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Background: Schizophrenia is a severe and often difficult to treat psychiatric illness. In many patients, negative symptoms dominate the clinical picture. Meta-analysis has suggested moderate, but significant effects of high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) on these symptoms. For treatment of depression a much shorter protocol - intermittent theta burst stimulation (iTBS) - has shown to be non-inferior to conventional high-frequency rTMS. This randomized, sham-controlled, rater-blinded clinical trial assesses the effects of conventional HF-rTMS as well as of iTBS of the left dorsolateral prefrontal cortex in comparison with sham., Methods: The study will be conducted at two psychiatric university hospitals in Germany and at two in the Czech Republic. Assuming an effect size of 0.64 to be detected with a power of 80%, the calculated sample size is 90 patients. Primary outcome will be the difference in the Scale for the Assessment of Negative Symptoms (SANS) score between each active arm and the sham arm at end of treatment.In addition, the trial investigates effects on depressive symptoms, cognitive performance and cigarette smoking. Recording magnetic resonance imaging (MRI) and electroencephalography (EEG) data will serve to assess whether treatment success can be predicted by neural markers and is related to specific neurobiological changes., Discussion: This is a clinical trial directly comparing 10 Hz-rTMS and iTBS in a sham-controlled manner in treating negative symptoms of schizophrenia. If successful, this would present an interesting treatment option for a chronic and severe condition that can be applied at most psychiatric hospitals and only takes up a few minutes per day., Trial Registration Number: This trial has been registered at clinicaltrials.gov, Identifier: NCT04318977., Data Dissemination: Results from the trial shall be published in peer-reviewed journals and presented at meetings and conferences., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BL receives royalties from Springer for edited books, received research grants from the company Neuromod as well as from national (DFG, BMBF) and European institutions (EU), received speaker's honoraria and advisory panel payments from Neuromod, Desyncra, Decibel Tx and Servier. SS received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant [agreement number 722046]. TH has had travel expenses paid for by Nexstim plc. All other authors have no competing interests or disclaimers to declare., (© 2022 The Authors. Published by Elsevier Inc.)

In the present modern times we see that the population is gradually ageing and along with it also the incidence of civilisation diseases, including those of the cardiovascular system, is increasing. As the care of these patients develops, so does the number of surgeries of invasive cardiology. Even though the development in this area is still dynamic, the techniques are improving and new technologies are appearing. However, these invasive methods are still associated with certain risks for the patient. In terms of vascular surgery, the most frequent complications are iatrogenic pseudo-aneurysms and large haematomas. The objective of the present study was to evaluate the development in the incidence of pseudo-aneurysms (PSA) appearing after punctures of femoral artery due to coronarography or PTCA, to verify the hypothesis that the ratio of the number of PSA to the total number of invasive-cardiologic diagnostic and therapeutic surgeries is decreasing and to indicate possible solutions of complications associated with catheterisation. The study presents a retrospective account of the number of invasive surgeries conducted at the 1st Department of Medicin - Cardioangiology of the St. Anne's University Hospital Brno (UH) from 1996 to 2004. Summarised are numbers of PSA's conducted in this period by surgeons of the 2nd Department of Surgery of the St. Anne's University Hospital Brno (Tab. 1, Fig. 5, Ref. 17).

We reported recently that roscovitine arrested human MCF-7 cancer cells at G2-M phase of the cell cycle and concomitantly induced apoptosis. After roscovitine treatment, the level of wild-type p53 protein strongly increased and p53 was accumulated in the nucleus. Here, we raised the question of which pathway would be involved in roscovitine-induced apoptosis in MCF-7 cells, which are known to be caspase-3-deficient, and whether roscovitine-mediated activation of p53 protein might positively affect the execution of cell death. Roscovitine induced a depolarization of mitochondrial potential beginning at 6 hours posttreatment as evidenced by changes in J-aggregate formation and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Interestingly, roscovitine stimulated a site-specific phosphorylation of wild-type p53 protein in a time-dependent manner. p53 protein was specifically phosphorylated at Ser46. P-Ser46-activated wild-type p53 tumor suppressor up-regulated p53AIP1 protein, its downstream target known to mediate the depolarization of mitochondria. The onset of phosphorylation of p53 at Ser46 preceded the up-regulation of p53AIP1 protein and the depolarization of mitochondrial potential. We compared the kinetics of roscovitine-mediated p53 activation between caspase-3-deficient parental MCF-7 cells and cells reconstituted with caspase-3. The kinetics and the extent of p53 protein activation in caspase-3-proficient cells differed from those observed in caspase-3-deficient parental cells. Remarkably, roscovitine failed to induce phosphorylation at Ser46 in caspase-3-reconstituted MCF-7 cells. Our results indicate that, depending on the status of caspase-3 in MCF-7 cells, different apoptotic pathways were initialized.

