Nile Red Staining Research Articles

Inverse molecular docking reveals a novel function of thymol: Inhibition of fat deposition induced by high-dose glucose in Caenorhabditis elegans.

As a natural product isolated from thyme oil in thyme, thymol (2‐isopropyl‐5‐methylphenol) harbors antiviral, antioxidant, and other properties, and thus could be potentially used for the treatment of various diseases. However, the function of thymol has not been comprehensively studied. Here, we applied an inverse molecular docking approach to identify unappreciated functions of thymol. Potential targets of thymol in humans were identified by the server of DRAR‐CPI, and targets of interest were then assessed by GO and KEGG pathway analysis. Subsequently, homologous proteins of these targets in Caenorhabditis elegans were identified by Blast tool, and their three‐dimensional structures were achieved using Swiss‐Model workspace. Interaction between thymol and the targeted proteins in worms was verified using AutoDock 4.0. Analyses of the targets revealed that thymol could be potentially involved in the glycolysis/gluconeogenesis and fatty acid degradation pathways. To verify the activity of thymol on lipid deposition in vivo, the C. elegans model was established. The lipid content of nematodes induced by high‐dose glucose was determined by Oil Red O and Nile Red staining, and gene expression was assessed by qRT‐PCR. The results showed that thymol might lead to the acceleration of β‐oxidation by upregulating cpt‐1, aco, fabp, and tph‐1, causing the descent of lipid content in nematodes. Our findings indicated that thymol could be potentially used for the treatment of chronic metabolic diseases associated with increased fatty acid deposition.

PRL-1 overexpressed placenta-derived mesenchymal stem cells suppress adipogenesis in Graves\u2019 ophthalmopathy through SREBP2/HMGCR pathway

BackgroundGraves’ ophthalmopathy (GO) is a disorder, in which orbital connective tissues get in inflammation and increase in volume. Stimulants such as thyroid-stimulating hormone (TSH), insulin-like growth factor 1(IGF-1), IL-1, interferon γ, and platelet-derived growth factor cause differentiation into adipocytes of orbital fibroblasts (OFs) in the orbital fat and extraocular muscles. Human placental mesenchymal stem cells (hPMSCs) are known to have immune modulation effects on disease pathogenesis. Some reports suggest that hPMSCs can elicit therapeutic effects, but to date, research on this has been insufficient. In this study, we constructed PRL-1 overexpressed hPMSCs (hPMSCsPRL-1) in an attempt to enhance the suppressive function of adipogenesis in GO animal models.MethodsIn order to investigate the anti-adipogenic effects, primary OFs were incubated with differentiation medium for 10 days. After co-culturing with hPMSCsPRL-1, the characteristics of the OFs were analyzed using Nile red stain and quantitative real-time polymerase chain reaction. We then examined the in vivo regulatory effectiveness of hPMSCsPRL-1 in a GO mouse model that immunized by leg muscle electroporation of pTriEx1.1Neo-hTSHR A-subunit plasmid. Human PMSCsPRL-1 injection was performed in left orbit. We also analyzed the anti-adipogenic effects of hPMSCsPRL-1 in the GO model.ResultsWe found that hPMSCsPRL-1 inhibited adipogenic activation factors, specifically PPARγ, C/EBPα, FABP4, SREBP2, and HMGCR, by 75.1%, 50%, 79.6%, 81.8%, and 87%, respectively, compared with naïve hPMSCs in adipogenesis-induced primary OFs from GO. Moreover, hPMSCsPRL-1 more effectively inhibited adipogenic factors ADIPONECTIN and HMGCR by 53.2% and 31.7%, respectively, than hPMSCs, compared with 15.8% and 29.8% using steroids in the orbital fat of the GO animal model.ConclusionOur findings suggest that hPMSCsPRL-1 would restore inflammation and adipogenesis of GO model and demonstrate that they could be applied as a novel treatment for GO patients.

An Insight into the Growing Concerns of Styrene Monomer and Poly(Styrene) Fragment Migration into Food and Drink Simulants from Poly(Styrene) Packaging.

