Production Of Droplets Research Articles

CO2-Laser-Micromachined, Polymer Microchannels with a Degassed PDMS slab for the Automatic Production of Monodispersed Water-in-Oil Droplets.

In our recent study, we fabricated a pump/tube-connection-free microchip comprising top and bottom polydimethylsiloxane (PDMS) slabs to produce monodispersed water-in-oil droplets in a fully automated, fluid-manipulation fashion. All microstructures required for droplet production were directly patterned on the surfaces of the two PDMS slabs through CO2-laser micromachining, facilitating the fast fabrication of the droplet-production microchips. In the current extension study, we replaced the bottom PDMS slab, which served as a microfluidic layer in the microchip, with a poly(methyl methacrylate) (PMMA) slab. This modification was based on our idea that the bottom PDMS slab does not contribute to the automatic fluid manipulation and that replacing the bottom PDMS slab with a more affordable and accessible, ready-to-use polymer slab, such as a PMMA, would further facilitate the rapid and low-cost fabrication of the connection-free microchips. Using a new PMMA/PDMS microchip, we produced water-in-oil droplets with high degree of size-uniformity (a coefficient of variation for droplet diameters of <5%) without a decrease in the droplet production rate (~270 droplets/s) as compared with that achieved via the previous PDMS/PDMS microchip (~220 droplets/s).

Sea Spray Impacts on Tropical Cyclone Olwyn Using a Coupled Atmosphere‐Ocean‐Wave Model

AbstractExtreme marine weather enhances the production of sea spray droplets which, in turn, affect the air‐sea fluxes. Despite the ability of sea spray to modulate air‐sea interaction processes, it remains under‐represented in tropical cyclone (TC) forecasting modeling. In this study, impacts of sea spray on the atmospheric and oceanic environments of TC Olwyn are investigated. This is achieved by high‐resolution nested simulations using an air‐sea‐wave model coupled with a sea spray production model. We compared two different sea spray models: a wave‐Reynolds‐number‐dependent model, and a wave‐steepness‐dependent spray model. By including a sea spray model, the root mean square error in the simulated TC Olwyn 10 m wind and significant wave height are significantly reduced by up to 30% and 26%, respectively, in comparison to the baseline simulation without spray. This improvement is because sea spray decreases air‐sea heat fluxes at larger radii of TC, and decreases air‐sea heat fluxes at smaller radii of TC. In contrast to the simulated results without sea spray, sea spray increases the air‐sea heat (sensible + latent) energy transfer and intensifies TC Olwyn. Sea spray also triggers stronger upper‐ocean mixing and strengthens the TC‐induced sea surface temperature cooling. Our results thus imply that sea spray critically impacts the atmosphere and ocean conditions during extreme marine weather and thus needs to be considered explicitly in TC forecasting modeling.

Production of cholesterol-like molecules impacts Escherichia coli robustness, production capacity, and vesicle trafficking

The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories.

Gradient Droplet Arrays by Acceleration‐Mode Dip‐Coating

AbstractDroplet microarray technology is of great interest in biology and chemistry as it allows for significant reactant savings and massive parallelization of experiments. Upon scaling down the footprint of each droplet in an array, it becomes increasingly challenging to produce the array drop‐by‐drop. Therefore, techniques for parallelized droplet production are developed, e.g., dip‐coating of biphilic substrates. However, it is in general difficult to tailor the characteristics of individual droplets, such as size and content, without updating the substrate. Here, the method of dip‐coating of uniformly patterned biphilic substrates in so‐called “acceleration‐mode” to produce droplet arrays featuring gradients in droplet height for fixed droplet footprint is developed. The results herein present this method applied to produce drops with base diameters varying over orders of magnitude, from as high as 6 mm to as small as 50 µm; importantly, the experimentally measured power‐law‐dependency of volume on capillary‐number matches analytical theory for droplet formation on heterogenous substrates though the precise quantitative values likely differ due to 2D substrate patterning. Gradient characteristics, including average droplet volume, steepness of the gradient, and its monotonicity, can all be tuned by changing the dip‐coating parameters, thus providing a robust method for high‐throughput screening applications and experiments.

