Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Distribution of Pb, Sr, and U isotopic signature and multielement composition of sediment in Lake Balaton (Hungary) at a sediment trap deep dredged 40 years ago near Balatongyörök-search for routes of recent pollution.

We studied the Pb, Sr, and U isotopic composition and the concentration of toxic metal elements in sediment core samples collected in Lake Balaton at a sediment trap that was deep dredged in 1979, to analyze their changes in the last 40 years. Pb isotopic composition profiles of cores taken from the sediment trap showed different 206Pb/207Pb ratios ranging from 1.206 ± 0.002 at the bottom of the core (phase 1) compared to 1.185 ± 0.002 at the top of the core (phase 2). Phase 2 is the fraction reflecting isotopic signatures of the latest 40 years. At 80-100-cm depth, a transition zone was observed. Pb concentration together with Zn, Sb, Cu, Cd, and Fe showed elevated, 2-4 times higher values in the top phase of the sediment. The calculated Pb isotopic composition of pollutant Pb fraction was 1.177 ± 0.005 in the case of the 206Pb/207Pb and 2.456 ± 0.004 for 208Pb/207Pb, which shows good agreement with literature data for lead ores in Poland and Germany, but it is distinct from literature data for leaded fuel concerning Middle and Eastern Europe. The marked difference in the Pb signatures of phases enabled the construction of a sediment deposition rate map. U and Mo showed a characteristic concentration peak positioned exactly at the depth of the Pb signature transition. The isotopic signature of U based on 234U and 235U also showed a similar pattern. We suggest that the deposition of U and Mo can be related to cyanobacterial blooms in Lake Balaton in the late 1970s and early1980s.

Mechanisms of Antiviral Immune Evasion of SARS-CoV-2.

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is characterized by a delayed interferon (IFN) response and high levels of proinflammatory cytokine expression. Type I and III IFNs serve as a first line of defense during acute viral infections and are readily antagonized by viruses to establish productive infection. A rapidly growing body of work has interrogated the mechanisms by which SARS-CoV-2 antagonizes both IFN induction and IFN signaling to establish productive infection. Here, we summarize these findings and discuss the molecular interactions that prevent viral RNA recognition, inhibit the induction of IFN gene expression, and block the response to IFN treatment. We also describe the mechanisms by which SARS-CoV-2 viral proteins promote host shutoff. A detailed understanding of the host-pathogen interactions that unbalance the IFN response is critical for the design and deployment of host-targeted therapeutics to manage COVID-19.

Microcystin-LR, a Cyanobacterial Toxin, Induces DNA Strand Breaks Correlated with Changes in Specific Nuclease and Protease Activities in White Mustard (Sinapis alba) Seedlings.

There is increasing evidence for the induction of programmed cell death (PCD) in vascular plants by the cyanobacterial toxin microcystin-LR (MC-LR). Our aim was to detect the occurrence of PCD-related DNA strand breaks and their possible connections to specific nuclease and protease activities. DNA breaks were studied by the deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method in the photoperiodically grown dicot model of white mustard (Sinapis alba). In-gel nuclease and protease activity assays showed changes in the activities of specific isoenzymes during treatments with MC-LR. Strand breaks occurred both in the developing root epidermis and cortex. Several isoenzyme activities were related to these breaks, for example: an increase in the activity of neutral 80-75 kDa, acidic high MW (100-120 kDa) and, most importantly, an increase in the activity of neutral 26-20 kDa nucleases, all of them having single-stranded DNA cleaving (SSP nuclease) activities. Increases in the activities of alkaline proteases in the 61-41 kDa range were also detected and proved to be in relation with MC-LR-induced PCD. This is one of the first pieces of evidence on the correlation of PCD-related DNA strand breaks with specific hydrolase activities in a model dicot treated with a cyanobacterial toxin known to have environmental importance.

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression.

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.

The Wilms' tumour 1 gene as a factor in non-syndromic hypospadias: evidence and controversy.

Hypospadias is one of the most frequent congenital anomalies of the male external genitalia. Its pathogenesis is due to largely unknown or poorly understood genetic factors and is further complicated by environmental-intrauterine-risk factors. One of the genes currently in focus by molecular biologists and clinicians studying syndromic forms of hypospadias is the Wilms' tumour 1 (WT1) gene. There is controversy over whether WT1 defects are also responsible for isolated hypospadias. In this review, we briefly cover the role of WT1 as a transcription factor and discuss proposed pathogenic pathways leading to hypospadias, outlining possible directions for research. We assess available evidence on the gene's mutations and polymorphisms recently suggested in the background of the disease, and examine the putative role of WT1-associated proteins. We also review relevant aspects of genome-wide association studies carried out so far, and raise some points to consider in future efforts.

