Demand-Driven Multi-Target Sample Preparation on Resource-Constrained Digital Microfluidic Biochips

Microfluidic lab-on-chips offer promising technology for the automation of various biochemical laboratory protocols on a minuscule chip. Sample preparation (SP) is an essential part of any biochemical experiments, which aims to produce dilution of a sample or a mixture of multiple reagents in a certain ratio. One major objective in this area is to prepare dilutions of a given fluid with different concentration factors, each with certain volume, which is referred to as the demand-driven multiple-target (DDMT) generation problem. SP with microfluidic biochips requires proper sequencing of mix-split steps on fluid volumes and needs storage units to save intermediate fluids while producing the desired target ratio. The performance of SP depends on the underlying mixing algorithm and the availability of on-chip storage, and the latter is often limited by the constraints imposed during physical design. Since DDMT involves several target ratios, solving it under storage constraints becomes even harder. Furthermore, reduction of mix-split steps is desirable from the viewpoint of accuracy of SP, as every such step is a potential source of volumetric split error. In this article, we propose a storage-aware DDMT algorithm that reduces the number of mix-split operations on a digital microfluidic lab-on-chip. We also present the layout of the biochip with -storage cells and their allocation technique for . Simulation results reveal the superiority of the proposed method compared to the state-of-the-art multi-target SP algorithms.

Learning Processes in Hierarchical Pairs Regulate Entire Gene Expression in Cells

Expression of numerous genes is precisely controlled in a cell in various contexts. While genetic and epigenetic mechanisms contribute to this regulation, how each mechanism cooperates to ensure the proper expression patterns of whole gene remains unclear. Here, I theoretically show that the repetition of simple biological processes makes appropriate whole-gene expression only if the appropriateness of current pattern is roughly detectable. A learning pair model is developed, in which two factors autonomously approach the target ratio by repeating two stochastic processes; competitive amplification with a small addition term and decay depending on the difference between the current and target ratios. Furthermore, thousands of factors are self-regulated in a hierarchical-pair architecture, in which the activation degrees competitively amplify, while transducing the activation signal, and decay at four different probabilities. Changes in whole-gene expression during human early embryogenesis and hematopoiesis are reproduced in simulation using this epigenetic learning process in a single genetically-determined hierarchical-pair architecture of gene regulatory cascades. On the background of this learning process, I propose the law of biological inertia which means that a living cell basically maintains the expression pattern while renewing the contents.

Senolytics in a Model of Alzheimer's Disease

The therapeutic effects of senescent cell killing with senolytics in neurodegeneration mouse models poise this strategy as an intervention candidate for Alzheimer’s Disease (AD). However, it is unclear whether senolytic therapies for AD are translatable to human cells. To determine whether senolytics could be a viable therapeutic for AD, we have treated long-term mixed human neuron/astrocyte primary cultures with amyloid beta oligomers (ABO), which we have shown to induce a phenotype consistent with senescence in neurons. Fifteen days after ABO treatment, we administered Navitoclax (Nav) and the natural killer cell-line NK92, which are known to selectively kill senescent cells in the periphery. Following treatment, we assessed senescence markers in our cultures as well as senescent cell killing selectivity through cleaved Caspase 3 quantification. Our preliminary data show that Nav (8, 4, and 0.5uM) kills both control and ABO treated cells. NK92 cells (10 to 1 effector to target ratio) also kill some control cells, suggesting there is not a clear cut mechanism by which NK92 cells can distinguish senescent from non-senescent neurons or astrocytes. Although analysis of selective killing is ongoing, off-target killing indicates that we need more refined senolytic strategies to implement their safe human use.

CAR-NK Cells Targeting Sars-Cov-2 Glycosites As COVID-19 Treatment

Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6; Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6; Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3; Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3; Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK; 65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05; 78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03; n=6; Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. Figure 1 Figure 1. Disclosures Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding; BMS: Research Funding; Kiadis Pharma: Research Funding.

Genetic Analysis of Gorlin Syndrome Using a Multiplex PCR Gene Panel, and Its Potential Clinical Utility for Liquid Biopsy

Background: Gorlin syndrome is an autosomal dominant, rare disease caused by mutations in PTCH1, PTCH2, and SUFU with various symptoms in multiple organs making early diagnosis challenging. In this study, we generated a Gorlin syndrome gene panel that could help to overcome the difficulties in diagnosing Gorlin syndrome using a single test. Results: This gene panel is time- and cost-efficient and highly reliable with a high-quality score of 30, on-target ratio, and coverage depth, and could detect more mutations than whole-exome sequencing of the same patient. Although the current in silico prediction tools have a limited genetic database of gene mutations in rare hereditary diseases, five prediction tools were used to identify pathological mutations. Pathogenic gene mutations were detected not only in PTCH1 but also in PTCH2 in five out of 12 patients with Gorlin syndrome diagnosed based on clinical symptoms. Conclusions: Using this gene panel, we showed the same gene mutation in the patients and their asymptomatic relatives; hence, it has enabled a highly reliable genetic diagnosis of Gorlin syndrome at a low cost requiring only blood sample.

