conditions, MSCs expressed several lung epithelial markers (cytokeratins 5, 8, 14, 18, 19, pro-surfactant protein C, zonula occludens-1), detected using quantitative reverse transcriptase polymerase chain reaction and Western blot, and beta-catenin signaling was activated in MSCs. Treatment of MSCs with 10 to 20 mM lithium chloride activated the beta-catenin pathway and enhanced expression of epithelial markers, although this activation was transient. We conclude that A-549 cells can trigger epithelial differentiation of MSCs by a paracrine mechanism that may include activation of beta-catenin signaling.

C-kit expression in renal oncocytomas and chromophobe renal cell carcinomas.

C- kit encodes the membrane-bound tyrosine kinase KIT, whose expression has been identified in several types of human neoplasms. Recently, KIT has been reported to be a marker for chromophobe renal cell carcinoma (RCC) and renal angiomyolipoma. However, expression of this molecule has not been adequately studied in other renal tumors, particularly oncocytoma, which may morphologically resemble chromophobe RCC. In this study, we analyzed c- kit messenger RNA (mRNA) levels in 17 chromophobe RCCs and 20 renal oncocytomas obtained from complementary DNA (cDNA) microarrays. Furthermore, comprehensive immunohistochemical analysis of KIT protein using a monoclonal antibody was performed in 226 renal tumors including chromophobe RCC (n=40), oncocytoma (n=41), clear-cell RCC (n=40), renal angiomyolipoma (n=29), and papillary RCC (n=21) on tissue microarrays (TMAs) and was compared with immunostaining results from 25 chromophobe RCCs and 30 oncocytomas using standard sections. The staining intensity was semiquantitatively graded on a 3-tier scoring system. All chromophobe RCCs and oncocytomas showed significant overexpression of c- kit mRNA. The average increase of mRNA compared with normal kidney tissue was 7.4-fold for chromophobe RCCs and 7.4-fold for oncocytomas. Immunohistochemical expression of KIT was found in most chromophobe RCCs (95% in TMAs and 96% in conventional sections) and oncocytomas (88% in TMAs and 100% in conventional sections) but was infrequently observed in renal angiomyolipomas (17%), papillary RCCs (5%), and clear-cell RCCs (3%). Furthermore, the average KIT immunoreactivity in TMAs was stronger in chromophobe RCC (1.93) and oncocytoma (2.07) than in other subtypes of renal tumors tested, including angiomyolipomas (0.17), papillary RCCs (0.05), and clear-cell RCCs (0.03). In conclusion, we found a significant elevation of c- kit mRNA by cDNA expression microarrays and overexpression of KIT protein by immunohistochemistry not only in chromophobe RCCs but also in oncocytomas. In contrast, immunohistochemical expression of KIT was not detected in most other types of renal cell tumors evaluated. The differential expression of c- kit in these renal tumors may have diagnostic and therapeutic implications.

pRb-E2F signaling in life of mesenchymal stem cells: Cell cycle, cell fate, and cell differentiation.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various mesodermal lines forming fat, muscle, bone, and other lineages of connective tissue. MSCs possess plasticity and under special metabolic conditions may transform into cells of unusual phenotypes originating from ecto- and endoderm. After transplantation, MSCs release the humoral factors promoting regeneration of the damaged tissue. During last five years, the numbers of registered clinical trials of MSCs have increased about 10 folds. This gives evidence that MSCs present a new promising resource for cell therapy of the most dangerous diseases. The efficacy of the MSCs therapy is limited by low possibilities to regulate their conversion into cells of damaged tissues that is implemented by the pRb-E2F signaling. The widely accepted viewpoint addresses pRb as ubiquitous regulator of cell cycle and tumor suppressor. However, current publications suggest that basic function of the pRb-E2F signaling in development is to regulate cell fate and differentiation. Through facultative and constitutive chromatin modifications, pRb-E2F signaling promotes transient and stable cells quiescence, cell fate choice to differentiate, to senesce, or to die. Loss of pRb is associated with cancer cell fate. pRb regulates cell fate by retaining quiescence of one cell population in favor of commitment of another or by suppression of genes of different cell phenotype. pRb is the founder member of the "pocket protein" family possessing functional redundancy. Critical increase in the efficacy of the MSCs based cell therapy will depend on precise understanding of various aspects of the pRb-E2F signaling.

Induction of Wnt/ß-catenin signaling in mouse mesenchymal stem cells is associated with activation of the p130 and E2f4 and formation of the p130/Gsk3ß/ß-catenin complex.