The tumor suppressor p53 is a short-lived protein that under normal conditions is reduced to a barely detectable level. The stability of p53 protein is primarily regulated in normal non-transformed cells by two interplayers: Mdm2 and p14(ARF). Relocation of p53, Mdm2, and p14(ARF) to the nucleolus seems to regulate, at least partially, the steady-state of p53. Moreover, there are alternative pathways of the regulation of p53 stability in unstressed cells. Jun-N(amino)-terminal kinase (JNK) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in the regulation of the steady-state of wild-type (wt) p53 protein. However, in most human cervical carcinomas, which express the high-risk human papilloma viruses (HPVs) E6 protein, a complete switch from Mdm2 to HPV E6-mediated degradation of p53 occurs. Virally encoded E6 protein utilizes the cellular ubiquitin-protein ligase termed E6-associated protein (E6-AP) to target p53 protein for proteolytic degradation. We recently addressed the question of whether p53 protein can be generally reactivated by chemotherapy in HeLa cells despite the E6 activity. We observed an increase of cellular p53 after cisplatin (CP) treatment. p53 protein accumulated preferentially in the nucleoli. We checked the cellular level of E6 during CP therapy. Six hours after application of CP the expression of E6 protein was markedly reduced. This coincided with the increase of cellular p53 level and preceded the nucleolar accumulation of p53 protein, thereby indicating that repression of virally coded E6 protein by CP contributes to the restoration of p53 expression.

The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis., (Copyright 2003 Wiley-Liss, Inc.)

Cyclin-dependent kinases (CDKs) have recently raised considerable interest in view of their key role in the regulation of the cell cycle progression. In proliferating cells, distinct CDKs associated with specific cyclins coordinate in an orchestrated way the appropriate transition between different phases of the cell cycle. Mutations and/or aberrant expression of distinct CDKs and their regulatory components lead to uncontrolled proliferation and finally to carcinogenesis. However, in post-mitotic neurons, all CDKs with the exception of CDK5 are silent. CDK5, a proline-directed serine/threonine kinase exhibiting a close structural homology to the mitotic CDKs, binds to p35, the neuron-specific regulatory subunit of CDK5. CDK5 is very abundant in mature neurons and seems to regulate neurotransmitter release through phosphorylation and down-regulation of calcium channel activity. Therefore, the inhibition of CDKs in neurons after oxidative stress and in neurodegenerative disorders has a protective action. Selective CDKs inhibitors were developed as promising drugs for cancer therapy due to their ability to arrest cell cycle progression. The aim of this study was to compare the anti-proliferative effect of roscovitine (ROSC), a potent CDKs inhibitor, with that of cisplatin (CP) on human breast cancer MCF-7 cells. ROSC exerted stronger inhibitory effect on proliferation and cell cycle progression of MCF-7 than CP. Accumulation of G(2)/M arrested cells starting 6 h after onset of ROSC treatment coincided with a strong up-regulation of the p53. Reconstitution with caspase-3 sensitized MCF-7 cells to CP action. It implicates that ROSC inhibits more selectively and efficaciously the proliferation of human breast carcinoma cells.

An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.

