A fractionation-based protocol to investigate RNA solubility phase transition during Xenopus oocyte maturation.

While RNAs are soluble in vitro, their solubility may be altered when incorporated into some protein complexes inside the cell. The solubility phase transition of RNAs is thus indicative of changes in the function and activity of RNAs. Here, we present a protocol for the assessment of RNA solubility phase transition during Xenopus oocyte maturation. We describe steps for sample preparation, cell fractionation, RNA extraction, real-time PCR, and analysis of the obtained results. For complete details on the use and execution of this protocol, please refer to Hwang et al. (2023).1.

Solubility phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains largely unclear. In this work, we discover the dynamic solubility phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere, and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus identifies important remodeling mechanisms that act on RNAs to control vertebrate OET.

Phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains an open question. In this work, we discover the dynamic phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus uncovers novel remodeling mechanisms that act on RNAs to regulate vertebrate OET.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon the loss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

Precise modulation of embryonic development through optogenetics.

The past decade has witnessed enormous progress in optogenetics, which uses photo-sensitive proteins to control signal transduction in live cells and animals. The ever-increasing amount of optogenetic tools, however, could overwhelm the selection of appropriate optogenetic strategies. In this work, we summarize recent progress in this emerging field and highlight the application of opsin-free optogenetics in studying embryonic development, focusing on new insights gained into optical induction of morphogenesis, cell polarity, cell fate determination, tissue differentiation, neuronal regeneration, synaptic plasticity, and removal of cells during development.

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

Correlation of attention deficit hyperactivity disorder with gut microbiota according to the dietary intake of Korean elementary school students.

We investigated the impact of dietary patterns on the gut microbiota and concentration of short-chain fatty acids in the feces of Korean elementary school students. The dietary intake and ADHD assessment of 40 Korean elementary school students were analyzed using a dish-based semi-quantitative food frequency questionnaire. Analysis of gut microbiota and short-chain fatty acids composition were performed using the real-time polymerase chain reaction, metagenomics, and gas chromatography methods. The dietary patterns of participants were divided into four groups: healthy, processed food, fish and shellfish, and meat. The participants were also divided into two groups according to their ADHD scores: 0-30, control group; over 30, ADHD group. The ADHD score of the processed food group was significantly higher than that of the healthy group. The processed food and ADHD groups showed significantly higher abundance of harmful bacteria, such as the Enterobacter, Escherichia coli, and Clostridium strains, and markedly lower abundance of beneficial bacteria, such as the Bifidobacterium and Ruminococcus strains, than the control group. The heat maps of metagenomics indicated that each group was separated into distinct clusters, and the processed food and ADHD groups showed significantly lower α-diversity of gut microbiota than the control group. In these groups, the concentration of acetate or butyrate in the feces was significantly lower than that in the control group. These results may indicate that imbalanced diets can disturb the colonic microbial balance and are likely to become a potential risk factor for the prevalence of ADHD.

Combined Toxicological Effects of Di (2-Ethylhexyl) Phthalate and UV-B Irradiation through Endoplasmic Reticulum Stress-Tight Junction Disruption in Human HaCaT Keratinocytes.

Di (2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer, and human exposure to DEHP is widespread and frequent. However, information about the combined effect of DEHP and ultraviolet (UV)-B on the skin are still limited. We investigated the cytotoxic effects of DEHP and UV-B on HaCaT keratinocytes and evaluated the related underlying mechanisms involving endoplasmic reticulum (ER) stress signals and the disruption of junction complexes as an effective target for skin inflammation. Our results revealed that co-treatment with DEHP and UV-B irradiation alleviated the cell cytotoxicity and markedly decreased X-box binding protein 1 (XBP1), endoplasmic reticulum oxidoreductase 1 alpha (Ero1α), and C/EBP homologous protein (CHOP) whereas a single dose of 40 mJ/cm2 UV-B generated mild ER stress to slightly less or similar levels as that seen with DEHP. DEHP was also shown to inhibit tight junctions (TJs) after UV-B irradiation, increased apoptosis by altering apoptotic gene Bax and stress kinases, JNK, and p38 MAPK. Furthermore, exposure of HaCaT cells to DEHP and UV-B irradiation resulted in the marked suppression of the nuclear factor kappa B (NF-κB)/p65 signaling pathway. Taken together, our data suggest that nontoxic DEHP and UV-B irradiation regulated ER stress and epidermal TJ disruption with the induction of apoptosis activation and the secretion of proinflammatory cytokines such as interleukin 1 beta (IL-1ß) and IL-6 in human keratinocytes. Further investigation is needed to confirm the mechanisms implicated in its toxicity and determine the effects of exposure to DEHP and UV-B irradiation on markers involved in this study.

