Droplet Contents Research Articles

Electrokinetic Desalting and Salting of Water-in-Oil Droplets.

Droplet-based microfluidic platforms demand modifications to the droplet composition to facilitate reactions and analyses. However, limited techniques exist to modify the droplet contents post their generation. Here, ion transport across two ion-exchange membranes possessing distinct selectivity is employed to introduce ions into (salt) or extract ions from (desalt) water-in-oil droplets. The ion concentration distribution and transport mechanisms are visualized using a precipitation reaction and a charged fluorescent tracer. Furthermore, current measurements reveal characteristic regimes in desalting and salting modes and demonstrate that the rates of ion transport linearly correlate with applied voltage and the ionic strength of the droplets. Importantly, up to 98% desalting efficiency is achieved. This technique advances droplet-based sample preparation through the straightforward manipulation of droplet contents.

Knockdown of PROX1 promotes milk fatty acid synthesis by targeting PPARGC1A in dairy goat mammary gland

Goat milk is rich in various fatty acids that are beneficial to human health. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-seq analyses of goat mammary glands at different lactation stages revealed a novel lactation regulatory factor, Prospero homeobox 1 (PROX1). However, the mechanism whereby PROX1 regulates lipid metabolism in dairy goats remains unclear. We found that PROX1 exhibits the highest expression level during peak lactation period. PROX1 knockdown enhanced the expression of genes related to de novo fatty acid synthesis (e.g., SREBP1 and FASN) and triacylglycerol (TAG) synthesis (e.g., DGAT1 and GPAM) in goat mammary epithelial cells (GMECs). Consistently, intracellular TAG and lipid droplet contents were significantly increased in PROX1 knockdown cells and reduced in PROX1 overexpression cells, and we observed similar results in PROX1 knockout mice. Following PROX1 overexpression, RNA-seq showed a significant upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) expression. Further, PPARGC1A knockdown attenuated the inhibitory effects of PROX1 on TAG contents and lipid-droplet formation in GMECs. Moreover, we found that PROX1 promoted PPARGC1A transcription via the PROX1 binding sites (PBSs) located in the PPARGC1A promoter. These results suggest a novel target for manipulating the goat milk-fat composition and improving the quality of goat milk.

Nazo, the Drosophila homolog of the NBIA-mutated protein-c19orf12, is required for triglyceride homeostasis.

Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as Neurodegeneration with Brain Iron Accumulation (NBIA). Here, we uncover the physiological role of Nazo, the Drosophila melanogaster homolog of the NBIA-mutated protein-c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo is required for maintaining normal levels of Perilipin-2, an inhibitor of the lipase-Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin-2 rescues the nazo phenotype, suggesting that this defect, at least in part, may arise from diminished Perilipin-2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings potentially provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.

Chrysoeriol Improves the Early Development Potential of Porcine Oocytes by Maintaining Lipid Homeostasis and Improving Mitochondrial Function.

Our previous study established that chrysoeriol (CHE) can reduce reactive oxygen species (ROS) accumulation, apoptosis, and autophagy in vitro culture (IVC) of porcine embryos. However, the role of CHE in oocyte maturation and lipid homeostasis is unclear. Herein, we aimed to elucidate the effect of CHE on porcine oocyte competence in vitro maturation (IVM) and subsequent embryo development. The study chooses parthenogenetic activated porcine oocytes as the research model. The study revealed that the cumulus expansion index and related gene expressions are significantly elevated after supplementing 1 μM CHE. Although there were no significant differences in nuclear maturation and cleavage rates, the blastocyst formation rate and total cell numbers were significantly increased in the 1 μM CHE group. In addition, CHE improved the expression of genes related to oocyte and embryo development. ROS was significantly downregulated in all CHE treatment groups, and intracellular GSH (glutathione) was significantly upregulated in 0.01, 0.1, and 1 μM CHE groups. The immunofluorescence results indicated that mitochondrial membrane potential (MMP) and lipid droplet (LD), fatty acid (FA), ATP, and functional mitochondria contents significantly increased with 1 μM CHE compared to the control. Furthermore, CHE increased the expression of genes related to lipid metabolism, mitochondrial biogenesis, and β-oxidation.

