Large-scale mapping of mutations affecting zebrafish development

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease3,4, rapid transformation to acute myeloid leukemia (AML)5, resistance to conventional therapies6–8 and dismal outcomes9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations10. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)11. Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response

Spectroscopic investigations of single Photosystem I complexes from Cyanobacteria

Im Rahmen dieser Dissertation wurden die roten Chlorophyllpools in Photosystem I (PSI) der Cyanobakterien Thermosynechococcus elongatus (TS. elongatus) und Arthrospira platensis (A. platensis) einzelmolekülspektroskopisch mit Hilfe eines konfokalen Laserscanningmikroskops untersucht. Neben der Untersuchung der spektralen Eigenschaften der roten Chlorophylle in PSI, wurde das photophysikalische Verhalten der roten Chlorophylle untersucht und eine mögliche Zuordnung der roten Chlorophyllpools zu den Antennen-Chlorophyllen in PSI aus TS. elongatus durchgeführt. Fluoreszenzemissionsspektren von PSI aus TS. elongatus auf Einzelmolekülebene weisen im Gegensatz zu Bulkspektren, in denen nur eine einzige Fluoreszenzemissionsbande bei 730 nm detektiert wird, drei Fluoreszenzemissionsbanden zwischen 706 nm und 715 nm, 720 nm und 735 nm und zwischen 736 nm und 751 nm auf. Einzelmolekül-Fluoreszenzemissionsspektren zeigen, dass neben dem roten Chlorophyllpool Chl719 auch der rote Chlorophyllpool Chl708 fluoresziert. Die Fluoreszenzbande zwischen 736 nm und 751 nm ist aufgrund der dominanten Bande von Chl719 im Bulkspektrum nicht zu erkennen. PSI aus A. platensis besitzt im Ensemble zwei Fluoreszenzemissionsbanden bei 725 nm und 760 nm, deren Fluoreszenzintensität eine unterschiedliche Abhängigkeit von der Anregungsleistung zeigt. Diese Abhängigkeit kann durch ein einfaches Modell erklärt werden, das auf den Ratengleichungen der beteiligten Chlorophyllpools und ihrer elektronischen Zustände basiert. Photophysikalische Parameter wie die Intersystem Crossing Raten der roten Chlorophyllpools in PSI aus TS. elongatus konnten mittels Fluoreszenzkorrelationsspektroskopie ermittelt werden. Chlorophylle können mit einem Dreiniveauschema beschrieben werden, das aus einem Singulett-Grundzustand, dem ersten angeregten Singulettzustand und dem metastabilen Triplettzustand besteht. Diese Zustände sind über die Übergangsraten ki miteinander verbunden. Bei kontinuierlicher Beleuchtung zeigt die Fluoreszenzemission der einzelnen Moleküle Unterbrechungen im Fluoreszenzsignal, die durch Intersystem Crossing Übergänge hervorgerufen werden. Solche Fluktuationen der Fluoreszenzintensität können mit Hilfe einer Fluoreszenzintensitätskorrelationsfunktion zweiten Grades analysiert und anschließend mit einem monoexponentiellen Zerfall gefittet werden. Die Zerfallsrate wurde für mehrere Anregungsleistungen bestimmt. Aus dieser Abhängigkeit kann die Populationsrate k23 und die Depopulationsrate k31 des Triplettzustands ermittelt werden. Im Vergleich zu Chlorophyll a in Lösung ist die Populationsrate k23 der roten Chlorophyllpools mit ungefähr 5,85 MHz stark verringert, wohingegen die Depopulationsrate k31 stark erhöht ist. Durch den Einbau der Chlorophylle in eine Proteinmatrix wird die Bildung des Triplettzustands der Chlorophylle somit verringert. Die Vermeidung des Triplettzustands ist essentiell für den photosynthetischen Organismus, da eine Bildung von Singulett-Sauerstoff verringert wird, welcher extrem schädlich für organische Moleküle ist. Eine Fluoreszenzpolarisationsanalyse der einzelnen Banden ermöglicht die Zuordnung der roten Chlorophyllpools zu Antennen-Chlorophyllen in PSI aus TS. elongatus, da die Orientierung der roten Chlorophyllpools durch die Lage der Übergangsdipolmomente bestimmt ist. Die Fluoreszenzemission verschiedener Chlorophyllpools in PSI wurde mittels eines Polarisators, der vor dem Spektrometer installiert war, auf lineare Polarisation untersucht. Linear polarisiertes Fluoreszenzlicht kann von einem einzelnen Emitter, von mehreren stark gekoppelten Emittern, die ein neues Quantensystem bilden, oder von mehreren Emittern, die kollinear zueinander ausgerichtet sind, erzeugt werden. Die Fluoreszenzintensitätsmodulation als Folge linearer Polarisation hat eine cos2-Abhängigkeit bei Rotation des Polarisators und kann nach Integration der einzelnen Banden mit einer cos2-Funktion gefittet werden. Aufgrund der Tatsache, dass die PSI-Komplexe isotrop in der Probe vorliegen, liefert eine einzelne Messung der Fluoreszenzmodulation einer Bande keine eindeutige Orientierung. Eine statistische Analyse der relativen Orientierung von zwei Fluoreszenzbanden ist notwendig, um eine eindeutige Orientierung, gegeben durch den Winkel zwischen zwei roten Chlorophyllpools, zu erhalten. Simulationen und eine anschließende Analyse der relativen Orientierungen von zwei Übergangsdipolmomenten von isotrop verteilten PSI-Komplexen zeigen, dass eine Bestimmung der Orientierung in dieser Art und Weise möglich ist. Durch die Berechnung der Phasenverschiebung zweier Fluoreszenzbanden und anschließendem Vergleich dieser Werte mit