Optoelectronic Tweezers Micro-Well System for Highly Efficient Single-Cell Trapping, Dynamic Sorting, and Retrieval.

Single-cell arrays have emerged as a versatile method for executing single-cell manipulations across an array of biological applications. In this paper, an innovative microfluidic platform is unveiled that utilizes optoelectronic tweezers (OETs) to array and sort individual cells at a flow rate of 20 µL min-1. This platform is also adept at executing dielectrophoresis (DEP)-based, light-guided single-cell retrievals from designated micro-wells. This presents a compelling non-contact method for the rapid and straightforward sorting of cells that are hard to distinguish. Within this system, cells are individually confined to micro-wells, achieving an impressive high single-cell capture rate exceeding 91.9%. The roles of illuminating patterns, flow velocities, and applied electrical voltages are delved into in enhancing the single-cell capture rate. By integrating the OET system with the micro-well arrays, the device showcases adaptability and a plethora of functions. It can concurrently trap and segregate specific cells, guided by their dielectric signatures. Experimental results, derived from a mixed sample of HepG2 and L-O2 cells, reveal a sorting accuracy for L-O2 cells surpassing 91%. Fluorescence markers allow for the identification of sequestered, fluorescence-tagged HepG2 cells, which can subsequently be selectively released within the chip. This platform's rapidity in capturing and releasing individual cells augments its potential for future biological research and applications.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

Spatiotemporal modulation of nitric oxide and Notch signaling by hemodynamic-responsive Trpv4 is essential for ventricle regeneration.

Zebrafish have a remarkable ability to regenerate injured hearts. Altered hemodynamic forces after larval ventricle ablation activate the endocardial Klf2a-Notch signaling cascade to direct zebrafish cardiac regeneration. However, how the heart perceives blood flow changes and initiates signaling pathways promoting regeneration is not fully understood. The present study demonstrated that the mechanosensitive channel Trpv4 sensed the altered hemodynamic forces in injured hearts and its expression was regulated by blood flow. In addition to mediating the endocardial Klf2a-Notch signal cascade around the atrioventricular canal (AVC), we discovered that Trpv4 regulated nitric oxide (NO) signaling in the bulbus arteriosus (BA). Further experiments indicated that Notch signaling primarily acted at the early stage of regeneration, and the major role of NO signaling was at the late stage and through TGF-ß pathway. Overall, our findings revealed that mechanosensitive channels perceived the changes in hemodynamics after ventricle injury, and provide novel insights into the temporal and spatial coordination of multiple signaling pathways regulating heart regeneration.

Ercc2/Xpd deficiency results in failure of digestive organ growth in zebrafish with elevated nucleolar stress.

Mutations in ERCC2/XPD helicase, an important component of the TFIIH complex, cause distinct human genetic disorders which exhibit various pathological features. However, the molecular mechanisms underlying many symptoms remain elusive. Here, we have shown that Ercc2/Xpd deficiency in zebrafish resulted in hypoplastic digestive organs with normal bud initiation but later failed to grow. The proliferation of intestinal endothelial cells was impaired in ercc2/xpd mutants, and mitochondrial abnormalities, autophagy, and inflammation were highly induced. Further studies revealed that these abnormalities were associated with the perturbation of rRNA synthesis and nucleolar stress in a p53-independent manner. As TFIIH has only been implicated in RNA polymerase I-dependent transcription in vitro, our results provide the first evidence for the connection between Ercc2/Xpd and rRNA synthesis in an animal model that recapitulates certain key characteristics of ERCC2/XPD-related human genetic disorders, and will greatly advance our understanding of the molecular pathogenesis of these diseases.

Knockout of Shelterin subunit genes in zebrafish results in distinct outcomes.

As the core component of telomeres, the Shelterin complex interacts with telomerase and the CST complex and plays a crucial role in maintaining telomere structure. Perturbation of Shelterin subunits results in telomere damage and subsequent genomic instability, which leads to aging as well as multiple human diseases. Recently, zebrafish have been widely utilized to model human diseases. To establish appropriate zebrafish models of Shelterin-related human disorders, we generated knockout zebrafish of the Shelterin subunit genes acd, pot1, tinf2, terf1 and pinx1 using the CRISPR/Cas9 technology and analyzed the effects of gene deficiency on zebrafish development in detail. We discovered that tinf2, terf1 and pinx1 homozygous mutants could grow to adulthood normally, whereas acd and pot1 homozygous mutant larvae died between 12 and 15 dpf without obvious abnormalities. A few acd-/- mutants survived to adulthood and displayed several premature aging-like phenotypes, including male sterility, cachectic dwarfism and reduced lifespan. Overall, our study established a variety of telomere-deficient zebrafish mutant strains and provided novel animal models for further exploring the relationship between telomeres and aging as well as the pathogenesis of human diseases associated with telomere deficiency.

