Controllable Fabrication of Highly Ordered Spherical Microcavity Arrays by Replica Molding of In Situ Self-Emulsified Droplets.

Ordered spherical hollow micro- and nanostructures hold great appeal in the fields of cell biology and optics. However, it is extremely challenging for standard lithography techniques to achieve spherical micro-/nanocavities. In this paper, we describe a simple, cost-effective, and scalable approach to fabricate highly ordered spherical microcavity arrays by replica molding of in situ self-emulsified droplets. The in situ self-emulsion involves a two-step process: discontinuous dewetting-induced liquid partition and interfacial tension-driven liquid spherical transformation. Subsequent replica molding of the droplets creates spherical microcavity arrays. The shapes and sizes of the microcavities can be easily modulated by varying the compositions of the droplet templates or utilizing an osmotically driven water permeation. To demonstrate the utility of this method, we employed it to create a spherical microwell array for the mass production of embryoid bodies with high viability and minimal loss. In addition, we also demonstrated the optical functions of the generated spherical microcavities by using them as microlenses. We believe that our proposed method will open exciting avenues in fields ranging from regenerative medicine and microchemistry to optical applications.

3D co-culture of macrophages and fibroblasts in a sessile drop array for unveiling the role of macrophages in skin wound-healing.

Three-dimensional (3D) heterotypic multicellular spheroid models play important roles in researches of the proliferation and remodeling phases in wound healing. This study aimed to develop a sessile drop array to cultivate 3D spheroids and simulate wound healing stage in vitro using NIH-3T3 fibroblasts and M2-type macrophages. By the aid of the offset of surface tension and gravity, the sessile drop array is able to transfer cell suspensions to spheroids via the superhydrophobic surface of each microwell. Meanwhile, each microwell has a cylinder hole at its bottom that provides adequate oxygen to the spheroid. It demonstrated that the NIH-3T3 fibroblast spheroid and the 3T3 fibroblast/M2-type macrophage heterotypic multicellular spheroid can form and maintain physiological activities within nine days. In order to further investigate the structure without destroying the entire spheroid, we reconstructed its 3D morphology using transparent processing technology and the Z-stack function of confocal microscopy. Additionally, a nano antibody-based 3D immunostaining assay was used to analyze the proliferation and differentiation characteristics of these cells. It found that M2-type macrophages were capable of promoting the differentiation of 3T3 fibroblast spheroid. In this study, a novel, inexpensive platform was constructed for developing spheroids, as well as a 3D immunofluorescence method for investigating the macrophage-associated wound healing microenvironment.

Controlled Rehydration of Dried Reagents for Robust Multiplex Digital PCR.

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).

Nanogap Electrode-Enabled Versatile Electrokinetic Manipulation of Nanometric Species in Fluids.

Noninvasive manipulation of nanoscopic species in liquids has attracted considerable attention due to its potential applications in diverse fields. Many sophisticated methodologies have been developed to control and study nanoscopic entities, but the low-power, cost-effective, and versatile manipulation of nanometer-sized objects in liquids remains challenging. Here, we present a dielectrophoretic (DEP) manipulation technique based on nanogap electrodes, with which the on-demand capturing, enriching, and sorting of nano-objects in microfluidic systems can be achieved. The dielectrophoretic control unit consists of a pair of swelling-induced nanogap electrodes crossing a microchannel, generating a steep electric field gradient and thus strong DEP force for the effective manipulation of nano-objects microfluidics. The trapping, enriching, and sorting of nanoparticles and DNAs were performed with this device to demonstrate its potential applications in micro/nanofluidics, which opens an alternative avenue for the non-invasive manipulation and characterization of nanoparticles such as DNA, proteins, and viruses.

Identification of Genes Involved in Resistance to High Exogenous 20-Hydroxyecdysone in Spodoptera litura.

To prevent their ingestion by phytophagous insects, plants produce secondary metabolites as defensive weapons. Conversely, insects need to counter these metabolites to survive. Different species, though they are closely related, can evolve distinct strategies to resist plant-derived factors. However, the mechanism under this high divergence resistance is still unclear at a molecular level. In this study, we focus on how Spodoptera litura (Lepidoptera; Noctuidae) detoxifies phytoecdysteroids, a class of metabolites capable of disrupting the normal development of insects. Firstly, we find that the S. litura show resistance to artificial foods containing a high level of 20-hydroxyecdysone (20E), the major form of phytoecdysteroids, without any adverse effects on growth and development. Furthermore, a comparative transcriptomic analysis between S. litura and another noctuid insect (Helicoverpa armigera) was performed. Almost all known ecdysteroid degradation pathways including 3-epimerization, 22-phosphorylation, 22-esterification, and 26-hydroxylation were upregulated in the midgut of 20E treated S. litura larvae, whereas only 22-esterification and 26-hydroxylation were enhanced in H. armigera larvae. In summary, though both species belong to the Noctuidae family, they evolved two different strategies to tolerate a high dosage of ingested 20E.

Semisupervised Consistent Projection Metric Learning for Person Reidentification.

Person reidentification is a hot topic in the computer vision field. Many efforts have been paid on modeling a discriminative distance metric. However, existing metric-learning-based methods are a lack of generalization. In this article, the poor generalization of the metric model is argued as the biased estimation problem that the independent identical distribution hypothesis is not valid. The verification experimental result shows that there is a sharp difference between the training and test samples in the metric subspace. A semisupervised consistent projection metric-learning method is proposed to ease the biased estimation problem by learning a consistent constrained metric subspace in which the identified pairs are forced to follow the distribution of the positive training pairs. First, a semisupervised method is proposed to generate potential matching pairs from the k -nearest neighbors of test samples. The potential matching pairs are used to estimate the distances' distribution center of the positive test pairs. Second, the metric subspace is improved by forcing this estimation to be close to the center of the positive training pairs. Finally, extensive experiments are conducted on five datasets and the results demonstrate that the proposed method reaches the best performance, especially on the rank-1 identification rate.