INTRODUCTION: Melatonin (MEL), a pineal hormone, is well known as a potent antioxidant in a variety of ischemia-reperfusion models. Recent studies have assumed a pivotal role of reactive oxygen species (ROS) in the development of apoptosis. There are few pieces of information concerning a possible protective role of MEL against apoptosis in ischemia-reperfusion injury of myocardium. METHODS: We conducted an in vitro experiment: (1) to study the effect of MEL in the model of isolated and perfused working rat heart; (2) to evaluate the antioxidant capacity of MEL by a simple fluorescence test; and (3) to analyze the extent of apoptosis inhibition by MEL. Four groups of male Wistar rat were used: (a) group 'MEL 50 muM' (n=8); (b) group 'ischemia 30 min' (n=8); (c) group 'controls' (n=8); and (d) group 'controls+MEL 50 muM' (n=8). The perfusion medium was an oxygenated Krebs-Henseleit buffer (KHB). Hearts in groups (a) and (b) underwent 30 min of global normothermic ischemia and 45 min of reperfusion; 3 min before ischemia the hearts of group (a) received KHB with MEL 50 muM (and MEL 50 muM was also present in KHB solution during reperfusion). Hearts of group (c) were only perfused by KHB, and hearts of group (d) perfused by KHB+MEL 50 muM throughout the experiment. Registered were basic hemodynamic parameters: coronary, aortic, cardiac output and heart rate. At the end of each experiment, a left ventricle samples were taken for in situ detection of apoptosis using a TUNEL in-situ detection kit (POD) and quantitative analysis was performed. Malonedialdehyde concentrations were evaluated from heart homogenate to determine the severity of oxidative damage. To study the antioxidant capacity of MEL, a fluorescence test with allophycocyanin as an indicator was performed. A peroxyl radical generator, 2,2'-azobis(2-amidinopropan)-4-hydrochloride (AAPH) was used, and the antioxidant effect of MEL was expressed in oxygen-radical absorbing capacity (ORAC) units. RESULTS: Treatment by MEL resulted in a significant improvement of hemodynamic parameters and reduction of postischemic arrhythmias during reperfusion. All hearts in group 'ischemia 30 min' developed fatal ventricular fibrillations. MEL significantly reduced the incidence of apoptotic cells (14+/-4.3%; **P<0.01) vs. group 'ischemia 30 min' (58+/-2.1%). No apoptotic cells were detected in both control groups (c) and (d). In the fluorescence test, MEL exhibited a significant dose-dependent protective effect against peroxyl radical; MEL also reduced significantly the level of lipoperoxidation (MDA; *P<0.05). Analysis of hemodynamic parameters in both control groups (c) and (d) did not show any significant differences; the presence of MEL 50 muM in KHB solution did not have any important influence on cardiac performance in this type of experiment. CONCLUSION: We confirmed the previously reported beneficial effects of MEL against ischemia-reperfusion injury, presumably via its antioxidant properties. A significant suppression of apoptosis and the peroxyl radical scavenging properties of MEL in our study could contribute to the hypothesis of a close link between oxidative stress and apoptosis promotion.

The aim of the therapy of human malignancies is the inhibition of cell proliferation and/or induction of apoptosis. We studied the kinetics of the morphological and biochemical changes in HeLa cells during chemotherapy by cisplatin (CP). Apoptosis was evaluated by scoring of cells exhibiting changes characteristic for early and late stages of apoptosis as determined by Hoechst 33258 staining and by examination of positive reaction for activated caspase-3. Expression and intracellular localization of distinct proteins was analyzed by immunoblotting of subcellular fractions and segregation of nucleoli by immunocytochemistry. Chromatin fragmentation characteristic for apoptosis was observed in single cells after 3h cisplatin. A strong cytoplasmic accumulation of cytochrome C detected by immunoblotting 6h post-treatment was accompanied by an activation of caspase-9. Neither inhibition of cell division nor blocking of DNA replication preceded the onset of apoptosis. Our results show that after short treatment by CP, cell proliferation and apoptosis concomitantly occurred.