A systematic exploration of ginsenoside Rg5 reveals anti-inflammatory functions in airway mucosa cells

BackgroundHyperactivated airway mucosa cells overproduce mucin and cause severe breathing complications. Here, we aimed to identify the effects of saponins derived from Panax ginseng on inflammation and mucin overproduction. MethodsNCI–H292 cells were pre-incubated with 16 saponins derived from P. ginseng, and mucin overproduction was induced by treatment with phorbol 12-myristate 13-acetate (PMA). Mucin protein MUC5AC was quantified by enzyme-linked immunosorbent assay, and mRNA levels were analyzed using quantitative polymerase chain reaction (qPCR). Moreover, we performed a transcriptome analysis of PMA-treated NCI–H292 cells in the absence or presence of Rg5, and differential gene expression was confirmed using qPCR. Phosphorylation levels of signaling molecules, and the abundance of lipid droplets, were measured by western blotting, flow cytometry, and confocal microscopy. ResultsGinsenoside Rg5 effectively reduced MUC5AC secretion and decreased MUC5AC mRNA levels. A systematic functional network analysis revealed that Rg5 upregulated cholesterol and glycerolipid metabolism, resulting in the production of lipid droplets to clear reactive oxygen species (ROS), and modulated the mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways to regulate inflammatory responses. Rg5 induced the accumulation of lipid droplets and decreased cellular ROS levels, and N-acetyl-l-cysteine, a ROS inhibitor, reduced MUC5AC secretion via Rg5. Furthermore, Rg5 hampered the phosphorylation of extracellular signal-regulated kinase and p38 proteins, affecting the NF-κB signaling pathway and pro-inflammatory responses. ConclusionRg5 alleviated inflammatory responses by reducing mucin secretion and promoting lipid droplet-mediated ROS clearance. Therefore, Rg5 may have potential as a therapeutic agent to alleviate respiratory disorders caused by hyperactivation of mucosa cells.

Hydrogels for Single-Cell Microgel Production: Recent Advances and Applications.

Single-cell techniques have become more and more incorporated in cell biological research over the past decades. Various approaches have been proposed to isolate, culture, sort, and analyze individual cells to understand cellular heterogeneity, which is at the foundation of every systematic cellular response in the human body. Microfluidics is undoubtedly the most suitable method of manipulating cells, due to its small scale, high degree of control, and gentle nature toward vulnerable cells. More specifically, the technique of microfluidic droplet production has proven to provide reproducible single-cell encapsulation with high throughput. Various in-droplet applications have been explored, ranging from immunoassays, cytotoxicity assays, and single-cell sequencing. All rely on the theoretically unlimited throughput that can be achieved and the monodispersity of each individual droplet. To make these platforms more suitable for adherent cells or to maintain spatial control after de-emulsification, hydrogels can be included during droplet production to obtain “microgels.” Over the past years, a multitude of research has focused on the possibilities these can provide. Also, as the technique matures, it is becoming clear that it will result in advantages over conventional droplet approaches. In this review, we provide a comprehensive overview on how various types of hydrogels can be incorporated into different droplet-based approaches and provide novel and more robust analytic and screening applications. We will further focus on a wide range of recently published applications for microgels and how these can be applied in cell biological research at the single- to multicell scale.

Assessment of the risk of disease transmission during fine-needle aspiration cytology under ultrasound guidance by visualization of droplet and aerosol formation.

Fine-needle aspiration cytology (FNAC) under ultrasound guidance is clinically useful, but there is a risk of spreading infection by generating droplets of contaminated fluids during the procedure. Risk assessment to better control infection remains to be established. The aim of this study was to estimate infection risks during FNAC by visualization of droplet production and deposition using a simulation model. The simulation comprised a puncture needle, a device for holding the needle, and a fluid specimen containing fluorescent particles as a model. Simulating each step of FNAC (removal of the inner and outer cylinder and transferring the specimen onto a glass slide), the generation and deposition of droplets were visualized using a laser. After removal of the inner cylinder, an aerosol of droplets in the air surrounding the needle was observed. After removal of the outer cylinder, several large droplets precipitating onto the circumjacent surface were observed. From the beginning of transferring the specimen, a large amount of sizeable droplets first moving away and then precipitating was observed, followed by the production of a cluster of fine droplets drifting and spreading through the air. Here, the generation of droplets at each step of FNAC, precipitation of large droplets onto the circumjacent surface, and drifting and spreading through the air of fine droplets was visualized. These results emphasize the need for precautions to prevent the transmission of infectious agents during FNAC.