Acanthopanax senticosus ameliorates steatohepatitis through HNF4 alpha pathway activation in mice

Non-alcoholic fatty liver disease is a common liver disease worldwide, and is associated with dysregulation of lipid metabolism, leading to inflammation and fibrosis. Acanthopanax senticosus Harms (ASH) is widely used in traditional medicine as an adaptogen food. We examined the effect of ASH on steatohepatitis using a high-fat diet mouse model. Mice were fed a choline-deficient, l-amino acid-defined, high-fat diet with ASH extract (ASHE). After 6 weeks, liver RNA transcriptome sequencing (RNA-Seq) was performed, followed by Ingenuity Pathway Analysis (IPA). Our findings revealed that mice fed a high-fat diet with 5% ASHE exhibited significantly reduced liver steatosis. These mice also demonstrated alleviated inflammation and reduced fibrosis in the liver. IPA of RNA-Seq indicated that hepatocyte nuclear factor 4 alpha (HNF4 alpha), a transcription factor, was the activated upstream regulator (P-value 0.00155, z score = 2.413) in the liver of ASHE-fed mice. Adenosine triphosphate binding cassette transporter 8 and carboxylesterase 2, downstream targets of HNF4 alpha pathway, were upregulated. Finally, ASHE-treated HepG2 cells exposed to palmitate exhibited significantly decreased lipid droplet contents. Our study provides that ASHE can activate HNF4 alpha pathway and promote fat secretion from hepatocytes, thereby serving as a prophylactic treatment for steatohepatitis in mice.

Reaction acceleration at the surface of a levitated droplet by vapor dosing from a partner droplet.

Chemical reactions in micrometer-sized droplets can be accelerated by up to six orders of magnitude. However, this acceleration factor (ratio of rate constants relative to bulk) drops to less than 10 for millimeter-sized droplets due to the reduction in surface/volume ratio. To enhance the acceleration in millimeter-sized droplets, we use a new synthesis platform that directly doses reagent vapor onto the reaction droplet surface from a second levitated droplet. Using Katritzky transamination as a model reaction, we made quantitative measurements on size-controlled vapor-dosed droplets, revealing a 31-fold increase in reaction rate constants when examining the entire droplet contents. This enhancement is attributed to a greater reaction rate constant in the droplet surface region (estimated as 105 times greater than that for the bulk). The capability for substantial reaction acceleration in large droplets highlights the potential for rapid synthesis of important chemicals at useful scales. For example, we successfully prepared 23 pyridinium salts within minutes. This efficiency positions droplets as an exceptional platform for rapid, in situ catalyst synthesis. This is illustrated by the preparation of pyridinium salts as photocatalysts and their subsequent use in mediation of amine oxidation both within the same droplet.

Effects of ultra-high pressure assisted extraction on the structure, antioxidant and hypolipidemic activities of Porphyra haitanensis polysaccharides

Porphyra haitanensis polysaccharides (PHPs) have potential antioxidant and hypolipidemic activities, but still need improvement. Therefore, ultra-high pressure (UHP) assisted extraction was applied to modify the structure, antioxidant, and hypolipidemic activities of PHPs. UHP assisted extraction increased the total sugar, uronic acid, and 3,6-anhydro-ʟ-galactose contents of PHP, which increased by 15.85 %–16.12 %, 18.95 %–24.32 %, 20.54 %–23.66 % with 500–600 MPa UHP, respectively. Meanwhile, UHP modified PHP became more rough and porous than native PHP. Besides, UHP assisted extracted PHP showed better in vitro antioxidant and hypolipidemic abilities. Especially, 500–600 MPa UHP increased 72.43 %–86.42 % 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacities, 12.32 %–12.82 % pancreatic lipase inhibitory ability, and 14.93 %–15.23 % glycocholate binding abilities of PHP (p < 0.05). Moreover, UHP assisted extracted PHP greatly decreased the lipid droplet and triglyceride contents of 3T3-L1 adipocytes (p < 0.05). Our findings can provide the theoretical basis for the high value utilization of Porphyra haitanensis and its polysaccharides with UHP modification.