Parallel Manipulation and Flexible Assembly of Micro-Spiral via Optoelectronic Tweezers.

Micro-spiral has a wide range of applications in smart materials, such as drug delivery, deformable materials, and micro-scale electronic devices by utilizing the manipulation of electric fields, magnetic fields, and flow fields. However, it is incredibly challenging to achieve a massively parallel manipulation of the micro-spiral to form a particular microstructure in these conventional methods. Here, a simple method is reported for assembling micro-spirals into various microstructures via optoelectronic tweezers (OETs), which can accurately manipulate the micro-/bio-particles by projecting light patterns. The manipulation force of micro-spiral is analyzed and simulated first by the finite element simulation. When the micro-spiral lies at the bottom of the microfluidic chip, it can be translated or rotated toward the target position by applying control forces simultaneously at multiple locations on the long axis of the micro-spiral. Through the OET manipulation, the length of the micro-spiral chain can reach 806.45 µm. Moreover, the different parallel manipulation modes are achieved by utilizing multiple light spots. The results show that the micro-spirulina can be manipulated by a real-time local light pattern and be flexibly assembled into design microstructures by OETs, such as a T-shape circuit, link lever, and micro-coil pairs of devices. This assembly method using OETs has promising potential in fabricating innovative materials and microdevices for practical engineering applications.

Magnetically Actuated Cell-Robot System: Precise Control, Manipulation, and Multimode Conversion.

Border-nearing microrobots with self-propelling and navigating capabilities have promising applications in micromanipulation and bioengineering, because they can stimulate the surrounding fluid flow for object transportation. However, ensuring the biosafety of microrobots is a concurrent challenge in bioengineering applications. Here, macrophage template-based microrobots (cell robots) that can be controlled individually or in chain-like swarms are proposed, which can transport various objects. The cell robots are constructed using the phagocytic ability of macrophages to load nanomagnetic particles while maintaining their viability. The robots exhibit high position control accuracy and generate a flow field that can be used to transport microspheres and sperm when exposed to an external magnetic field near a wall. The cell robots can also form chain-like swarms to transport a large object (more than 100 times the volume). This new insight into the manipulation of macrophage-based cell robots provides a new concept by converting other biological cells into microrobots for micromanipulation in biomedical applications.

Reducing the Guidewire Friction for Endovascular Interventional Surgery by Radial Micro-Vibration.

Guidewires for endovascular interventional surgery are inevitably affected by high frictional resistance because of direct contact with the vascular wall, which greatly reduces the operation efficiency and safety. This article presents a method of applying radial ultrasonic microvibration at the proximal end of a conventional passive guidewire to reduce the frictional resistance. The proposed method theoretically reduced the frictional resistance by reducing the friction coefficient, actual contact area, and the net friction time between the guidewire and vascular wall. The effectiveness of the proposed method was experimentally demonstrated in designed simulations of the blood vessel environment, where the influences of the vibration amplitude on the drag reduction effect were considered. The results indicated that vibrating the guidewire at the resonant frequency with the designed device clearly reduced the drag with a maximum frictional reduction rate of 85.19%. At the resonant frequency, the change in frictional resistance showed a linear negative correlation with the applied vibration amplitude. The proposed method offers a new approach to improving the efficiency and safety of vascular interventional surgery.

Optimization of Nanoparticles for Smart Drug Delivery: A Review.

Nanoparticle delivery systems have good application prospects in the treatment of various diseases, especially in cancer treatment. The effect of drug delivery is regulated by the properties of nanoparticles. There have been many studies focusing on optimizing the structure of nanoparticles in recent years, and a series of achievements have been made. This review summarizes the optimization strategies of nanoparticles from three aspects-improving biocompatibility, increasing the targeting efficiency of nanoparticles, and improving the drug loading rate of nanoparticles-aiming to provide some theoretical reference for the subsequent drug delivery of nanoparticles.

Interaction between positive and negative dielectric microparticles/microorganism in optoelectronic tweezers.

