User-Loaded SlipChip for Equipment-Free Multiplexed Nanoliter-Scale Experiments

This paper describes a microfluidic approach to perform multiplexed nanoliter-scale experiments by combining a sample with multiple different reagents, each at multiple mixing ratios. This approach employs a user-loaded, equipment-free SlipChip. The mixing ratios, characterized by diluting a fluorescent dye, could be controlled by the volume of each of the combined wells. The SlipChip design was validated on an ∼12 nL scale by screening the conditions for crystallization of glutaryl-CoA dehydrogenase from Burkholderia pseudomallei against 48 different reagents; each reagent was tested at 11 different mixing ratios, for a total of 528 crystallization trials. The total consumption of the protein sample was ∼10 µL. Conditions for crystallization were successfully identified. The crystallization experiments were successfully scaled up in well plates using the conditions identified in the SlipChip. Crystals were characterized by X-ray diffraction and provided a protein structure in a different space group and at a higher resolution than the structure obtained by conventional methods. In this work, this user-loaded SlipChip has been shown to reliably handle fluids of diverse physicochemical properties, such as viscosities and surface tensions. Quantitative measurements of fluorescent intensities and high-resolution imaging were straighforward to perform in these glass SlipChips. Surface chemistry was controlled using fluorinated lubricating fluid, analogous to the fluorinated carrier fluid used in plug-based crystallization. Thus, we expect this approach to be valuable in a number of areas beyond protein crystallization, especially those areas where droplet-based microfluidic systems have demonstrated successes, including measurements of enzyme kinetics and blood coagulation, cell-based assays, and chemical reactions

Human Factors Analysis of Air Traffic Safety Based on HFACS-BN Model

traffic control (ATC) performance is important to ensure flight safety and the sustainability of aviation growth. To better evaluate the performance of ATC, this paper introduces the HFACS-BN model (HFACS: Human factors analysis and classification system BN: Bayesian network), which can be combined with the subjective information of relevant experts and the objective data of accident reports to obtain more accurate evaluation results. The human factors of ATC in this paper are derived from screening and analysis of 142 civil and general aviation accidents/incidents related to ATC human factors worldwide from 1980 to 2019, among which the most important 25 HFs are selected to construct the evaluation model. The authors designed and implemented a questionnaire survey based on the HFACS framework and collected valid data from 26 frontline air traffic controllers (ATCO) and experts related to ATC in 2019. Combining the responses with objective data, the noisy MAX model is used to calculate the conditional probability table. The results showed that, among the four levels of human factors, unsafe acts had the greatest influence on ATC Performance (79.4%), while preconditions for safe acts contributed the least (40.3%). The sensitivity analysis indicates the order of major human factors influencing the performance of ATC. Finally, this study contributes to the literature in terms of methodological development and expert empirical analysis, providing data support for human error management intervention of ATC in aviation safety. Document type: Articl

Permanence and existence of periodic solution of a discrete periodic Lotka–Volterra competition system with feedback control and time delays

In this paper, we consider a discrete predator–prey system with feedback and time delays. By applying the theory of difference inequality, as well as analysis technique, sufficient conditions are obtained for the permanence of the system. And by applying Mawhin's coincidence degree theory, we obtain the existence of the positive periodic solutions

Multiparameter Screening on SlipChip Used for Nanoliter Protein Crystallization Combining Free Interface Diffusion and Microbatch Methods

This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 µL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoylCoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 Å resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays

Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for “digital” (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12 000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, 1 000 000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA

Complex Function by Design Using Spatially Pre-Structured Synthetic Microbial Communities: Degradation of Pentachlorophenol in the Presence of Hg(II)