Is surgical plume developing during routine LEEPs contaminated with high-risk HPV? A pilot series of experiments.

INTRODUCTION: Growing evidence shows a causal role of high-risk humane papillomavirus (HPV) infections in the development of head and neck cancer. A recent case report shows two patients suffering from tonsillar cancer without any risk factors apart from their work as gynecologists doing laser ablations and loop electrosurgical excision procedures (LEEP). The aim of the present investigation is to evaluate whether surgical plume resulting from routine LEEPs of HSIL of the cervix uteri might be contaminated with the DNA of high-risk HPV. MATERIALS AND METHODS: The prospective pilot study is done at the Department of Gynecology and Obstetrics of the University of Lübeck, Germany. The primary outcome was defined as HPV subtype in resected cone and in surgical plume resulting from LEEPs of HSIL of the cervix uteri. Plume resulting from LEEPs was analyzed using a Whatman FTA Elute Indicating Card which was placed in the tube of an exhaust suction device used to remove the resulting aerosols. For detection of HPV and analysis of its subtype, the novel EUROArray HPV test was performed. Resected cones of LEEPs were evaluated separately for HPV subtypes. RESULTS: Four samples of surgical plume resulting from routine LEEPs indicated contamination with high-risk HPV and showed the same HPV subtype as identified in the resected cones. CONCLUSION: Surgical plume resulting from routine LEEPs for HSIL of the cervix uteri has the risk of contamination with high-risk HPV. Further investigations of infectiousness of surgical plume are necessary for evaluation of potential hazards to involved healthcare professionals.

Excisions of severe cervical dysplasia: Are there mandatory diameters of the cone that need to be considered?

OBJECTIVE: To achieve optimal depth for negative margin cones after loop electrosurgical excision procedures (LEEP) for cervical dysplasia. MATERIAL AND METHODS: Retrospective cohort analysis of LEEP cones of 201 patients with cervical dysplasia during a four-year period. Analysed cones were divided into two different groups: cones with negative margins without dysplasia, and cones with margins positive for dysplasia. In order to determine the cut-off value of the depth of the resected cones, receiver operating characteristic (ROC) analysis was performed. RESULTS: Negative margins were found in 71.0% (n=49) of all cones, whereas positive margins were reported in 29.0% (n=20). Negative margin cones were achieved in 100% with a cone depth of ≥20 mm. A resection depth between 10-19.9 mm led to 73.0% negative margin cones. Calculation of cone volume shows for 2.0 cm3, a sensitivity of 79% and a specificity of 64%. Statistical analysis using an ROC model showed p=0.002. CONCLUSION: Forth greatest safety of patients, cone depths from LEEPs for cervical dysplasia should be ≥20 mm to achieve negative margins.

Nucleotide Transition 390C-T in the Wilms' Tumor 1 Gene: A Risk Factor of Hypospadias?

INTRODUCTION: The gene Wilms' tumor 1 (WT1) encodes a unique transcription factor. Its defects are known to cause a wide range of complex genitourinary malformations and may contribute to non-syndromic forms of hypospadias. MATERIALS AND METHODS: We performed WT1 mutation analysis and copy number analysis of WT1-interacting protein in 13 Hungarian patients diagnosed with isolated hypospadias. RESULTS: Sequencing of WT1 revealed a high frequency of heterozygosity for transition 390C-T (5 heterozygotes out of 13 patients, including 2 brothers). WT1-interacting protein had a normal copy number in all patients. CONCLUSION: Nucleotide substitution 390C-T may play a role in the pathogenesis of non-syndromic hypospadias. The genotype-phenotype correlation should be confirmed by a larger-scale analysis.

C-Section Prevalence Among Obese Mothers and Neonatal Hypoglycemia: a Cohort Analysis of the Department of Gynecology and Obstetrics of the University of Lübeck.

