Sorting Platform Research Articles

Accelerated sunlight-driven conversion of industrial flue gas into biofuels by microfluidic high-throughput screening towards improving photosynthesis in microalgae under fluctuating light

Sunlight-driven microalgal conversion of CO2 is a promising carbon neutral strategy for the sustainable production of various value-added products. However, its real-world application is challenging due to the irregularity of sunlight which reduces photosynthetic efficiency. Here, we report that microdroplet-based screening of photosynthesis-augmented microalgae mutant under fluctuating light accelerates mass production of CO2-derived algal biofuel in natural sunlight. Random insertional Chlamydomonas reinhardtii mutants were cultivated in single-layered microdroplet photobioreactors to be effectively applied to light conditions mimicking natural sunlight without self-shading. High-density microdroplets containing fast-growing mutants were efficiently selected at the single-droplet level by a centrifugation-assisted droplet sorting platform for high-throughput screening. Consequently, we isolated a mutant exhibiting cell growth 1.85-fold that of the wild-type even under continuous and rapid alteration in light intensity, which can serve as a cell stressor. After whole-genome resequencing, we found disruption in the SNF2 gene encoding ATP-dependent chromatin remodeling complexes, which reveal its importance for the regulation of chlorophyll biosynthesis and accumulation of reactive oxygen species under fluctuating light. After a 1.6 ton-scale field culture utilizing natural sunlight and industrial CO2, the mutant showed increased biomass productivity, CO2 fixation rate, and calorific value, a crucial parameter of fuel performance, by 34.38%, 45.07%, and 16.82%, respectively. Our results indicate that improving photosynthesis of microalgae in fluctuating light environments elucidates the mechanisms responsible for enhancing the sunlight utilization, allowing industrial-scale biological CO2 conversion.

A sensor combination based automatic sorting system for waste washing machine parts

In order to solve the difficulties encountered in E-wastes disposal automation, this work investigates the sensor-based sorting of waste washing machine parts. To satisfy the post-processing, we divided the parts into 6 categories (Piece, Cover, Base, Shell, Spin tub and Drain hose) based on the shape differences and used object recognition algorithm to classify them. In the recycling terminal, parts were divided based on types of materials. Piece and Cover contained acrylonitrile butadiene styrene (ABS) parts and polystyrene (PS) parts, while Base, Shell, Spin tub and Drain hose were polypropylene (PP) parts only. Therefore near-infrared (NIR) spectroscopy was applied for sorting ABS, PS and PP. Algorithm for object recognition and NIR spectroscopy both reached high precision. In object recognition, algorithm of YOLOv5 and its improved models, including focal loss and channel attention mechanism, were tested. YOLOv5 with channel attention achieved the best 98.7% mean Average Precision (mAP) and the overall over 98% Average Precision (AP). In the task of NIR spectroscopy, principal component analysis (PCA) coupled with support vector machine (SVM) reached the overall 97.8% classification precision. Finally, a sorting platform based on our device and algorithm was designed.

Ultrathroughput immunomagnetic cell sorting platform.

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.

Research of Comprehensive Operation Vehicle for Apple Picking

Aiming at the defects of low mechanization, single function, poor integration of machinery, agronomy and high damage rate in apple picking process, a comprehensive operation equipment for apple picking was developed. Around the standardized apple cultivation mode with dwarf and dense planting, intensive and efficient, the research of multi-link mechanized operation technology method based on the integration of agricultural machinery and agronomy is carried out. The structure of apple picking comprehensive operation equipment is designed, including the orchard traffic system, chassis support device, operation platform, platform lifting device, apple collection device, apple conveying system, automatic sorting, bagging device and fruit box rotation platform, which solves the key technical problems of mechanization, such as adaptability of trafficability, extension of operation space and low loss of fruit delivery and fruit collection and placement. The damage of apples is reduced by adopting flexible collection, conveying device and the quality of apples is improved by adopting apple collection, automatic sorting and packing system to realize the second sorting and packing of apples. The design of the device can realize the integrated service of apple from picking to sealing and selling, eliminating the intermediate labour-intensive and time-consuming links, the production efficiency of apples is improved and the production cost is reduced. It laid a foundation for the realization of the whole mechanization of apple production.