Naturally occurring microbes perform a variety of useful functions, with more complex processes requiring multiple functions performed by communities of multiple microbes. Synthetic biology via genetic engineering may be used to achieve desired multiple functions, e.g. multistep chemical and biological transformations, by adding genes to a single organism, but this is sometimes not possible due to incompatible metabolic requirements or not desirable in certain applications, especially in medical or environmental applications. Achieving multiple functions by mixing microbes that have not evolved to function together may not work due to competition of microbes, or lack of interactions among microbes. In nature, microbial communities are commonly spatially structured. Here, we tested whether spatial structure can be used to create a community of microbes that can perform a function they do not perform individually or when simply mixed. We constructed a core–shell fiber with Sphingobium chlorophenolicum, a pentachlorophenol (PCP) degrader, in the core layer and Ralstonia metallidurans, a mercuric ion (Hg(II)) reducer, in the shell layer as a structured community using microfluidic laminar flow techniques. We applied a mixture of PCP and Hg(II) to either a simple well-mixed culture broth (i.e. the unstructured community) or the spatially structured core–shell fibers. We found that without spatial structure, the community was unable to degrade PCP in the presence of Hg(II) because S. chlorophenolicum is sensitive to Hg(II). In contrast, with spatial structure in a core–shell fiber system, S. chlorophenolicum in a core layer was protected by R. metallidurans deposited in a shell layer, and the community was able to completely remove both PCP and Hg(II) from a mixture. The appropriate size of the core–shell fiber was determined by the Damköhler number—the timescale of removal of Hg(II) was on the same order of the timescale of diffusion of Hg(II) through the outer layer when the shell layer was on the order of B200 mm. Ultimately, with the ease of a child putting together ‘Legos’ to build a complex structure, using this approach one may be able to put together microorganisms to build communities that perform functions in vitro or even in vivo, e.g. as in a ‘‘microbiome on a pill’’

Argyres-Douglas Theories, Chiral Algebras and Wild Hitchin Characters

We use Coulomb branch indices of Argyres-Douglas theories on S1×L(k,1) to quantize moduli spaces M_H of wild/irregular Hitchin systems. In particular, we obtain formulae for the "wild Hitchin characters" -- the graded dimensions of the Hilbert spaces from quantization -- for four infinite families of M_H, giving access to many interesting geometric and topological data of these moduli spaces. We observe that the wild Hitchin characters can always be written as a sum over fixed points in M_H under the U(1) Hitchin action, and a limit of them can be identified with matrix elements of the modular transform STkS in certain two-dimensional chiral algebras. Although naturally fitting into the geometric Langlands program, the appearance of chiral algebras, which was known previously to be associated with Schur operators but not Coulomb branch operators, is somewhat surprising

Research on measuring method of shot exit time for muzzle vibration analysis

The measurement of shot exit time is very important to measuring data analysis of muzzle vibration. Optoelectronic method, which is based on the optical-electrical conversion theory and timed with the muzzle flame, is a general method of measuring the shot exit time for the major caliber gun. However, it has not been identified that the shot exit time corresponds to which characteristic point in the voltage pulse time signal curve obtained by the optoelectronic method. In this paper, by combining the high speed digital photography and the optoelectronic method, both the triggering time signal of the high speed digital photography and the voltage signal output by the optoelectronic sensor were obtained simultaneously. Then by comparing the voltage time curve of the optoelectronic sensor with the photographs obtained by the digital photography, the relationship between the characteristic points in the signal curve and the shot exit time was identified. It has been shown that, the voltage pulse beginning time is the same with the muzzle flame beginning, which is later than the time when the projectile band leaves from muzzle

Experimental research on influence of ground vibration on gun measurement instrument support

In order to know well the vibration law on influence of ground vibration on instrument support in gun firing process, a measuring method for the vibration response of the instrument support based on the principle of acceleration measurement is presented. The instrument support is arranged on the ground. When the gun fires, the acceleration response of the instrument support is measured. The vibration velocity and vibration displacement responses of the support can be obtained by the integral operation of the acceleration. The results show that when the gun fires with single shot and the period of the projectile in-bore motion, the vibration response of the instrument support caused by ground vibration is very small, and it can be ignored. When the projectile moves outside the bore, the ground vibration has a significant influence on the vibration of the instrument support. The vibration response of the instrument support in the range of muzzle shock wave is mainly caused by the muzzle shock wave