INTRODUCTION: Heart transplantation is often accompanied by multiple functional alterations, especially in reperfusion period. These are probably related to the reactive oxygen species (ROS) formation catalyzed by transition metals such as iron and copper, and thus the preservation time of the donor hearts is limited. Metabolic protection of the heart grafts is a permanent objective of numerous experiments. Recently, an iron chelator deferoxamine (DFX) was proposed as antioxidant agent for storage solutions in heart grafts. Oxidative stress is also known to mediate the apoptotic cell death in different tissues during ischemia-reperfusion. METHODS: The aim of this study was to evaluate a possible role of DFX in prevention of apoptosis using in vitro model of isolated working rat heart and cold cardioplegia. Two groups of rats were evaluated: (a) group 'DFX 50 &mgr;M' (n=8) and (b) group 'controls' (n=8). Isolated rat hearts were perfused by Krebs-Henseleit buffer (KHB) for 30 min, arrested by cardioplegic solution and stored for 4 h in B21 solution at 4 degrees C. Then, the hearts were reperfused by KHB for 45 min. DFX was added to the cardioplegic and storage solutions and in KHB in reperfusion. Basic functional parameters were evaluated: coronary, aortic, cardiac outputs and heart rate. At the end of reperfusion period a tissue samples were taken from left ventricle and in situ detection of apoptotic cells was performed using an ApopTag kit. RESULTS: DFX significantly reduced the occurrence of apoptotic cells in myocardium (*P<0.05). Hearts treated by 50 &mgr;M of DFX showed also a better recovery of the cardiac output (***P<0.001). The presence of DFX in KHB, cardioplegic and storage solution reduced also the incidence of postischemic arrhythmias and fibrillation's but without statistical significance. CONCLUSIONS: Our results give evidence of the protective potential of DFX during cold ischemia and reperfusion, presumably due to its antioxidant properties. The significant decrease of apoptosis in hearts treated by DFX could be considered as an existence of close link between oxidative stress and apoptotic death promotion in ischemia-reperfusion injury.

We previously reported that therapy of human cervical carcinoma HeLa cells with CP induced segregation of nucleoli and changes of nuclei characteristic of apoptosis. We raised the question of whether p53 can be reactivated by chemotherapy in HeLa cells despite the presence of HPV-encoded E6 activity. Cellular levels of p53 protein increased after CP treatment, reaching a maximum after 6 hr. p53 protein accumulated preferentially in the nucleoli, with a peak after 15 hr. CP-induced nucleolar targeting of p53 appears to be selective because p73, another member of the p53 gene family, accumulated primarily in nuclei in response to CP. Monitoring of the intranuclear distribution of Hdm-2, a negative regulator of p53, revealed this protein in the nucleoli of untreated controls translocated into chromatin during CP therapy. Interestingly, p14(ARF) showed an inverse intranuclear redistribution. Proteasome inhibitors were not able to mimic the effect of CP on p53 levels. Since the reduced stability of wild-type p53 protein in HeLa cells is a consequence of its enhanced ubiquitination by virally encoded E6 protein, resulting in its accelerated degradation, we checked the cellular level of E6 during CP therapy. Six hours after application of CP, E6 protein expression was markedly reduced. This coincided with the increase of cellular p53 and preceded the nucleolar accumulation of p53 protein, indicating that repression of virally coded E6 protein by CP contributes to the restoration of p53 expression., (Copyright 2002 Wiley-Liss, Inc.)

Activation of the p53 tumour suppressor protein by distinct forms of stress leads to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. The cyclin-dependent kinase inhibitor roscovitine has been shown to induce nuclear accumulation of wild-type p53 in human untransformed and tumour-derived cells. We analyzed the response of different human tumour cell lines to roscovitine treatment with respect to their p53 status. Striking induction of wild-type p53 protein and dramatic enhancement of p53-dependent transcription, coinciding with p21WAF1 induction, was observed in wildtype, but not mutant, p53-bearing tumour cells after treatment with roscovitine. The transcriptional activity of p53 was substantially higher in roscovitine-treated cells than in cells irradiated with ultraviolet C or ionizing radiation, even though all these agents induced a similar amount of p53 accumulation. These results highlight the therapeutic potential of roscovitine as an anticancer drug, especially in tumours retaining a functional wild-type p53 pathway.

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.