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Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling cascade occurs in a variety of human malignancies, where it sustains tumor cell proliferation and survival. Pharmacologic blockade of this pathway exerts antineoplastic activity by triggering apoptosis and/or cell-cycle arrest. Pituitary adenomas show activation of the PI3K/AKT/mTOR pathway, but only a fraction of them respond in vitro to the antiproliferative action of rapamycin and RAD001 (mTOR inhibitors), possibly because of the described negative feedback loop on AKT which reactivates the signaling cascade. Rats affected by the multiple endocrine neoplasia-like syndrome (MENX) develop pituitary adenomas showing increased activated AKT. In this study, we comparatively investigated the antitumor potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235 and the single mTOR inhibitor RAD001 on rat pituitary adenoma cells in primary culture. NVP-BEZ235 inhibits the PI3K pathway both upstream and downstream of AKT, thereby preventing the negative feedback loop. NVP-BEZ235 was more effective than RAD001 in reducing cell viability of pituitary adenomas. Consistently, NVP-BEZ235 treatment decreased Akt and S6 phosphorylation and triggered apoptosis. Because MENX is caused by a germline loss-of-function mutation in the cell-cycle inhibitor p27Kip1, we investigated the relationship between this defect and response to NVP-BEZ235 treatment. The levels of p27Kip1 positively correlate with the response to NVP-BEZ235 treatment. Combined treatment with NVP-BEZ235 and the proteasome inhibitor bortezomib, which increases p27Kip1 amount, shows synergistic antiproliferative effects on pituitary adenoma cells. Our data suggest that NVP-BEZ235 may represent an effective therapeutic modality for pituitary adenomas and that p27Kip1 levels represent a potential predictor of response to dual PI3K/mTOR inhibition. Mol Cancer Ther; 10(8); 1450–9. ©2011 AACR.

PURPOSE: The long-term effect of low and moderate doses of ionizing radiation on the lens is still a matter of debate and needs to be evaluated in more detail. MATERIAL AND METHODS: We conducted a detailed histological analysis of eyes from B6C3F1 mice cohorts after acute gamma irradiation (60Co source; 0.063 Gy/min) at young adult age of 10 weeks with doses of 0.063, 0.125 and 0.5 Gy. Sham irradiated (0 Gy) mice were used as controls. To test for genetic susceptibility heterozygous Ercc2 mutant mice were used and compared to wild type mice of the same strain background. Mice of both sexes were included in all cohorts. Eyes were collected 4 hours, 12, 18 and 24 months after irradiation. For a better understanding of the underlying mechanisms, metabolomics analyses were performed in lenses and plasma samples of the same mouse cohorts at 4 and 12 hours as well as 12, 18 and 24 months after irradiation. For this purpose, a targeted analysis was chosen. RESULTS: This analysis revealed histological changes particularly in the posterior part of the lens that rarely can be observed by using Scheimpflug imaging, as we reported previously. We detected a significant increase of posterior subcapsular cataracts 18 and 24 months after irradiation with 0.5 Gy (odds ratio 9.3; 95%-confidence interval 2.1 - 41.3) independent of sex and genotype. Doses below 0.5 Gy (i.e. 0.063 and 0.125 Gy) did not significantly increase the frequency of posterior subcapsular cataracts at any time point. In lenses, we observed a clear effect of sex and aging but not of irradiation or genotype. While metabolomics analyses of plasma from the same mice showed only a sex effect. CONCLUSIONS: This paper demonstrates a significant radiation-induced increase in the incidence of posterior subcapsular cataracts, which could not be identified using Scheimpflug imaging as the only diagnostic tool.

To identify the mutation causing an autosomal dominant congenital nuclear cataract in a south Indian family by whole exome sequencing and to characterize further phenotypically the same in a zebra ...

βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens before it was detected in various brain regions of the mouse, including the hippocampus and the cerebral cortex. Mutations in the mouse Crybb2 gene lead to alterations of sensorimotor gating measured as prepulse inhibition (PPI) and reduced hippocampal size, combined with an altered number of parvalbumin-positive GABAergic interneurons. Decreased PPI and alterations of parvalbumin-positive interneurons are also endophenotypes that typically occur in schizophrenia. To verify the results found in mice, we genotyped 27 single nucleotide polymorphisms (SNPs) within the CRYBB2 gene and its flanking regions and investigated different schizophrenia typical endophenotypes in a sample of 510 schizophrenia patients and 1322 healthy controls. In the case-control study, no association with schizophrenia was found. However, 3 of the 4 investigated haplotype blocks indicated a decreased CRYBB2 mRNA expression. Two of these blocks were associated with poorer antisaccade task performance and altered working memory-linked functional magnetic resonance imaging signals. For the two haplotypes associated with antisaccade performance, suggestive evidence was found with visual memory and in addition, haplotype block 4 showed a nominally significant association with reduced sensorimotor gating, measured as P50 ratio. These results were not schizophrenia-specific, but could be detected in a combined sample of patients and healthy controls. This is the first study to demonstrate the importance of βB2-crystallin for antisaccade performance and memory function in humans and therefore provides implications for βB2-crystallin function in the human brain.

We have shown previously that a single radiation event (0.063, 0.125 or 0.5 Gy, 0.063 Gy/min) in adult mice (age 10 weeks) can have delayed dose-dependent effects on locomotor behavior 18 months postirradiation. The highest dose (0.5 Gy) reduced, whereas the lowest dose (0.063 Gy) increased locomotor activity at older age independent of sex or genotype. In the current study we investigated whether higher doses administered at a higher dose rate (0.5, 1 or 2 Gy, 0.3 Gy/min) at the same age (10 weeks) cause stronger or earlier effects on a range of behaviors, including locomotion, anxiety, sensorimotor and cognitive behavior. There were clear dose-dependent effects on spontaneous locomotor and exploratory activity, anxiety-related behavior, body weight and affiliative social behavior independent of sex or genotype of wild-type and Ercc2S737P heterozygous mice on a mixed C57BL/6JG and C3HeB/FeJ background. In addition, smaller genotype- and dose-dependent radiation effects on working memory were evident in males, but not in females. The strongest dose-dependent radiation effects were present 4 months postirradiation, but only effects on affiliative social behaviors persisted until 12 months postirradiation. The observed radiation-induced behavioral changes were not related to alterations in the eye lens, as 4 months postirradiation anterior and posterior parts of the lens were still normal. Overall, we did not find any sensitizing effect of the mutation towards radiation effects in vivo.

The posttranscriptional modifications (PTM) of the Histone H3 family play an important role in ocular system differentiation. However, there has been no study on the nature of specific Histone H3 subtype carrying these modifications. Fortuitously, we had previously identified a dominant small-eye mutant Aey69 mouse with a mutation in the H3.2 encoding Hist2h3c1 gene (Vetrivel et al., 2019). In continuation, in the present study, the role of Histone H3.2 with relation to the microphtalmic Aey69 has been elaborated. Foremost, a transgenic mouse line expressing the fusion protein H3.2-GFP was generated using Crispr/Cas9. The approach was intended to confer a unique tag to the Hist2h3c1 gene which is similar in sequence and encoded protein structure to other histones. The GFP tag was then used for ChIP Seq analysis of the genes regulated by H3.2. The approach revealed ocular specific H3.2 targets including Ephrin family genes. Altered enrichment of H3.2 was found in the mutant Aey69 mouse, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered enrichment on Ephrin signaling was further analysed by QPCR and immunohistochemistry. This study identifies Hist2h3c1 encoded H3.2 as an important epigenetic player in ocular development. By binding to specific regions of ocular developmental factors Histone H3.2 facilitates the function of these genes for successful early ocular development.