The Lipid Metabolism as Target and Modulator of BOLD-100 Anticancer Activity: Crosstalk with Histone Acetylation.

The leading first-in-class ruthenium-complex BOLD-100 currently undergoes clinical phase-II anticancer evaluation. Recently, BOLD-100 is identified as anti-Warburg compound. The present study shows that also deregulated lipid metabolism parameters characterize acquired BOLD-100-resistant colon and pancreatic carcinoma cells. Acute BOLD-100 treatment reduces lipid droplet contents of BOLD-100-sensitive but not -resistant cells. Despite enhanced glycolysis fueling lipid accumulation, BOLD-100-resistant cells reveal diminished lactate secretion based on monocarboxylate transporter 1 (MCT1) loss mediated by a frame-shift mutation in the MCT1 chaperone basigin. Glycolysis and lipid catabolism converge in the production of protein/histone acetylation substrate acetyl-coenzymeA (CoA). Mass spectrometric and nuclear magnetic resonance analyses uncover spontaneous cell-free BOLD-100-CoA adduct formation suggesting acetyl-CoA depletion as mechanism bridging BOLD-100-induced lipid metabolism alterations and histone acetylation-mediated gene expression deregulation. Indeed, BOLD-100 treatment decreases histone acetylation selectively in sensitive cells. Pharmacological targeting confirms histone de-acetylation as central mode-of-action of BOLD-100 and metabolic programs stabilizing histone acetylation as relevant Achilles' heel of acquired BOLD-100-resistant cell and xenograft models. Accordingly, histone gene expression changes also predict intrinsic BOLD-100 responsiveness. Summarizing, BOLD-100 is identified as epigenetically active substance acting via targeting several onco-metabolic pathways. Identification of the lipid metabolism as driver of acquired BOLD-100 resistance opens novel strategies to tackle therapy failure.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 μg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 μg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Multiparametric, Label-Free Analysis of Microfluidic Droplets via a Dynamic Phase Grating Approach.

Droplet-based microfluidic systems possess many fundamental advantages as a platform for the analysis of chemical and biological species. However, whereas on-chip operations have rapidly developed over the past decades, approaches for analyzing target molecules within droplets have largely remained limited to methods requiring bulky and expensive instrumentation. In this work, we describe a droplet analysis approach whereby the droplet train itself is the sensing construct. Specifically, the droplet train is interrogated as a transmission phase grating, allowing high-throughput, label-free, solution-phase, and multi-parametric analysis of droplet contents. Importantly, three distinct properties of generated droplets can be simultaneously extracted using this conceptually simple and experimentally straightforward measurement approach. Under constant droplet generation conditions, measurement of droplet viscosity is achieved by monitoring changes in zero order to first order peak separation in the far-field diffraction pattern, with a sensitivity of 2.28 × 10-4 cSt per μm change in peak separation. In parallel, measurement of droplet refractive index (RI) is achieved by measuring changes in the ratio of the zero order to first order peak intensity, with a sensitivity of 2.14 × 10-4 RI units per unit change in a diffracted peak intensity ratio. Finally, droplet generation frequency is determined from the time-varying oscillation of the peak height ratio, yielding comparable results to an expensive high-speed camera commonly used for droplet imaging. Importantly, the experimental strategy for this approach is straightforward and does not require expensive instrumentation; therefore, it may find utility in affordable and portable analysis approaches applied to diverse droplet microfluidic assays.

Myc derived circRNA promotes triple-negative breast cancer progression via reprogramming fatty acid metabolism