미세액적 제조를 위한 다층 액적 분할 미세유체 시스템 개발

In this study, we present the multilayered symmetrical droplet splitting microfluidic system for preparation of microspheres. The microfluidic device was fabricated by conventional photolithography and PDMS casting. Multiple layers of microfluidic channels for symmetrical droplet splitting were stacked and integrated into a device. Each layer was designed to obtain 16 microdroplets from one droplet by droplet splitting. The droplet size was controlled with flow rate of dispersed phase (DI-water) and continuous phase (Mineral Oil with 3 wt.% SPAN80) by using a syringe pump. The droplet splitting behavior and production rate were analyzed by high-speed camera and inverted microscope in one layer of the microfluidic device. Additionally, the droplet size and size distribution were observed in each layer of the microfluidic device. The droplet size could be controlled by flow control of two phase flows with high uniformity of droplet size less than 5% coefficient of variation.

Importance of the Precautionary Principle With Regard to the Risk of Exposure to Aerosols Containing Viral Loads of SARS-CoV-2 Present in Feces: In Perspective.

In COVID-19 infection, the emissions of droplets and aerosols produced by the respiratory tract of contaminated subjects may represent a high risk of spreading the SARS-COV-2 virus in the environment. Thus, studies have shown that there is, at least, another source of droplets and aerosols in which viral particles of SARS-COV-2 can be found. It happens after flushing of toilet to dispose of the stools of a patient who has contracted COVID-19. The presence of viral particles of SARS-COV-2 in the stool could be linked to the concentration of angiotensin-converting enzyme 2 (ACE2) found on the surface of intestinal cells. Therefore, there is a reason to wonder whether the emission of viral particles by activating a toilet flush could represent an important potential risk of contamination for health care workers. To investigate this hypothesis, we have correlated different studies on the production of droplets and aerosols as well as the presence of viral particles following flush of toilet. This pooling of these studies led to the following conclusion: the precautionary principle should be applied with regard to the potential risk represented by viral particles of SARV-COV-2 in the stool when flushing the toilet.

Curbing High‐Fat Induced Perturbations in Aortic Perivascular Adipose Tissue in Adolescent Male Sprague‐Dawley Rats: Soil as Therapy?

Directly attached to the outer wall of blood vessels, perivascular adipose tissue (PVAT) is a unique variant of fat that is an important paracrine modulator of vascular physiology. Due to the proximity and endocrine‐like characteristics of this tissue, PVAT can contribute to the regulation vascular contractility and endothelial function. PVAT can also assume several properties of brown adipose tissue, including increased vascularization, small lipid droplet formation, mitochondrial density, and production of cardiometabolic signaling intermediates. Importantly, these properties promote the metabolic activity of this tissue which appear to be associated with improved vascular health. However, such properties may be diminished by chronic deleterious stimuli such as a high‐fat diet (HFD) and disease conditions like metabolic syndrome. Under such pressures, PVAT may serve a more insidious role in vascular dysfunction and promote the development of cardiovascular disease. Chronic diet‐induced changes in the vasculature and associated tissues like PVAT may be occurring early in the life cycle, promoting degeneration and disease‐associated remodeling. Currently, examination of these tissues early in life remain relatively unexplored and may offer important insight into the identification of prevention strategies and therapeutic targets. Previously, our lab identified increased expression of genes in the aorta of HFD fed adolescent Sprague‐Dawley rats, highlighting potential pathways and signaling intermediates that may impact the development of early vascular dysfunction. Moreover, we have demonstrated that an organic mineral complex (OMC) derived from plant and soil fractions prevented symptoms of metabolic syndrome in these animals. OMC is a complex of minerals, trace elements, organic acids (particularly fulvic acid), nitrates, and various microbial degradation products from plant and animal origins. The objective of the current study is to investigate the effects of a HFD and OMC on the expression of key proteins in aortic PVAT that play a role in adipose beiging, metabolism, and mitochondrial biogenesis, and are thus expected to provide critical insight into early mitigation of vascular disease progression. Six‐week‐old male Sprague‐Dawley rats (n = 36) were divided into two dietary groups: Chow or HFD fed for 10 weeks. Animals were further divided (n = 6/group) and administered 0, 0.6, or 3.0 mg/mL OMC in their drinking water. After the 10‐week intervention PVAT surrounding the aorta was extracted from the animals and Western blot analyses were performed using antibodies for the proteins UCP1, SDHA, PI3K, GLUT4, and b‐actin (as a loading control). Overall, we hypothesize that OMC supplementation will minimize decreases in these key proteins involved in health associated PVAT metabolism and function.