Mouse mutants are a long-lasting, valuable tool to identify genes underlying eye diseases, because the absence of eyes, very small eyes and severely affected, cataractous eyes are easily to detect without major technical equipment. In mice, actually 145 genes or loci are known for anophthalmia, 269 for microphthalmia, and 180 for cataracts. Approximately, 25% of the loci are not yet characterized; however, some of the ancient lines are extinct and not available for future research. The phenotypes of the mutants represent a continuous spectrum either in anophthalmia and microphthalmia, or in microphthalmia and cataracts. On the other side, mouse models are still missing for some genes, which have been identified in human families to be causative for anophthalmia, microphthalmia, or cataracts. Finally, the mouse offers the possibility to genetically test the roles of modifiers and the role of SNPs; these aspects open new avenues for ophthalmogenetics in the mouse.

Malaria ist eine der haufigsten Infektionskrankheiten weltweit; sie wird durch Plasmodien hervorgerufen, die durch Stiche der Anopheles-Mucken auf Menschen ubertragen werden. Viele Versuche, die Krankheit zu bekampfen, hatten bisher nicht die gewunschten Erfolge.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min-1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken postirradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time postirradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (

To identify the mutation causing an autosomal dominant congenital nuclear cataract in a south Indian family by whole exome sequencing and to characterize further phenotypically the same in a zebra fish model.A six-generation family (DKEC1) with several affected members registered at the Regional Institute of Ophthalmology (RIO), Chennai was documented to have congenital nuclear cataract. Detailed clinical history and blood samples were collected from all available family members. Genomic DNA of the proband was subjected to whole exome sequencing. Sequence variations suggestive of putative mutations were further confirmed by bidirectional sequencing and restriction site analysis. Functional analysis of the mutantA unique variation viz., c.382 G T in the coding region of theThis study identified a novel

Ionizing radiation is widely known to induce various kinds of lens cataracts, of which posterior subcapsular cataracts (PSCs) have the highest prevalence. Despite some studies regarding the epidemiology and biology of radiation-induced PSCs, the mechanism underscoring the formation of this type of lesions and their dose dependency remain uncertain. Within the current study, our team investigated the in vivo characteristics of PSCs in B6C3F1 mice (F1-hybrids of BL6 × C3H) that received 0.5–2 Gy γ-ray irradiation after postnatal day 70. For purposes of assessing lenticular damages, spectral domain optical coherence tomography was utilized, and the visual acuity of the mice was measured to analyze their levels of visual impairment, and histological sections were then prepared in to characterize in vivo phenotypes. Three varying in vivo phenotype anterior and posterior lesions were thus revealed and correlated with the applied doses to understand their marginal influence on the visual acuity of the studied mice. Histological data indicated no significantly increased odds ratios for PSCs below a dose of 1 Gy at the end of the observation time. Furthermore, our team demonstrated that when the frequencies of the posterior and anterior lesions were calculated at early time points, their responses were in accordance with a deterministic model, whereas at later time points, their responses were better described via a stochastic model. The current study will aid in honing the current understanding of radiation-induced cataract formation and contributes greatly to addressing the fundamental questions of lens dose response within the field of radiation biology.

Die wesentlichen Grundzuge der molekularen Genstruktur und -funktion sind an Prokaryoten aufgeklart worden. Neben Genen von Escherichia coli (E. coli) haben hierfur besonders extrachromosomale genetische Elemente (Plasmide) und Bakteriophagen eine wichtige Rolle gespielt. Die Untersuchung der Bakterien- und Phagengene hat nicht nur den Schlussel fur den genetischen Code geliefert, sondern auch grundlegende Einsichten in die Feinstruktur und die Regulation von Genen im Stoff wechsel ergeben. Die Bakterien- und Phagengenetik ist daher eine wichtige Grundlage unseres heutigen Verstandnisses der Molekulargenetik.

Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.