Myc is a well-known proto-oncogene that is frequently amplified and activated in breast cancer, especially in triple-negative breast cancer (TNBC). However, the role of circular RNA (circRNA) generated by Myc remains unclear. Herein, we found that circMyc (hsa_circ_0085533) was remarkably upregulated in TNBC tissues and cell lines, which was attributed to gene amplification. Genetic knockdown of circMyc mediated by lentiviral vector significantly inhibited TNBC cell proliferation and invasion. Importantly, circMyc increased cellular triglycerides, cholesterols and lipid droplet contents. CircMyc was detected in both cytoplasm and nucleus, cytoplasmic circMyc could directly bind to HuR protein, facilitating the binding of HuR to SREBP1 mRNA, resulting in increasing SREBP1 mRNA stability. Nuclear circMyc bound to Myc protein, facilitating the occupation of Myc on SREBP1 promoter, leading to increasing SREBP1 transcription. As a result, the elevated SREBP1 increased the expression of its downstream lipogenic enzymes, enhancing lipogenesis and TNBC progression. Moreover, the orthotopic xenograft model showed that depletion of circMyc markedly inhibited lipogenesis and reduced tumor size. Clinically, high circMyc was closely related to larger tumor volume, later clinical stage and lymph node metastasis, functioning as an adverse prognostic factor. Collectively, our findings characterize a novel Myc-derived circRNA controlling TNBC tumorigenesis via regulation of metabolic reprogramming, implying a promising therapeutic target.

Numerical modeling and quantification of droplet mixing using mechanowetting

Capillary forces are often found in nature to drive fluid flow, and methods have been developed aimed to exploiting these forces in microfluidic systems to move droplets or mix droplet contents. Mixing of small fluid volumes, however, is challenging due to the laminar nature of the flow. Here, we show that mechanowetting, i.e., the capillary interaction between droplets and deforming surfaces, can effectively mix droplet contents. By concentrically actuating the droplet, vortex-like flow patterns are generated that promote effective mixing. To quantify the degree of mixing, we introduce two strategies that are able to determine mixer performance independent of the initial solute distribution within a droplet, represented by single scalars derived from a matrix-based method. We compare these strategies to existing measures and demonstrate the full decoupling from the initial condition. Our results can be used to design efficient mixers, featuring mechanowetting as a new enabling technology for future droplet mixers.

Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes

Triacylglycerols (TAGs) are a major component of intramuscular fat. Diacylglycerol O-acyltransferase 2(DGAT2) expression determines the rate of TAG synthesis. The purpose of this study was to investigate the role of DGAT2 in the differentiation of Yanbian cattle preadipocytes and lipid metabolism-related signalling pathways. Bovine preadipocytes were infected with overexpression and interfering adenovirus vectors of DGAT2. The effects on the differentiation of Yanbian cattle preadipocytes were examined using molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. DGAT2 overexpression significantly increased (p < 0.05) intracellular TAG, adiponectin, and lipid droplet (LD) contents. Moreover, it upregulated (p < 0.05) peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α, and fatty acid binding protein 4 mRNA expression. In contrast, DGAT2 knockdown reduced intracellular TAG and LD content and downregulated (p < 0.05) C/EBPβ, mannosyl (alpha-1,3-)-glycoproteinbeta-1,2-N-acetylglucosaminyltransferase, lipin 1,1-acylglycerol-3-phosphate O-acyltransferase 4, and acetyl-CoA carboxylase alpha mRNA expression. Between DGAT2-overexpressing preadipocytes and normal cells, 208 DEGs were identified, including 106 upregulated and 102 downregulated genes. KEGG pathway analysis revealed DEGs mainly enriched in PPAR signalling and AMP-activated protein kinase pathways, cholesterol metabolism, and fatty acid biosynthesis. These results demonstrated that DGAT2 regulated preadipocyte differentiation and LD and TAG accumulation by mediating the expression of adipose differentiation-, lipid metabolism-, and fatty acid synthesis-related genes.

Interfacial Tension Driven Open Droplet Microfluidics

AbstractDroplet microfluidics enables compartmentalized reactions in small scales and is utilized for a variety of applications across chemical analysis, material science, and biology. While droplet microfluidics is a successful technology, barriers include high “activation energy” to develop custom applications and complex peripheral equipment. These barriers limit the adoption of droplet microfluidics in labs or prototyping environments. This work demonstrates for the first time an open channel droplet microfluidic system that autonomously generates droplets at low capillary numbers. Hundreds of droplets are produced in a run using only an open channel, pipettes, and a commercially available carrier fluid. Conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting are demonstrated. The open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, a new method of droplet sorting and transfer unique to open systems is demonstrated. This platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power‐free open droplet microfluidic systems and provides design rules for similar channel designs.

Fatty acid composition and metabolic partitioning of α-linolenic acid are contingent on life stage in human CD3+ T lymphocytes.