Poly(styrene) (PS) has been heavily utilised in disposable food packaging due to its insulating properties, optical translucency, and long-shelf life. Despite these desirable characteristics, (PS) poses toxicity concerns to human’s health through styrene monomer leaching into foodstuffs. Environmental and marine hazards are another growing concerns due to improper and/or absence of recycling strategies and facilities. This preliminary work aims to investigate the effect of temperature, food composition and contact times on the migration of the styrene monomer from poly(styrene) food contact materials into food simulants. Poly(styrene) cups showed a relatively low level of styrene migration with the highest being 0.110 µg/mL, whereas food containers showed a much higher level of styrene leaching with up to 6 µg/mL. This could be due to an increase in the hydrophobicity of the simulants’ characteristics from low to high fat content and the increase in the testing temperatures from 5 °C to 70 °C. ANOVA statistical analysis is used to compare the means of three or more groups of data, whereas t-test analysis is used to compare means of two groups. This was carried out on each individual sample to determine the significance of changing the temperature, simulant type, or both on the level of migration observed in the results. All significant values were tested at 95% confidence level p < 0.05, concluding that fat content and high temperatures were found to significantly increase the level of styrene migration. Nile Red staining method was used to demonstrate that particulate poly(styrene), as well as styrene monomer, migrated into tested food simulants from typical containers, which is becoming a cause for concern as evidence of microplastic ingestion increases.

Upregulated monocyte expression of PLIN2 is associated with early arterial injury in children with overweight/obesity

Background and aimsPerilipin 2 (PLIN2) regulates intracellular lipid metabolism in macrophages, and thus, plays a role in atherosclerosis. Aim of the study was to evaluate whether PLIN2 dysregulation is involved in the onset of preclinical atherosclerosis in children with overweight/obesity and to explore dysregulation mechanisms. MethodsSixty-three children with overweight/obesity and 21 normal weight children (controls) of the same age and sex were enrolled. Carotid intima media thickness (cIMT) was evaluated; mRNA expression of PLIN2 and proteasome subunits (PSMD3, PSMC4) was determined by Real Time PCR, and protein expression of PLIN2, LAMP2A and Hsc70 by Western blot analysis; fluorimetric assay was used to measure proteasome chymotrypsin like activity. We performed transient LAMP2A downregulation by siRNA and quantified intracellular lipids in monocytes by Nile Red staining and flow cytometry analysis. ResultsPLIN2 protein levels were significantly higher in children with overweight/obesity and correlated with cIMT after adjusting for confounders. Accordingly, monocytes of children with overweight/obesity showed a higher intracellular amount of lipids compared with controls. mRNA expression of the regulatory subunits PSMC4 and PSMD3 and proteasome activity were lower in children with overweight/obesity, while expression of LAMP2A and Hsc70 proteins, which belong to the chaperone-mediated autophagy (CMA) pathway, was not different, suggesting that PLIN2 dysregulation in monocytes was due to an impairment of proteasome efficiency and was not CMA related. ConclusionPLIN2 was overexpressed in monocytes of children with overweight/obesity and could contribute to the onset of arteropathy. Our data suggest that proteasome impairment could contribute to PLIN2 overexpression.

Coffea arabica pulp aqueous extract attenuates oxidative stress and hepatic lipid accumulation in HepG2 cells

Coffea arabica pulp aqueous extract (CPE) contains several bioactive compounds including chlorogenic acid (CGA), caffeine, catechin and epicatechin. These constituents exhibit several biological activities including antibacterial, anti-inflammatory, antioxidant, and lipid-lowering activities. Therefore, it was hypothesized that polyphenol-rich CPE could exert hepatic lipid-lowering effects on a human hepatocyte cell line. The present study investigated the effect of CPE against oxidative stress and hepatic lipid accumulation in hepatocellular carcinoma (HepG2) cells. The major constituents in CPE were identified using nuclear magnetic resonance imaging, and the effect of CPE and its major constituents, CGA and caffeine, on intracellular oxidative stress and hepatic lipid accumulation were quantified using fluorescent 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and Nile red staining followed by flow cytometry. Total triglyceride (TG) and cholesterol (TC) contents and secretion were determined. Genes involving hepatic lipid metabolism and transporters were evaluated using real-time PCR. CPE and CGA exhibited antioxidant capacity against ABTS+, DPPH and hydrogen peroxide-induced oxidative stress without any cytotoxic effects in HepG2 cells. Intracellular lipid contents, TG and TC, were decreased and correlated with cholesterol secretion by CGA, caffeine and CPE. These findings were related mainly through downregulating peroxisome proliferator-activated receptor γ (PPAR γ), a lipogenic gene, and upregulating PPARα, a lipolytic gene. Correspondingly, CPE increased cholesterol efflux transporter ABCG5/8, decreased free fatty acid (FFA) uptake transporter fatty acid translocase (FAT/CD36) and strongly alleviated FFA-induced hepatic lipid accumulation. Taken together, CPE has effective antioxidant and hepatic lipid-lowering effects and could potentially be developed as a nutraceutical product to prevent dyslipidaemia-induced hepatic steatosis.

