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Background and Aim: The 1H-perimidne, as novel source carbene ligand, is well known for its anti-fungal, anti-microbial or anti-tumor activities. Here, we aimed to study the acute toxicity in Wistar rat of 2-(2,3-dihydro-1H-perimidin-2-yl)-6-methoxyphenol, a new heterocyclic 1H- perimidine synthetized in our laboratory. Materials and Methods: Five groups of males Wistar rats were intraperitoneally injected with 7 mg/kg, 40 mg/kg, 90 mg/kg, 130 mg/kg and 150 mg/kg dissolved in dimethyl sulfoxide (DMSO), and followed during 14 days. We noted any clinical signs of acute toxicity as body weight loss, salivation, tremor, convulsion among others, as well as food consumption and water intake level. Results: The DL50 of the new 2-(2,3-dihydro-1H-perimidin-2-yl)-6-methoxyphenol was estimated to 65 mg/kg. The No Observed Adverse Effect Level (NOAEL) dose was 7 mg/kg because it did not caused mortality, clinical signs of acute toxicity, and not affected feed and water intake behavior. However, significant abnormalities as inflammation and necrosis were observed at doses-effects dependent in liver, when compared to NOAEL dose and vehicle. Conclusion: The new heterocyclic 2-(2,3-dihydro-1H-perimidin-2-yl)-6-methoxyphenol is considered as high toxicity grade product. From NOAEL dose, subsequent biological, toxicological, pharmacological and neurobehavioral studies are needed before using for clinical trials.

From (2,3-dihydro-1H-perimidin-2-yl)-phenyl, the substitution of OH group in ortho or para position on the phenyl ring, allows us to synthesize the studied compounds. These three compounds have been characterized by conventional spectroscopic methods (NMR and MS). The interest of this work is to review the antioxidant activity of our compounds. The antioxidant activity screening carried out according to FRAP and DPPH methods revealed significant anti-free radical properties for compounds 1 and 2 even at low concentrations. In contrast to the compound 2, compound 3 for which the OH group is substituted in para position has the lowest activity in both cases. Therefore the para position seems to be the least sensitive position to increase the antioxidant activity of this pharmacophore.

Fluoropyrimidine-based regimens are among the most commonly used chemotherapy combinations for the treatment of solid tumors. Several genetic polymorphisms that are implicated with fluoropyrimidine anabolism and catabolism have been associated with the development of life-threatening toxicities. Uridine triacetate is an FDA-approved antidote for 5-fluorouracil or capecitabine overdose and early-onset, life-threatening toxicity within 96 h of last chemotherapy dose. To date, it is not accessible for Greek patients as per the current summary of product characteristic's time restrictions. We report and discuss the course and outcome of capecitabine toxicity in a 66-year-old female colorectal cancer patient with heterozygous dihydropyrimidine dehydrogenase deficiency. This paper highlights the difficulty in timely access of this lifesaving medication for Greek and possibly other European patients.

Early intervention methods for children with Autism Spectrum Disorders, increasingly turn to multidimensional approaches with a parallel focus on interprofessional collaboration in order the interventions to be effective. Multidimensional approaches efficiently support the complex system of family interaction and function affecting positively the child’s development. Early and efficient interventions are widely recognized to lead to long-term positive socio-cognitive results for children with Autism Spectrum Disorders and their families. The purpose of this research is to delve into professionals’, special educators ’and therapists’, opinions on their collaboration with parents and other professionals as they apply early intervention programs to children with ASD. Semi-structured interviews of 15 professionals, experts on early intervention in children with ASD, from different regions of Greece, were analyzed using qualitative research methods. Part of the data analysis that presented in this paper demonstrates clearly that the professionals’ and special educators’ opinions emphasize the need for multidimensional approaches. The analysis also highlights the obstacles that restrict collaboration with parents and other professionals. The material resources of state support have also been suggested as factors that interact with family resources that affect the child’s developmental characteristics.

Elevated, tumor-derived extracellular vesicle (tdEV) and circulating tumor cell (CTC) loads in metastatic cancer are associated with poor clinical outcome. Herein, we investigate whether endothelium-derived extracellular vesicles (edEVs) can be detected in the blood of metastatic colorectal cancer (mCRC) patients, and whether those vesicles associate with prognosis. The open-source ACCEPT (Automated CTC Classification, Enumeration, and Phenotyping) software was used to enumerate edEVs, tdEVs, and other objects from digitally stored CellSearch images acquired after CTC and circulating endothelial cell (CEC) enrichment from the blood of 395 mCRC patients before the initiation of a new therapy. Patients had participated in the prospective phase III CAIRO2 study. The presence of edEVs was found 5- to 10-fold higher than CECs. The hazard ratio (HR) (95% CI) of progression-free survival (PFS) for increased CTCs (&ge, 3 in 7.5 mL), tdEVs (&ge, 40 in 7.5 mL), and edEVs (&ge, 287 in 4.0 mL.) was 1.4 (1.1&ndash, 1.9), 2.0 (1.5&ndash, 2.6), and 1.7 (1.2&ndash, 2.5), respectively. The HR of Overall Survival (OS) for increased CTCs, tdEVs and edEVs was 2.2 (1.7&ndash, 3.0), 2.7 (2.0&ndash, 3.5), and 2.1 (1.5&ndash, 2.8), respectively. There was no cut-off value for CECs, leading to a dichotomization of patients with a significant HR. Only tdEVs remained a significant predictor of OS in the final multivariable model.