Immune function changes across the life course; the fetal immune system is characterised by tolerance while that of seniors is less able to respond effectively to antigens and is more pro-inflammatory than in younger adults. Lipids are involved centrally in immune function but there is limited information about how T cell lipid metabolism changes during the life course. We investigated whether life stage alters fatty acid composition, lipid droplet content and α-linolenic acid (18:3ω-3) metabolism in human fetal CD3+ T lymphocytes and in CD3+ T lymphocytes from adults (median 41 years) and seniors (median 70 years). Quiescent fetal T cells had higher saturated (SFA), monounsaturated fatty acid (MUFA), and ω-6 polyunsaturated fatty acid (PUFA) contents than adults or seniors. Activation-induced changes in fatty acid composition differed between life stages. The principal metabolic fates of [13C]18:3ω-3 were constitutive hydroxyoctadecatrienoic acid synthesis and β-oxidation and carbon recycling into SFA and MUFA. These processes declined progressively across the life course. Longer chain ω-3 PUFA synthesis was a relatively minor metabolic fate of 18:3ω-3 at all life stages. Fetal and adult T lymphocytes had similar lipid droplet contents, which were lower than in T cells from seniors. Variation in the lipid droplet content of adult T cells accounted for 62% of the variation in mitogen-induced CD69 expression, but there was no significant relationship in fetal cells or lymphocytes from seniors. Together these findings show that fatty acid metabolism in human T lymphocytes changes across the life course in a manner that may facilitate the adaptation of immune function to different life stages.

A Periodic Wetting Surface Driven by Triboelectric Nanogenerator for Efficient Postimpact Droplet Collection

AbstractThe phenomenon of droplet impact is widely observed in nature and industrial processes. No matter the intrinsic wettability (hydrophobic, hydrophilic, and superhydrophobic) of the surface, the postimpact droplets tend to rebound or splash. The droplet impact dynamics have been well investigated but the inhibition of the rebounding or splashing of the postimpact droplet requires further exploration. This paper proposes a periodic wetting surface (PWS) driven by a triboelectric nanogenerator (TENG) for efficient postimpact droplet collection. With the high‐voltage electric signal generated from a rotating freestanding triboelectric nanogenerator (RF‐TENG), the wettability of an interdigital electrodes‐based electrowetting‐on‐dielectric (EWOD) surface can be changed periodically. The droplet impacts on the PWS show oscillating sliding motion that compensates the kinetic energy for droplet rebounding or splashing. As compared to pure hydrophobic or hydrophilic surfaces, the PWS shows the best droplet collection performance. The effects of the electric signal of the RF‐TENG such as open‐circuit voltage (0.44–3.00 kV) and frequency (0–60 Hz), droplet contents such as ions type and concentration of the droplet on the postimpact droplet collection efficiency are discussed. Finally, a practical application is demonstrated by the TENG driven PWS for real‐time and high‐efficiency collection of multiple postimpact droplets.

Early-life perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) exposure cause obesity by disrupting fatty acids metabolism and enhancing triglyceride synthesis in Caenorhabditis elegans

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are widely used and considered as emerging persistent pollutants, posing a potential threat to the aquatic ecosystem due to their metabolic toxicity. However, the effects of early-life PFOA and PFOS exposure on metabolic disruption and underlying mechanisms are not fully understood. Therefore, we investigated the effects of early-life PFOA or PFOS exposure on lipid accumulation, feeding behaviors, fatty acids composition, and possible genetic regulation using the nematode Caenorhabditis elegans as an in vivo model. Our results showed that low concentrations of PFOA and PFOS (0.1 and 1 μM) induced obesity in C. elegans, which was not due to the increased feeding rate. The altered fatty acid composition illustrated the decrease of saturated fatty acids and the increase of polyunsaturated fatty acids. Furthermore, the mutant assay and mRNA levels revealed that fatty acid desaturation related genes mdt-15, nhr-49, fat-6 as well as fatty acid (fasn-1) and triglyceride (TG) (dgat-2) synthesis related genes, were associated with the increased body fat, TG, and lipid droplet (LD) contents in C. elegans exposed to PFOA and PFOS. Hence, this present study provides the genetic regulatory information of PFOA and PFOS induced metabolic disruption of lipid metabolism and obesity.