Asymbiotic germination of Vanilla planifolia in relation to the timing of seed collection and seed pretreatments

BackgroundVanilla planifolia is an important tropical orchid for production of natural vanilla flavor. Traditionally, V. planifolia is propagated by stem cuttings, which produces identical genotype that are sensitive to virulent pathogens. However, propagation with seed germination of V. planifolia is intricate and unstable because the seed coat is extremely hard with strong hydrophobic nature. A better understanding of seed development, especially the formation of impermeable seed coat would provide insights into seed propagation and conservation of genetic resources of Vanilla.ResultsWe found that soaking mature seeds in 4% sodium hypochlorite solution from 75 to 90 min significantly increased germination. For the culture of immature seeds, the seed collection at 45 days after pollination (DAP) had the highest germination percentage. We then investigated the anatomical features during seed development that associated with the effect of seed pretreatment on raising seed germination percentage. The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. Seeds at 60 DAP and subsequent stages germinated poorly. As the seed approached maturity, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope at maturity. On toluidine blue O staining, the wall of outer seed coat stained greenish blue, indicating the presence of phenolic compounds. As well, on Nile red staining, a cuticular substance was detected in the surface wall of the embryo proper and the innermost wall of the inner seed coat.ConclusionWe report a reliable protocol for seed pretreatment of mature seeds and for immature seeds culture based on a defined time schedule of V. plantifolia seed development. The window for successful germination of culturing immature seed was short. The quick accumulation of lignin, phenolics and/or phytomelanins in the seed coat may seriously inhibit seed germination after 45 DAP. As seeds matured, the thickened and lignified seed coat formed an impermeable envelope surrounding the embryo, which may play an important role in inducing dormancy. Further studies covering different maturity of green capsules are required to understand the optimal seed maturity and germination of seeds.

Lipid accumulation on optimized condition through biomass production in green algae

Microalgae, mainly green algae, are well known as sources of lipids for food, feed, and energy. As our institution owned microalgae collections, we performed microalgae screening to find candidate/s, producing high lipid content. In this research, microalgae were screened by various media, and then the medium was optimized. Larger scale cultivation was done in order to find out the yield of the biomass production and lipid content. The condition with phosphate buffer was applied to increase the lipid accumulation. The lipid content was analyzed by Nile red staining. As a result, Chlamydomonas reinhardtii and Chlorella vulgaris showed high biomass production in the Tris-Acetate-Phosphate (TAP) medium. Chlamydomonas reinhardtii at four times concentration and C. vulgaris at the initial concentration of TAP medium demonstrated the most increased biomass production. The maximum biomass production and lipid content was 0.9 g/l and 31% for C. reinhardtii and 1.7 g/l and 27% for C. vulgaris, respectively. Under starvation, the lipid content was increased up to 52% for C. reinhardtii and 34% for C. vulgaris, which were higher than their control. From this study, the TAP medium was found to have the most increased biomass production, and starvation enhanced lipid accumulation.

