Fabrication of Perfluoropolyether Microfluidic Devices Using Laser Engraving for Uniform Droplet Production.

A perfluoropolyether (PFPE)-based microfluidic device with cross-junction microchannels was fabricated with the purpose of producing uniform droplets. The microchannels were developed using CO2 laser engraving. PFPE was chosen as the main material because of its excellent solvent resistance. Polyethylene glycol diacrylate (PEGDA) was mixed with PFPE to improve the hydrophilic properties of the inner surface of the microchannels. The microchannels of the polydimethylsiloxane microfluidic device had a blackened and rough surface after laser engraving. By contrast, the inner surface of the microchannels of the PFPE-PEGDA microfluidic device exhibited a smooth surface. The lower power and faster speed of the laser engraving resulted in the development of microchannels with smaller dimensions, less than 30 µm in depth. The PFPE and PFPE-PEGDA microfluidic devices were used to produce uniform water and oil droplets, respectively. We believe that such a PFPE-based microfluidic device with CO2-laser-engraved microchannels can be used as a microfluidic platform for applications in various fields, such as biological and chemical analysis, extraction, and synthesis.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Microfluidic Bioreactor with Fibrous Micromixers for In Vitro mRNA Transcription.

A new type of microfluidic bioreactor with fibrous micromixers for the ingredient mixing and a long macrochannel for the in vitro transcription reaction was fabricated for the continuous production of mRNA. The diameter of the fibrous microchannels in the micromixers was tuned by using an electrospun microfibrous disc with different microfiber diameters. The micromixer with a larger diameter of fibrous microchannels exhibited a better mixing performance than the others. The mixing efficiency was increased to 0.95 while the mixture was passed through the micromixers, suggesting complete mixing. To demonstrate the continuous production of mRNA, the ingredients for in vitro transcription were introduced into the perfluoropolyether microfluidic bioreactor. The mRNA synthesized by the microfluidic bioreactor had the same sequence and in vitro/in vivo performances as those prepared by the bulk reaction. The continuous reaction in the microfluidic bioreactor with efficient mixing performance can be used as a powerful platform for various microfluidic reactions.

Advances in small molecule two-photon fluorescent trackers for lipid droplets in live sample imaging.

Two-photon fluorescent trackers for monitoring of lipid droplets (LDs) would be highly effective for illustrating the critical roles of LDs in live cells or tissues. Although a number of one-photon fluorescent trackers for labeling LDs have been developed, their usability remains constrained in live sample imaging due to photo damage, shallow imaging depth, and auto-fluorescence. Recently, some two-photon fluorescent trackers for LDs have been developed to overcome these limitations. In this mini-review article, the advances in two-photon fluorescent trackers for monitoring of LDs are summarized. We summarize the chemical structures, two-photon properties, live sample imaging, and biomedical applications of the most recent representative two-photon fluorescent trackers for LDs. Additionally, the current challenges and future research trends for the two-photon fluorescent trackers of LDs are discussed.

Cancer-Targeted Azo Dye for Two-Photon Photodynamic Therapy in Human Colon Tissue.

Inappropriate cancer management can be prevented by simultaneous cancer diagnosis, treatment, and real-time assessment of therapeutic processes. Here, we describe the design of a two-photon (TP) photosensitizer (PS), ACC-B, for high temporal and spatioselective near-infrared cancer therapy. ACC-B consisting of a biotin unit significantly enhanced the cancer sensitivity of the PS. Upon TP irradiation, ACC-B generated reactive oxygen species (ROS) through the type I photodynamic therapy (PDT) process and triggered highly selective cancer ablation. In addition, fluorescence microscopy images revealed that ACC-B-loaded live human colon tissues showed a marked difference in ACC-B uptake between normal and cancer tissues, and this property was used for real-time imaging. Upon 770 nm TP treatment, ACC-B generated ROS efficiently in live colon cancer tissues with high spatial selectivity. During PDT, ACC-B can provide in situ spatioselective visualization of cellular behavior and molecular information for therapeutic assessment in specific regions.

Dysregulation of the Acrosome Formation Network by 8-oxoguanine (8-oxoG) in Infertile Sperm: A Case Report with Advanced Techniques.

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10-7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility.

Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases.

Quantitative analysis of the bilateral coordination and gait asymmetry using inertial measurement unit-based gait analysis.

Inertial measurement unit (IMU)-based gait analysis can be used to quantitatively analyze the bilateral coordination and gait asymmetry (GA). The purpose of this study was to investigate changes in bilateral coordination and GA due to gait speed using an IMU based gait analysis and identify spatiotemporal factors affecting bilateral coordination and GA. Eighty healthy adults (40 men and 40 women) participated in the study. The mean age was 26.2 years, and the mean body mass index was 22.8 kg/m2. Three different walking speeds (80%, 100%, and 120% of preferred walking speed) on a treadmill were applied for 1 min of continuous level walking using a shoe-type IMU-based gait analysis system. The phase coordination index (PCI) and GA were calculated on three different walking speeds. Several variables (gait speed, height, body mass index, cadence, and step length) were analyzed as possible factors affecting the PCI and GA. Bilateral coordination and GA improved during fast walking (p = 0.005 and p = 0.019, respectively) and deteriorated during slow walking (p<0.001 and p = 0.008, respectively), compared with the participants' preferred walking speeds. The correlation analysis revealed that PCI was negatively correlated with step length at each walking condition and lower gait speed was negatively correlated with PCI and GA during slow walking. Both bilateral coordination and GA had a negative linear relationship with gait speed, showing an improvement in the fast walking condition and deterioration in the slow walking condition. Step length was the factor associated with the change in the bilateral coordination.

Addition of amino acid moieties to lapatinib increases the anti-cancer effect via amino acid transporters.

Anti-cancer agents delivered to cancer cells often show multi-drug resistance (MDR) due to expulsion of the agents. One way to address this problem is to increase the accumulation of anti-cancer agents in cells via amino acid transporters. Thus, val-lapatinib and tyr-lapatinib were newly synthesized by adding valine and tyrosine moieties, respectively, to the parent anti-cancer agent lapatinib without stability issues in rat plasma. Val-lapatinib and tyr-lapatinib showed enhanced anti-cancer effects versus the parent lapatinib in various cancer cell lines, including human breast cancer cells (MDA-MB-231, MCF7) and lung cancer cells (A549), but not in non-cancerous MDCK-II cells. A glutamine uptake study revealed that both val-lapatinib and tyr-lapatinib, but not the parent lapatinib, inhibited glutamine transport in MDA-MB-231 and MCF7 cells, suggesting the involvement of amino acid transporters. In conclusion, val-lapatinib and tyr-lapatinib have enhanced anti-cancer effects, likely due to an increased uptake of the agents into cancer cells via amino acid transporters. The present data suggest that amino acid transporters may be an effective drug delivery target to increase the uptake of anti-cancer agents, leading to one method of overcoming MDR in cancer cells.