Retromer dependent changes in cellular homeostasis and Parkinson's disease.

To date, mechanistic treatments targeting the initial cause of Parkinson's disease (PD) are limited due to the underlying biological cause(s) been unclear. Endosomes and their associated cellular homeostasis processes have emerged to have a significant role in the pathophysiology associated with PD. Several variants within retromer complex have been identified and characterised within familial PD patients. The retromer complex represents a key sorting platform within the endosomal system that regulates cargo sorting that maintains cellular homeostasis. In this review, we summarise the current understandings of how PD-associated retromer variants disrupt cellular trafficking and how the retromer complex can interact with other PD-associated genes to contribute to the disease progression.

High-Throughput Functional Screening of Antigen-Specific T Cells Based on Droplet Microfluidics at a Single-Cell Level.

The lack of an efficient method for the identification of tumor antigen-specific T cell receptors (TCRs) impedes the development of T cell-based cancer immunotherapies. Here, we introduce a droplet-based microfluidic platform for function-based screening and sorting of tumor antigen-specific T cells with high throughput. We built a reporter cell line by co-transducing the TCR library and reporter genes at the downstream of TCR signaling, and reporter cells fluoresced upon functionally binding with antigens. We co-encapsulated reporter cells and antigen-presenting cells in droplets to allow for stimulation on a single-cell level. Functioning reporter cells specific against the antigen were identified in the microfluidic channel based on the fluorescent signals of the droplets, which were immediately sorted out using dielectrophoresis. We validated the reporter system and sorting results using flow cytometry. We then performed single-cell RNA sequencing on the sorted cells to further validate this platform and demonstrate the compatibility with genetic characterizations. Our platform provides a means for precise and efficient T cell immunotherapy, and the droplet-based high-throughput TCR screening method could potentially facilitate immunotherapeutic screening and promote T cell-based anti-tumor therapies.

The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site.

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

Selective Single-Cell Sorting Using a Multisectorial Electroactive Nanowell Platform.

Current approaches in targeted patient treatments often require the rapid isolation of specific rare target cells. Stream-based dielectrophoresis (DEP) based cell sorters have the limitation that the maximum number of sortable cell types is equivalent to the number of output channels, which makes upscaling to a higher number of different cell types technically challenging. Here, we present a microfluidic platform for selective single-cell sorting that bypasses this limitation. The platform consists of 10 000 nanoliter wells which are placed on top of interdigitated electrodes (IDEs) that facilitate dielectrophoresis-driven capture of cells. By use of a multisectorial design formed by 10 individually addressable IDE structures, our platform can capture a large number of different cell types. The sectorial approach allows for fast and straightforward modification to sort complex samples as different cell types are captured in different sectors and therefore removes the need for individual output channels per cell type. Experimental results obtained with a mixed sample of benign (MCF-10A) and malignant (MDA-MB-231) breast cells showed a target to nontarget sorting accuracy of over 95%. We envision that the high accuracy of our platform, in addition to its versatility and simplicity, will aid clinical environments where reliable sorting of varying complex samples is essential.

Correlative nanophotonic approaches to enlighten the nanoscale dynamics of living cell membranes.

Dynamic compartmentalization is a prevailing principle regulating the spatiotemporal organization of the living cell membrane from the nano- up to the mesoscale. This non-arbitrary organization is intricately linked to cell function. On living cell membranes, dynamic domains or ‘membrane rafts' enriched with cholesterol, sphingolipids and other certain proteins exist at the nanoscale serving as signaling and sorting platforms. Moreover, it has been postulated that other local organizers of the cell membrane such as intrinsic protein interactions, the extracellular matrix and/or the actin cytoskeleton synergize with rafts to provide spatiotemporal hierarchy to the membrane. Elucidating the intricate coupling of multiple spatial and temporal scales requires the application of correlative techniques, with a particular need for simultaneous nanometer spatial precision and microsecond temporal resolution. Here, we review novel fluorescence-based techniques that readily allow to decode nanoscale membrane dynamics with unprecedented spatiotemporal resolution and single-molecule sensitivity. We particularly focus on correlative approaches from the field of nanophotonics. Notably, we introduce a versatile planar nanoantenna platform combined with fluorescence correlation spectroscopy to study spatiotemporal heterogeneities on living cell membranes at the nano- up to the mesoscale. Finally, we outline remaining future technological challenges and comment on potential directions to advance our understanding of cell membrane dynamics under the influence of the actin cytoskeleton and extracellular matrix in uttermost detail.