Zebrafish model of Gestational Diabetes

One in seven births in the US are affected by Gestational Diabetes Mellitus (GDM). In addition to the acute risk of death and complications for the fetus, GDM exposure in utero leads to an increase in the development of cardiovascular diseases, obesity, insulin resistance, type 2 diabetes and metabolic syndrome later in life. In utero fetal exposure to hyperglycemia leads to Non-Alcoholic Fatty Liver Disease (NAFLD) in adulthood, later in life, characterized by increased lipid droplets in hepatocytes. Although the consequences of GDM in infants have been documented, the cellular, molecular and physiological mechanisms underlying the link between fetal hyperglycemia and metabolic consequences later in life is poorly understood. We recently developed a model of fetal hyperglycemia in zebrafish embryo, by exposure of zebrafish embryos to high glucose (4.5% or 5% w/v) for 24 hours. Glucose level was detected in the fish homogenate by glucometer. Body composition was detected by EcoMRI. Gene expression of glycolytic and lipogenic markers (Glucokinase, Hexokinase, Pyruvate Kinase, Acetyl CoA Carboxylase, Fatty Acid Synthase) was quantified by RT-qPCR. Adiposity was assessed by Nile red staining and liver lipid droplet accumulation was performed to evaluate NAFLD. Additionally, the enzymes involved in Triglyceride and Cholesterol synthesis (Acetyl-CoA and Malonyl-CoA) were assessed by ELISA. Our data demonstrate that zebrafish embryos exposed to high glucose and raised to adulthood under normal glycemic conditions (normal fish diet) have significant increase in body mass index (826.6±26.34 Vs. 598.8± 33.89) g/m2and fat mass (12.07± 2.92 Vs 3.4± 0.4) g, develop obesity (by size), have increased adiposity and develop NAFLD, however these adults are normoglycemic. At the cellular and molecular level, our results show a differential increase in glycolytic and lipogenic gene expression. Moreover, there was significant increases in Acetyl-CoA (2.5-fold) and Malonyl-CoA (1.4-fold) levels that was associated with increased cholesterol (57.92± 0.7 Vs 34.8±1.01) uM and Triglyceride (4.1± 0.75 Vs 0.98±0.27) levels in embryos exposed to high glucose compared to control group. Together our results show that fetal exposure to glucose leads to metabolic consequences later in life including obesity and NAFLD under normal diet. We also concluded from our results that fetal hyperglycemia increases glycolysis and lipogenesis. Moreover, our project demonstrates that the zebrafish is an excellent system to model fetal hyperglycemia by focusing on direct glucose exposure of the embryo without the need of modifying the glucose levels of the mother. Experiments utilizing both genetic and pharmacological approaches are in progress to investigate if the molecular changes observed during fetal life under glucose exposure are persistent in the adults.

Role of m 6A Reader YTHDC2 in Differentiation of Human Bone Marrow Mesenchymal Stem Cells

To study the regulatory effect of YTH domain-containing protein 2 (YTHDC2), a member of N 6-methyladenosine (m 6A) readers, on the osteogenic or adipogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). YTHDC2 expression was knocked down by small interfering RNA (siRNA) in vitro. Osteogenic differentiation and adipogenic differentiation of hBMSCs were induced after YTHDC2 knockdown in order to study the changes in the differentiation phenotype of hBMSCs. Alkaline phosphatase staining (ALP staining) and alizarin red S staining were performed to examine osteogenic activity and calcium-nodular formation. Nile red staining was performed to examine lipid-droplet formation. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of osteogenesis and adipogenesis-related genes. RNA-sequencing was performed to identify the transcriptome changes after YTHDC2 knockdown and to explore the potential regulatory mechanism by which YTHDC2 regulated the diferentiation of hBMSCs. In this study, we found that siRNA-induced YTHDC2 knockdown resulted in increased ALP activity and calcium-nodular formation of hBMSCs during osteogenic differentiation, and significantly upregulated the expression of osteogenesis-related genes. In addition, the lipid-droplet formation capacity of hBMSCs was decreased during adipogenic differentiation. The expression of adipogenesis-related genes was significantly down-regulated. Gene-set enrichmen analysis of RNA-seq data showed that YTHDC2 was significantly correlated with ribosome function and mRNA-translation-related signaling pathways. The findings indicate that YTHDC2 knockdown can promote the osteogenic differentiation of hBMSCs and inhibit the adipogenic differentiation. YTHDC2 knockdown may cause changes in ribosome function.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

Anti-Bacterial Properties of Cannabigerol Toward Streptococcus mutans.

