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Nanoscale Apr 2021Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in...
Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.
Topics: Coronavirus Nucleocapsid Proteins; Cryoelectron Microscopy; Molecular Weight; Phosphoproteins; Protein Structure, Secondary; SARS-CoV-2
PubMed: 33889923
DOI: 10.1039/d1nr00388g -
Electrophoresis Dec 2011The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity... (Review)
Review
The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.
Topics: Animals; Electrophoresis, Capillary; Electrophoresis, Microchip; Glycoproteins; Humans; Mass Spectrometry; Polysaccharides
PubMed: 22180203
DOI: 10.1002/elps.201100342 -
ACS Sensors Oct 2018Rapid detection of bacteria and their susceptibility to specific antibiotics plays a vital role in microbial infection treatments. Antimicrobial susceptibility testing...
Rapid detection of bacteria and their susceptibility to specific antibiotics plays a vital role in microbial infection treatments. Antimicrobial susceptibility testing (AST) is a common measure to select effective drugs. However, the conventional practices, such as broth dilution, E-test, and disk diffusion, in clinical applications require a long turnaround time (∼3 days), thereby compromising treatments and increasing mortality. This study presents self-powered sensors for on-site microorganism monitoring and rapid AST based on functionalized microbeads. The microbead sensors are driven by Brownian motion, rendering external power unnecessary. Fluorescent microbeads ( d = 2 μm) were coated with vancomycin to capture bacteria. The growth and responses of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus under antibiotic treatment were evaluated. The method showed stable selective binding despite the presence of some interferential substances, such as proteins and cells. Diffusivity change was strongly related to bacterial concentration. Accordingly, the diffusivity values of microbeads bound with motile and nonmotile bacteria exhibited specific patterns because of extra motility from microbes and increased particle diameter. Only a drop of microbead-bacteria suspension (∼5 μL) was needed in a microchip for each measurement. The microchip provided a steady environment for measurement over a few hours. By distinguishing the slope of the last four data points in the temporal diffusivity curve, bacterial susceptibility or resistance to specific antibiotics could be determined within a time frame of 2 h. The study provides insights into saving more lives by using a fast and robust AST technique in future clinical practice.
Topics: Anti-Bacterial Agents; Escherichia coli; Magnetics; Microbial Sensitivity Tests; Microchip Analytical Procedures; Microspheres; Staphylococcus aureus; Vancomycin
PubMed: 30221509
DOI: 10.1021/acssensors.8b00790 -
Micromachines Sep 2019The rapid and reliable detection of chemical and biological agents in the field is important for many applications such as national security, environmental monitoring,...
The rapid and reliable detection of chemical and biological agents in the field is important for many applications such as national security, environmental monitoring, infectious diseases screening, and so on. Current commercially available devices may suffer from low field deployability, specificity, and reproducibility, as well as a high false alarm rate. This paper reports the development of a portable lab-on-a-chip device that could address these issues. The device integrates a polymer multiplexed microchip system, a contactless conductivity detector, a data acquisition and signal processing system, and a graphic/user interface. The samples are pre-treated by an on-chip capillary electrophoresis system. The separated analytes are detected by conductivity-based microsensors. Extensive studies are carried out to achieve satisfactory reproducibility of the microchip system. Chemical warfare agents soman (GD), sarin (GB), O-ethyl S-[2-diisoproylaminoethyl] methylphsophonothioate (VX), and their degradation products have been tested on the device. It was demonstrated that the device can fingerprint the tested chemical warfare agents. In addition, the detection of ricin and metal ions in water samples was demonstrated. Such a device could be used for the rapid and sensitive on-site detection of both chemical and biological agents in the future.
PubMed: 31527486
DOI: 10.3390/mi10090617 -
Blood Advances Nov 2020Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their...
Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombin-activatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent anti-fibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI- or factor XIII-deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its cross-linkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.
Topics: Animals; Blood Coagulation; Blood Platelets; Carboxypeptidase B2; Fibrin Clot Lysis Time; Fibrinolysis; Mice
PubMed: 33166409
DOI: 10.1182/bloodadvances.2020002923 -
Scientific Reports Oct 2015Alignment of microchips with receptors is an important process step in the construction of integrated micro- and nanosystems for emerging technologies, and facilitating...
Alignment of microchips with receptors is an important process step in the construction of integrated micro- and nanosystems for emerging technologies, and facilitating alignment by spontaneous self-assembly processes is highly desired. Previously, capillary self-alignment of microchips driven by surface tension effects on patterned surfaces has been reported, where it was essential for microchips to have sufficient overlap with receptor sites. Here we demonstrate for the first time capillary self-transport and self-alignment of microchips, where microchips are initially placed outside the corresponding receptor sites and can be self-transported by capillary force to the receptor sites followed by self-alignment. The surface consists of hydrophilic silicon receptor sites surrounded by superhydrophobic black silicon. Rain-induced microscopic droplets are used to form the meniscus for the self-transport and self-alignment. The boundary conditions for the self-transport have been explored by modeling and confirmed experimentally. The maximum permitted gap between a microchip and a receptor site is determined by the volume of the liquid and by the wetting contrast between receptor site and substrate. Microscopic rain applied on hydrophilic-superhydrophobic patterned surfaces greatly improves the capability, reliability and error-tolerance of the process, avoiding the need for accurate initial placement of microchips, and thereby greatly simplifying the alignment process.
PubMed: 26450019
DOI: 10.1038/srep14966 -
Journal of Pharmaceutical Analysis Aug 2020Researches on detection of human papillomavirus (HPV) high-risk samples were carried out by polymerase chain reaction (PCR) coupled with microchip electrophoresis (MCE)....