Background Tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) in the blood of metastatic cancer patients associate with poor outcomes. In this study, we explored the human epidermal growth factor receptor 2 (HER2) expression on CTCs and tdEVs of metastatic breast cancer patients. Methods Blood samples from 98 patients (CLCC-IC-2006-04 study) were originally processed with the CellSearch® system using the CTC kit and anti-HER2 as an additional marker in the staining cocktail. CTCs and tdEVs were automatically enumerated from the generated CellSearch images using the open-source ACCEPT software. Results CTCs and tdEVs were subdivided based on their cytokeratin (CK) and HER2 phenotype into CK+HER2−, CK−HER2+, and CK+HER2+. The inclusion of anti-HER2 increased the percentage of informative samples with ≥ 1 detectable CTC from 89 to 95%. CK− CTCs and tdEVs correlated equally well with the clinical outcome as CK+ CTCs and tdEVs. Inter- and intra-patient heterogeneity was found for the CTC/tdEV phenotypes, and the presence of 2 or 3 classes of CTCs/tdEVs was associated with worse prognosis compared to a uniform CTC/tdEV phenotype present (1 class). The use of ≥ 7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity using the HER2 amplification status of the primary tumor as a classification variable. Conclusions HER2 can be detected on CTCs and tdEVs not expressing CK, and these CK− CTCs/tdEVs have similar clinical relevance to CTCs and tdEVs expressing CK. tdEVs perform better than CTCs in predicting the HER2 status of the primary tissue. CTC and tdEV heterogeneity in the blood of patients is inversely associated with overall survival.

A qualitative, action research study has been conducted to investigate cooperative learning strategies for children with ASD and typical development during educational robotic activities. The participants were 4 children, 2 with ASD and 2 with typical development, around the ages of 12-14. The sessions were held at “School for all: Tokei Maru” in Thessaloniki, once a week, for 3 months. Observation protocols, researcher's diary, and interviews were used for data collection. Children with ASD, although they had a strong motivation to participate in robotic activities, exhibited disruptive behaviors. The cooperative learning strategy was not effective. Self-regulation strategies have proven helpful in limiting the disrupting behaviors of children with ASD and in supporting cooperative learning.

After a general introduction in Chapter 1, we showed in Chapters 2 and 3, that large tdEVs in blood of metastatic prostate, breast, colorectal and non-small cell lung cancer patients have equivalent prognostic power as CTCs. Importantly, patients with metastatic prostate, breast and colorectal cancer with favorable CTC counts could be further stratified using tdEV counts implying that a subset of patients with favorable CTCs and unfavorable tdEV have a relatively poor outcome and may benefit from a more aggressive treatment similarly to the patients with unfavorable CTC counts. In Chapter 4, we identified large leukocyte-derived extracellular vesicles (ldEVs) in blood of healthy individuals and metastatic cancer patients after immunomagnetic EpCAM enrichment and fluorescent labelling with the CellSearch system. Whereas tdEVs were 20-fold more frequent as compared to CTCs in metastatic cancer patients, the frequency of ldEVs were 5-fold less frequent as compared to leukocytes coming along with the enrichment in both patients and controls. Fluorescence microscopy imaging of whole blood showed the presence of ldEVs in a 3-fold lower frequency as compared to leukocytes suggesting that the “fragmentation” of leukocytes into ldEVs is not caused by processing of blood samples in the CellSearch system and thus, are actually present in blood. In Chapter 5, protocols were developed to image CTCs and tdEVs of castration-resistant prostate cancer patients isolated by the CellSearch system and CTCs isolated using 5 μm filters by scanning electron microscopy (SEM). SEM images of CellSearch enriched CTCs and tdEVs were obtained, but detailed morphologic information was obscured by the presence of ferrofluid, whereas in case of filtration the cells were clearly deformed by the pressure the cells undergo, while entering the filter holes. Interestingly, using SEM many microparticles were observed with similar morphology and size as large tdEVs (EpCAM+, CK+, CD45-, DAPI-) but not detected by fluorescence microscopy. Whether they originate from the tumor or not remains to be further investigated. In Chapter 6, the addition of the HER2 antibody in the CellSearch assay revealed the presence of HER2+, CK-, CD45- CTCs and tdEVs in the blood of breast cancer patients and had a similar association with poor clinical outcome as the CK+, CD45- CTCs. The larger frequency of tdEVs allowed the assessment of the presence of HER2 in a larger portion of patients and encourage the examination of more treatment targets on tdEVs. Importantly, these results pave the path towards a more rational and objective choice of patients who will or will not be subjected to HER2 targeting therapies. In Chapter 7, we compared the presence of CTCs and tdEVs before initiation of therapy and after the first cycle of therapy in CRPC, mBC and mCRC patients to evaluate the effect of therapy on CTCs versus tdEVs. The association between CTCs and tdEVs with overall survival was similar before the initiation of therapy but after the first cycle of therapy, CTCs outperformed tdEVs in mCRC implying that tdEV secretion is dependent on the treatment and possibly the cancer type. The distinction of different tdEV classes using a deep learning approach encourages us to determine the ones that rise or decline after the administration of an effective therapy in order to improve the evaluation of therapy responses and the patient treatment monitoring. In Chapter 8, we investigated whereas we can detect endothelium-derived EVs (edEVs) in the CellSearch image datasets acquired from the CD146 enriched blood samples of metastatic colorectal cancer (mCRC) patients. Circulating endothelial cells (CECs) are significantly elevated in the blood circulation of cancer patients compared to healthy individuals; their presence is however not associated with better or poorer clinical outcome. The CEC number is biased by venipuncture procedure as endothelial cells are released due to the vacuum and enter the collection tube. edEVs should not be influenced; so we explored their presence through ACCEPT analysis and revealed that edEVs are detected at 5- to 10- fold higher frequencies compared to CECs. Moreover, their counts correlated with the clinical outcome of the patients. Importantly, the final multivariate Cox regression model included both tdEVs and edEVs as significant independent prognostic markers of the overall survival of mCRC patients. The elevated edEV counts denote either their active role in promoting tumor angiogenesis or/and their passive secretion because of the growth of the tumor. If the former hypothesis is correct, then edEVs could serve as a promising biomarker to predict patients that could benefit from anti-angiogenic treatments. Whether edEVs could serve as a more informative diagnostic tool in cardiovascular diseases remains to be investigated. In Chapter 9, three different (des)biotin liposomes were compared in terms of their fusion with cells from cancer cell lines and blood from healthy individuals. DOPC liposomes of similar size distribution containing 20 mol% DOPE-desbiotin, 20 mol% DOPE-biotin or 5 mol% chol-EG3-biotin were prepared by extrusion. Their mean hydrodynamic diameter was around 100 nm. Leukocyte subpopulations, platelets and different cancer cell lines were incubated with the different liposome systems, stained with fluorophore-tagged streptavidin and their fusion with liposomes was assessed by flow cytometry and immunofluorescence microscopy. The chol-EG3-biotin liposomes achieved the highest biotin incorporation into the cell membrane for all different cell types, followed by DOPE-biotin liposomes. DOPE-desbiotin liposomes did not show considerable fusion with any cell type. The highest liposome uptake was found in cancer cells followed by the monocytes indicating a relationship between available cell surface and liposome uptake.