Literaturdaten, konnten mögliche Kandidaten für die roten Chlorophyllpools Chl708 und Chl719 in der Antenne gefunden werden. Chl708 besteht demnach aus dem Chlorophyll B11 und dem Trimer A2-A3-A4, Chl719 aus den Chlorophyllen A31-A32-B06-B07.In the framework of this thesis, the red chlorophyll pools in Photosystem I (PSI) isolated from the cyanobacteria Thermosynechococcus elongatus (TS. elongatus) and Arthrospira platensis (A. platensis), were investigated by single molecule spectroscopy using a confocal laser scanning microscope. Besides studying the spectral properties of the red chlorophylls in single PSI complexes, the photophysical behaviour of the red chlorophylls were investigated and a possible assignment of the red chlorophyll pools to antenna chlorophylls in PSI of TS. elongatus was performed. Fluorescence emission spectra of PSI from TS. elongatus on single molecule level reveal three fluorescence emission bands between 706 nm and 715 nm, 720 nm and 735 nm and between 736 nm and 751 nm in contrary to bulk spectra, where only one fluorescence emission band at 730 nm is detectable. Single molecule fluorescence spectra showed that in addition to the red chlorophyll pool Chl719, the red chlorophyll pool Chl708 is able to fluoresce. The fluorescence emission band between 736 nm and 751 nm is not visible in a bulk spectrum, because of the dominant band of Chl719. Fluorescence emission spectra of a bulk sample of PSI from A. platensis show two bands at 725 nm and at 760 nm. The fluorescence intensity of these bands shows a different dependency of the excitation intensity. This can be explained by a simple model based on the rate equations of the corresponding molecules and their electronic states. Photophysical parameters like the intersystem crossing rates of the red chlorophyll pools in PSI of TS. elongatus are studied using fluorescence correlation spectroscopy. With this method, it is possible to unravel dynamic molecular events based on statistical analysis of the fluctuations of the fluorescence originated by the pigments. Chlorophylls can be described in terms of a three-level system including ground and excited singlet states and a metastable triplet state, which are connected by the transition rates ki. Under continuous illumination, the fluorescence emission of single molecules shows interruptions in the fluorescence signal related to intersystem crossing events. Such fluctuations of the fluorescence intensity can be analyzed using a second order fluorescence intensity correlation function. The correlation function of a correlated signal can be fitted with a single-exponential decay. The decay rate was determined for several excitation powers. From this dependency the population rate k23 and the depopulation rate k31 of the triplet state can be extracted. In comparison to chlorophyll a in solution, the population rate k23 of the triplet state of the red chlorophyll pools with around 5.85 MHz is highly decreased, whereas the depopulation rate k31 of the triplet state is highly increased. Therefore, by embedding the chlorophylls in a protein matrix, the formation of the triplet state of the chlorophylls is minimized. The prevention of the triplet state is essential for the photosynthetic organism, because it inhibits the production of singlet oxygen, which is extremely destructive to organic molecules. Fluorescence polarization analysis of the different fluorescence bands allows a possible assignment of the red chlorophyll pools to the antenna chlorophylls for PSI from TS. elongatus, based on the known crystal structure. The orientation of the red chlorophylls can be specified by the orientation of the transition dipole moment. The fluorescence light of distinct chlorophyll pools in trimeric and monomeric PSI complexes was tested for linear polarization using a rotating polarizer in front of the spectrometer. Linearly polarized fluorescence light can originate either from a single emitter, or several emitters, which are strongly coupled and thus form a new quantum system, or several emitters, which are orientated collinearly. The fluorescence intensity modulation shows a cos2-dependency on the rotation angle of the polarizer. The modulation was analyzed by integration of the individual bands followed by non-linear curve fitting applying a cos2-function. Due to the fact that the PSI complexes are randomly oriented in the sample, a single measurement of one fluorescing band does not result in a unique orientation which can be identified using structural data. A statistical analysis of the relative orientation of two fluorescing bands is necessary to obtain a unique orientation corresponding to the angle between two chlorophyll pools. Simulations and subsequent analysis of the relative orientation of two transition dipole moments in an isotropic distributed sample showed that a determination of the orientation is possible. By evaluating the phase shifts of two fluorescence bands and comparison with literature data, candidates for the red chlorophyll pools Chl708 and Chl719 were found. Chl708 consists of the chlorophyll B11 and the trimer A2-A3-A4 and Chl719 of the chlorophylls A31-A32-B06-B07