Die Zelle als ein Grundbaustein aller Organismen wurde bereits 1665 durch Robert Hooke (1635–1703) bei seinen Untersuchungen an Pflanzen beschrieben; er fuhrte auch die Bezeichnung cell ein. Diese Beobachtungen waren mithilfe eines einfachen Mikroskops gemacht worden (Abb. 5.1). Obwohl in der Folge Nehemiah Grew (1614–1712) und Antoni van Leeuwenhoek (1632–1723) die mikroskopische Feinstruktur von Tieren und Pflanzen in vielen Details studierten, setzte das mangelhafte Auflosungsvermogen der fruhen Mikroskope solchen Studien enge Grenzen. Erst die Verbesserungen der optischen Qualitat, insbesondere durch die Korrektur spharischer und chromatischer Aberrationen, erlaubten es, Feinheiten im Bau tierischer Gewebe zu erkennen.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2–13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function., International Journal of Molecular Sciences, 21 (24), ISSN:1422-0067

Purpose: The increasing use of low-dose ionizing radiation in medicine requires a systematic study of its long-term effects on the brain, behaviour and its possible association with neurodegenerative disease vulnerability. Therefore, we analysed the long-term effects of a single low-dose irradiation exposure at 10 weeks of age compared to medium and higher doses on locomotor, emotion-related and sensorimotor behaviour in mice as well as on hippocampal glial cell populations. Materials and methods: We determined the influence of radiation dose (0, 0.063, 0.125 or 0.5 Gy), time post-irradiation (4, 12 and 18 months p.i.), sex and genotype (wild type versus mice with Ercc2 DNA repair gene point mutation) on behaviour. Results: The high dose (0.5 Gy) had early-onset adverse effects at 4 months p.i. on sensorimotor recruitment and late-onset negative locomotor effects at 12 and 18 months p.i. Notably, the low dose (0.063 Gy) produced no early effects but subtle late-onset (18 months) protective effects on sensorimotor recruitment and exploratory behaviour. Quantification and morphological characterization of the microglial and the astrocytic cells of the dentate gyrus 24 months p.i. indicated heightened immune activity after high dose irradiation (0.125 and 0.5 Gy) while conversely, low dose (0.063 Gy) induced more neuroprotective features. Conclusion: This is one of the first studies demonstrating such long-term and late-onset effects on brain and behaviour after a single radiation event in adulthood.

Bestimmte erbliche Eigenschaften konnen durch Infektion von Mausen mit abgetoteten Erregern ubertragen werden. Die chemische Analyse der ubertragenen Substanz zeigte, dass es sich um Desoxyribonukleinsaure (DNA) handelt. Der chemische Aufbau der DNA ist sehr einfach. Sie besteht aus einem Ruckgrat aus Zuckermolekulen (Desoxyribose), die durch Phosphodiesterbrucken miteinander verknupft sind. An der Desoxyribose befi nden sich heterozyklische Basen. Insgesamt gibt es in der DNA nur vier verschiedene Basen (Adenin, Thymin, Guanin und Cytosin).

Alle bisher besprochenen Eigenschaften der DNA stehen in Einklang mit den Anforderungen an eine chemische Verbindung, die die erbliche Information eines Organismus beherbergt. Dennoch haben wir eine entscheidende Frage bisher nicht gestellt: Welche molekulare Eigenschaft der DNA befahigt sie, die grose Vielfalt der Erscheinungsformen von Lebewesen in sich zu vergegenwartigen? In der DNA-Struktur gibt es ja praktisch nur eine variable chemische Komponente: die an das gleichformige Zucker-Phosphat-Ruckgrat seitlich angefugte Base. Aber auch die hierbei mogliche Variabilitat erscheint uns zunachst, wie schon Miescher vor mehr als 100 Jahren feststellte, sehr wenig geeignet, die Vielfalt lebender Erscheinungen zu erklaren, da sich im Allgemeinen nur vier verschiedene Basen in der DNA miteinander abwechseln. Immerhin fallt uns die Vorstellung, dass nur vier unterschiedliche Einzelelemente eine sehr grose Menge unterschiedlichster Information verschlusseln konnen, heute viel leichter, da es uns aus der Informatik gelaufig ist, dass schon zwei unterschiedliche Elemente – z. B. »0« und »1« – sehr viel Information aufzunehmen vermogen, wenn sie in geeigneter Form gruppiert werden. Genau das ist durch die organischen Basen in der DNA moglich: Durch die Vielfalt der Moglichkeiten der Basenreihenfolge im DNA-Strang wird die zur Existenz eines Organismus erforderliche Information in der DNA festgelegt.