Interactions With Plant Defences Isolate Sympatric Populations of an Herbivorous Mite

Host plant specialisation can promote evolutionary divergence between herbivore populations associated with different plant species. While the mechanisms by which specialist species exploit their hosts have been studied widely across taxa, less is known about the mechanisms that allow intraspecific variants to arise and to be maintained across spatial and temporal scales. To understand whether adaptations to plant defences against herbivory contribute to the co-existence of genetically distinct populations of an herbivore, we investigate the interaction between honeysuckle (Lonicera periclymenum) and sympatric specialist and generalist populations of the spider miteTetranychus urticae. We found that mite folivory induces the production of sticky droplets on honeysuckle, which have a defensive role: they increase mite mortality directly, and potentially indirectly by increasing the arrestment of a predator. We show that droplet induction and the preference to feed on honeysuckle depend on mite genotype, where the generalist avoids this host and the specialist suppresses droplet production. These traits are heritable and dominant in F1 hybrids between generalists and specialists. Selection pressure from honeysuckle and differences in host preference likely reduce the opportunity of mating encounters on this host. We propose that the interplay between selection from host plant defences and ecological barriers to hybridisation contribute to the persistence of genetically distinct populations of a single species in sympatry.

LPIN1 Induces Gefitinib Resistance in EGFR Inhibitor-Resistant Non-Small Cell Lung Cancer Cells.

Simple SummaryHere, we attempted to identify targets that could be used to overcome resistance toward epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). To accomplish this, we chose LPIN1 among the candidate targets that were identified from a previously performed genome-wide RNAi screening assay and validated it as a key factor regulating gefitinib resistance in EGFR-mutant NSCLC cells. We confirmed that LPIN1 depletion increased gefitinib sensitivity in drug-resistant H1650 NSCLC cells, as well as patient-derived YL05 lung cancer cells. Moreover, we found that LPIN1 expression was induced following gefitinib treatment, and activities of protein kinase C delta and nuclear factor kappa B, and lipid droplet formation were induced in an LPIN1-dependent manner. Additionally, we validated that targeting LPIN1 synergistically retarded tumor growth in an in vivo mouse xenograft model.Drug resistance limits the efficacy of targeted therapies, including tyrosine kinase inhibitors (TKIs); however, a substantial portion of the drug resistance mechanisms remains unexplained. In this study, we identified LPIN1 as a key factor that regulates gefitinib resistance in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) cells. Unlike TKI-sensitive HCC827 cells, gefitinib treatment induced LPIN1 expression and increased diacylglycerol concentration in TKI-resistant H1650 cells, followed by the activation of protein kinase C delta and nuclear factor kappa B (NF-κB) in an LPIN1-dependent manner, resulting in cancer cell survival. Additionally, LPIN1 increased the production of lipid droplets, which play an important role in TKI drug resistance. All results were recapitulated in a patient-derived EGFR-mutant NSCLC cell line. In in vivo tumorigenesis assay, we identified that both shRNA-mediated depletion and pharmaceutical inhibition of LPIN1 clearly reduced tumor growth and confirmed that gefitinib treatment induced LPIN1 expression and LPIN1-dependent NF-κB activation (an increase in p-IκBα level) in tumor tissues. These results suggest an effective strategy of co-treating TKIs and LPIN1 inhibitors to prevent TKI resistance in NSCLC patients.

Preparation of core-shell microcapsules based on microfluidic technology for the encapsulation, protection and controlled delivery of phycocyanin

Phycocyanin (PC) is a naturally active protein with extensive potential physiological functions. However, the poor stability and low bioavailability of PC have limited its development in nutraceuticals. Therefore, we developed an efficient microfluidic device to fabricate core-shell microcapsules for PC entrapment and colonic delivery. Microfluidics utilizes microchannels and fluid flow rates to shear mutually incompatible fluids to form dispersed emulsion droplets, providing an opportunity to investigate the structure and size of emulsions and precisely control the production of emulsion droplets. By introducing intermolecular interactions, PC was effectively immobilized in the cavity of the inner core sodium alginate (SA) gel, ensuring a high encapsulation efficiency of up to 98% for SA-E microcapsules. The release effects of microcapsules with different wall thicknesses were evaluated by simulating gastrointestinal juice digestion experiments. The results revealed that the microcapsules with 20–30 μm wall thickness were more stable and had superior release performance in gastrointestinal fluid compared with the microcapsules with 10 and 50 μm wall thickness. In addition, after the simulated digestion process in artificial gastrointestinal fluid, the PC embedded in SA-E microcapsules still remained the same structure and exhibited good antioxidant and immunomodulatory activities. The enhanced stability and bioavailability of PC suggested that SA-E microcapsules can be used for PC entrapment and colonic delivery.

