The recent observation of vector sequences in the semen of men undergoing clinical gene therapy for hemophilia has highlighted the need to evaluate the risk of inadvertent germ-line transduction in a clinically relevant animal model. In the present study, we used three different approaches to investigate whether the germ line is at risk of inadvertent alteration following in utero retroviral gene transfer in the clinically relevant, random-bred sheep model. First, we conducted breeding studies. All organs from the 10 resultant offspring were devoid of proviral DNA, suggesting that the germ line had not been altered. As a second approach, we performed PCR on gradient-enriched, forensically purified sperm cells from in utero-transduced rams. The purified sperm cells from 6 of 19 of these rams were PCR positive for provirus, providing compelling evidence that the germ line had been transduced. As a third approach, we performed immunohistochemistry on sections of the testis from in utero-transduced sheep. Numerous somatic cells and very low levels of germ cells within the male reproductive tissues were transduced. In conclusion, our analysis on over 3 x 10(9) sperm cells suggests that the direct-injection approach employed in these studies may result in the inadvertent transduction of very low numbers of male germ cells.

BackgroundIncreasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic).MethodsWe exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate.ResultsAfter functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 – 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 – 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05).ConclusionsAnti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-756) contains supplementary material, which is available to authorized users.

Surface activities of the first Mars mission crew, as suggested in phase I of the NASA HEDS reference mission, are discussed in this paper. The HEDS reference mission calls for a two phased approach. In phase I, humans supported by robotic systems will explore the Martian surface, collect and analyze geologic, geophysical, and meteorological data, search for potential permanent base sites, and conduct technology verification experiments. In phase II, a Mars base site will be selected, and the building of a permanent human base will be initiated. In this report two complementary architectures are portrayed. First, a permanent base for 3-6 people consisting of an ISRU unit, two nuclear power systems, a green house, and inflatable habitats and laboratories, built inside adobe structures. Second, a reusable, and resupplyable methane propelled very long range type traverse vehicle capable of collecting and analyzing data, and repairing and deploying scientific payloads during its planned 150 days 4800 km traverse. The very long range traverse vehicle will carry smaller rovers, crawlers, blimps, and an air drill capable of quickly reaching depths beyond 100m. The report presents a global vision of human activities on the surface of Mars at a programmatic level. It consists of several vignettes called "concept architectures" We speculate that these activities will facilitate a phase I Mars exploration architecture.

Purpose: Cancer stem cells (CSCs) are a putative source of resistance and relapse following conventional anti-proliferative therapies. We hypothesized that anti-proliferative, but not anti-angiogenic, tyrosine kinase inhibitors would enrich for CSCs in models of soft tissue sarcoma. Experimental Design: We exposed multiple human sarcoma cell lines to sorafenib and pazopanib and assessed cell viability and expression of CSC markers. We exposed mice harboring lung/liver Ewing's sarcoma metastases to daily sorafenib and evaluated survival and CSC expression. We exposed dissociated primary sarcoma tumors to overnight sorafenib and pazopanib. We used tissue microarray (TMA) to evaluate the frequency and intensity of CSC markers in clinical sarcoma samples after neoadjuvant therapy with sorafenib. Results: Viability following sorafenib exposure in vitro ranged from 1 - 20% with a corresponding 2 - 8.2 fold increase in ALDHbright cells from baseline (P&lt;0.05), while negligible effects from pazopanib were observed. In vivo, sorafenib treatment improved median survival by 10 days (P&lt;0.05), but increased the population of ALDHbright cells 2.5 - 2.8 fold (P&lt;0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib with a corresponding increase in ALDHbright cells (P&lt;0.05). Again, negligible effects from pazopanib were observed. TMA analysis of clinical sarcoma specimens demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen following neoadjuvant therapy including sorafenib (P&lt;0.05). Conclusions: Sorafenib, but not pazopanib, exerts a direct anti-proliferative effect on sarcoma cells with a corresponding enrichment in sarcoma CSCs. Sustained anti-sarcoma therapeutic effects may require targeting of the CSC population in combination with cytotoxic treatments. Note: This abstract was not presented at the meeting. Citation Format: Robert J. Canter, Erik Ames, Stephanie Mac, Steve K. Grossenbacher, Mingyi Chen, Joe Tellez, Arta M. Monjazeb, William J. Murphy. Tyrosine kinase inhibition with sorafenib but not pazopanib enriches for sarcoma cancer stem cells in diverse models of soft tissue sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1929. doi:10.1158/1538-7445.AM2014-1929

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