The book presents the method and results of the Structured Inclusive Judo Program (SI _Judo) specifically designed to include children or adults with Autistic Spectrum Disorder (ASD) in basic Gokyo Judo lessons. It is important to note that Senpai of ASD children in Judo lessons were members of therapeutic community who were practicing in Judo during their therapy. The SI_ Judo has been implemented for 6 months, at a once weekly frequency, in the informal learning environment of “School for All: Tokei Maru” of Include in Thessaloniki Greece, with positive effects on specific psychosocial and psychomotor skills for ASD participants. The effects of SI_Judo ​​ascertained after SI-Judo educational intervention to two ASD children. According to the research methodology, the effects of SI_Judo ​​had been investigated by interviews and questionnaires of parents of ASD children, Senpai calendars and independent and participating observation protocols. According to parents interviews “SI_Judo was a window in their children world” or Si _Judo supported the ASD child to find his position in family and local community”. According the results of Si_Judo the program ​​is suggested as an alternative intervention not only for ASD children or adults but for people of vulnerable groups. Judo is categorized as a sport that exercises the mind along with the body. As it has been defined by Jigoro Kano, Judo is based on the principle of maximum efficiency and as such, functions positively at many levels at the same time. According to results of the SI_judo, Judo could be approached as a kind of intervention to enhance ASD children psychosocial and psychomotor skills. The SI_Judo program “Learn How to fall, Stand Up and Demand” were funded by “Support Points” of big Foundations in Greece.

Background Circulating tumour cells (CTCs) in blood associate with overall survival (OS) of cancer patients, but they are detected in extremely low numbers. Large tumour-derived extracellular vesicles (tdEVs) in castration-resistant prostate cancer (CRPC) patients are present at around 20 times higher frequencies than CTCs and have equivalent prognostic power. In this study, we explored the presence of tdEVs in other cancers and their association with OS. Methods The open-source ACCEPT software was used to automatically enumerate tdEVs in digitally stored CellSearch® images obtained from previously reported CTC studies evaluating OS in 190 CRPC, 450 metastatic colorectal cancer (mCRC), 179 metastatic breast cancer (MBC) and 137 non-small cell lung cancer (NSCLC) patients before the initiation of a new treatment. Results Presence of unfavourable CTCs and tdEVs is predictive of OS, with respective hazard ratios (HRs) of 2.4 and 2.2 in CRPC, 2.7 and 2.2 in MBC, 2.3 and 1.9 in mCRC and 2.0 and 2.4 in NSCLC, respectively. Conclusions tdEVs have equivalent prognostic value as CTCs in the investigated metastatic cancers. CRPC, mCRC, and MBC (but not NSCLC) patients with favourable CTC counts can be further prognostically stratified using tdEVs. Our data suggest that tdEVs could be used in clinical decision-making.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 μm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (

Studies of changes in taste threshold, perceived intensity, liking, and preference during pregnancy were reviewed, because such changes have the potential to negatively impact nutrient intake in pregnant women. Medline and Web of Science were searched; eligibility was based on inclusion, exclusion, and quality criteria. Fourteen articles were included: 5 reported taste thresholds, 8 taste intensity, and 13 liking/preferences. Articles addressed sweet, salty, sour, and bitter, not umami. Results of self-reported changes suggested that many women experienced some sort of alteration in taste during pregnancy. Studies with real stimuli demonstrated that the only consistent finding for taste thresholds was that pregnant women showed higher thresholds for bitterness in their first trimester. For taste intensity, no consistent differences between pregnant women and controls were observed. However, over the course of pregnancy, salty intensity seemed to decrease, the intensity of other tastes did not change. During pregnancy, higher saltiness was liked more and salty snacks were consumed more, particularly in the second and/or third trimester. Drinks with lower sweetness were preferred and intake of sweet snacks was highest in the second trimester. Preference for sour and bitter did not seem to be affected. Self-reports suggested that many women experienced some taste changes during pregnancy, while changes based on studies with real stimuli were limited. Many women experienced a higher threshold for bitter perception in the first trimester, a preference for sweet snacks in the second trimester, and higher saltiness appetite in the second and third trimester of pregnancy, which may have nutritional consequences.

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4&prime, 6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes.

Summary Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory/metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) member with pleiotropic effects in inflammation and cancer. In response to proinflammatory stimuli, endothelial Tpl2 deletion alters tight junction claudin-5 protein expression through inhibition of JNK signaling and lysosomal degradation activation, resulting in reduced vascular permeability and immune cell infiltration. This results in significantly attenuated disease scores in experimental autoimmune encephalomyelitis and fewer tumor nodules in a hematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or small interfering RNA (siRNA)-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish an endothelial-specific role for Tpl2 and highlight the therapeutic potential of blocking the endothelial-specific Tpl2 pathway in chronic inflammatory and metastatic diseases.

Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory / metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tpl2, a MAP3 kinase with pleiotropic effects in inflammation and cancer. In response to pro-inflammatory stimuli, endothelial Tpl2 deletion alters Tight Junction (TJ) claudin-5 protein expression and attenuates the increase in vascular permeability, inflammatory cell infiltration and accumulation of plasma proteins. This results in significantly attenuated disease scores in Experimental Autoimmune Encephalomyelitis and significantly less tumor nodules in a haematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or siRNA-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish a novel endothelial-specific role for Tpl2 in the regulation of TJ-mediated endothelial permeability and highlight the therapeutic potential of Tpl2 blockade in chronic inflammatory and metastatic diseases.

