Flow cytometry and cell sorting

While flow cytometry is a critical single cell analytical technique in biomedical science, the technology of flow cytometry associated cell sorting is equally important. Physical separation of cells analyzed by flow cytometry was recognized as an important goal even in the field’s beginning, and many of the earliest cytometers were also cell sorters. Isolation of cells based on flow cytometric analysis has formed the foundation of immune cell differentiation and development and continues to grow importance as techniques for genomic and proteomic analysis expand. This brief review will describe both the historical development and current state of cell sorting. The multiple mechanisms for cell sorters will be covered, and critical aspects of cell sorting will be discussed. Newer technologies for cell sorting including microfluidic technologies will also be considered

Rigging skeletal perissodactyl and artiodactyl ungulate limbs using analytic inverse kinematic-based solutions for a feature film production environment

The goal of this thesis is to develop and construct a repeatable, scalable, and portable rigging solution for the skeletal limbs of ungulates, maximizing functionality while streamlining intuitive interface controls for a feature film production pipeline. The research presents a methodology for breaking down character reference materials commonly available to feature film productions like artwork, anatomical drawings, photographs, and client provided performance criteria. It then presents a modular methodology and approach for successfully evaluating and applying the character reference to the construction of skeletal limbs using ungulates as the primary example. Each limb is broken down into modules that more easily translate into the digital world. The methodology then further defines how to combine and apply digital rigging tools such as constraints and inverse and forward kinematic techniques in a layered and modular way in order to achieve a robust character rig. The resulting ungulate limb rig provides an efficient, intuitive, and robust solution capable of replicating the given performance criteria as well as an example of a scalable approach applicable to non-ungulates. In application of the repeatable modular approach presented, huge efficiency gains have been realized in feature film production pipelines. Animation studios are under increasing pressure to create larger quantities of work, at higher quality, with shorter timetables, and smaller relative budgets. This methodology successfully meets those criteria

Investigating the Use of Destination Math in an Urban School District

Destination Math was a program utilized by Xcellence ISD. A determination if usage of the software had a significant positive effect on math performance was needed. In this study, the researcher created a student database that included the usage data from the comprehensive mathematics software program, Destination Math, and the math residual value, an added value statistic that was derived from the math scores of the 2006 Texas Assessment of Knowledge and Skills (TAKS) test. An analysis was performed to determine if time spent using the Destination Math software resulted in differences between student usage level groups in regard to the math residual value (MRV). The researcher also looked at the usage levels of teachers and campuses to determine if there were differences in the MRV for different classifications of usage. Certain student classifications were added as independent variables. Since Destination Math was offered in Spanish, it was theorized that the program might be beneficial to students who were designated as Limited English Proficient (LEP). Therefore, this student classification was included as an independent variable. Because research existed that provided evidence that some software contained a gender bias, the student classification of gender was also included as an independent variable. The population for this study included every student in Xcellence ISD in grades 3-11 who took the math portion of the TAKS test during the week of April 18-21, 2006. Altogether, 3177 students were included in the data analysis for this study. This represented 53% of the tested population. Students with no usage data reported by Destination Math were eliminated from the study. While there are a number of cited studies that document score gains with Destination Math, this researcher did not find that residual math scores were significantly different among teacher or campus usage groups. While there was a significant difference between the student usage groups, High users exhibited negative MRVs. The results of this study are consistent with another large quantitative study that involved Destination Math. This researcher feels that there are an ample number of studies that provide evidence that Destination Math can have a positive effect on student math performance. However, the program should not be purchased with the intent to improve significantly the residual math scores

Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity

<p>Abstract</p> <p>Background</p> <p>Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined.</p> <p>Methods</p> <p>We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice.</p> <p>Results</p> <p>Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using <it>ex vivo </it>generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation.</p> <p>Conclusion</p> <p>Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer <it>in vivo </it>that involves inhibition of the mTOR pathway at doses that also suppress host immune responses.</p