Evaluating a Novel P300-Based Real-Time Image Ranking BCI

Brain–computer interfaces (BCIs) establish communication between a human brain and a computer or external devices by translating the electroencephalography (EEG) signal into computer commands. After stimulating a sensory organ, a positive deflection of the EEG signal between 250 and 700 ms can be measured. This signal component of the event-related potential (ERP) is called “P300.” Numerous studies have provided evidence that the P300 amplitude and latency are linked to sensory perception, engagement, and cognition. Combining the advances in technology, classification methods, and signal processing, we developed a novel image ranking system called the Unicorn Blondy Check. In this study, the application was tested on 21 subjects using three different visual oddball paradigms. Two consisted of female faces and gray-scale images, while the third test paradigm consisted of familiar and unfamiliar faces. The images were displayed for a duration of 150 ms in a randomized order. The system was trained using 50 trials and tested with 30 trials. The EEG data were acquired using the Unicorn Hybrid Black eight-channel BCI system. These synchronized recordings were analyzed, and the achieved classification accuracies were calculated. The EEG signal was averaged over all participants and for every paradigm separately. Analysis of the EEG data revealed a significant shift in the P300 latency dependent on the paradigm and decreased amplitude for a lower target to non-target ratio. The image ranking application achieved a mean accuracy of 100 and 95.5% for ranking female faces above gray-scale images with ratios of 1:11 and 5:11, respectively. In the case of four familiar faces to 24 unfamiliar faces, 86.4% was reached. The obtained results illustrate this novel system’s functionality due to accuracies above chance levels for all subjects.

Extending a Euclidean Model of Ratio and Proportion

This paper is written for mathematics educators and researchers engaged at the elementary and middle school levels and interested in exploring ideas and representations for introducing students to ratio and proportion and for making a smooth transition from multiplication and division by whole numbers to their counterparts with fractions. Book V of Euclid’s Elements offers a scenario for deciding whether two ratios of magnitudes, embodied as a pair of line segments, are equal based on whether the ratios of magnitudes, when multiplied by the same whole numbers, n and m, each yield common products. This test of proportion can be performed using an educational software application where students are presented with a target ratio of commensurable magnitudes, A:B, and challenged to produce a selected ratio, C:D, that behaves like the target ratio under the critical conditions. The selected ratio is automatically constructed such that C:D = m:n, on the basis of a lattice point (n, m) chosen by the student. By adding partitive and Euclidean division to Euclid’s model, five new scenarios with similar goals are proposed. Representations in the Euclidean plane, on a number line, and in the Cartesian plane provide feedback that students may use to help identify a ratio of whole numbers corresponding to the targe ratio of magnitudes. The representations serve to highlight fractions as members of equivalence classes. The model remains to be investigated with teachers and students.

Optimization Transmission Efficiency with Driver Intention for Automotive Continuously Variable Transmission Under Slip Mode

This study proposes and experimentally validates an optimal integrated system to control the automotive continuously variable transmission (CVT) to achieve its expected transmission efficiency range. The control system framework consists of top and bottom layers. In the top layer, a driving intention recognition system is designed on the basis of fuzzy control strategy to determine the relationship between the driver intention and CVT target ratio at the corresponding time. In the bottom layer, a new slip state dynamic equation is obtained considering slip characteristics and its related constraints, and a clamping force bench is established. Innovatively, a joint controller based on model predictive control (MPC) is designed taking internal combustion engine torque and slip between the metal belt and pulley as optimization dual targets . A cycle is attained by solving the optimization target to achieve optimum engine torque and the input slip in real-time . Moreover, the new controller provides good robustness. Finally, performance is tested by actual CVT vehicles. Results show that compared with traditional control, the proposed control improves vehicle transmission efficiency by approximately 9.12%–9.35% with high accuracy.

Perspective of Local Government on the Performance Assessment of District Sports and Leisure Centers

This paper addresses the problem of performance management of operate-transfer (OT) project finance for public sports and leisure centers (SLC) from the perspective of local government. This study contributes to use an evolutionary theory of competitive advantage and mixed-methods, including a modified Delphi method to develop the efficiency-based performance model (EPM) under OT project finance for the public SLCs. The total-factor framework disaggregating the efficiency into an innovative output surplus target ratio (OSTR) provides local governments with a contracted period to manage the SLCs through further specific improvement advice. This study further proposed the four-quadrant matrix formulated by long-term efficiency and short-term profitability to identify the benchmark and improvement directions. The empirical results indicated that there are fifteen SLCs located in the benchmark quadrant. This study provides policy makers in the local governments with a scientific reference to keep or drop the current operating private enterprise in the next concession period. The most underperforming SLCs could follow this proposed quadrant analysis and OSTR index, utilizing their internal resources to develop more attractive and reasonable-price exercise courses for participant growth.

Targeting 5α-reductase with 99mTc labeled dutasteride derivatives for prostate imaging

To assess the suitability of 99mTc labeled 5α-reductase (5α-Rds) inhibitors for non-invasive targeting of prostate cancer (PCa) using Male Sprague Dawely Rat (MSDR) model. In this work, dutasteride (Cpd-1) a 5α-Rds inhibitor was derivatized to its dithiocarbamate analogue (Cpd-2) for subsequent synthesis of 99mTc(CO)3-dutasteride dithiocarbamate (Cpd-3) using tricarbonyl technique. To determine the structure of Cpd-3, for the first time a reference Re(CO)3-dutasteride dithiocarbamate (Cpd-4) was synthesized and characterized with NMR, ESIMS, HPLC and elemental analysis. HPLC was used to establish the identity of Cpd-3 using Cpd-4 as a reference standard. The suitability of Cpd-3 as a new 5α-Rds targeting agent was investigated, both in vitro and vivo. The Cpd-3 has shown ≥98 % in vitro stability at room temperature and was remained ≥90 % stable up to 6 h. In serum Cpd-3 has revealed an analogous behavior with a small decrease in stability after 16 h. High uptake (26.25 ± 1.10 %, after 4 h of i.v.) of Cpd-3 was observed in the prostate (target tissue) of MSDR model with reasonably good target to non-target ratio. Blocking the target site with excess Cpd-2 considerably decreased the uptake of Cpd-3 to 4.10 ± 0.75 % in PCa. High in vitro stability in saline and serum and in vivo uptake in prostate of MSDR model plausibly attracts the opportunity of using Cpd-3 as a novel radiopharmaceutical for non invasive targeting of prostate.