Streptococcus mutans (S. mutans) is a gram-positive facultatively anaerobic bacterium and the most common pathogen associated with tooth caries. The organism is acid tolerant and can undergo physiological adaptation to function effectively in acid environments such as carious dental plaque. Some cannabinoids have been found to have potent anti-microbial activity against gram-positive bacteria. One of these is the non-psychoactive, minor phytocannabinoid Cannabigerol (CBG). Here we show that CBG exhibits anti-bacterial activities against S. mutans. CBG halts the proliferation of planktonic growing S. mutans, which is affected by the initial cell density. High-resolution scanning electron microscopy showed that the CBG-treated bacteria become swollen with altered membrane structures. Transmission electron microscopy provided data showing that CBG treatment leads to intracellular accumulation of membrane structures. Nile red, DiOC2(3) and laurdan staining demonstrated that CBG alters the membrane properties, induces membrane hyperpolarization, and decreases the membrane fluidity. CBG-treated bacteria showed increased propidium iodide uptake and reduced calcein AM staining, suggesting that CBG increases the membrane permeability and reduces the metabolic activity. Furthermore, CBG prevented the drop in pH caused by the bacteria. In summary, we present here data showing the mechanisms by which CBG exerts its anti-bacterial effect against S. mutans.

Anandamide alters the membrane properties, halts the cell division and prevents drug efflux in multidrug resistant Staphylococcus aureus

Antibiotic resistance is a serious public health problem throughout the world. Overcoming methicillin and multidrug-resistant Staphylococcus aureus (MRSA/MDRSA) infections has become a challenge and there is an urgent need for new therapeutic approaches. We have previously demonstrated that the endocannabinoid Anandamide (AEA) can sensitize MRSA to antibiotics. Here we have studied the mechanism of action using a MDRSA clinical isolate that are sensitized by AEA to methicillin and norfloxacin. We found that AEA treatment halts the growth of both antibiotic-sensitive and antibiotic-resistant S. aureus. The AEA-treated bacteria become elongated and the membranes become ruffled with many protrusions. AEA treatment also leads to an increase in the percentage of bacteria having a complete septum, suggesting that the cell division is halted at this stage. The latter is supported by cell cycle analysis that shows an accumulation of bacteria in the G2/M phase after AEA treatment. We further observed that AEA causes a dose-dependent membrane depolarization that is partly relieved upon time. Nile red staining of the bacterial membranes indicates that AEA alters the membrane structures. Importantly, 4′-6-diamidino-2-phenylindole (DAPI) accumulation assay and ethidium bromide efflux (EtBr) assay unveiled that AEA leads to a dose-dependent drug accumulation by inhibiting drug efflux. In conclusion, our study demonstrates that AEA interferes with cell division, alters the membrane properties of MDRSA, and leads to increased intracellular drug retention, which can contribute to the sensitization of MDRSA to antibiotics.

Preparation of biological samples for microplastic identification by Nile Red

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 μm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94–100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97–100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

Selection of microplastics by Nile Red staining increases environmental sample throughput by micro-Raman spectroscopy

Nile Red staining enables visual identification and quantification of fluorescent particles as a proxy to microplastics at low cost and high throughput, including those of small sizes (≥2 μm), when preceded by proper natural organic matter removal, but providing no chemical characterization. On the other hand, micro-spectroscopy methods allow chemical characterization of particles based on their spectra, essential for polymer identification, but are costly and time-consuming. This work addresses the combination of both Nile Red staining with micro-Raman spectroscopy for the identification of microplastics. Besides being useful for quantification, Nile Red staining can be advantageously used as an objective criterion for pre-selection of particles for micro-Raman spectroscopy, producing little interference. The use of the 442 nm laser in micro-Raman spectroscopy induces Nile Red luminescence thus allowing to target the specific suspected microplastics when using an orange filter, reducing the number of particles subjected to identification and improving sample throughput. Staining dyes could also be used for mapping suspected microplastics before targeted analysis by micro-Raman spectroscopy. Thus, coupling Nile Red with micro-Raman spectroscopy can be useful to improve time efficiency while using this equipment.