Changes in sweet and bitter taste perception during pregnancy have been reported in a limited number of studies leading, however, to inconclusive results. The current study aimed to investigate possible differences in perceived intensity and liking of sweetness and bitterness between pregnant and nonpregnant women. Forty-six pregnant and 45 nonpregnant women evaluated taste intensity and liking of five samples of each of four different products: two sweet (cake and apple + berry juice) and two bitter (salad and grapefruit juice). Product samples varied in sweetness and bitterness, respectively. Pregnant women completed also a self-administered questionnaire on changes in sweet and bitter taste perception due to pregnancy. Perceived intensity of sweetness and bitterness was not different between pregnant and nonpregnant women for any of the products. However, the liking of the least sweet apple + berry juice was significantly higher, and the optimal preferred sugar content was significantly lower in pregnant compared to nonpregnant women. With regards to self-report, pregnant women who reported higher sensitivity in sweet or bitter taste did not have significantly different intensity scores from those with no self-reported changes. The apple + berry juice sample highest in sugar content was liked less by pregnant women who reported higher sensitivity toward sweet taste compared to those who reported no change. This study provides evidence that pregnant women may prefer lower sugar content in a juice mixture compared to nonpregnant women. Future research should focus on the possible occurrence of this phenomenon in other beverages and foods, as well.

The objective of this study was to evaluate a waste, waste cooking oil (WCO) as substrate for carotene production by Blakeslea trispora in shake flask culture. WCO was found to be a useful substrate for carotene production. B. trispora formed only pellets during fermentation. The oxidative stress in B. trispora induced by hydroperoxides and BHT as evidenced by increase of the specific activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly the production of carotenes. The highest concentration of carotenes (2021 ± 75 mg/l or 49.3 ± 0.2 mg/g dry biomass) was obtained in culture grown in WCO (50.0 g/l) supplemented with CSL (80.0 g/l) and BHT (4.0 g/l). In this case the carotenes produced consisted of β-carotene (74.2%), γ-carotene (23.2%), and lycopene (2.6%). The external addition in the above medium glucose, Span 80, yeast extract, casein acid hydrolysate, l-asparagine, thiamine. HCl, KH2PO4, and MgSO4·7H2O did not improve the production of carotenes.

Tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) in blood of metastatic cancer patients associate with poor outcome. In this study, we explored the human epidermal growth factor receptor 2 (HER2) expression on CTCs and tdEVs of metastatic breast cancer patients. Blood samples from 98 patients (IC 2006-04 study) were originally processed with the CellSearch® system using the CTC kit and anti-HER2 as an additional marker in the staining cocktail. CTCs and tdEVs were automatically enumerated from the generated CellSearch image datasets using the open-source ACCEPT software. The HER2 status of the tissue was assessed by fluorescence in situ hybridization (FISH). The inclusion of anti-HER2 increased the percentage of informative samples with ≥1 detectable CTC and tdEV from 89% and 99% to 95 and 100%, respectively. CTCs and tdEVs were subdivided based on their cytokeratin (CK) and HER2 phenotype into CK+HER2-, CK-HER2+ and CK+HER2+. Inter- and intra- patient heterogeneity was found regarding the phenotype of CTCs and tdEVs with the majority of patients having all different subclasses present. CK- CTCs and tdEVs correlated equally well with clinical outcome as CK+ CTCs and tdEVs. Use of ≥7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity, whereas the respective use of ≥23% HER2+CK+ CTCs leads to 65% sensitivity and 66% specificity. HER2 can be detected on CTCs and tdEVs not expressing CK, which have similar clinical relevance to CTCs and tdEVs expressing CK. tdEVs perform better than CTCs in predicting the HER2 status of the tissue. Funded by the NWO Applied and Engineering Sciences Cancer-ID project #14190, the EUFP7 CTCTrap project #305341 and the EU IMI CANCER-ID project # 115749-1. Citation Format: Afroditi Nanou, Leonie L. Zeune, Francois-Clement Bidard, Jean-Yves Pierga, Leon W. Terstappen. HER2 expression on tumor-derived extracellular vesicles and circulating tumor cells in metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5372.

Self-awareness and self-regulation yoga techniques lead to stress management, reducing anxiety and well-being of individuals with Autism Spectrum Disorders. The current study seeks to investigate the degree of the autonomous involvement of an 11-year-old girl with Autism Spectrum Disorder in the self-awareness and self-regulation yoga exercises. The research was designed as an educational intervention. The intervention was applied once a week for 3 months, and took place at the sensory room of “School for all: Tokei Maru” at Include in Thessaloniki. Research diaries and observation protocols, by participant and external observers, were used for data collection before, during and after the intervention. According to the research findings, girl’s degree and quality of involvement improved during the intervention, so that she managed to use specific techniques autonomously. Research findings cannot be generalized, but they are encouraging in developing effective self-awareness and self-regulation programs for children with Autism Spectrum Disorder in future research.

During joint action tasks, expectations for outcomes of one's own and other's actions are collectively monitored. Recent evidence suggests that trait empathy levels may also influence performance monitoring processes. The present study investigated how outcome expectation and empathy interact during a turn-taking piano duet task, using simultaneous electroencephalography (EEG) recording. During the performances, one note in each player's part was altered in pitch to elicit the feedback-related negativity (FRN) and subsequent P3 complex. Pianists memorized and performed pieces containing either a similar or dissimilar sequence as their partner. For additional blocks, pianists also played both sequence types with an audio-only computer partner. The FRN and P3a were larger in response to self than other, while P3b occurred only in response to self, suggesting greater online monitoring of self- compared to other-produced actions during turn-taking joint action. P3a was larger when pianists played a similar sequence as their partner. Finally, as trait empathy level increased, FRN in response to self decreased. This association was absent for FRN in response to other. This may reflect that highly-empathetic musicians during joint performance could use a strategy to suppress exclusive focus on self-monitoring.