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.Peer reviewe

Aquatic community response to volcanic eruptions on the Ecuadorian Andean flank: evidence from the palaeoecological record

Aquatic ecosystems in the tropical Andes are under increasing pressure from human modification of the landscape (deforestation and dams) and climatic change (increase of extreme events and 1.5 °C on average temperatures are projected for AD 2100). However, the resilience of these ecosystems to perturbations is poorly understood. Here we use a multi-proxy palaeoecological approach to assess the response of aquatic ecosystems to a major mechanism for natural disturbance, volcanic ash deposition. Specifically, we present data from two Neotropical lakes located on the eastern Andean flank of Ecuador. Laguna Pindo (1°27.132′S–78°04.847′W) is a tectonically formed closed basin surrounded by a dense mid-elevation forest, whereas Laguna Baños (0°19.328′S–78°09.175′W) is a glacially formed lake with an inflow and outflow in high Andean Páramo grasslands. In each lake we examined the dynamics of chironomids and other aquatic and semi-aquatic organisms to explore the effect of thick (> 5 cm) volcanic deposits on the aquatic communities in these two systems with different catchment features. In both lakes past volcanic ash deposition was evident from four large tephras dated to c.850 cal year BP (Pindo), and 4600, 3600 and 1500 cal year BP (Baños). Examination of the chironomid and aquatic assemblages before and after the ash depositions revealed no shift in composition at Pindo, but a major change at Baños occurred after the last event around 1500 cal year BP. Chironomids at Baños changed from an assemblage dominated by Pseudochironomus and Polypedilum nubifer-type to Cricotopus/Paratrichocladius type-II, and such a dominance lasted for approximately 380 years. We suggest that, despite potential changes in the water chemistry, the major effect on the chironomid community resulted from the thickness of the tephra being deposited, which acted to shallow the water body beyond a depth threshold. Changes in the aquatic flora and fauna at the base of the trophic chain can promote cascade effects that may deteriorate the ecosystem, especially when already influenced by human activities, such as deforestation and dams, which is frequent in the high Andes

Open Science principles for accelerating trait-based science across the Tree of Life.

Synthesizing trait observations and knowledge across the Tree of Life remains a grand challenge for biodiversity science. Species traits are widely used in ecological and evolutionary science, and new data and methods have proliferated rapidly. Yet accessing and integrating disparate data sources remains a considerable challenge, slowing progress toward a global synthesis to integrate trait data across organisms. Trait science needs a vision for achieving global integration across all organisms. Here, we outline how the adoption of key Open Science principles-open data, open source and open methods-is transforming trait science, increasing transparency, democratizing access and accelerating global synthesis. To enhance widespread adoption of these principles, we introduce the Open Traits Network (OTN), a global, decentralized community welcoming all researchers and institutions pursuing the collaborative goal of standardizing and integrating trait data across organisms. We demonstrate how adherence to Open Science principles is key to the OTN community and outline five activities that can accelerate the synthesis of trait data across the Tree of Life, thereby facilitating rapid advances to address scientific inquiries and environmental issues. Lessons learned along the path to a global synthesis of trait data will provide a framework for addressing similarly complex data science and informatics challenges

miR-155 augments CD8(+) T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic gamma(c) cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic gamma c cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.112922Ysciescopu

The Astropy Problem

The Astropy Project (http://astropy.org) is, in its own words, "a community effort to develop a single core package for Astronomy in Python and foster interoperability between Python astronomy packages." For five years this project has been managed, written, and operated as a grassroots, self-organized, almost entirely volunteer effort while the software is used by the majority of the astronomical community. Despite this, the project has always been and remains to this day effectively unfunded. Further, contributors receive little or no formal recognition for creating and supporting what is now critical software. This paper explores the problem in detail, outlines possible solutions to correct this, and presents a few suggestions on how to address the sustainability of general purpose astronomical software