Butyrate modulates mucin secretion and bacterial adherence in LoVo cells via MAPK signaling.

Short-chain fatty acids contribute to normal bowel function and prevent bacterial infections. In particular, butyrate is a promising candidate that plays an important role in regulating the functional integrity of the gastrointestinal tract by stimulating mucin secretion. We investigated whether butyrate treatment modulates mucin secretion and bacterial adherence in LoVo cells. In addition, the possible signaling pathways were also examined in connection with the upregulation of mucin secretion. The results showed that butyrate induced mucin secretion in LoVo cells, resulting in the inhibition of Escherichia coli adhesion by increasing the adherence of Lactobacillus acidophilus and Bifidobacterium longum. The gene expression analysis suggests that mitogen-activated protein kinase (MAPK) signaling pathways including Cdc42-PAK pathway appears to be involved in stimulating mucin secretion. More importantly, butyrate induced the increased actin expression and polymerization in LoVo cells, which could be attributable to the Cdc42-PAK signaling pathway, implicated in actin cytoskeleton and mucin secretion. Our results provide a molecular basis in modulating bacterial adherence and the MAPK signaling pathway for the improved homeostasis of colonic epithelial cells.

Associations between Dietary Intake and Attention Deficit Hyperactivity Disorder (ADHD) Scores by Repeated Measurements in School-Age Children.

Attention deficit hyperactivity disorder (ADHD) is a common psychiatric disorder in school-age children and adolescents. However, the reported associations between ADHD and single nutrient intake are inconsistent. The aim of the study was to investigate the relationships between dietary intake changes and the prevalence of ADHD over time with repeat measurements using data from the Children Health and Environment Research (CHEER). To assess changes over time, we used data obtained in 2006 and 2008 (Phases 1 and 2). In this study, there were 2899 children aged 8 years or older in Phase 1 and 2120 children aged 9 years or older in Phase 2 from Korea, and the ADHD scores and dietary intake of 1733 children in Phases 1 and 2 were used in the final analysis. The YN group refers to children whose disease had improved in Phase 2, and the NY group refers to children diagnosed with ADHD in Phase 2. A notable within-group result was the increase in vegetable protein (p = 0.03) in the YN group. A between-group comparison showed that significant changes in nutrient intake could be confirmed most in the NY group, and the YN group tended to have a lower nutrient intake than the NY group. In the correlation of changes in nutrient intake and three subtypes (combined, AD, and HD), the total fat (p = 0.048) and animal protein (p = 0.099) showed a positive correlation with the prevalence of AD. Vegetable iron (p = 0.061 and p = 0.044, respectively), zinc (p = 0.022 and p = 0.007, respectively), vegetable protein (p = 0.074), and calcium (p = 0.057) had inhibitory effects on ADHD and its subtype. In conclusion, management of dietary and nutritional status should be considered to ameliorate ADHD and its subtypes in school-age children, and these relationships require further exploration in other settings.

Quality Characteristics of Substitute Meat Patties Developed Using Aruncus dioicus var. kamtschaticus Hara.

We developed a vegetable alternative to meat patties using Aruncus dioicus var. kamtschaticus Hara (A. dioicus) and used it to generate basic data for the alternative meat market by comparing nutritional and microbiological components with commercially available vegetable and meat patties. Nutrient analysis, microbiological analysis, chromaticity, and texture analysis were performed on substitute meat patties (SMPs) with A. dioicus and commercially available vegetable and animal patties. Among sugars, the contents of fructose and maltose were respectively high in commercial meat patties (CMPs) and SMPs. SMPs were low in saturated and trans-fat, and high in ω-3 fatty acids. The contents (in descending order) of leucine > phenylalanine > threonine > isoleucine were high in SMPs and commercial vegetable patties (CVPs). Qualitative and quantitative findings of Escherichia coli (E. coli) and Staphylococcus aureus were all negative. Our SMPs had high lightness (L*), low redness (a*), and low yellowness (b*). The hardness, chewiness, and resilience of our SMPs were lower than those of other vegetable and animal patties. Considering our results, the method of manufacturing SMPs developed in the present study allows meat to be flavored without significant nutritional differences compared with commercially available CMPs. Our findings provide a base for studies on future meat alternatives.

