Centrosome misorientation reduces stem cell division during ageing

Asymmetric division of adult stem cells generates one self- renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. Here we show that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells ( GSCs) are oriented within their niche and this ensures asymmetric division. We found that GSCs containing misoriented centrosomes accumulate with age and that these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re- enter the cell cycle on correction of centrosome orientation. On the basis of these findings, we propose that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. We also show that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia.University of Michigan ; March of Dimes Basil O'Conner Starter Scholar Research Award ; Searle Scholar Program ; NIH [P01 DK53074, R01GM072006]We thank C. Gonzalez, D. McKearin, N. Rusan, M. Peifer and the Bloomington Stock Center for fly stocks; R. Lehmann, C. Field and the Developmental Studies Hybridoma Bank for antibodies; M. Kiel and D. Nakada for help with X-ray irradiation; and S. Morrison and T. Mahowald for comments on the manuscript. This research was supported by a University of Michigan start-up fund, March of Dimes Basil O'Conner Starter Scholar Research Award and the Searle Scholar Program (to Y.M.Y.), and NIH grants P01 DK53074 (to M.T.F.) and R01GM072006 (to A.J.H.).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62879/1/nature07386.pd

A short update on the structure of drug binding sites on neurotransmitter transporters

<p>Abstract</p> <p>Background</p> <p>The dopamine (DAT), noradrenalin (NET) and serotonin (SERT) transporters are molecular targets for different classes of psychotropic drugs. Cocaine and the SSRI (<it>S</it>)-citalopram block neurotransmitter reuptake competitively, but while cocaine is a non-selective reuptake inhibitor, (<it>S</it>)-citalopram is a selective SERT inhibitor.</p> <p>Findings</p> <p>Here we present comparisons of the binding sites and the electrostatic potential surfaces (EPS) of DAT, NET and SERT homology models based on two different LeuT_Aatemplates; with a substrate (leucine) in an occluded conformation (PDB id <ext-link ext-link-id="2a65" ext-link-type="pdb">2a65</ext-link>), and with an inhibitor (tryptophan) in an open-to-out conformation (PDB id <ext-link ext-link-id="3f3a" ext-link-type="pdb">3f3a</ext-link>). In the occluded homology models, two conserved aromatic amino acids (tyrosine and phenylalanine) formed a gate between the putative binding pockets, and this contact was interrupted in the open to out conformation. The EPS of DAT and NET were generally negative in the vestibular area, whereas the EPS of the vestibular area of SERT was more neutral.</p> <p>Conclusions</p> <p>The findings presented here contribute as an update on the structure of the binding sites of DAT, NET and SERT. The updated models, which have larger ligand binding site areas than models based on other templates, may serve as improved tools for virtual ligand screening.</p

Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη−/−/POLζ−/− cells from the chicken DT40 cell line. POLζ−/− cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη−/−/POLζ−/− cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ−/− cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells

Non-PEGylated liposomes for convection-enhanced delivery of topotecan and gadodiamide in malignant glioma: initial experience

Convection-enhanced delivery (CED) of highly stable PEGylated liposomes encapsulating chemotherapeutic drugs has previously been effective against malignant glioma xenografts. We have developed a novel, convectable non-PEGylated liposomal formulation that can be used to encapsulate both the topoisomerase I inhibitor topotecan (topoCED™) and paramagnetic gadodiamide (gadoCED™), providing an ideal basis for real-time monitoring of drug distribution. Tissue retention of topoCED following single CED administration was significantly improved relative to free topotecan. At a dose of 10 μg (0.5 mg/ml), topoCED had a half-life in brain of approximately 1 day and increased the area under the concentration–time curve (AUC) by 28-fold over free topotecan (153.8 vs. 5.5 μg day/g). The combination of topoCED and gadoCED was found to co-convect well in both naïve rat brain and malignant glioma xenografts (correlation coefficients 0.97–0.99). In a U87MG cell assay, the 50% inhibitory concentration (IC50) of topoCED was approximately 0.8 μM at 48 and 72 h; its concentration–time curves were similar to free topotecan and unaffected by gadoCED. In a U87MG intracranial rat xenograft model, a two-dose CED regimen of topoCED co-infused with gadoCED greatly increased median overall survival at dose levels of 0.5 mg/ml (29.5 days) and 1.0 mg/ml (33.0 days) vs. control (20.0 days; P < 0.0001 for both comparisons). TopoCED at higher concentrations (1.6 mg/ml) co-infused with gadoCED showed no evidence of histopathological changes attributable to either agent. The positive results of tissue pharmacokinetics, co-convection, cytotoxicity, efficacy, and lack of toxicity of topoCED in a clinically meaningful dose range, combined with an ideal matched-liposome paramagnetic agent, gadoCED, implicates further clinical applications of this therapy in the treatment of malignant glioma

