Magnetographic array for the capture and enumeration of single cells and cell pairs

We present a simple microchip device consisting of an overlaid pattern of micromagnets and microwells capable of capturing magnetically labeled cells into well-defined compartments (with accuracies >95%). Its flexible design permits the programmable deposition of single cells for their direct enumeration and pairs of cells for the detailed analysis of cell-cell interactions. This cell arraying device requires no external power and can be operated solely with permanent magnets. Large scale image analysis of cells captured in this array can yield valuable information (e.g., regarding various immune parameters such as the CD4:CD8 ratio) in a miniaturized and portable platform.The emergent need for point-of-care devices has spurred development of simplified platforms to organize cells across well-defined templates.¹ These devices employ passive microwells, immunospecific adhesive islands, and electric, optical, and acoustic traps to manipulate cells.^2–6 In contrast, magnetic templating can control the spatial organization of cells through its ability to readily program ferromagnetic memory states.⁷ While it has been applied to control the deposition of magnetic beads,^8–13 it has not been used to direct the deposition of heterogeneous cell pairs, which may help provide critical insight into the function of single cells.^14,15 As such, we developed a simple magnetographic device capable of arraying single cells and pairs of cells with high fidelity. We show this magnetic templating tool can use immunospecific magnetic labels for both the isolation of cells from blood and their organization into spatially defined wells.We used standard photolithographic techniques to fabricate the microchips (see supplementary material¹⁶). Briefly, an array of 10 × 30 μm cobalt micromagnets were patterned by a photolithographic liftoff process and overlaid with a pattern of dumbbell-shaped microwells formed in SU-8 photoresist (Fig. 1(a)). The micromagnets were designed to produce a predominantly vertical field in the microwells by aligning the ends of the micromagnet at the center of each well of the dumbbell. These features were deposited across an area of ≈400 mm² (>50 000 well pairs per microchip) (Fig. 1(b)). Depending on the programmed magnetization state with respect to the external field, magnetic beads or cells were attracted to one pole and repelled by the other pole of each micromagnet, leading to a biased deposition (Fig. 1(c)).¹²Open in a separate windowFIG. 1.Magnetographic array for single cell analysis. (a) SEM image of the dumbbell-shaped well pairs for capturing magnetically labelled cells. (b) Photograph of the finished device. (c) An array of well pairs displaying a pitch of 60 × 120 μm before (top) and 10 min after the deposition of magnetic beads (bottom).To demonstrate the capability of the array to capture cells into a format amenable for rapid image processing, we organized CD3+ lymphocytes using only hand-held permanent magnets. We isolated CD3+ lymphocytes from blood via positive selection using anti-CD3 magnetic nanoparticles (EasySep™, STEMCELL Technologies) with purities confirmed by flow cytometry (97.8%; see supplementary material¹⁶). We then stained 1 × 10⁶ CD3+ cells with anti-CD8 Alexa-488 and anti-CD4 Alexa-647 (5 μl of each antibody in 100 μl for 20 min; BD Bioscience) to determine the CD4:CD8 ratio, a prognostic ratio for assessing the immune system.^17,18Variably spaced neodymium magnets (0.5 in. × 0.5 in. × 1 in.; K&J Magnetics, Inc.) were fixed on either side of the microchip to generate a tunable magnetic field (0–400 G; Fig. 2(a)). Using this setup, fluorescently labeled cells were deposited, and the populations of CD4+ and CD8+ cells were indiscriminately arrayed, imaged, and enumerated using ImageJ. The resulting CD4:CD8 ratio of 1.84 ± 0.18 (Fig. 2(b)) was confirmed by flow cytometry with a high correlation (5.4% difference; Fig. 2(c)), indicating the magnetographic microarray can pattern cells for the rapid and accurate assessment of critical phenotypical parameters without complex equipment (e.g., function generators or flow cytometers).Open in a separate windowFIG. 2.CD8 analysis of CD3+ lymphocytes. (a) Photograph of the magnetographic device activated by permanent magnets (covered with green tape). The CD4:CD8 ratio determined by the (b) magnetographic microarray and (c) and (d) flow cytometry was 1.84 and 1.74, respectively.More complex operations, such as the programmed deposition of cell pairs, can be achieved by leveraging the switchable, bistable magnetization of the micromagnets for the detailed studies of cell-cell interactions (Figs. 3(a)–3(d)).¹² For these studies, a 200 G horizontal field generated from an electromagnetic coil was used to magnetize the micromagnets.¹⁹ We then captured different concentrations of magnetic beads as surrogates for cells (8.4 μm polystyrene, Spherotech, Inc.) and found that higher bead concentrations did not affect the capture accuracy (>95%; see supplementary material¹⁶).Open in a separate windowFIG. 3.Programmed pairing of magnetic beads and CD3+ lymphocytes. (a) Schematic of the magnetographic cell pair isolations. (b) Polarized micromagnets isolate cells of one type to one side in a vertical magnetic field and then cells of a second type to the other side when the field is reversed. (c) Fluorescent image of magnetically trapped green stained (top) and red stained (bottom) cell pairs. (d) SEM image of magnetically labeled cells in the microwells. (e) Capture accuracy of magnetic bead pairs. (Each color (and shape) represents the field strength of the reversed field.) (f) Change in the capture accuracy (loss) of initially captured beads after reversing the magnetic field. The capture accuracy of (g) magnetically labeled cell pairs and (h) the second magnetically labeled cell (for (e)–(h): n = 5; time starts from the deposition of the second set of cells or beads).The opposite side of each micromagnet was then populated with the second (yellow fluorescent) bead by reversing the direction of the applied magnetic field. We tested several field strengths (i.e., 10, 25, 40, or 55 G) to optimize the conditions for isolating the desired bead in the opposite well without ejecting the first bead. If the field strength was too large, the previously deposited beads could be ejected from their wells due to the repulsive magnetic force overcoming gravity.¹² As shown in Figure 3(e), increasing the field strength from 10 to 25 G significantly increased the capture accuracy at 60 min from the deposition of the second bead (p < 0.01), but increases from 25 to 55 G did not affect the capture accuracy (p > 0.10). As shown in Figure 3(f), higher field strengths (i.e., 40 and 55 G) resulted in lower capture accuracies compared to lower field strengths (i.e., 10 and 25 G) (p < 0.01), which was primarily due to ejection of the initially captured beads when the micromagnets reversed their polarity.We then arranged pairs of membrane dyed (calcein AM, Invitrogen; PKH26, Sigma) magnetically labeled CD3+ lymphocytes. First, red stained cells (150 μl of 2 × 10⁴ cells/ml) were deposited on the microchip in the presence of 250 G vertical magnetic field. After 20 min, the field was reversed (i.e., to 40, 55, and 70 G) and green stained cells (150 μl of 2 × 10⁴ cells/ml) were deposited on the microchip with images taken in 10 min intervals. Fluorescence images were overlaid (Fig. 3(c)) and the capture accuracy of cell pairs was determined (ImageJ).As seen in Figure 3(g), the capture accuracy of pairs of CD3+ lymphocytes was lower than that of magnetic beads (Fig. 3(e)). However, as shown in Figure 3(h), the second set of cells (green fluorescent) exhibited an average capture accuracy of 91.8% ± 1.9%. This indicates that the lower capture accuracy of cell pairs was either due to the ejection of initially captured (red fluorescent) cells or the migration of initially captured cells through the connecting channel, resulting from their relatively high deformability compared to magnetic beads.In summary, we developed a simple device capable of organizing magnetic particles, cells, and pairs of cells into well-defined compartments. A major advantage of this system is the use of specific magnetic labels to both isolate cells and program their deposition. While the design of this device does not enable dynamic control of the spacing between captured cell pairs as does some dielectrophoresis-based devices,²⁰ it can easily capture cells with high fidelity using only permanent magnets and has clinical relevance in the assessment of immune parameters. These demonstrations potentiate a relatively simple and robust device where highly organized spatial arrangement of cells facilitates rapid and accurate analyses towards a functional and low-cost point-of-care device.     