The Association between Self-Rated Health Status, Psychosocial Stress, Eating Behaviors, and Food Intake According to the Level of Sunlight Exposure in Korean Adults.

Sunlight exposure has been reported to have various beneficial effects on human health. This study investigated the relationship between self-rated health status, psychosocial stress, eating behaviors, and food intake according to sunlight exposure in 948 adults. Sunlight exposure was classified as less than one hour, less than three hours, and greater than three hours. Of the participants, 49.2% had fewer than three hours of daily exposure to sunlight. Regarding participants exposed to sunlight for less than one hour, the largest response was that they did not engage in outdoor activities on weekdays or weekends, and the rate of being outdoors in the shade on sunny days was the highest in this group at 42.7%. Furthermore, the participants exposed to sunlight for less than one hour had a lower health response than the other two groups, and there were significantly more participants classified in the stress risk group. Regarding eating habits, those with less than an hour of exposure to sunlight frequently ate fried foods, fatty foods, added salt, and snacks, and had significantly lower total dietary scores or three regular meals. Additionally, their frequency of consumption of cereals, milk and dairy products, orange juice, and pork was also significantly lower than the other groups. Thus, it is necessary to provide sufficient guidelines for adequate sunlight exposure and food intake because participants with low sunlight exposure may have low vitamin D synthesis and insufficient food intake.

A study on the nutritional and biochemical analysis of Selaginella tamariscina powder.

This study reports the various nutritional components of Selaginella tamariscina, which is traditionally used in folk or Chinese medicine. The iron nutrient content in S. tamariscina powder was 0.94 ± 0.06 mg/100 g powder, whereas selenium was present in a small amount, which showed strong antioxidant power. The total phenolic content of S. tamariscina powder was 8.65-11.61 mg gallic acid equivalents/g. S. tamariscina showed antioxidant activity in 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The ferric reducing antioxidant power of S. tamariscina powder was higher in the ethanol extract. Additionally, the ethanol extract demonstrated antimicrobial activity against Bacillus subtilis KCTC 2189. The level of high-density lipoprotein-cholesterol in the blood of ICR mice was significantly higher in the HF 20% + S. tamariscina 20% group than in the other groups (p < 0.05). The present study demonstrates that S. tamariscina, an abundantly existing plant, possesses antimicrobial, antioxidant, and anticytotoxic activities. S. tamariscina powder has potential as a functional food.

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development.

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.

Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis.

Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ∼130 µm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.

Daily walnut intake improves metabolic syndrome status and increases circulating adiponectin levels: randomized controlled crossover trial.

BACKGROUND/OBJECTIVES: Several previous studies have investigated whether regular walnut consumption positively changes heart-health-related parameters. The aim of this study was to investigate the effects of daily walnut intake on metabolic syndrome (MetS) status and other metabolic parameters among subjects with MetS. SUBJECTS/METHODS: This study was a two-arm, randomized, controlled crossover study with 16 weeks of each intervention (45 g of walnuts or iso-caloric white bread) with a 6 week washout period between interventions. Korean adults with MetS (n = 119) were randomly assigned to one of two sequences; 84 subjects completed the trial. At each clinic visit (at 0, 16, 22, and 38 weeks), MetS components, metabolic parameters including lipid profile, hemoglobin A1c (HbA1c), adiponectin, leptin, and apolipoprotein B, as well as anthropometric and bioimpedance data were obtained. RESULTS: Daily walnut consumption for 16 weeks improved MetS status, resulting in 28.6%-52.8% reversion rates for individual MetS components and 51.2% of participants with MetS at baseline reverted to a normal status after the walnut intervention. Significant improvements after walnut intake, compared to control intervention, in high-density lipoprotein cholesterol (HDL-C) (P = 0.028), fasting glucose (P = 0.013), HbA1c (P = 0.021), and adiponectin (P = 0.019) were observed after adjustment for gender, age, body mass index, and sequence using a linear mixed model. CONCLUSION: A dietary supplement of 45 g of walnuts for 16 weeks favorably changed MetS status by increasing the concentration of HDL-C and decreasing fasting glucose level. Furthermore, consuming walnuts on a daily basis changed HbA1c and circulating adiponectin levels among the subjects with MetS. This trial is registered at ClinicalTrials.gov as NCT03267901.

Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development.