Precise Control of Microcapillary Size in Microcapillary Films by Liquid‐Assisted Extrusion

AbstractMicrocapillary films (MCFs) are plastic films with an array of microcapillaries, which have been widely applied in microfluidics and bioanalytical techniques. However, the precise control of microcapillary size is still a problem hindering the industrial applications of MCFs because the high size uniformity of microcapillaries ensures the production of homogeneous droplets and the robust performance of immunoassays. Here, a novel approach is provided to precisely adjust the size of microcapillaries by liquid‐assisted extrusion and to investigate the combined effects of extrusion, drawing and liquid injection on the microcapillary size. Compared with gas‐assisted extrusion, the size uniformity is improved by at least 5% for 89% experiments and 10% for 42% experiments. Therefore, this method facilitates MCFs’ applications in droplet microfluidics and biotechnologies.

Experimental Investigation of Droplet Generation by Post-Breaking Plunger Waves

Understanding the droplet cloud and spray dynamics is important for the study of the ocean surface and marine boundary layer. The role that the wave energy and the type of wave breaking play in the resulting distribution and dynamics of droplets are yet to be understood. The aim of this work was to generate violent plunging breakers in the laboratory and analyze the spray production post-breaking, i.e. after the crest of the wave impacts in the free surface. The droplet sizes and their dynamics were measured with imaging techniques and the effect of different wind speeds on the droplet production was also considered. It was found that the mean radius increases with the wave energy content and the number of large droplets (radius > 1 mm) in the vertical direction increases with the presence of wind. Furthermore, the normalized distribution of droplet sizes is consistent with the distribution of ligament-mediated spray formation. Also, indications of turbulence affecting the droplet dynamics at wind speeds of 5 m/s were found. The amount of large droplets (radius > 1 mm) found in this work was larger than reported in field studies.

Distribution of liquid-liquid two-phase flow and droplet dynamics in asymmetric parallel microchannels

The numbering-up of microchannels in parallel is usually engineered to achieve high-throughput production of droplets, and the flow distribution of fluids and uniformity of droplets in parallel microchannels are core issues for droplet formation. In this work, the distribution of liquid–liquid two-phase flow and droplet dynamics in asymmetric parallel microchannels are investigated by using a high speed camera, and the factors affecting the stability and uniformity of droplets are explored. The dynamic behavior of droplet swarm in the rear cavity and its feedback effect on droplet generation at T-junctions are revealed. The microfluidic devices with various configurations are designed to investigate its effects on flow distribution. In addition, based on the principle of hydrodynamic resistance for the liquid-liquid two-phase flow in microchannels, the prediction for the distribution of liquid-liquid two-phase flow under slug flow is proposed.

The Small-Scale Instability of the Air–Water Interface Responsible for the Bag-Breakup Fragmentation

Abstract The “bag breakup” fragmentation is the dominant mechanism for spume droplet production in high winds, which substantially affects the ocean–atmosphere exchange processes. The amount of droplets ejected from the surface, as well as their typical sizes, is prescribed by the surface wind velocity and fetch. The corresponding empirical correlations were obtained only for the limited parameters of the laboratory environment. The applicability range can be extended through the construction of a theoretical model that describes the initiation of the bag-breakup fragmentation, estimates the fragmenting liquid volume prescribing the droplet sizes, and determines the dependence on the wind parameters. This paper presents such a model. First, we conducted linear stability analysis of small-scale disturbances at the water surface under a high wind; this showed that the small-scale ripples (about 1 cm) propagating against the wind in the surface wind drift following the reference frame grew fast due to the Kelvin–Helmholtz instability, when the wind friction velocity exceeded the threshold of about 1 m s−1. Given the weak dispersion, the nonlinear stage of evolution was addressed using the Riemann simple wave equation modified to describe the increasing disturbances. The analytical solution for the equation suggested the scaling of the volume of liquid undergoing the bag-breakup fragmentation and its dependence on in agreement with the laboratory data. Using the scaling, we calculated the statistics of the bag-breakup fragmentation based on the lognormal size distribution of the fragmenting objects. Significance Statement The “bag breakup” fragmentation is the dominant mechanism for generating spray in hurricane winds. The parameters of spray droplets substantially affect the exchange processes between the ocean and the atmosphere and, thereby, the development of sea storms. The rapid process of spray generation can only be studied in laboratory environments using sophisticated experimental techniques. To apply the laboratory data to field conditions, we need a theoretical model that describes the threshold for fragmentation initiation, the fragmenting liquid volume, which scales the size and number of spray droplets, their dependence on wind parameters, etc. In the present work, we suggest a simple analytical model of the bag-breakup initiation, verify it in the laboratory experiment, and suggest the statistical description of the fragmentation events.