INTRODUCTION: Data from the World Health Organization (WHO) demonstrates an increasing prevalence of obesity in Western countries. This study investigates the influence of obesity on the mode of delivery and the occurrence of hypoglycemia in newborns. MATERIALS AND METHODS: A retrospective analysis of all deliveries at the Department of Gynecology and Obstetrics of the University of Lübeck, Germany was conducted over a period of eleven years with the primary outcome as non-elective C-sections and hypoglycemia of newborns from obese mothers. Patients were divided into six subgroups according to WHO weight classifications as follows: control group body mass index (BMI) 18.5 - 24.9 kg/m 2 , n = 7712; general obesity BMI ≥ 25 kg/m 2 , n = 4227; overweight BMI 25 - 29.9 kg/m 2 , n = 2628; obesity I° BMI 30 - 34.9 kg/m 2 , n = 1017; obesity II° BMI 35 - 39.9 kg/m 2 , n = 370; obesity III° BMI ≥ 40 kg/m 2 , n = 212. RESULTS: Analysis of the primary outcome shows an increased incidence of non-elective C-sections with an elevated BMI (general obesity vs. control group: 20.5 vs. 15.9%, p < 0.001; OR 1.3; 95% CI 1.2 - 1.4) and elevated rates of neonatal hypoglycemia in newborns of obese mothers (general obesity vs. control group: 0.6 vs. 0.3%, p < 0.05; OR 1.8; 95% CI 1.0 - 3.0). CONCLUSIONS: Obesity is an essential obstetric risk factor. Obese women face an increased risk of non-elective C-sections, and newborns of obese mothers suffer from elevated rates of hypoglycemia.

Do ART patients face higher C-section rates during their stage of delivery? A German monocenter experience.

PURPOSE: The mode of delivery depends on multiple parameters. After assisted reproductive technology (ART), previous studies have shown elevated C-section rates but few studies differentiated between elective and emergency operations and different protocols of cryopreservation. Because these studies did not use multiparity as exclusion criteria which reduces confounding with previous pregnancies, aim of this study is to compare mode of delivery of different techniques of ART using data of primiparae only [1, 2]. METHODS: Retrospective analysis of patient data treated at the university hospital of Luebeck in a period of 12 years. Patients were divided in different groups according to their way of conception: spontaneous conception and conception after ART. The group of ART was further divided into: (a) a group of fresh transferred embryos (IVF/ICSI), (b) vitrification and (c) slow freezing. Exclusion criteria were defined as: multiparity, delivery <24. + 0 p.m., incomplete data and treatment outside university of Luebeck. Main parameter of this study was mode of delivery which was divided into spontaneous delivery or C-section. C-sections were further differentiated into elective or emergency C-sections. RESULTS: The group of fresh transferred embryos and slow freezing showed higher risks for elective and emergency C-sections (elective C-sections odds ratio 2.0, CI 95% 1.6-2.6, emergency C-sections odds ratio 1.4, CI 95% 1.1-1.9). Moreover, all groups of ART show enhanced risk of significant perinatal bleeding. CONCLUSION: Patients after ART treatment suffer from higher C-section rates during their stage of delivery.

Limbal and Conjunctival Epithelial Cell Cultivation on Contact Lenses-Different Affixing Techniques and the Effect of Feeder Cells.

OBJECTIVES: Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells. METHODS: Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells. RESULTS: Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate. CONCLUSIONS: Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.

Cellular Effects of Cylindrospermopsin (Cyanobacterial Alkaloid Toxin) and its Potential Medical Consequences.

Cylindrospermopsin (CYN) is a tricyclic guanidino alkaloid toxin produced by several cyanobacterial genera. It alters cellular functioning in eukaryotes, including animal and plant organisms. Over the past decades, more and more evidence shows its potential hazardous effects on animal and human health. In this review, we give a critical survey and interpretation of data currently available on its biochemical and consequently, cellular effects. CYN is considered to be a cytotoxin. Several reports suggest that it is a potent inhibitor of eukaryotic protein synthesis, though the exact mechanisms are not completely understood. Here we show that the biochemical changes induced by CYN are complex, possibly involving multiple modes of action. Glutathione metabolism and pyrimidine nucleotide synthesis is affected besides the proposed protein synthesis inhibition. Biochemical alterations lead to the following cellular/subcellular alterations both in animals and plants: (i) changes in cell division rates due to perturbations in chromatin and cytoskeleton; (ii) perturbations of structure and functioning of endomembranes including endoplasmic reticulum; (iii) general metabolic alterations leading to genotoxicity and programmed cell death/apoptosis. The underlying mechanisms and possible health consequences are discussed.

Cylindrospermopsin induces biochemical changes leading to programmed cell death in plants.