Assessment of the mechanism of drug resistance in Trichophyton mentagrophytes in response to various substances

BackgroundTrichophyton mentagrophyte (TM), a zoonotic pathogen, has been endangering public health due to emerging drug resistance. Although increased attention is paid to this issue, there is very limited research available on drug resistance in TM. In this study, we studied the gene and proteomic changes, morphological changes, cellular fat localization, fat content changes, and biofilm of TM treated with different substances.ResultsThe TM growth curve showed a positive correlation with the concentration of Fenarimol (FE), genistein (GE), clotrimazole (KM), and Miconazole nitrate salt (MK). The morphology of TM cells changed in different degrees after treatment with different substances as observed by TEM and SEM. The results showed that under KM and berberine hydrochloride (BB) treatment, a total of 3305 differentially expressed genes were detected, with the highest number in the KM-treated group (578 up-regulated and 615 down-regulated). A total of 847 proteins and 1850 peptides were identified in TM proteomics. Nile red staining showed that the fat content of TM was significantly higher in the BB-, ethidium bromide- (EB), FE-, KM-, Adriamycin hydrochloride- (YA), and MK-treated group compared to the control group. Results of the biofilm thickness showed that it gradually increased under treatment with specific concentrations of KM or BB, which may be related to the up-regulation of ERG25 and CYP related gene proteins.ConclusionsIt is suggested that in order to effectively deal with dermatomycosis caused by TM, it is necessary to inhibit the expression of ERG25 and CYP related genes and fat metabolism, which can result in the inhibition of the production of biofilm by the fungus and solve the problem of fungal drug resistance in clinical settings.

Berberine regulates lipid metabolism via miR-192 in porcine oocytes matured in vitro.

BackgroundThe berberine (Ber) is an isoquinoline alkaloid compound extracted from Rhizoma coptidis and has the effect that reduces adipose. MicroRNA‐192 (miR‐192) is related to fat metabolism. However, the relevant mechanism of berberine on lipid metabolism during in vitro maturation (IVM) of porcine oocytes remains unclear.ObjectivesIn this study, we investigated the molecular mechanism by which berberine promotes the IVM and lipid metabolism of porcine oocytes via miR‐192.MethodsBer was added to IVM medium of porcine oocytes. MiR‐192 agomir, miR‐192 antagomir and negative control fragment were microinjected into the cytoplasm of oocytes without Ber. Rates of oocyte IVM and embryonic development in each group were observed. The content of lipid droplets in IVM oocytes in each group was analyzed by Nile red staining. Expression levels of miR‐192 and FABP3, SREBF1 and PPARG, were detected by qPCR and western blotting. The target genes of miR‐192 were determined by luciferase reporter assays.Results and ConclusionsWe found that Ber significantly increased the rate of oocytes IVM and blastocyst development, and decreased the area and numbers of lipid droplets in IVM oocytes. Ber significantly increased the expression of miR‐192 in IVM oocytes, and significantly decreased the expression of SREBF1 and PPARG, which were target genes of miR‐192. This study indicates that Ber promotes lipid metabolism in porcine oocytes by activating the expression of miR‐192 and down‐regulating SREBF1 and PPARG, thus, improving IVM of porcine oocytes.

Amphiphilic peptide-based MMP3 inhibitors for intra-articular treatment of knee OA

Since 2007, Metalloproteases (MMPs) have been considered potential targets for treating osteoarthritis (OA), for which the primary pathogenic event is the extensive degeneration of articular cartilage. MMP3 is an enzyme critical for these degenerative changes. However, problems of selectivity, low bioavailability and poor metabolic profile during clinical trials of MMPs inhibitors (MMPIs) led to limited beneficial effect and thus did not justify further pursuit of the clinical studies. In a previous work, a new alkyl derivative of hyaluronic acid (HA), HYADD4®, previously approved as intra-articular treatment for knee OA, was studied in vitro and in vivo as MMP3I. Molecular simulation studies confirmed the interaction between the alkyl side chain of this HA derivative and the additional S1′ pocket of MMP3. However, the high MW and the polar HA backbone of HYADD4® imply a high desolvation energy cost, which can potentially decrease its inhibitory potency. In this study, a new class of MMP3Is based on a small peptide backbone (CGV) chemically derivatized with an alkyl chain was developed through interactive cycles of design, synthesis and screening, accompanied by computational evaluation and optimization. Two MMP3Is, e(I) and l(II), were selected because of their effective inhibitory activity (3.2 and 10.2 µM, respectively) and water solubility. Both MMPIs showed a broad range of inhibitory effects against almost all the MMPs tested. In an in vitro model of inflammatory OA, e(I) was the most effective MMPI: at the concentration of 93 µM, it reversed inflammatory outcomes. Moreover, because of its amphiphilic structure, the e(I) MMPI promoted stable micellar formulation at concentrations higher than 0.2 mg/mL in water. The findings were confirmed by TEM and Nile red staining analysis. Based on these results, the e(I) MMPI can be considered a good candidate for the intra-articular treatment of OA, and the micellar formulation of this peptide in an aqueous buffer can potentially increase the bioavailability and, thus, the efficacy of the MMPIs.