Researches on detection of human papillomavirus (HPV) high-risk samples were carried out by polymerase chain reaction (PCR) coupled with microchip electrophoresis (MCE). Herein, we introduced a simple, rapid, automated method for detecting high-risk samples HPV16 and HPV18. In this research, general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection, then type-specific primers were further used to evaluate the specificity of MCE method. The results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18, and also enabled simultaneous detection of multiplex samples. This MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing results. The inherent capability of MCE facilitated HPV detection conducted in a small chip with automated, high throughput, massive parallelized analysis. We envision that MCE method will definitely pave a way for clinical diagnosis, and even on-site screening of cervical cancer.
PubMed: 32923006
DOI: 10.1016/j.jpha.2020.04.003 -
Optimizing Binding Site Spacing in Fluidic Self-Assembly for Enhanced Microchip Integration Density.Micromachines Feb 2024This manuscript presents a comprehensive study on the assembly of microchips using fluidic self-assembly (FSA) technology, with a focus on optimizing the spacing between...
This manuscript presents a comprehensive study on the assembly of microchips using fluidic self-assembly (FSA) technology, with a focus on optimizing the spacing between binding sites to improve yield and assembly. Through a series of experiments, we explored the assembly of microchips on substrates with varying binding site spacings, revealing the impact of spacing on the rate of undesired chip assembly across multiple sites. Our findings indicate a significant reduction in incorrect assembly rates as the spacing increases beyond a critical threshold of 140 μm. This study delves into the mechanics of chip alignment within the fluid medium, hypothesizing that the extent of the alloy's grip on the chips at different spacings influences assembly outcomes. By analyzing cases of undesired assembly, we identified the relationship between binding site spacing and the area of chip contact, demonstrating a decrease in the combined left and right areas of chips as the spacing increases. The results highlight a critical spacing threshold, which, when optimized, could significantly enhance the efficiency and precision of microchip assembly processes using FSA technology. This research contributes to the field of microcomponent assembly, offering insights into achieving higher integration densities and precision in applications, such as microLED displays and augmented reality (AR) devices.
PubMed: 38542547
DOI: 10.3390/mi15030300 -
Scientific Reports Jun 2021Mechanical forces are pervasive in the inflammatory site where dendritic cells (DCs) are activated to migrate into draining lymph nodes. For example, fluid shear stress...
Mechanical forces are pervasive in the inflammatory site where dendritic cells (DCs) are activated to migrate into draining lymph nodes. For example, fluid shear stress modulates the movement patterns of DCs, including directness and forward migration indices (FMIs), without chemokine effects. However, little is known about the effects of biomechanical forces on the activation of DCs. Accordingly, here we fabricated a microfluidics system to assess how biomechanical forces affect the migration and activity of DCs during inflammation. Based on the structure of edema, we proposed and experimentally analyzed a novel concept for a microchip model that mimicked such vascular architecture. The intensity of shear stress generated in our engineered chip was found as 0.2-0.6 dyne/cm by computational simulation; this value corresponded to inflammation in tissues. In this platform, the directness and FMIs of DCs were significantly increased, whereas the migration velocity of DCs was not altered by shear stress, indicating that mechanical stimuli influenced DC migration. Moreover, DCs with shear stress showed increased expression of the DC activation markers MHC class I and CD86 compared with DCs under static conditions. Taken together, these data suggest that the biomechanical forces are important to regulate the migration and activity of DCs.
Topics: Animals; B7-2 Antigen; Biomechanical Phenomena; Bone Marrow; Bone Marrow Cells; Cell Movement; Cell Separation; Chemokines; Computer Simulation; Dendritic Cells; Edema; Flow Cytometry; Inflammation; Lymph Nodes; Mice; Mice, Inbred C57BL; Microfluidics; Shear Strength; Stress, Mechanical
PubMed: 34103554
DOI: 10.1038/s41598-021-91117-2 -
"State of the Mewnion": Practices of Feral Cat Care and Advocacy Organizations in the United States.Frontiers in Veterinary Science 2021Over the last several decades, feral cats have moved from the fringes to the mainstream in animal welfare and sheltering. Although many best practice guidelines have...
Over the last several decades, feral cats have moved from the fringes to the mainstream in animal welfare and sheltering. Although many best practice guidelines have been published by national non-profits and veterinary bodies, little is known about how groups “in the trenches” actually operate. Our study sought to address that gap through an online survey of feral cat care and advocacy organizations based in the United States. Advertised as “The State of the Mewnion,” its topics included a range of issues spanning non-profit administration, public health, caretaking and trapping, adoptions of friendly kittens and cats, veterinary medical procedures and policies, data collection and program efficacy metrics, research engagement and interest, and relationships with wildlife advocates and animal control agencies. Respondents from 567 organizations participated, making this the largest and most comprehensive study on this topic to date. Respondents came primarily from grassroots organizations. A majority reported no paid employees (74.6%), served 499 or fewer feral cats per year (75.0%), engaged between 1 and 9 active volunteers (54.9%), and did not operate a brick and mortar facility (63.7%). Some of our findings demonstrate a shared community of practice, including the common use of a minimum weight of 2.0 pounds for spay/neuter eligibility, left side ear tip removals to indicate sterilization, recovery holding times after surgery commonly reported as 1 night for male cats and 1 or 2 nights for females, requiring or recommending to adopters of socialized kittens/cats that they be kept indoor-only, and less than a quarter still engaging in routine testing of cats for FIV and FeLV. Our survey also reveals areas for improvement, such as most organizations lacking a declared goal with a measurable value and a time frame, only sometimes scanning cats for microchips, and about a third not using a standardized injection site for vaccines. This study paints the clearest picture yet available of what constitutes the standard practices of organizations serving feral and community cats in the United States.
PubMed: 34970620
DOI: 10.3389/fvets.2021.791134