// Afroditi Nanou 1 , Frank A.W. Coumans 2 , Guus van Dalum 3 , Leonie L. Zeune 1, 4 , David Dolling 6 , Wendy Onstenk 5 , Mateus Crespo 6 , Mariane Sousa Fontes 6, 7 , Pasquale Rescigno 6, 7 , Gemma Fowler 6 , Penny Flohr 6 , Christoph Brune 4 , Stefan Sleijfer 5 , Johann S. de Bono 6, 7 and Leon W.M.M. Terstappen 1 1 Department of Medical Cell BioPhysics, MIRA Institute, University of Twente, Enschede, the Netherlands 2 Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, the Netherlands 3 Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University, Dusseldorf, Germany 4 Department of Applied Mathematics, MIRA Institute and Faculty of EEMCS, University of Twente, Enschede, the Netherlands 5 Department of Medical Oncology, Erasmus MC – Cancer Institute, Rotterdam, The Netherlands 6 Division of Clinical Studies, The Institute of Cancer Research, London, United Kingdom 7 Prostate Cancer Targeted Therapies Group, The Royal Marsden NHS Foundation Trust, London, United Kingdom Correspondence to: Afroditi Nanou, email: a.nanou@utwente.nl Leon W.M.M. Terstappen, email: l.w.m.m.terstappen@utwente.nl Keywords: circulating tumor cells (CTCs); tumor-derived Extracellular Vesicles (tdEVs); cytokeratin 18 (CK18); caspase-cleaved cytokeratin 18 (ccCK18); castration-resistant prostate cancer (CRPC) Received: November 07, 2017 Accepted: March 17, 2018 Published: April 10, 2018 ABSTRACT Purpose: The presence of Circulating Tumor Cells (CTCs) in Castration-Resistant Prostate Cancer (CRPC) patients is associated with poor prognosis. In this study, we evaluated the association of clinical outcome in 129 CRPC patients with CTCs, tumor-derived Extracellular Vesicles (tdEVs) and plasma levels of total (CK18) and caspase-cleaved cytokeratin 18 (ccCK18). Experimental Design: CTCs and tdEVs were isolated with the CellSearch system and automatically enumerated. Cut-off values dichotomizing patients into favorable and unfavorable groups of overall survival were set on a retrospective data set of 84 patients and validated on a prospective data set of 45 patients. Plasma levels of CK18 and ccCK18 were assessed by ELISAs. Results: CTCs, tdEVs and both cytokeratin plasma levels were significantly increased in CRPC patients compared to healthy donors (HDs). All biomarkers except for ccCK18 were prognostic showing a decreased median overall survival for the unfavorable groups of 9.2 vs 21.1, 8.1 vs 23.0 and 10.0 vs 21.5 months respectively. In multivariable Cox regression analysis, tdEVs remained significant. Conclusions: Automated CTC and tdEV enumeration allows fast and reliable scoring eliminating inter- and intra- operator variability. tdEVs provide similar prognostic information to CTC counts.

The L-type Ca2+ channel Cav1.2 controls heart rate and neuronal excitability. Qian et al . found that enhancement of Cav1.2 channel activity in the brain by β-adrenergic receptor (βAR) signaling required phosphorylation of Ser1928, whereas in the heart, this site was dispensable for βAR-mediated regulation. In contrast to those from wild-type mice, hippocampal neurons from mice, in which Ser1928 of Cav1.2 was mutated to alanine, did not exhibit increased L-type calcium channel activity in response to β-adrenergic stimulation. Phosphorylation of Ser1928 involved signaling through the β2AR, but not through the β1AR, and this phosphorylation event enabled a particular form of long-term potentiation, a process linked to learning and memory. These results were in marked contrast to βAR-mediated regulation of Cav1.2 activity in cardiomyocytes, which involved β1AR and was independent of Ser1928. This differential regulation in the heart and brain implies that tissue-specific therapeutics could be identified.

Circulating Tumor Cell (CTC) counts, as determined by the CellSearch® system, associate with poor overall survival (OS) in castration-resistant prostate cancer (CRPC), breast, colorectal and non-small cell lung (NSCLC) cancer patients. The cut-offs used to discriminate between patients with favorable and unfavorable prognoses are 5 CTCs / 7.5 mL of blood for breast and CRPC, 3 for colorectal and 1 for NSCLC. The low numbers of CTCs frequently limit the accurate discrimination of patients into favorable or unfavorable prognosis groups. We previously reported the presence of CK+, DNA-, CD45- tumor-derived extracellular vesicles (tdEVs) in ~20x higher frequencies in CRPC patients and showed their equivalence to CTC counts in predicting clinical outcome. In this study, we explored the presence of tdEVs in the blood of metastatic CRPC, breast, colorectal, and NSCLC patients, determined the association of tdEVs with OS and whether they aid in stratifying patients with favorable CTC counts. Digitally stored CellSearch® system images obtained from previously reported studies from 190 CRPC, 450 colorectal, 179 breast and 117 NSCLC patients before the initiation of a new treatment were used to enumerate tdEVs automatically using the open-source ACCEPT software. tdEV counts highly correlated with CTC counts in all cancer types (Spearman’s Rho tests p Citation Format: Afroditi Nanou, Leonie L. Zeune, Sanne de Wit, Craig M. Miller, Cornelis J. Punt, Harry J. Groen, Daniel F. Hayes, Johann S. de Bono, Leon W. Terstappen. Tumor-derived extracellular vesicles in blood of metastatic breast, colorectal, prostate, and non-small cell lung cancer patients associate with worse survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4464.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no effective treatment to date. Despite its multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of antioxidant genes that are downregulated in the presence of mutant SOD1 may serve as a therapeutic strategy for motor neuronal protection. Lentiviral vectors expressing either PRDX3 or NRF2 genes were tested in the motor neuronal-like NSC34 cell line, and in the ALS tissue culture model, NSC34 cells expressing the human SOD1(G93A) mutation. The NSC34 SOD1(G93A) cells overexpressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40 and 50% respectively compared with controls, whereas cell survival was increased by 30% in both cases. The neuroprotective potential of those two genes was further investigated in vivo in the SOD1(G93A) ALS mouse model, by administering intramuscular injections of adenoassociated virus serotype 6 (AAV6) expressing either of the target genes at a presymptomatic stage. Despite the absence of a significant effect in survival, disease onset or progression, which can be explained by the inefficient viral delivery, the promising in vitro data suggest that a more widespread CNS delivery is needed.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by iron ions during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD). The addition of 1.0 mM of FeCl3 to the medium was associated with a mild oxidative stress as evidenced by remarkable increase of the specific activities of SOD and CAT. On the other hand, the addition 5.0 mM of FeCl3 caused a strong oxidative stress resulting in a drastic decrease in carotene concentration. The oxidative stress in B. trispora changed the composition of the carotenes and caused a significant increase of γ-carotene ratio. The highest concentration of carotenes (115.0 ± 3.5 mg/g dry biomass) was obtained in the basal medium without the addition of FeCl3 after 8 days of fermentation. In this case, the carotenes consisted of β-carotene (46.3 %), γ-carotene (40.1 %), and lycopene (13.6 %). The addition of 1.0 mM of FeCl3 into the medium did not change the concentration of carotenes. But, the composition of carotenes was changed with a drastic increase of γ-carotene ratio (61.6 %) and a decrease in β-carotene and lycopene ratio (31.2 and 7.2 %, respectively).