A video-based analysis of situations bearing the risk of respiratory disease transmission during football matches

We aimed to analyze the number and type of contacts involving the risk of respiratory disease transmission during football match play. We analysed 50 matches from different playing levels. Two reviewers evaluated the contacts of all players in each match. We focused on between-player contacts, crowding, actions with potentially increased aerosol and droplet production and within-player hand-to-head contacts. We categorized the duels with direct contact into frontal and other ones and measured contact duration. The number of between-player contacts were similar between playing levels (median 28.3 [IQR 22.6, 33] contacts per player-hour). Frontal contacts summed up to 8% of all contacts. Contacts involving the head occurred less than once per player and match with none lasting longer than 3 s. Crowding included between two and six players and the duration was mostly less than 10 s. Aerosol and droplet producing activities were three to four times more frequent in adult compared to youth players. Our results suggest that the risk of respiratory pathogen transmission is low during football matches. This conclusion is based on the finding that most close contact situations are of short duration and on the fact that it is an outdoor sport.

Dysregulated m6A modification promotes lipogenesis and development of non-alcoholic fatty liver disease and hepatocellular carcinoma

Type 2 diabetes mellitus (DM2) is associated closely with non-alcoholic fatty liver disease (NAFLD) by affecting lipid metabolism, which may lead to non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). N6-methyladenosine (m6A) RNA methylation is an important epigenetic regulation for gene expression and is related to HCC development. We developed a new NAFLD model oriented from DM2 mouse, which spontaneously progressed to histological features of NASH, fibrosis, and HCC with high incidence. By RNA sequencing, protein expression and methylated RNA immunoprecipitation (MeRIP)-qPCR analysis, we found that enhanced expression of ACLY and SCD1 in this NAFLD model and human HCC samples was due to excessive m6A modification, but not elevation of mature SREBP1. Moreover, targeting METTL3/14 invitro increases protein level of ACLY and SCD1 as well as triglyceride and cholesterol production and accumulation of lipid droplets. m6A sequencing analysis revealed that overexpressed METTL14 binds to mRNA of ACLY and SCD1 and alters their expression pattern. Our findings demonstrate a new NAFLD mouse model that provides a study platform for DM2-related NAFLD and reveals a unique epitranscriptional regulating mechanism for lipid metabolism via m6A-modified protein expression of ACLY and SCD1.

High-Aspect-Ratio Microfluidic Channel with Parallelogram Cross-Section for Monodisperse Droplet Generation.

Droplet-based microfluidics has been widely used as a potent high-throughput platform due to various advantages, such as a small volume of reagent consumption, massive production of droplets, fast reaction time, and independent control of each droplet. Therefore, droplet microfluidic systems demand the reliable generation of droplets with precise and effective control over their size and distribution, which is critically important for various applications in the fields of chemical analysis, material synthesis, lab-on-a-chip, cell research, diagnostic test, and so on. In this study, we propose a microfluidic device with a high-aspect-ratio (HAR) channel, which has a parallelogram cross-section, for generating monodisperse droplets. The HAR channel was fabricated using simple and cheap MEMS processes, such as photolithography, anisotropic wet etching, and PDMS molding, without expensive equipment. In addition, the parallelogram cross-section channel structure, regarded as a difficult shape to implement in previous fabrication methods, was easily formed by the self-alignment between the silicon channel and the PDMS mold, both of which were created from a single crystal silicon through an anisotropic etching process. We investigated the effects of the cross-sectional shape (parallelogram vs. rectangle) and height-to-width ratio of microfluidic channels on the size and uniformity of generated droplets. Using the developed HAR channel with the parallelogram cross-section, we successfully obtained smaller monodisperse droplets for a wider range of flow rates, compared with a previously reported HAR channel with a rectangular cross-section.