Optoelectronic tweezers (OET) is a noncontact micromanipulation technology for controlling microparticles and cells. In the OET, it is necessary to configure a medium with different electrical properties to manipulate different particles and to avoid the interaction between two particles. Here, a new method exploiting the interaction between different dielectric properties of micro-objects to achieve the trapping, transport, and release of particles in the OET system was proposed. Besides, the effect of interaction between the micro-objects with positive and negative dielectric properties was simulated by the arbitrary Lagrangian-Eulerian (ALE) method. In addition, compared with conventional OET systems relying on fabrication processes involving the assembly of photoelectric materials, a contactless OET platform with an iPad-based wireless-control interface was established to achieve convenient control. Finally, this platform was used in the interaction of swimming microorganisms (positive-dielectric properties) with microparticles (negative-dielectric properties) at different scales. It showed that one particle could interact with 5 particles simultaneously, indicating that the interaction can be applied to enhance the high-throughput transportation capacities of the OET system and assemble some special microstructures. Owing to the low power, microorganisms were free from adverse influence during the experiment. In the future, the interaction of particles in a simple OET platform is a promising alternative in micro-nano manipulation for controlling drug release from uncontaminated cells in targeted therapy research.

Bone formation recovery with gold nanoparticle-induced M2 macrophage polarization in mice.

The prevention of fractures induced by inflammatory bone disease remains a clinical challenge. This is because of a lack of bone formation to fill in the bone defects, which are believed to be due in part to persistent inflammation caused by the imbalance of M1 over M2 macrophages. In this study, gold nanoparticles (AuNPs) were synthesized to shift the balance of macrophages at the site of bone damage to improve osteanagenesis in a mouse model of LPS-induced inflammatory bone erosion. Specifically, the AuNPs treatment improved bone structure and increased bone mineral density (BMD) by ~14% compared with model group. Macrophages recruited by LPS treatment were reduced by ~11% after AuNPs injection. Compared to LPS treatment only, the percentage of M2 macrophages increased threefold by AuNPs, while the proportion of M1 macrophages decreased by 59%. This promoted the regeneration of bone matrix proteins in the bone defect site, which finally leads to increased bone mass and improved bone structure in model mice. These data suggest that AuNPs could be a novel candidate therapeutic for inflammatory bone disease rather than a drug carrier.

Postoperative evaluation of tumours based on label-free acoustic separation of circulating tumour cells by microstreaming.

Metastatic tumour recurrence caused by circulating tumour cells (CTCs) after surgery is responsible for more than 90% of tumour-related deaths. A postoperative evaluation system based on the long-term dynamic detection of CTCs helps in guiding the postoperative treatment of tumours in real time and preventing metastases and recurrence of tumours after treatment. In this study, a simple, rapid, and low-cost postoperative evaluation system was established based on the number of CTCs captured by a label-free acoustic separation device from whole blood samples of mice, of which breast tumours were surgically removed, and tumour metastasis was successfully predicted. First, an acoustofluidic device with a custom-designed bottom microcavity array was fabricated to induce highly localised acoustic microstreaming by applying acoustic vibration. Second, experiments of capturing 'defined' cells (artificially mixed individual 4T1 cancer cells into normal blood) based on optimal acoustic streaming were performed. The separation device exhibited a high capture efficiency (>96%). Further applications of capturing the 'true' CTCs derived from postoperative mice were successfully developed to predict tumour prognosis based on the number of captured CTCs. Finally, the prediction was verified through long-term observation of mice with excised tumours. The acoustofluidic device can efficiently capture CTCs and precisely predict tumour metastasis in a low-cost and non-invasive manner. This will help clinicians monitor patients that underwent surgical resection of tumours over a long period of time and facilitate optimal treatment strategies in a timely manner.

A Versatile Optoelectronic Tweezer System for Micro-Objects Manipulation: Transportation, Patterning, Sorting, Rotating and Storage.

Non-contact manipulation technology has a wide range of applications in the manipulation and fabrication of micro/nanomaterials. However, the manipulation devices are often complex, operated only by professionals, and limited by a single manipulation function. Here, we propose a simple versatile optoelectronic tweezer (OET) system that can be easily controlled for manipulating microparticles with different sizes. In this work, we designed and established an optoelectronic tweezer manipulation system. The OET system could be used to manipulate particles with a wide range of sizes from 2 µm to 150 µm. The system could also manipulate micro-objects of different dimensions like 1D spherical polystyrene microspheres, 2D rod-shaped euglena gracilis, and 3D spiral microspirulina. Optical microscopic patterns for trapping, storing, parallel transporting, and patterning microparticles were designed for versatile manipulation. The sorting, rotation, and assembly of single particles in a given region were experimentally demonstrated. In addition, temperatures measured under different objective lenses indicate that the system does not generate excessive heat to damage bioparticles. The non-contact versatile manipulation reduces operating process and contamination. In future work, the simple optoelectronic tweezers system can be used to control non-contaminated cell interaction and micro-nano manipulation.

Identification of novel candidate genes in heterotaxy syndrome patients with congenital heart diseases by whole exome sequencing.

