Reagent-driven reconfiguration/optimization of a 16-parameter BD FACSARIA II SORP to allow accurate detection of violet- and UV-excited Sirigen dyes

Background. Modern flow cytometers can detect emission from a variety of commercially available fluorescent reagents. However, accurate detection of novel dyes is often difficult due to the lack of readily available quality assurance tools, for instrument manufacturer QC and optimization protocols often become available secondary to new fluorescent reagents. Aims. For standard QC and instrument calibration of the FACSARIA, BD provides a standard 'CS&T' method that includes three-peak beads and a dedicated software module. While this method allows tracking instrument state over time, it does not accurately access PMT performance on a significant part of the spectrum for violet-or UV-excited dyes. In order to ensure accurate detection of reagents within all 16 channels of the Boston University Flow Core 16-color, 4-laser BD FACSARIA SORP, we created and performed an novel optimization process that allows simultaneous accommodation of as many as nine polymeric Sirigen dyes, including those emitting in both long-wavelength violet-and UV-laser excited channels. Methods and Results. Firstly, the electronic noise of all PMTs was assessed and information was collected on rSD of non-stained cells within 100-800V range. The derived basal PMT values then provided a starting point for voltage optimization of instrument-specific panels. These values differed greatly from CS&T-deduced PMT voltages for abovementioned channels, for some the CS&T calculation of optimal PMT voltage was not possible due to a poor resolution of CS&T peaks at certain wavelengths. Several PMTs were identified with sub-par performance and were consequently replaced. Our testing of multiple commercial compensation beads found that the majority demonstrated prohibitively high backgrounds; the eBioscience UltraComp beads performed best and were therefore our reagent of choice. For instrument performance tracking, we also compared multi-peak beads from several manufacturers and found Spherotech Ultra Rainbow beads to be the sole bead type with satisfactory resolution of all peaks on long-wavelength UV channels. Finally, we developed an ergonomic protocol for facility users that includes an experiment template and electronic tables for data processing. With that protocol, a user can: (1) finely tune PMT voltages to accommodate a specific panel, (2) determine antibody concentrations for compensation control preparations, and (3) associate these optimized settings with multi-peak bead target values. Such preliminary setup allows quick panel-specific instrument calibration for each experimental run. This approach was successfully applied to several 16-color panels used in our Core facility and resulted in vastly improved reproducibility of acquired data over months of use. Conclusions. Synchronizing cutting-edge reagent technologies with existing instrument QC and maintenance methodology requires development of mix-and-match solutions not necessarily provided by the instrument manufacturer. Creating a user-friendly, accurate QC and calibration protocol that accommodates novel reagents allows dramatic expansion of our userbase's experimental capabilities

Візуальні репрезентації трансформації маскулінності (Visual representation of transformation of masculinity)

Актуальність цього дослідження можна аргументувати такими тезами: а) розвиток чоловічих досліджень пов’язаний із становленням культури постмодернізму, в якій трансформація маскулінності розглядається не як соціокультурна девіація, а як один із варіантів “нормального” розвитку соціокультурної сфери; б) все більше з’являється візуальних репрезентацій андрогінних образів, і це пов’язано з тим, що суспільство стало нав’язувати сучасному чоловікові якості, які традиційно вважалися жіночими. (The main ideas of this study are: a) the development of male research associated with the formation of postmodern culture in which the transformation of masculinity is not seen as sociocultural deviation, but as one of the following “normal” development of socio-cultural sphere. b) It appears more visual representations androgyny’s images, and this is due to the fact that society has imposed as modern men, which are traditionally considered feminine

Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy

Adhesion to extracellular matrix (ECM) is crucially important for survival of normal epithelial cells as detachment from ECM triggers specific apoptosis known as anoikis. As tumor cells lose the requirement for anchorage to ECM, they rely on cell-cell adhesion 'multicellular aggregation' for survival. Multicellular aggregation of tumor cells also significantly determines the sensitivity of tumor cells to the cytotoxic effects of chemotherapeutics. In this report, we demonstrate that expression of immunoglobulin containing and proline-rich receptor-1 (IGPR-1) is upregulated in human primary colon cancer. Our study demonstrates that IGPR-1 promotes tumor multicellular aggregation, and interfering with its adhesive function inhibits multicellular aggregation and, increases cell death. IGPR-1 supports colon carcinoma tumor xenograft growth in mouse, and inhibiting its activity by shRNA or blocking antibody inhibits tumor growth. More importantly, IGPR-1 regulates sensitivity of tumor cells to the chemotherapeutic agent, doxorubicin/adriamycin by a mechanism that involves doxorubicin-induced AKT activation and phosphorylation of IGPR-1 at Ser220. Our findings offer novel insight into IGPR-1's role in colorectal tumor growth, tumor chemosensitivity, and as a possible novel anti-cancer target.Grant support from: R01 CA175382/CA/NCI NIH HHS/United States, R21 CA191970/CA/NCI NIH HHS/United States, and R21 CA193958/CA/NCI NIH HHS/United State

Cloud-based highly parallel execution of t-SNE and SPADE with metaclustering for analysis and visualization of large single-cell datasets

The use of machine learning techniques, in particular unsupervised clustering and dimensionality reduction algorithms, is quickly becoming a standard workflow for identifying and visualizing biological populations from within high-dimensional data. These methods allow researchers to approach data analysis without the bias and subjectivity that has traditionally been standard in the field. Algorithms have context-dependent strengths and weaknesses. Across algorithms, an inability to scale computation to large datasets is a common theme. Most algorithms are designed and distributed to run on individual computers where memory and CPU are quickly exhausted by large datasets. Even when high-performance compute resources are available, algorithms often don't scale to large datasets as a fundamental property of their design. If they do, it might result in an untenable increase in runtime or diminished quality of results. t-SNE and SPADE are two well-published algorithms that suffer problems as discussed above after datasets exceed a number of observations on the order of 1 million. This study introduces an alternative approach to the use of SPADE and t- SNE whereby a dataset is divided and distributed across numerous compute nodes in the cloud to process independently in parallel. The results of each computation are then combined in a metaclustering step for final visualization and analysis. The improvement in execution speed as a function of degree of parallelization is established. The method is validated against a non-parallel analysis of the same dataset to establish concordance of identified populations. The workflow is executed on Cytobank for portability to other researchers

Performance and development trends of small businesses in the agro-industrial complex

The article reviews key economic indicators of the development of small businesses in Russian agro-industrial complex (in terms of farm enterprises) over the period of their establishment and development up to the present day. The over-time contribution of farm enterprises to the total output of agricultural produce is shown; their work performance is analyzed. The article provides the performance review as well as the role of small businesses in the agro-industrial complex of the Saratov Oblast. It is demonstrated that the increase of state support for small businesses is required in the modern context. Research results of the performance of farm enterprises in the Saratov Oblast are presented. It is concluded that there is a required farm enlargement in order to increase the competitive ability and performance of small businesses. The article also provides survey results of rural population of the Saratov Oblast in order to determine the concerns of job loss in the mid-term

Variability of Winter Wheat Quality Features in Northern Trans-Urals

The studies were carried out in laboratory conditions on the basis of the Agrotechnological Institute of Northern Trans-Ural State Agricultural University. The studies include 5 varieties of winter wheat grown in 2009-2011 in three agroclimatic zones of Tyumen Region: sub-regional (Nizhnetavdinsky state crop testing site), northern forest steppe (Yalutorovsky state crop testing site), southern forest steppe (Berduzhsky state crop testing site). Bashkirskaya variety – 10 (45 g) demonstrated the highest potential in the formation of a 1000 grain weight. The same variety has the highest range of variability (11.7 g) and the variability coefficient (10.9%) indicates average variability. Other varieties showed minor variability of characteristic (V = 8.7-9.9%). Winter wheat varieties varied in terms of average hardness, at the same time the level of values of all varieties (66-77%) corresponded to the standards of high classes according to GOST. Novosibirskayaaya 32 (61-93%) and Bashkirskaya 10 (60-86%) varieties were the most stable in forming the required value of characteristic. The average gluten content in a grain of winter wheat varieties was corresponded to the standards of the third class according to GOST. Novosibirskaya 32 variety (V = 8.5%) had minor variability of the characteristic, while other varieties demonstrated the average variability. A significant proportion of the effect of the “variety” factor on the variability of such features as a 1000 grain weight (56.5%), grain-unit (50.5%) and hardness (45.1%) was established. The amount of gluten depended more on the growing environment (35.5%) and interaction of factors (39.3%). The quality of gluten was much influenced by the growing environment (52.2%)