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants

Remission of Behcet's disease with anti-tumor necrosis factor monoclonal antibody therapy: a case report

BACKGROUND: Behcet's disease (BD) is a chronic relapsing multisystem inflammatory disorder with mucocutaneous, ocular, articular, vascular, gastrointestinal and central nervous system manifestations. Tumor necrosis factor (TNF)-alpha is believed to play a pivotal role in BD. Therapeutic blockade of the activity of TNF has been successfully given in a short course of therapy with favorable effects in patients with BD refractory to conventional immunosuppressive drugs. We aimed to find out whether a 12-month treatment with infliximab, a chimeric monoclonal antibody to TNF-alpha, had any beneficial effect in reducing relapses of a patient with long-standing BD refractory to conventional immunosuppressive drugs. CASE PRESENTATION: A 54 year-old-woman with a 35-year history of BD with orogenital ulcerations, arthritis in the right knee and retinal lesions compatible with vasculitis received infliximab, 5 mg/kg by a two-hour intravenous infusion. Symptoms improved within 24 hours and eight days later the genital and oral ulcers healed as well as the arthritis in the right knee subsided. The retinal infiltrates completely resolved within 10 days. The infusions were repeated at weeks 2, 6, 14, 22 and then every 8 weeks. The patient was able to return to her domestic daily life. No exacerbation of the mucocutaneous ocular or arthritic symptoms occurred during the treatment period. CONCLUSIONS: Previous studies have suggested that infliximab given in a short course of treatment is effective in inducing remission of severe mucocutaneous, gastrointestinal and ocular manifestations of BD. Our patient received a 12-month infliximab treatment showing a favorable effect on remission of BD manifestations. The long-term infliximab treatment appears as a new therapeutic option for patients with active BD who failed to respond to conventional immunosuppressive agents

Defining strawberry shape uniformity using 3D imaging and genetic mapping

Strawberry shape uniformity is a complex trait, influenced by multiple genetic and environmental components. To complicate matters further, the phenotypic assessment of strawberry uniformity is confounded by the difficulty of quantifying geometric parameters ‘by eye’ and variation between assessors. An in-depth genetic analysis of strawberry uniformity has not been undertaken to date, due to the lack of accurate and objective data. Nonetheless, uniformity remains one of the most important fruit quality selection criteria for the development of a new variety. In this study, a 3D-imaging approach was developed to characterise berry shape uniformity. We show that circularity of the maximum circumference had the closest predictive relationship with the manual uniformity score. Combining five or six automated metrics provided the best predictive model, indicating that human assessment of uniformity is highly complex. Furthermore, visual assessment of strawberry fruit quality in a multi-parental QTL mapping population has allowed the identification of genetic components controlling uniformity. A “regular shape” QTL was identified and found to be associated with three uniformity metrics. The QTL was present across a wide array of germplasm, indicating a potential candidate for marker-assisted breeding, while the potential to implement genomic selection is explored. A greater understanding of berry uniformity has been achieved through the study of the relative impact of automated metrics on human perceived uniformity. Furthermore, the comprehensive definition of strawberry shape uniformity using 3D imaging tools has allowed precision phenotyping, which has improved the accuracy of trait quantification and unlocked the ability to accurately select for uniform berries

Toxic risk of stereotactic body radiotherapy and concurrent helical tomotherapy followed by erlotinib for non-small-cell lung cancer treatment - case report

<p>Abstract</p> <p>Background</p> <p>Stereotactic body radiation therapy (SBRT) applied by helical tomotherapy (HT) is feasible for lung cancer in clinical. Using SBRT concurrently with erlotinib for non-small cell lung cancer (NSCLC) is not reported previously.</p> <p>Case Presentation</p> <p>A 77-year-old man with stage III NSCLC, received erlotinib 150 mg/day, combined with image-guided SBRT via HT. A total tumor dose of 54 Gy/9 fractions was delivered to the tumor bed. The tumor responded dramatically and the combined regimen was well tolerated. After concurrent erlotinib-SBRT, erlotinib was continued as maintenance therapy. The patient developed dyspnea three months after the combined therapy and radiation pneumonitis with interstitial lung disease was suspected.</p> <p>Conclusions</p> <p>Combination SBRT, HT, and erlotinib therapy provided effective anti-tumor results. Nonetheless, the potential risks of enhanced adverse effects between radiation and erlotinib should be monitored closely, especially when SBRT is part of the regimen.</p