Changes in the expression and subcellular distribution of galectin-3 in clear cell renal cell carcinoma

Abstract Background Clear cell renal cell carcinoma, a solid growing tumor, is the most common tumor in human kidney. Evaluating the usefulness of β-galactoside binding galectin-3 as a diagnostic marker for this type of cancer could open avenues for preventive and therapeutic strategies by employing specific inhibitors of the lectin. To study a putative correlation between the extent of galectin-3 and the development of clear cell renal cell carcinoma, we monitored the quantity and distribution of this lectin in tissue samples from 39 patients. Methods Galectin-3 concentrations in normal, intermediate and tumor tissues were examined by immunofluorescence microscopy and on immunoblots with antibodies directed against galectin-3 and renal control proteins. The cell nuclei were isolated to determine quantities of galectin-3 that were transferred into this compartment in normal or tumor samples. Results Immunofluorescence data revealed a mosaic pattern of galectin-3 expression in collecting ducts and distal tubules of normal kidney. Galectin-3 expression was significantly increased in 79% of tumor samples as compared to normal tissues. Furthermore, we observed an increase in nuclear translocation of the lectin in tumor tissues. Conclusions Our data indicate that changes in the cellular level of galectin-3 correlate with the development of clear cell renal cell carcinoma, which is in line with previously published data on this specific type of tumor. In most of these studies the lectin tends to be highly expressed in tumor tissues. Furthermore, this study suggests that the increase in the proportion of galectin-3 affects the balance from a cytosolic distribution towards translocation into the nucleus.</p

Changes in the expression and subcellular distribution of galectin-3 in clear cell renal cell carcinoma.

International audienceUNLABELLED: ABSTRACT: BACKGROUND: Clear cell renal cell carcinoma, a solid growing tumor, is the most common tumor in human kidney. Evaluating the usefulness of β-galactoside binding galectin-3 as a diagnostic marker for this type of cancer could open avenues for preventive and therapeutic strategies by employing specific inhibitors of the lectin. To study a putative correlation between the extent of galectin-3 and the development of clear cell renal cell carcinoma, we monitored the quantity and distribution of this lectin in tissue samples from 39 patients. METHODS: Galectin-3 concentrations in normal, intermediate and tumor tissues were examined by immunofluorescence microscopy and on immunoblots with antibodies directed against galectin-3 and renal control proteins. The cell nuclei were isolated to determine quantities of galectin-3 that were transferred into this compartment in normal or tumor samples. RESULTS: Immunofluorescence data revealed a mosaic pattern of galectin-3 expression in collecting ducts and distal tubules of normal kidney. Galectin-3 expression was significantly increased in 79% of tumor samples as compared to normal tissues. Furthermore, we observed an increase in nuclear translocation of the lectin in tumor tissues. CONCLUSIONS: Our data indicate that changes in the cellular level of galectin-3 correlate with the development of clear cell renal cell carcinoma, which is in line with previously published data on this specific type of tumor. In most of these studies the lectin tends to be highly expressed in tumor tissues. Furthermore, this study suggests that the increase in the proportion of galectin-3 affects the balance from a cytosolic distribution towards translocation into the nucleus

Author Correction: Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes (Nature Medicine, (2020), 26, 10, (1549-1556), 10.1038/s41591-020-1008-z)

In the version of this article initially published, one name (Harold K. Elias) was omitted from the author list. The correct section of the author list is “...Matteo Giovanni Della Porta37,38, Harold K. Elias31, Alexandra G. Smith39...”. The error has been corrected in the HTML and PDF versions of the article