Effects of pre-competitional rapid weight loss on nutrition,vitamin status and oxidative stress in elite boxers

Dietary intake, vitamin status and oxidative stress were evaluated in 17 elite male boxers. Ten of them frequently reduced body weight rapidly before competitions (Weight Loss Group) and 7 did not practice rapid weight loss (Control Group). Food record checklists, blood samples for determination of vitamin status and plasma glutathione levels were obtained during a week of weight maintenance, a pre-competition week and a post-competition week. The average dietary intakes in both groups were 33 ± 8 kcal·kg^?1, 3.7 ± 1.1 g·kg^?1 carbohydrates, 1.5 ± 0.4 g·kg^?1 protein, 1.2 ± 0.4 g·kg^?1 fat and 2.2 ± 1.0 L water per day (excluding pre-competition week in Weight Loss Group). Energy (18 ± 7 kcal·kg^?1), carbohydrate (2.2 ± 0.8 g·kg^?1), protein (0.8 ± 0.4 g·kg^?1), fat (0.6 ± 0.3 g·kg^?1) and water (1.6 ± 0.6 L) consumption (P-values <0.001) and intakes of most vitamins (P-values < 0.05) were significantly reduced during the pre-competition week in the Weight Loss Group. In both groups, the intakes of vitamins A, E and folate were below recommended values throughout the three periods; however, blood vitamin and plasma glutathione levels did not change significantly. Our findings indicate a low-caloric and low-carbohydrate diet in elite boxers, regardless of participating in rapid weight loss or not. Apparently, the pre-competitional malnutitrition in the Weight Loss Group did not induce alterations in the vitamin and glutathione status.     