In the present study we provide cytological and biochemical evidence that the cyanotoxin cylindrospermopsin (CYN) induces programmed cell death (PCD) symptoms in two model vascular plants: the dicot white mustard (Sinapis alba) and the monocot common reed (Phragmites australis). Cytological data include chromatin fragmentation and the increase of the ratio of TUNEL-positive cells in roots, the latter being detected in both model systems studied. The strongest biochemical evidence is the elevation of the activity of several single-stranded DNA preferring nucleases-among them enzymes active at both acidic and alkaline conditions and are probably directly related to DNA breaks occurring at the initial stages of plant PCD: 80 kDa nucleases and a 26 kDa nuclease, both having dual (single- and double-stranded nucleic acid) specificity. Moreover, the total protease activity and in particular, a 53-56 kDa alkaline protease activity increases. This protease could be inhibited by PMSF, thus regarded as serine protease. Serine proteases are detected in all organs of Brassicaceae (Arabidopsis) having importance in differentiation of specialized plant tissue through PCD, in protein degradation/processing during early germination and defense mechanisms induced by a variety of biotic and abiotic stresses. However, knowledge of the physiological roles of these proteases and nucleases in PCD still needs further research. It is concluded that CYN treatment induces chromatin fragmentation and PCD in plant cells by activating specific nucleases and proteases. CYN is proposed to be a suitable molecule to study the mechanism of plant apoptosis.

Vitrified-warmed embryo transfer is associated with mean higher singleton birth weight compared to fresh embryo transfer.

OBJECTIVE: To test for differences in birth weight between singletons born after IVF with fresh embryo transfer vs. vitrified-warmed 2PN embryo transfer (vitrification protocol). DESIGN: Retrospective analysis of 464 singleton live births after IVF or ICSI during a 12 year period. SETTING: University hospital. INTERVENTIONS: Fresh embryo transfer, vitrified-warmed 2PN embryo transfer (vitrification protocol). MAIN OUTCOME MEASURES: Birth weight standardized as a z-score, adjusting for gestational week at delivery and fetal sex. As a reference, birth weight means from regular deliveries from the same hospital were used. Multivariate regression analysis was used to investigate the relationship between the dependent variable z-score (fetal birth weight) and the independent predictor variables maternal age, weight, height, body mass index, RDS prophylaxis, transfer protocol, number of embryos transferred, indication for IVF treatment and sperm quality. RESULTS: The mean z-score was significantly lower after fresh transfer (-0.11±92) as compared to vitrification transfer (0.72±83) (p<0.001). Multivariate regression analysis indicated that only maternal height and maternal body mass index, but not type of cryopreservation protocol, was a significant predictor of birth weight. CONCLUSIONS: In this analysis focusing on 2PN oocytes, vitrified-warmed embryo transfer is associated with mean higher birth weight compared to fresh embryo transfer. Maternal height and body mass index are significant confounders of fetal birth weight and need to be taken into account when studying birth weight differences between ART protocols.

Microcystin-LR induces mitotic spindle assembly disorders in Vicia faba by protein phosphatase inhibition and not reactive oxygen species induction.

We aimed to reveal the mechanisms of mitotic spindle anomalies induced by microcystin-LR (MCY-LR), a cyanobacterial toxin in Vicia faba, a well-known model in plant cell and molecular biology. MCY-LR inhibits type 1 and 2A phosphoserine/threonine specific protein phosphatases (PP1 and PP2A) and induces reactive oxygen species (ROS) formation. The cytoskeleton is one of the main targets of the cyanotoxin during cytopathogenesis. Histochemical-immunohistochemical and biochemical methods were used. A significant number of MCY-LR induced spindle alterations are described for the first time. Disrupted, multipolar spindles and missing kinetochore fibers were detected both in metaphase and anaphase cells. Additional polar microtubule (MT) bundles, hyperbundling of spindle MTs, monopolar spindles, C-S- shaped, additional and asymmetric spindles were detected in metaphase, while midplane kinetochore fibers were detected in anaphase cells only. Several spindle anomalies induced mitotic disorders, i.e. they occurred concomitantly with altered sister chromatid separation. Alterations were dependent on the MCY-LR dose and exposure time. Under long-term (2 and mainly 6 days') exposure they were detected in the concentration range of 0.1-20µgmL(-1) MCY-LR that inhibited PP1 and PP2A significantly without significant ROS induction. Elevated peroxidase/catalase activities indicated that MCY-LR treated V. faba plants showed efficient defense against oxidative stress. Thus, although the elevation of ROS is known to induce cytoskeletal aberrations in general, this study shows that long-term protein phosphatase inhibition is the primary cause of MCY-LR induced spindle disorders.