Proteins p130 and E2f4, members of the retinoblastoma protein (pRb) family/E2F transcription factor family, are the key elements in regulation of cell cycle and differentiation. The functional role of the p130/E2f4 in mesenchymal stem cells (MSC) is unclear. We demonstrate here that activation of the Wnt/ß-catenin pathway in mouse MSC is associated with accumulation of active forms of the p130, E2f4, and ß-catenin but does not result in inhibition of cell cycle progression. The levels and phosphorylation patterns of p130, E2f4, and ß-catenin in MSC do not change during cell cycle progression. This is different from control T98G glyoblastoma cells that accumulated differently phosphorylated forms of the p130 in quiescence, and under active proliferation. In MSC, synchronized at G0/G1 and S cell cycle phases, the p130 and ß-catenin physically interact each other, whereas Gsk3ß was associated and co-precipitated with both p130 and ß-catenin. Our results indicate that Wnt/ß-catenin and pRb signal pathways interact with each other and form common p130/Gsk3ß/ß-catenin complex during MSC cycle progression. Physiological relevance of such complex may be associated with coupling of the cell cycle and differentiation in MSC, which is related to a wide differentiation potential of these stem cells.

Evidence of temporary airway epithelial repopulation and rare clonal formation by BM-derived cells following naphthalene injury in mice.

The goal of the study was to investigate participation of bone marrow (BM) cells in the process of airway epithelial restoration after naphthalene-induced injury. We transplanted sex-mismatched green fluorescent protein (GFP) -tagged BM-derived cultured plastic-adherent mesenchymal stem cells into 5Gy-irradiated C57BL/6 recipients. After 1 month of recovery, experimental animals were subjected to 250 mg/kg naphthalene IP. Animals were killed at 2-30 days after naphthalene. By immunofluorescence, immunohistochemistry, and by in situ hybridization for the Y-chromosome, we observed patches of donor-derived cells in the large and small conducting airways, mostly at 2-6 days after injury. GFP(+) cells in the epithelium of airways were positive for pancytokeratin and some other epithelial markers. Although rare, GFP(+) cells formed clear isolated patches of the bronchial epithelium, consistent with clonal formation; as some cells were also positive for proliferating cell nuclear antigen, a marker of proliferating cells. After day 12, only occasional GFP(+) cells were present in the epithelium. These data confirm that bone marrow-derived cultured mesenchymal cells can participate in the recovery of the injured airway epithelium after naphthalene-induced injury with minimal long-term engraftment.

Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice.

Recent in vivo and in vitro work suggests that mesenchymal stem cells (MSC) have anti-inflammatory properties. In this study, we tested the effect of administering MSC directly into the airspaces of the lung 4 h after the intrapulmonary administration of Escherichia coli endotoxin (5 mg/kg). MSC increased survival compared with PBS-treated control mice at 48 h (80 vs 42%; p < 0.01). There was also a significant decrease in excess lung water, a measure of pulmonary edema (145 +/- 50 vs 87 +/- 20 microl; p < 0.01), and bronchoalveolar lavage protein, a measure of endothelial and alveolar epithelial permeability (3.1 +/- 0.4 vs 2.2 +/- 0.8 mg/ml; p < 0.01), in the MSC-treated mice. These protective effects were not replicated by the use of further controls including fibroblasts and apoptotic MSC. The beneficial effect of MSC was independent of the ability of the cells to engraft in the lung and was not related to clearance of the endotoxin by the MSC. MSC administration mediated a down-regulation of proinflammatory responses to endotoxin (reducing TNF-alpha and MIP-2 in the bronchoalveolar lavage and plasma) while increasing the anti-inflammatory cytokine IL-10. In vitro coculture studies of MSC with alveolar macrophages provided evidence that the anti-inflammatory effect was paracrine and was not cell contact dependent. In conclusion, treatment with intrapulmonary MSC markedly decreases the severity of endotoxin-induced acute lung injury and improves survival in mice.

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion.

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

Tularemia outbreak, Bulgaria, 1997-2005.

The 1997-2005 tularemia outbreak in Bulgaria affected 285 people. Ten strains were isolated from humans, a tick, a hare, and water. Amplified fragment length polymorphism typing of the present isolates and of the strain isolated in 1962 suggests that a new genetic variant caused the outbreak.

Cell cycle-related transformation of the E2F4-p130 repressor complex.

During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.