Facilitation and inactivation of P/Q-type calcium (Ca(2+)) currents through the regulation of voltage-gated Ca(2+) (CaV) 2.1 channels by Ca(2+) sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca(2+) entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10-30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50-100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.

The carotene production from a synthetic medium sterilized at initial pH 11.0 by Blakeslea trispora in a bubble column reactor was investigated. Under the above conditions the fungus formed compact aggregates (pellets). As a consequence, a mild oxidative stress in B. trispora was occurred indicated by the specific activities of superoxide dismutase (SOD) and catalase (CAT). The oxidative stress resulted in a significant increase in carotene production and a change in the biosynthesis of carotenes. The highest concentration of carotenes (85.0 mg/g dry biomass) was obtained after 8 days of fermentation. In this case, the proportion of β-carotene, γ-carotene, and lycopene was 52.42%, 45.43%, and 2.15%, respectively. Bubble column reactor has a potential for carotene production from synthetic medium sterilized at initial pH 11.0.

The oxidative stress and the morphological changes in Blakeslea trispora induced by enhanced aeration during carotene production in a bubble column reactor was investigated. Enhanced aeration caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by increase of the specific activities of superoxide dismutase (SOD) and catalase (CAT). The oxidative stress in B. trispora resulted in a significant increase in carotene production. The highest concentration of carotenes (55.0 ± 2.5 mg/g dry biomass) was obtained at aeration rate of 4 vvm. Very high oxidative stress in B. trispora caused a change in the biosynthesis of carotenes resulting an increase in γ-carotene concentration. The maximum proportion of β-carotene (91.68%), γ-carotene (44.67%), and lycopene (11.54% of total carotenes) was observed at aeration rates of 4, 5, and 5 vvm, respectively. The external addition of butylated hydroxytoluene (BHT) and hydrogen peroxide in the medium did not improve the production of carotenes.

The present study investigated the influence of electromagnetic fields (EMF), similar to that emitted by mobile phones, on brain activity. Ten women and ten men, matched for age and educational level, performed a short memory task (Wechsler test), with simultaneous Electroencephalogram (EEG) recordings at 15 scalp electrodes, both without (baseline) and with exposure to an 1,800 MHz signal. The EEG energy was found concentrated at the four basic EEG bands [α (8–13 Hz), β (14–30 Hz), δ (0–4 Hz), and θ (5–7 Hz)]. The analysis revealed that in the presence of EMF, the energy of the β band was significantly greater for females than for males at the majority of the electrodes. Since beta oscillation is associated with the shift of attention during the perception, these findings may indicate that the particular EMF (1,800 MHz signal) exerts an influence on this brain activity, which appears to be gender-related.

Na+-activated K+ channels (K(Na)) exist in different types of neurones and their activation has been shown to depend on Na+ influx via voltage-activated channels. However, one major route for Na+ influx into neurones is through ionotropic receptors and its role in activating K(Na) is still unclear. We have examined whether Na+ influx induced by activation of AMPA receptors can activate K(Na) in lamprey spinal cord neurones. Our results showed that the application of AMPA induced not only the characteristic inward current but also produced an outward current outlasting the activation of the receptors. This outward current was mediated by K+ and was abolished when Na+ was substituted with Li+. The AMPA-mediated K(Na) current was completely blocked by quinidine but was not modulated by increased intracellular Cl- concentration or ATP. Thus, Na+ influx via AMPA receptor channels activates K(Na) with properties similar to Slack channels. The AMPA-activated K(Na) may act as an inherent negative feedback mechanism to regulate the homeostasis of excitation.

The effects of electromagnetic fields (EMF) emitted by cellular phones on human electroencephalogram (EEG) were studied during an auditory memory task. The experimental method and the experimental setup are introduced as a credible measurement method of EEG. 19 normal subjects (10 women and 9 men) performed the memory task both with and without exposure to a 900 MHz signal, emitted by a dipole antenna placed near the subjects' head. The energy of the EEG signals was calculated at the time domain. A Fourier transform of the EEG signals was done and the EEG energy was also calculated at the frequency domain. As the Parseval's theorem anticipates the energies were identical. The EEG energy was found concentrated at the four basic bands (α (8–13 Hz), β(14–30 Hz), δ (0–4 Hz) and θ(5–7 Hz)). The primary concern of the present study was the gender related influence of EMF on the spectral energy of EEG. The results show evidence of a strong gender—radiation interaction effect on the EEG energy and on the peak amplitudes within each of the four rhythms. Without radiation the spectral power of males is greater than of females, while under exposure the situation is reversed. Under the influence of EMF the spectral power of the males EEG is decreased while that of the females is increased. In conclusion both the baseline EEG and the changes effected to the EEG power spectrum under the influence of EMF seem to be gender dependent.