Reversible Curvature Reversal of Monolithic Liquid Crystal Elastomer Film and Its Smart Valve Application.

Beyond a traditional stimuli-responsive soft actuator that shows a single motion by a stimulus, multidirectional actuation reversal with a single stimulus is highly required in applications such as shape morphing sensors and soft robotics. Liquid crystal elastomers (LCEs) are one of the most attractive candidates for the soft actuator due to their capability of stimuli-responsive shape changing in 3D, which is programmable with local orientation of LC mesogens. Here, a simple but effective method to fabricate monolithic LCE actuators that are capable of reversible curvature reversal in bending and twisting deformation by a single stimulus-heat-is reported. The curvature reversal of the LCE film can be programmed by means of asymmetric crosslinking density along the thickness and the orientation of the LC mesogens. The curvature reversal of the monolithic LCE film exhibits highly reversible (more than 100 times) and fast actuation (≈3-5 s) by heating and cooling, allowing new concept of a practical application using LCE material: a self-regulated smart valve that is capable of qualitatively sorting liquids by temperature. It is believed that this system is potentially applied to a self-regulated sorting platform for various endothermic and exothermic chemical or biological reactions.

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System.

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome’s cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 “pods” that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

Localized strain profile in surface electrode array for programmable composite multiferroic devices

We investigate localized in-plane strains on the microscale, induced by arrays of biased surface electrodes patterned on piezoelectrics. Particular focus is given to the influence that adjacent electrode pairs have on one another to study the impact of densely packed electrode arrays. We present a series of X-ray microdiffraction studies to reveal the spatially resolved micrometer-scale strain distribution. The strain maps with micrometer-scale resolution highlight how the local strain profile in square regions up to 250 × 250 μm2 in size is affected by the surface electrodes that are patterned on ferroelectric single-crystal [Pb(Mg1/3Nb2/3)O3]x-[PbTiO3]1−x. The experimental measurements and simulation results show the influence of electrode pair distance, positioning of the electrode pair, including the angle of placement, and neighboring electrode pair arrangements on the strength and direction of the regional strain. Our findings are relevant to the development of microarchitected strain-mediated multiferroic devices. The electrode arrays could provide array-addressable localized strain control for applications including straintronic memory, probabilistic computing platforms, microwave devices, and magnetic-activated cell sorting platforms.

Microfluidics for Peptidomics, Proteomics, and Cell Analysis.

Microfluidics is the advanced microtechnology of fluid manipulation in channels with at least one dimension in the range of 1–100 microns. Microfluidic technology offers a growing number of tools for manipulating small volumes of fluid to control chemical, biological, and physical processes relevant to separation, analysis, and detection. Currently, microfluidic devices play an important role in many biological, chemical, physical, biotechnological and engineering applications. There are numerous ways to fabricate the necessary microchannels and integrate them into microfluidic platforms. In peptidomics and proteomics, microfluidics is often used in combination with mass spectrometric (MS) analysis. This review provides an overview of using microfluidic systems for peptidomics, proteomics and cell analysis. The application of microfluidics in combination with MS detection and other novel techniques to answer clinical questions is also discussed in the context of disease diagnosis and therapy. Recent developments and applications of capillary and microchip (electro)separation methods in proteomic and peptidomic analysis are summarized. The state of the art of microchip platforms for cell sorting and single-cell analysis is also discussed. Advances in detection methods are reported, and new applications in proteomics and peptidomics, quality control of peptide and protein pharmaceuticals, analysis of proteins and peptides in biomatrices and determination of their physicochemical parameters are highlighted.

HrpB4 from Xanthomonas campestris pv. vesicatoria acts similarly to SctK proteins and promotes the docking of the predicted sorting platform to the type III secretion system.