Automated analysis of 16-color polychromatic flow cytometry data maps immune cell populations and reveals a distinct inhibitory receptor signature in systemic sclerosis

Background. The phenotypic profiles of both peripheral blood and tissue-resident immune cells have been linked to the health status of individuals with infectious and autoimmune diseases, as well as cancer. In light of the promising clinical trial results of agents that block the Inhibitory Receptor (IR) Programmed Death 1 (PD-1) axis, novel flow cytometric panels that simultaneously measure multiple IRs on several immune cell subsets could provide the distinct IR signatures to target in combinational therapies for many disease states. Also, due to the paucity of human samples, larger (14+ color) ‘1-tube’ panels for immune cell characterization ex vivo are of a high value in translational studies. Development of fluorescent-based panels offer several advantages as compared with analogous mass cytometric methods, including the ability to sort multiple populations of interest from the sample for further study. However, automated platforms of multi-dimensional single cell analysis that allow objective and comprehensive population characterization are severely underutilized on data generated from large polychromatic panels. Methods. A 16-color flow cytometry (FCM) panel was developed and optimized for the simultaneous characterization and purification of multiple human immune cell populations on a 4- laser BD FACSARIA II cell sorter. FCM data of samples obtained from healthy subjects and individuals with systemic sclerosis (SSc) were loaded into Cytobank cloud, then compensated and analyzed with SPADE clustering algorithm. The viSNE algorithm was also employed to compress the data into a 2D map of phenotypic space that was subsequently clustered using SPADE. For comparison, the FCM data were also analyzed manually using FlowJo software. Results. Our novel 16-color panel recognizes CD3, CD4, CD8, CD45RO, CD25, CD127, CD16, CD56, γδTCR, vα24, PD-1, LAG-3, CTLA-4, and TIM-3; it also contains a CD1d-tetramer and a live-dead dye (with CD19 and CD14 included as a combined dump channel). This panel allows combinational IR signatures to be determined from CD4+ T, CD8+ T, Natural Killer (NK), invariant Natural Killer (iNKT), and gamma delta (γδ) immune cell subsets within one sample. We have successfully identified all subsets of interest using automatic SPADE and viSNE algorithms integrated into Cytobank services, and demonstrated a distinctive phenotype of IR distribution on healthy versus systemic sclerosis subject groups. Conclusions. Methods of automatic analysis that were originally developed for processing multi-dimensional mass cytometry can be applied to polychromatic FCM datasets and provide robust results, including subset identification and distinct IR signatures in healthy compared to diseased subject groups

Performance and development trends of small businesses in the agro-industrial complex

The article reviews key economic indicators of the development of small businesses in Russian agro-industrial complex (in terms of farm enterprises) over the period of their establishment and development up to the present day. The over-time contribution of farm enterprises to the total output of agricultural produce is shown; their work performance is analyzed. The article provides the performance review as well as the role of small businesses in the agro-industrial complex of the Saratov Oblast. It is demonstrated that the increase of state support for small businesses is required in the modern context. Research results of the performance of farm enterprises in the Saratov Oblast are presented. It is concluded that there is a required farm enlargement in order to increase the competitive ability and performance of small businesses. The article also provides survey results of rural population of the Saratov Oblast in order to determine the concerns of job loss in the mid-term