Heterotaxy syndrome (HS) involves dysfunction of multiple systems resulting from abnormal left-right (LR) body patterning. Most HS patients present with complex congenital heart diseases (CHD), the disability and mortality of HS patients are extremely high. HS has great heterogeneity in phenotypes and genotypes, which have rendered gene discovery challenging. The aim of this study was to identify novel genes that underlie pathogenesis of HS patients with CHD. Whole exome sequencing was performed in 25 unrelated HS cases and 100 healthy controls; 19 nonsynonymous variants in 6 novel candidate genes (FLNA, ITGA1, PCNT, KIF7, GLI1, KMT2D) were identified. The functions of candidate genes were further analyzed in zebrafish model by CRISPR/Cas9 technique. Genome-editing was successfully introduced into the gene loci of flna, kmt2d and kif7, but the phenotypes were heterogenous. Disruption of each gene disturbed normal cardiac looping while kif7 knockout had a more prominent effect on liver budding and pitx2 expression. Our results revealed three potential HS pathogenic genes with probably different molecular mechanisms.

On-chip rotational manipulation of microbeads and oocytes using acoustic microstreaming generated by oscillating asymmetrical microstructures.

The capability to precisely rotate cells and other micrometer-sized biological samples is invaluable in biomedicine, bioengineering, and biophysics. We propose herein a novel on-chip cell rotation method using acoustic microstreaming generated by oscillating asymmetrical microstructures. When the vibration is applied to a microchip with our custom-designed microstructures, two different modes of highly localized microvortices are generated that are utilized to precisely achieve in-plane and out-of-plane rotational manipulation of microbeads and oocytes. The rotation mechanism is studied and verified using numerical simulations. Experiments of the microbeads are conducted to evaluate the claimed functions and investigate the effects of various parameters, such as the frequency and the driving voltage on the acoustically induced flows. Accordingly, it is shown that the rotational speed and direction can be effectively tuned on demand in single-cell studies. Finally, the rotation of swine oocytes is involved as further applications. By observing the maturation stages of M2 after the exclusion of the first polar body of operated oocytes, the proposed method is proved to be noninvasive. Compared with the conventional approaches, our acoustofluidic cell rotation approach can be simple-to-fabricate and easy-to-operate, thereby allowing rotations irrespective of the physical properties of the specimen under investigation.

The complete mitochondrial genome of Electrona carlsbergi (Myctophiformes, Myctophidae) with phylogenetic consideration.

The complete mitochondrial genome of Electrona carlsbergi was obtained, which was 18,282 bp in size and including 13 protein-coding genes, 2 ribosomal RNAs, 23 transfer RNAs and 1 control region. The overall nucleotide composition is 27.92% for A, 24.66% for T, 30.90% for C and 16.52% for G. Among 23 tRNA genes, 8 tRNAs were encoded on the L-strand. Further, the phylogenetic tree, which based on complete mtDNA sequences, revealed that the E. carlsbergi was genetically closest to species E. antarctica and Krefftichthys anderssoni. This study could provide a basic data for the studies on evolution for low temperature adaptability, stock evaluation and conservation genetics.

The complete mitochondrial genome of Chionodraco rastrospinosus (Notothenioidei: Channichthyidae) with phylogenetic consideration.

In this study, the complete mitochondrial genome of Chionodraco rastrospinosus was obtained, which was 17598 bp including 2 ribosomal RNAs, 13 protein-coding genes, 22 transfer RNAs, and a non-coding control region. The length of D-loop was 1332 bp and its contents of A, T, C, and G were 30.3%, 27.6%, 26.8%, and 15.3%. The complete mtDNA sequences of C. rastrospinosus and other 14 species were used to reconstruct the phylogenetic tree, suggested that C. rastrospinosus was closest to two species of Chionodraco. The study would provide a basic data for further research on population structure, conservation genetics and molecular evolution of C. rastrospinosus.

The complete mitochondrial genome of Neopagetopsis ionah (Channichthyidae, neopagetopsis) with phylogenetic consideration.

In this study, the complete mitochondrial genome of Neopagetopsis ionah was obtained, which was 17,634 bp including two ribosomal RNAs, 13 protein-coding genes, 22 transfer RNAs, and a non-coding control region. In 13 protein-coding genes, there types of initiation codon (ATC, ATG, and GTG) and four types of stop codons (TAA, TAG, TA, and T) were identified. Among the 22 transfer RNAs, eight tRNAs were encoded by L-strand. The length of D-loop was 1519 bp and its contents of A, T, C, and G were 26.9%, 27.6%, 17.5%, and 30%, respectively. The complete mtDNA sequences of N. ionah and other 13 species were used to reconstruct the phylogenetic tree suggested that N. ionah was closest to some species of Channichthyidae. The study would provide a basic data for further research on population structure, conservation genetics and molecular evolution of N. ionah.