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates bacterial effector proteins into plant cells. At least nine membrane-associated and cytoplasmic components of the secretion apparatus are homologous to corresponding Sct (secretion and cellular translocation) proteins from animal pathogens, suggesting a similar structural organisation of T3S systems in different bacterial species. T3S in X. campestris pv. vesicatoria also depends on non-conserved proteins with yet unknown function including the essential pathogenicity factor HrpB4. Here, we show that HrpB4 localises to the cytoplasm and the bacterial membranes and interacts with the cytoplasmic domain of the inner membrane (IM) ring component HrcD and the cytoplasmic HrcQ protein. The analysis of HrpB4 deletion derivatives revealed that deletion of the N- or C-terminal protein region affects the interaction of HrpB4 with HrcQ and HrcD as well as its contribution to pathogenicity. HrcQ is a component of the predicted sorting platform, which was identified in animal pathogens as a dynamic heterooligomeric protein complex and associates with the IM ring via SctK proteins. HrcQ complex formation was previously shown by fluorescent microscopy analysis and depends on the presence of the T3S system. In the present study, we provide experimental evidence that the absence of HrpB4 severely affects the docking of HrcQ complexes to the T3S system but does not significantly interfere with HrcQ complex formation in the bacterial cytoplasm. Taken together, our data suggest that HrpB4 links the predicted cytoplasmic sorting platform to the IM rings of the T3S system.

The Shigella Type III Secretion System: An Overview from Top to Bottom.

Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention.

Molecular Targets and Strategies for Inhibition of the Bacterial Type III Secretion System (T3SS); Inhibitors Directly Binding to T3SS Components.

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS’s complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.

A 3D hydrodynamic flow-focusing device for cell sorting

Optical-based microfluidic cell sorting has become increasingly attractive for applications in life and environmental sciences due to its ability of sophisticated cell handling in flow. The majority of these microfluidic cell sorting devices employ two-dimensional fluid flow control strategies, which lack the ability to manipulate the position of cells arbitrarily for precise optical detection, therefore resulting in reduced sorting accuracy and purity. Although three-dimensional (3D) hydrodynamic devices have better flow-focusing characteristics, most lack the flexibility to arbitrarily position the sample flow in each direction. Thus, there have been very few studies using 3D hydrodynamic flow focusing for sorting. Herein, we designed a 3D hydrodynamic focusing sorting platform based on independent sheath flow-focusing and pressure-actuated switching. This design offers many advantages in terms of reliable acquisition of weak Raman signals due to the ability to precisely control the speed and position of samples in 3D. With a proof-of-concept demonstration, we show this 3D hydrodynamic focusing-based sorting device has the potential to reach a high degree of accuracy for Raman activated sorting.

Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography

Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.

The Structures of SctK and SctD from Pseudomonas aeruginosa Reveal the Interface of the Type III Secretion System Basal Body and Sorting Platform

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components – the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to “pods” (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.

Ionic strength controls long-term cell-surface interactions – A QCM-D study of S. cerevisiae adhesion, retention and detachment

Understanding microbial adhesion and retention is crucial for controlling many processes, including biofilm formation, antimicrobial therapy as well as cell sorting and cell detection platforms. Cell detachment is inextricably linked to cell adhesion and retention and plays an important part in the mechanisms involved in these processes. Physico-chemical and biological forces play a crucial role in microbial adhesion interactions and altering the medium ionic strength offers a potential means for modulating these interactions. Real-time studies on the effect of ionic strength on microbial adhesion are often limited to short-term bacterial adhesion. Therefore, there is a need, not only for long-term bacterial adhesion studies, but also for similar studies focusing on eukaryotic microbes, such as yeast. Hereby, we monitored, in real-time, S. cerevisiae adhesion on gold and silica as examples of surfaces with different surface charge properties to disclose long-term adhesion, retention and detachment as a function of ionic strength using quartz crystal microbalance with dissipation monitoring. Our results show that short- and long-term cell adhesion levels in terms of mass-loading increase with increasing ionic strength, while cells dispersed in a medium of higher ionic strength experience longer retention and detachment times. The positive correlation between the cell zeta potential and ionic strength suggests that zeta potential plays a role on cell retention and detachment. These trends are similar for measurements on silica and gold, with shorter retention and detachment times for silica due to strong short-range repulsions originating from a high electron-donicity. Furthermore, the results are comparable with measurements in standard yeast culture medium, implying that the overall effect of ionic strength applies for cells in nutrient-rich and nutrient-deficient media.