Introducing a new scoring system for pap smear in the detection of high-grade squamous intraepithelial lesions in pregnancy (The Luebeck Score).

PURPOSE: To construct a scoring system for pap smears to objectify cytological appraisal and to enhance the accuracy and comparability of pap smear interpretation in pregnancy. MATERIALS AND METHODS: For development of a scoring system for cell appraisal of pap smears the style of the Modified Masood's Scoring Index for appraisal of cells from fine needle aspirations of breast lesions was used. Cohort analysis of n = 54 dysplastic pap smears for polymorphology of cells, anisonucleosis, structure of the nucleus, signs of tissue destruction, nucleus/plasma relation and signs of tumordiathesis. Each criteria was classified into three stages: The first with little evidence for dysplasia (one point), second stage with sporadic evidence (two points) or third stage with frequent evidence (three points). To further evaluate if pregnancy associated cells changes interfere with this scoring system we compared the results of pregnant and non-pregnant women. Histological result was used as an indicator of correctness of the score. RESULTS: Statistical analysis showed a good correlation of the scoring system with histological results. Especially in pregnancy statistical analysis shows promising results (sensitivity 86.67 %, Specificity 100 %, receiver operating characteristic analysis p ≤ 0.05). CONCLUSION: The Luebeck Score seems to be a useful approach for appraisal of pap smears in pregnancy. Further studies containing high numbers of cases are needed for further evaluation of potential benefits of the scoring system compared to conventional evaluation of pap smears.

The Effects of Microcystins (Cyanobacterial Heptapeptides) on the Eukaryotic Cytoskeletal System.

Microcystins (MCYs) are cyanobacterial heptapeptides known for their high toxicity in eukaryotic cells and for their potential human health hazards. They are potent and specific inhibitors of type 1 and 2A, serine-threonine protein phosphatases (PP1 and PP2A) and as such, interfere with key cellular and metabolic events. Moreover, they induce oxidative stress involving reactive oxygen species (ROS) generation. Their cytoskeletal effects involve both mitotic and differentiated eukaryotic cells. The main objective of the present review is to summarize the most important cytoskeletal effects of MCY on human, animal and plant cells known to date and to give an insight into the cellular and molecular background of these alterations. Disruptions of microtubule (MTs), microfilament (MF) and intermediate filament (IF) organization have all been described, having consequences on cell shape, tissue integrity and functionality and mitotic division. Most of these subcellular changes are closely related to PP1 and PP2A inhibition and involve misfunctioning of cytoskeleton associated proteins. However, several cytoskeletal alterations are likely to be related to the induction of oxidative stress. MCY induced changes in MT, MF and IF assembly may have severe human health consequences. The main target of cyanotoxin in human/ animal cells is liver and cytoskeletal disruption alters structure and functioning of hepatocytes. However, many other cell types undergo alterations similar to those observed in hepatocytes. Both PP1/PP2A inhibition and ROS generation are involved and the activation of mitogen activated protein kinases (MAPKs) seems to play a crucial role in the molecular events leading to cytoskeletal disruption.

WT1 Expression in Adult Acute Myeloid Leukemia: Assessing its Presence, Magnitude and Temporal Changes as Prognostic Factors.

Expression of the gene Wilms tumor 1 (WT1) has been suggested as a marker of minimal residual disease in acute myeloid leukemia (AML), but literature data are not without controversy. Our aim was to assess the presence, magnitude and temporal changes of WT1 expression as prognostic factors. 60 AML patients were followed until death or the end of the 6-year observation period. Blood samples were taken at diagnosis, post-induction, during remission and in case of a relapse. Using quantitative real-time PCR, we determined WT1 expression from each sample, normalized it against the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and classified samples as negative, moderately positive or highly positive. We divided the patients into groups based on detected WT1 expression values, illustrated overall and disease-free survival on Kaplan-Meier curves, and compared differences between each group by the logrank test. Disappearance of WT1-positivity during chemotherapy had a favorable effect on survival. Interestingly, no difference was seen between the survivals of WT1-positive subgroups that expressed moderate or high levels of WT1 mRNA. A 1-log decrease in WT1 expression without becoming negative did not affect prognosis, either. Our results suggest that defining a cut-off value for WT1-positivity, rather than just using logarithmic figures of changes in gene expression, might have prognostic use in post-induction AML patients. We encourage further, larger-scale studies.