Innovation and Performance Improvement Integration

G2000's HR Execution Excellence—Retail Attendance System was one of the innovative projects to receive the ISPI Award of Excellence in 2016. It is a continuous improvement project that applies the concept of holistic human performance improvement using an ISPI human performance technology (HPT) model (ISPI, 2012) to streamline the front‐end and back‐end processes of our Retail Attendance System. As a result, it leads us to achieve one of our business goals: employment regulatory compliance. In our case, the project team was tasked with seeking solutions to ensure that the payroll process for retail staff could be performed accurately and in a timely manner. After applying the HPT model to conduct the gap analysis and identify the causes or factors that were limiting our performance, we integrated the concept of human‐centered design approach at the solution‐design phase of the project, to lead us to innovative solutions.     

Official and popular culture in the schooling process

This article analyses the schooling process in rural areas of Galicia (Spain) from the mid-nineteenth to the mid-twentieth century, considering educational initiatives promoted by both the state and the rural population. The former, which were governed by the official school culture, were driven by a moralistic approach; the latter, within the framework of popular school culture, were essentially aimed at literacy. The confrontation that arose between the two initiatives was finally resolved in favour of the official school. Our main goal here is to analyse how popular schools (ferrado schools) were perceived by official culture (school administrators and education experts) as well as by those who were directly involved as protagonists, i.e. the schoolteachers who ran these schools (escolantes) and their pupils.     

Growing through copying: The negative consequences of innovation on franchise network growth

We explore how more exactly copying a blueprint for establishing a franchise network in a new country influences franchising network growth. We test opposing hypotheses using panel data involving the transfer of franchising knowledge to 23 different countries, measuring the degree to which master licensees ‘copy exactly’ knowledge concerning how to grow a network in their country and the effect that their approach has on subsequent network growth. We conclude that a strategy of copying more exactly seems to enhance growth and that the benefits of more exactly replicating knowledge in the 1st year of a local network’s existence persist for several years. Thus, innovation, in this specific context, seems to hinder firm growth.     

From Dakar to Brasilia: monitoring UNESCO’s education goals

Active participation of Brazilian civil society, coupled with the 2007 education development plan launched by the Brazilian government, provides an interesting example of the influences of the Dakar Goals. The two domestic initiatives share the same name, spirit and direction proposed in Dakar 2000. Here we analyze changes in the Brazilian policies and indicators related to the Dakar Education Goals. Since they were promulgated, we note: (1) an increase in enrolment over the relevant period; (2) access to primary education was nearly universal by 2000; (3) numbers of over-aged youth and adult students declined considerably during the period, but access did not expand; (4) illiteracy has been falling at a rate which, if sustained, will enable us to meet the goal; (5) gender discrimination was not apparent in Brazil; (6) most pupil proficiency indicators have deteriorated progressively from what was already a low standard. In summary, quantitative indicators did improve over the period while most qualitative indicators worsened.     

Assimilation attitudes predict lower immigration-related self-efficacy among Israeli immigrant teachers

This study focuses on self-efficacy among teachers working in culturally diverse educational contexts. We put forward the notion of immigration-related self-efficacy and provide initial support for its relationship with the acculturation attitudes held by immigrant teachers. One hundred thirty-three teachers who immigrated to Israel from the Former Soviet Union participated in this study. We found that teachers tend to report high levels of self-efficacy in all the investigated aspects. According to our predictions, immigrant teachers endorsing more assimilative approaches report lower levels of self-efficacy vis-à-vis their immigrant students. Our results can contribute to a critical discussion on the place and roles of immigrant teachers in schools.     

Anthropometrics and electromyography as predictors for maximal voluntary isometric arm strength

BackgroundMuscular strength can be conceptually determined by two components: muscle activation and size. Muscle activation by the central nervous system can be measured by surface electromyography (sEMG). Muscular size reflects the amount of contractile protein within a skeletal muscle and can be estimated by anthropometric measurements. The purpose of this study was to determine the relative contributions of size parameters and muscle activation to the prediction of maximal voluntary isometric elbow flexion strength.MethodsA series of anthropometric measurements were taken from 96 participants. Torque and root-mean-square (RMS) of the sEMG from the biceps brachii were averaged across three maximal voluntary isometric contractions. A multiple linear regression analysis was performed based on a Pearson's correlation matrix.ResultsBody weight (BW) accounted for 39.1% and 27.3% in males and females, respectively, and was the strongest predictor of strength for males. Forearm length (L3) was the strongest predictor of strength in females (partial R² = 0.391). Elbow circumference (ELB) accounted for a significant (p < 0.05) amount of variance in males but not females. The addition of sEMG RMS as a third variable accounted for an average of 10.1% of the variance excluding the equation of BW and L3 in females. The strongest prediction equation included BW, L3, and ELB accounting for 55.6% and 58.5% of the variance in males and females, respectively.ConclusionAnthropometrics provide a strong prediction equation for the estimation of isometric elbow flexion strength. Muscle activation, as measured by sEMG activity, accounted for a significant (p < 0.05) amount of variance in most prediction equations, however, its contribution was comparable to an additional anthropometric variable.