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials.

Arginine Regulates Zygotic Genome Activation in Porcine Embryos Under Nutrition Restriction.

Arginine has a positive effect on pre-implantation development in pigs. However, the exact mechanism by which arginine promotes embryonic development is undefined. Here, single-cell RNA sequencing technology was applied to porcine in vivo pre-implantation embryos from the zygote to morula stage, it was found that that the expression of arginine metabolism-related genes clearly changed from the 2-cell stage to the 4-cell stage, when zygotic genome activation (ZGA) occurs in porcine embryos. Further analysis showed that arginine metabolism-related genes are significantly correlated with key ZGA genes. To determine the function of arginine in porcine embryos during ZGA, the in vitro fertilization embryos were cultured in PZM-3 medium (0.12 mM arginine, Control group), a modified PZM-3 medium (0 mM arginine, Block group) and a modified PZM-3 medium supplemented with arginine (0.12 mM arginine, Block + Arg group). The results showed that the 4-cell arrest rate was significantly increased in the Block group compared to the Control group (P < 0.05). The 4-cell arrest rate in the Block + Arg group was significantly decreased than that in the Block group (P < 0.05). Meanwhile, the expression of ZGA marker genes and SIRT1 protein in 4-cell embryos was significantly decreased in the Block group compared to the Control group, and their expression was significantly increased in the Block + Arg group. In addition, we observed that the glutathione (GSH), ATP levels, and lipid droplet contents were significantly increased, and the reactive oxygen species (ROS) level was decreased in the Block + Arg group compared to the Block group. Compared with Control group, spermine content in culture medium and the mRNA expression of ornithine decarboxylase1 (ODC1) of embryos in the Block group were significantly decreased (P < 0.05), and those in the Block + Arg group were significantly increased compared with the Block group (P < 0.05). Moreover, when difluoromethylornithine (an inhibitor of ODC1) was added to the modified PZM-3 medium supplemented with arginine, the effect of arginine on ZGA was inhibited. In summary, our findings demonstrated that arginine may regulate ZGA under nutrition restriction in porcine embryos by promoting polyamine synthesis.

MiR-146a-5p, targeting ErbB4, promotes 3T3-L1 preadipocyte differentiation through the ERK1/2/PPAR-γ signaling pathway

BackgroundMicroRNAs (MiRNAs) are known to participate in preadipocyte differentiation, but the manner in which miR-146a-5p participates in this process remains unclear. This study was performed to examine the participation of miR-146a-5p in 3T3-L1 cell differentiation.Material and MethodsmiR-146a-5p expression was upregulated and down-regulated to examine effects on 3T3-L1 cell differentiation. Bioinformatics analysis was performed to predict its target genes, and the signaling pathway it regulates was identified by qRT-PCR and Western blotting. The expression of miR-146a-5p in epididymal adipose tissue from obese mice and in an obese mouse adipose cell model was examined by qRT-PCR.Results3T3-L1 cells differentiated into mature adipocytes successfully, as verified by increased areas of intracellular lipid droplets and elevated expression of mature adipocyte markers, and these cells had elevated miR-146a-5p expression. The intracellular lipid droplet and triglyceride contents and the expression of mature adipocyte markers were significantly increased in miR-146a-5p–overexpressing 3T3-L1 cells and markedly decreased in miR-146a-5p–inhibited 3T3-L1 cells. ErbB4 was a predicted target gene of miR-146a-5p. In miR-146a-5p–overexpressing 3T3-L1 cells, ErbB4 expression and ERK1/2 phosphorylation were decreased and the expression of PPAR-γ was increased; the opposite was observed in miR-146a-5p–inhibited 3T3-L1 cells. In addition, miR-146a-5p expression was significantly increased in the mouse epididymal adipose tissue and adipose cell model.ConclusionsUpregulated miR-146a-5p expression was related to 3T3-L1 cell differentiation. MiR-146a-5p promoted 3T3-L1 cell differentiation by targeting ErbB4 and via the ERK1/2/PPAR-γ signaling pathway.