Rifampicin (also known as rifampin) is one of the most potent first-line antituberculosis drugs and an indispensable treatment option for isoniazid-resistant, rifampicin-sensitive tuberculosis (TB). Hypersensitivity reactions to rifampicin, mainly including fever, flu-like syndrome, rash, thrombocytopenia, acute renal failure, urticaria, and anaphylaxis, are considered rare but may occur among susceptible individuals and lead to premature discontinuation of the drug. Drug desensitization is a well-established procedure that may temporarily modify a patient’s immunologic response to the sensitizing agent, thus allowing for continuation of treatment. Desensitization to rifampicin has been previously described in rare case reports, almost invariably with successful results. We herein report a case of anaphylaxis during rapid oral desensitization to rifampicin in a male patient with active TB and a history of anaphylactic reactions to this agent. To the best of our knowledge, this is the first report in the English literature of a severe immediate-type reaction during desensitization to rifampicin. A 72-year-old man presented to the outpatient pulmonary clinic of “Sotiria” General Hospital, Athens, Greece, with fever, nonproductive cough, and weakness, lasting for 2 weeks. His medical history was significant for adult-onset diabetes mellitus and rheumatoid arthritis, treated with metformin and low-dose prednisone and intravenous infliximab, respectively. The patient was diagnosed with miliary TB and was started on anti-TB treatment with isoniazid (300 mg/day), rifampicin (600 mg/day), pyrazinamide (1500 mg/day), and ethambutol (1200 mg/day). Within 2 weeks after initiation of the above regimen, and 30 minutes after administration of the last dose of rifampicin, the patient developed fever and widespread urticaria; all drugs were subsequently discontinued and gradually reintroduced—after resolution of the cutaneous reaction—to identify the culprit drug. Urticaria, palmoplantar, and oropharyngeal pruritus were noted within 5 minutes after reintroduction of rifampicin and the drug was stopped; rifampicin was thereafter replaced with rifabutin. Ten days after initiation of rifabutin, the patient developed a maculopapular rash that affected the trunk and lower extremities. A skin biopsy was compatible with drug-induced eruption and rifabutin was discontinued. Approximately 3 years after his initial diagnosis of TB, the patient developed recurrent TB and was again started on a multidrug anti-TB regimen comprising isoniazid, rifabutin, pyrazinamide, and ethambutol. Twelve days after initiation of treatment a maculopapular rash appeared on the patient’s upper and lower extremities; rifabutin was again discontinued, followed by gradual resolution of the eruption, and the patient was referred to the Allergy Department of our hospital for further evaluation and management. At the time of the patient’s presentation to our department, skin prick test (SPT) and intradermal (ID) skin testing to rifampicin were performed, based on previous recommendations defining the highest nonirritant intradermal skin concentration of rifampicin (0.002 mg/mL). Histamine and saline were also used as positive and negative controls, respectively. SPT at a concentration of 2 mg/mL was negative, whereas a positive reaction (with a wheal of 15 17 mm and a flare of 42 44 mm) was shown on ID skin testing at a concentration of 0.002 mg/ mL. After obtaining the patient’s informed consent, oral desensitization to rifampicin was carried out in the intensive care unit. Premedication with antihistamines or steroids was not used (prednisone was discontinued for 3 days before the procedure) because these drugs may mask initial symptoms of anaphylaxis during desensitization, as previously emphasized, and the patient was not taking b-blockers or angiotensin-converting enzyme inhibitors. Increasing dosages of rifampicin were administered every 30 minutes, at a starting dose of 0.0002 mg followed by gradual dose escalation (Table I), as described in previously published desensitization protocols. Twenty minutes after administration of the 50 mg rifampicin dose, the patient started complaining of pruritus of the palms, soles, and groin followed by facial and trunk erythema, cough, generalized urticaria, hypotenstion, and sinus tachycardia followed by sinus bradycardia (without other ECG abnormalities). The desensitization procedure was discontinued, and the patient was treated with IV fluids, oxygen and nebulized salbutamol, intramuscular adrenaline, and intravenous methylprednisolone, dimetindene and ranitidine, with gradual recovery. Serum tryptase obtained at 1 hour after the onset of symptoms was 25 mg/L. Repeat serum tryptase, measured 1 week later, was found to be within normal limits (4 mg/L). Given the patient’s anaphylactic reaction during rapid desensitization, anti-IgE treatment and desensitization to rifampicin using slow dose increment over several days were recommended but the patient refused. Hypersensitivity reactions to anti-TB agents are relatively uncommon, encountered in approximately 4%-5% of the treated population, but may as well lead to withdrawal of the culprit drug and switching to an alternative agent. Rifampicin, in particular, is generally considered a well-tolerated drug with a low percentage of adverse events, especially when administered in usual therapeutic doses and outside the context of HIV infection. Delayed reactions, including a variety of cutaneous manifestations, are the commonest adverse reactions induced by rifampicin, whereas severe immediate reactions, such as anaphylaxis, are exceedingly rare.

The generation of activity in the central nervous system requires precise tuning of cellular properties and synaptic transmission. Neural networks in the spinal cord produce coordinated locomotor movements. Synapses in these networks need to be equipped with multiple mechanisms that regulate their operation over varying regimes to produce locomotor activity at different frequencies. Using the in vitro lamprey spinal cord, we explored whether Ca2+ influx via different routes in postsynaptic soma and dendrites and in presynaptic terminals can activate apamin-sensitive Ca2+-activated K+ (SK) channels and thereby shape synaptic transmission. We show that postsynaptic SK channels are tightly coupled to Ca2+ influx via NMDA receptors. Activation of these channels by synaptically induced NMDA-dependent Ca2+ transients restrains the time course of the synaptic current and the amplitude of the synaptic potential. In addition, presynaptic SK channels are activated by Ca2+ influx via voltage-gated channels and control the waveform of the action potential and the resulting Ca2+ dynamics in the axon terminals. The coupling of SK channels to different Ca2+ sources, pre- and postsynaptically, acts as a negative feedback mechanism to shape synaptic transmission. Thus SK channels can play a pivotal role in setting the dynamic range of synapses and enabling short-term plasticity in the spinal locomotor network.

Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis infection. Our aim was to analyze the epidemiology, clinical features, diagnostic approach, and outcome of tuberculous meningitis in childhood.During a 25-y period (1984-2008), 43 children, aged 7 months to 13 y, were hospitalized in the Paediatric Department of the referral centre for infectious diseases in Thessaloniki, Greece with the diagnosis of TBM. The patients were classified according to the clinical findings on admission as per the UK Medical Research Council (MRC) staging: stage I, 16/43 (37.2%) children; stage II, 16/43 (37.2%); and stage III 11/43 (25.6%).Twenty-seven of the 43 patients were Greek (63%) and none had been BCG-vaccinated. A family history of tuberculosis was identified in 18 cases (42%). 35 patients (81%) had a positive tuberculin skin test. An extrameningeal site of infection was identified in 14 children (33%); pulmonary tuberculosis in 14/43 patients (5/13 miliary tuberculosis) and spondylitis in 1. All patients were treated with anti-tuberculous drugs and 36 (84%) also received corticosteroids. Complications during hospitalization (coma, seizures, cranial nerve palsy, hydrocephalus) presented in 26 patients (60%). Two patients died (5%) and 6 (14%) had permanent neurological sequelae.TBM, although rare, remains a disease with significant morbidity and mortality. Early clinical diagnosis and appropriate treatment initiation are important for the prognosis.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by elevated dissolved oxygen concentrations during carotene production was investigated by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. Changes in the ratio of the volume of air (V(a)) over the medium and the volume of medium (V(m)) in the flask caused changes of the morphology of microorganism from clumps to pellets and increases in the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a significant increase in carotene production, and a maximum proportion of β-carotene (60%), γ-carotene (50%), and lycopene (10%) (as percentages of total carotenes) was observed at a ratio V(a)/V(m) of 15.7, 4.0 and 1.5, respectively. The highest concentration of carotenes (115.0mg/g dry biomass) was obtained in V(a)/V(m) ratio of 9.0.

The prognostic significance of thrombocytopenia in critically ill patients has not been thoroughly explored. Our study aimed at investigating the incidence of thrombocytopenia among ICU patients, its association with ICU-acquired infection and its clinical impact on ICU mortality.

Discrete neural networks in the central nervous system generate the repertoire of motor behavior necessary for animal survival. The final motor output of these networks is the result of the anatomical connectivity between the individual neurons and also their biophysical properties as well as the dynamics of their synaptic transmission. To illustrate how this processing takes place to produce coordinated motor activity, we have summarized some of the results available from the lamprey spinal locomotor network. The detailed knowledge available in this model system on the organization of the network together with the properties of the constituent neurons and the modulatory systems allows us to determine how the impact of specific ion channels and receptors is translated to the global activity of the locomotor circuitry. Understanding the logic of the neuronal and synaptic processing within the locomotor network will provide information about not only their normal operation but also how they react to disruption such as injuries or trauma.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by butylated hydroxytoluene (BHT) during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. The addition of exogenous BHT to the medium caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by remarkable increase of the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a fivefold increase of carotene production. The highest concentration of carotenes (125.0 mg/g dry biomass) was obtained in culture grown in medium supplemented with 20 mM of BHT.

Gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches. For neurodegenerative diseases, gene therapy is particularly attractive due to the restricted bioavailability of conventional therapeutic substances to the affected structures of the brain and progressive nature of these diseases. With the development of lentiviral vector systems, many issues have been addressed and new delivery routes to the nervous system have been identified. Lentiviral vectors can efficiently deliver genes to postmitotic neuronal cell types offering long-term expression, can be generated in high titers, and do not give immunological complications. Various animal studies have demonstrated the effectiveness of these vectors to deliver therapeutic genes into the nervous system, as well as to model human diseases. This chapter will describe the basic features of lentiviral vectors, the progress, and their applications as a therapeutic strategy to treat diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, Parkinson's disease, and Huntington's disease.

The present study introduces the concept of spectral power coherence (SPC), which reflects the pattern of coordination of the four basic EEG bands (delta, theta, alpha, and beta) at a specific location of the brain. The SPC was calculated for the pre-stimulus EEG signal during an auditory memory task under different electromagnetic field (EMF) conditions (900 MHz and 1800 MHz). The results showed that delta rhythm is less consequential in the overall cooperation between the bands than the higher frequency theta, alpha and beta rhythms. Additionally, it has been shown that the radiation effect on SPC is different for the two genders. In the absence of radiation males exhibit higher overall SPC than females. These differences disappear in the presence of 900 MHz and are reversed in the presence of 1800 MHz.

The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during beta-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of beta-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during beta-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased beta-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.

Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI-TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest-scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest-scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8,900 Da) was purified further and was characterized as the C-terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8,900-Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment of vitronectin as a new serum marker of HCC in patients with chronic liver diseases. (HEPATOLOGY 2005;41:40–47.)

The present study investigated the gender-related influence of electromagnetic fields (EMF), similar to that emitted by mobile phones, on brain activity. Ten women and nine men performed a short memory task (Wechsler test), both without (baseline) and with exposure to a 900 MHz signal. The EEG energy of the total waveform and the alpha, beta, delta and theta; rhythms were calculated from the recordings of 15 scalp electrodes. Baseline EEG energy of males was greater than that of females, while exposure to EMF decreased EEG energy of males and increased that of females. Memory performance was invariant to EMF exposure and gender influences. These findings indicate that EMF may exert a gender-related nfluence on brain activity.