Bundle sheath suberisation is required for C4 photosynthesis in a Setaria viridis mutant

C4 photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO2 concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C4 monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway. Here, Nile red staining, TEM, and GC/MS confirmed the alteration in suberin deposition in the BS cell wall of the mutant. We show that this has disrupted the suberin lamellae of BS cell wall and increased BS conductance to CO2 diffusion more than two-fold in the mutant. Consequently, BS CO2 partial pressure is reduced and CO2 assimilation was impaired in the mutant. Our findings provide experimental evidence that a functional suberin lamellae is an essential anatomical feature for efficient C4 photosynthesis in NADP-ME plants like S. viridis and have implications for engineering strategies to ensure future food security.

Ginsenoside Rb1 Alleviates Alcohol-Induced Liver Injury by Inhibiting Steatosis, Oxidative Stress, and Inflammation.

Alcoholic liver disease (ALD) has become a heavy burden on health worldwide. Ginsenoside Rb1 (GRb1), extracted from Panax quinquefolium L., has protective effects on many diseases, but the effect and mechanisms of GRb1 on ALD remain unknown. This study aimed to investigate the protective effects of GRb1 on ALD and to discover the potential mechanisms. Zebrafish larvae were exposed to 350 mM ethanol for 32 h to establish a model of acute alcoholic liver injury, and the larvae were then treated with 6.25, 12.5, or 25 μM GRb1 for 48 h. The human hepatocyte cell line was stimulated by 100 mM ethanol and meanwhile incubated with 6.25, 12.5, and 25 μM GRb1 for 24 h. The lipid changes were detected by Oil Red O staining, Nile Red staining, and triglyceride determination. The antioxidant capacity was assessed by fluorescent probes in vivo, and the expression levels of inflammatory cytokines were detected by immunohistochemistry, immunofluorescence, and quantitative real-time PCR. The results showed that GRb1 alleviated lipid deposition in hepatocytes at an optimal concentration of 12.5 μM in vivo. GRb1 reversed the reactive oxygen species accumulation caused by alcohol consumption and partially restored the level of glutathione. Furthermore, GRb1 ameliorated liver inflammation by inhibiting neutrophil infiltration in the liver parenchyma and downregulating the expression of nuclear factor-kappa B pathway-associated proinflammatory cytokines, including tumor necrosis factor-α and interleukin-1β. This study revealed that GRb1 has a protective effect on alcohol-induced liver injury due to its resistance to lipid deposition as well as antioxidant and anti-inflammatory actions. These findings suggest that GRb1 may be a promising candidate against ALD.

Microplastics and fibers from three areas under different anthropogenic pressures in Douro river.

Sources contributing to specific concentration of microplastics and fibers are still not completely understood. This study aimed at assessing the concentrations of microplastics (2-5000μm) and fibers (18-5667μm) in three areas of distinct influences in the Douro river, Porto, Portugal: (i) a countryside area; (ii) a wastewater treatment effluent release zone; and (iii) an area in proximity to a boat dock and maintenance station. Nile Red staining coupled with microscopy allowed the identification of small microplastics (≥2μm) with a median concentration of the three areas of 231MPL-1. Most were fragments (69%). Sizes <40μm were the most abundant (84%). Highest concentrations of microplastics were found near the boat dock/maintenance and lowest in the countryside area. Fibers were mostly natural (non-synthetic, 63%). Highest concentrations of fibers were found in the area influenced by the wastewater effluent, especially of synthetic fibers, and lowest in the countryside area. Concentration of all fibers and synthetic fibers was 46FL-1 and 6FL-1, respectively. High concentrations of microplastics and fiber contamination suggest that the wastewater treatment plant effluent and boat dock/maintenance are the likely sources originating hotspot areas.