In most species, early germline development occurs in the absence of transcription with germline determinants subject to complex translational and post-translational regulations. Here, we report for the first time that early germline development is influenced by dynamic regulation of the proteasome system, previously thought to be ubiquitously expressed and to serve 'housekeeping' roles in controlling protein homeostasis. We show that proteasomes are present in a gradient with the highest levels in the animal hemisphere and extending into the vegetal hemisphere of Xenopus oocytes. This distribution changes dramatically during the oocyte-to-embryo transition, with proteasomes becoming enriched in and restricted to the animal hemisphere and therefore separated from vegetally localized germline determinants. We identify Dead-end1 (Dnd1), a master regulator of vertebrate germline development, as a novel substrate of the ubiquitin-independent proteasomes. In the oocyte, ubiquitin-independent proteasomal degradation acts together with translational repression to prevent premature accumulation of Dnd1 protein. In the embryo, artificially increasing ubiquitin-independent proteasomal degradation in the vegetal pole interferes with germline development. Our work thus reveals novel inhibitory functions and spatial regulation of the ubiquitin-independent proteasome during vertebrate germline development.

Structural basis of the specific interaction of SMRT corepressor with histone deacetylase 4.

Modification of chromatin and related transcription factors by histone deacetylases (HDACs) is one of the major strategies for controlling gene expression in eukaryotes. The HDAC domains of class IIa HDACs repress the respective target genes by interacting with the C-terminal region of the silencing mediator for retinoid and thyroid receptor (SMRT) repression domain 3 (SRD3c). However, latent catalytic activity suggests that their roles as deacetylases in gene regulation are unclear. Here, we found that two conserved GSI-containing motifs of SRD3c are critical for HDAC4 binding. Two SMRT peptides including these motifs commonly form a ß-hairpin structure in the cleft and block the catalytic entry site of HDAC4. They interact mainly with class IIa HDAC-specific residues of HDAC4 in a closed conformation. Structure-guided mutagenesis confirmed critical interactions between the SMRT peptides and HDAC4 and -5 as well as the contribution of the Arg1369 residue in the first motif for optimal binding to the two HDACs. These results indicate that SMRT binding does not activate the cryptic deacetylase activity of HDAC4 and explain how class IIa HDACs and the SMRT-HDAC3 complex are coordinated during gene regulation.

Enigma Plays Roles in Survival of Thyroid Carcinoma Cells through PI3K/AKT Signaling and Survivin.

BACKGROUND/AIM: The aim of the present study was to assess the role of enigma protein in survival of thyroid carcinoma cells. MATERIALS AND METHODS: BCPAP and 8505C human thyroid carcinoma cells were used. Cell viability using CCK-8 assay, the percentage of dead cells using trypan blue assay, cytotoxic activity using cytotoxicity assay, cell growth rate and cell migration using wound-healing assay were performed. RESULTS: In enigma siRNA-transfected cells, cell viability, and the protein levels of AKT and survivin decreased. The percentage of dead cells, cytotoxic activity and cleaved poly (ADP-ribose) polymerase (PARP) protein levels increased. After transfection of p110α plasmid, the alterations in cell viability, the percentage of dead cells, cytotoxic activity, and protein levels of AKT, survivin and cleaved PARP were abrogated. Cell growth rate and cell migration were reduced with reduction of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) protein levels, as well as increased p53 and p21 protein levels. CONCLUSION: Enigma affects cell survival through modulation of phosphatidylinositol-3 kinase/AKT signaling and survivin, and regulates cell proliferation and migration via involvement of MMP-2, MMP-9, p53 and p21 in thyroid carcinoma cells.

Time and Intervention Effects of Daily Almond Intake on the Changes of Lipid Profile and Body Composition Among Free-Living Healthy Adults.

Favorable health benefits of almond have been shown in several previous studies. However, repeated measures, randomized, controlled trials to investigate the changes due to almond intake based on the time effects have not yet been reported. The current study was conducted to evaluate the effects of daily almond intake on changes in body composition and lipid profiles for 20 weeks with four measurements among healthy adults. Participants in the almond group showed favorable changes on blood lipid profiles, including levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein (non-HDL-C) after consuming 56 g of almond per day for 20 weeks compared with those at baseline. At week 20, subjects in the almond group showed significantly decreased TC, LDL-C, non-HDL-C, TG, body fat mass, and waist-hip ratio compared with those of the control group who consumed isocaloric control food. The mixed model also confirmed that there were significant time effects in several bioimpedance indicators (i.e., total body protein, fat-free mass, etc.) and all of the lipid profile parameters in the almond group. These results confirm the effects of lipid-lowering and modifying body composition of almond consumption. In addition, our results suggest that the measuring time points would be critical to capture the effects of dietary intervention.