A superhydrophobic chip integrated with an array of medium reservoirs for long-term hanging drop spheroid culture.

Hanging drop (HD) is one of the most popular methods used for forming three-dimensional (3D) cell spheroids. However, conventional hanging drop systems are only applicable for short-term spheroid culture due to their inconvenience in exchanging cell culture media. Here we present a medium-reservoir-integrated superhydrophobic (MRI-SH) chip for long-term HD spheroid cultures. The device consists of two main components: i) a patterned superhydrophobic (SH) surface containing an array of wettable spots which anchor arrays of droplets of cell suspension, and ii) an array of chambers that serve as medium reservoirs, both interconnected via an array of thru-holes. This configuration provides two distinct advantages over conventional HD configurations: i) the high wettability contrast of the SH pattern on the chip leads to the formation and adhesion of nearly spherical hanging droplets on its surface, which minimizes interactions between the liquid and the substrate; ii) the integrated chambers provide large volumes of medium to maintain longer culture durations. Using this device, spheroids of MHCC97H cells were successfully formed, and the cultured spheroids could maintain high viability for up to 30 days and exhibited enhanced spheroid morphology compared to those cultured in the conventional HD systems. STATEMENT OF SIGNIFICANCE: This paper presents a medium-reservoir-integrated superhydrophobic hanging drop (HD) platform for the long-term culture of spheroids with enhanced morphology. By monolithically integrating medium reservoirs and a patterned SH surface into a single device, this HD platform can not only produce high-quality spheroids, but also permit them to sustain high viability for up to 30 days without the need for tedious medium replenishment. We believe that such a platform will be valuable in a wide range of biological or biomedical applications, including tissue engineering, regenerative medicine, and drug discovery.

A hand-powered microfluidic system for portable and low-waste sample discretization.

In this work, we present a simple and equipment-free system for discretizing samples into tens of thousands of discrete volumes in tens of seconds. Unlike conventional sample discretization systems that require bulky syringe pumps, pressure controllers, or vacuum equipment, our system requires only a sheet of water-soluble film, a hand-operated syringe, and a microfluidic device containing a high-density microchamber array. In this system, the water-soluble film seals the device inlet to form a closed channel-chamber system, while the syringe is used to create a vacuum in the closed system. Benefitting from the high negative pressure created by syringe-vacuum and the dissolution-triggered gating mechanism of the sealing water-soluble film, the aqueous sample loaded into the device inlet can be rapidly partitioned into tens of thousands of isolated chambers without the need for any expensive pumping systems. We demonstrated the utility of this system by exploiting it for digital PCR. We believe that this simple discretization system will find broad applications, such as in digital bioassays, single-cell analysis, and point-of-care diagnostics.

The dynamic landscape of gene regulation during Bombyx mori oogenesis.

BACKGROUND: Oogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined. RESULTS: In this study, we obtained time-series transcriptome data and used these data to reveal the dynamic landscape of gene regulation during oogenesis. A total of 1932 genes were identified to be differentially expressed among different stages, most of which occurred during the transition from late vitellogenesis to early choriogenesis. Using weighted gene co-expression network analysis, we identified six stage-specific gene modules that correspond to multiple regulatory pathways. Strikingly, the biosynthesis pathway of the molting hormone 20-hydroxyecdysone (20E) was enriched in one of the modules. Further analysis showed that the ecdysteroid 20-hydroxylase gene (CYP314A1) of steroidgenesis genes was mainly expressed in previtellogenesis and early vitellogenesis. However, the 20E-inactivated genes, particularly the ecdysteroid 26-hydroxylase encoding gene (Cyp18a1), were highly expressed in late vitellogenesis. These distinct expression patterns between 20E synthesis and catabolism-related genes might ensure the rapid decline of the hormone titer at the transition point from vitellogenesis to choriogenesis. In addition, we compared landscapes of gene regulation between silkworm (Lepidoptera) and fruit fly (Diptera) oogeneses. Our results show that there is some consensus in the modules of gene co-expression during oogenesis in these insects. CONCLUSIONS: The data presented in this study provide new insights into the regulatory mechanisms underlying oogenesis in insects with polytrophic meroistic ovaries. The results also provide clues for further investigating the roles of epigenetic reconfiguration and circadian rhythm in insect oogenesis.

A color gamut description algorithm for liquid crystal displays in CIELAB space.

Because the accuracy of gamut boundary description is significant for gamut mapping process, a gamut boundary calculating method for LCD monitors is proposed in this paper. Within most of the previous gamut boundary calculation algorithms, the gamut boundary is calculated in CIELAB space directly, and part of inside-gamut points are mistaken for the boundary points. While, in the new proposed algorithm, the points on the surface of RGB cube are selected as the boundary points, and then converted and described in CIELAB color space. Thus, in our algorithm, the true gamut boundary points are found and a more accurate gamut boundary is described. In experiment, a Toshiba LCD monitor's 3D CIELAB gamut for evaluation is firstly described which has regular-shaped outer surface, and then two 2D gamut boundaries ( CIE-a*b* boundary and CIE-C*L* boundary) are calculated which are often used in gamut mapping process. When our algorithm is compared with several famous gamut calculating algorithms, the gamut volumes are very close, which indicates that our algorithm's accuracy is precise and acceptable.