Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 ...(LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.
Autophagy plays a dual role in tumorigenesis by functioning as both a tumor suppressor and promoter, depending on the stage of tumorigenesis. However, it is still unclear at what stage the role of ...autophagy changes during tumorigenesis. Herein, we investigated the differences in the basal levels and roles of autophagy in five cell lines at different stages of cell transformation. We found that cell lines at higher transformation stages were more sensitive to the autophagy inhibitors, suggesting that autophagy plays a more important role as the transformation progresses. Our ptfLC3 imaging analysis to measure Atg5/LC3-dependent autophagy showed increased autophagic flux in transformed cells compared to untransformed cells. However, the Cyto-ID analysis, which measures Atg5-dependent and -independent autophagic flux, showed high levels of autophagosome formation not only in the transformed cells but also in the initiated cell and Atg5 KO cell line. These results indicate that Atg5-independent autophagy may be more critical in initiated and transformed cell lines than in untransformed cells. Specially, we observed that transformed cells maintained relatively high basal autophagy levels under rapidly proliferating conditions but exhibited much lower basal autophagy levels at high confluency; however, autophagic flux was not significantly reduced in untransformed cells, even at high confluency. In addition, when continuously cultured for 3 weeks without passage, senescent cells were significantly less sensitive to autophagy inhibition than their actively proliferating counterparts. These results imply that once a cell has switched from a proliferative state to a senescent state, the inhibition of autophagy has only a minimal effect. Taken together, our results suggest that autophagy can be differentially regulated in cells at different stages of tumorigenesis under stressful conditions.
Gastric cancer (GC) is a leading cause of cancer-related mortality. Although microbes besides
may also contribute to gastric carcinogenesis, wild-type germ-free (GF) mouse models investigating the ...role of human gastric microbiota in the process are not yet available. We aimed to evaluate the histopathological features of GF mouse stomachs transplanted with gastric microbiota from patients with different gastric disease states and their relationships with the microbiota.
Microbiota profiles in corpus and antrum tissues and gastric fluid from 12 patients with gastric dysplasia or GC were analysed. Thereafter, biopsied corpus and antrum tissues and gastric fluid from patients (n=15 and n=12, respectively) with chronic superficial gastritis, intestinal metaplasia or GC were inoculated into 42 GF C57BL/6 mice. The gastric microbiota was analysed by amplicon sequencing. Histopathological features of mouse stomachs were analysed immunohistochemically at 1 month after inoculation. An independent set of an additional 15 GF mice was also analysed at 1 year.
The microbial community structures of patients with dysplasia or GC in the corpus and antrum were similar. The gastric microbiota from patients with intestinal metaplasia or GC selectively colonised the mouse stomachs and induced premalignant lesions: loss of parietal cells and increases in inflammation foci, in F4/80 and Ki-67 expression, and in CD44v9/GSII lectin expression. Marked dysplastic changes were noted at 1 year post inoculation.
Major histopathological features of premalignant changes are reproducible in GF mice transplanted with gastric microbiota from patients with intestinal metaplasia or GC. Our results suggest that GF mice are useful for analysing the causality of associations reported in human gastric microbiome studies.
Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 ...system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.
ATG5 knockout cells show morphological transformation and anchorage‐independent growth‐ ATG5 KO cells have a lower proportion of the apoptotic cell population as a way to escape cell death in response to PP2.
Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid) and include 9,10-EpOME and 12,13-EpOME. They are synthesized by cytochrome P450 ...monooxygenases (CYPs) and degraded by soluble epoxide hydrolase (sEH). Although EpOMEs are well known to play crucial roles in mediating various physiological processes in mammals, their role is not well understood in insects. This study chemically identified their presence in insect tissues: 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME in fat body of a lepidopteran insect, Spodoptera exigua. Injection of 9,10-EpOME or 12,13-EpOME into larvae suppressed the cellular immune responses induced by bacterial challenge. EpOME treatment also suppressed the expression of antimicrobial peptide (AMP) genes. Among 139 S. exigua CYPs, an ortholog (SE51385) to human EpOME synthase was predicted and its expression was highly inducible upon bacterial challenge. RNA interference (RNAi) of SE51385 prevented down-regulation of immune responses at a late stage (> 24 h) following bacterial challenge. A soluble epoxide hydrolase (Se-sEH) of S. exigua was predicted and showed specific expression in all development stages and in different larval tissues. Furthermore, its expression levels were highly enhanced by bacterial challenge in different tissues. RNAi reduction of Se-sEH interfered with hemocyte-spreading behavior, nodule formation, and AMP expression. To support the immune association of EpOMEs, urea-based sEH inhibitors were screened to assess their inhibitory activities against cellular and humoral immune responses of S. exigua. 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) was highly potent in suppressing the immune responses. The addition of AUDA to a pathogenic bacterium significantly increased bacterial pathogenicity by suppressing host immune defense. In sum, this study demonstrated that EpOMEs play a crucial role in facilitating anti-inflammatory responses in S. exigua.
The paradigm shift of information carriers from charge to spin has long been awaited in modern electronics. The invention of the spin‐information transistor is expected to be an essential building ...block for the future development of spintronics. Here, a proof‐of‐concept experiment of a magnetic skyrmion transistor working at room temperature, which has never been demonstrated experimentally, is introduced. With the spatially uniform control of magnetic anisotropy, the shape and topology of a skyrmion when passing the controlled area can be maintained. The findings will open a new route toward the design and realization of skyrmion‐based spintronic devices in the near future.
A proof‐of‐concept experiment of a magnetic skyrmion transistor is presented using spatially uniform voltage‐controlled magnetic anisotropy. A gate operation can control the translational motion of a magnetic skyrmion. Utilizing the gate‐controlled skyrmion motion technique, the authors expect various skyrmion logic devices to be demonstrated in the future.
Background Lysophosphatidic acid receptor 3 (LPAR3) is coupled to Galpha.sub.i/o and Galpha.sub.11/q signaling. Previously, we reported that LPAR3 is highly methylated in carcinogen-induced ...transformed cells. Here, we demonstrate that LPAR3 exhibits malignant transforming activities, despite being downregulated in transformed cells. Methods The LPAR3 knockout (KO) in NIH 3 T3 and Bhas 42 cells was established using the CRISPR/Cas9 system. Both RT-PCR and DNA sequencing were performed to confirm the KO of LPAR3. The cellular effects of LPAR3 KO were further examined by WST-1 assay, immunoblotting analysis, transwell migration assay, colony formation assay, wound scratch assday, in vitro cell transformation assay, and autophagy assay. Results In v-H-ras-transformed cells (Ras-NIH 3 T3) with LPAR3 downregulation, ectopic expression of LPAR3 significantly enhanced the migration. In particular, LPAR3 knockout (KO) in Bhas 42 (v-Ha-ras transfected Balb/c 3 T3) and NIH 3 T3 cells caused a decrease in cell survival, transformed foci, and colony formation. LPAR3 KO led to the robust accumulation of LC3-II and autophagosomes and inhibition of autophagic flux by disrupting autophagosome fusion with lysosome. Conversely, autolysosome maturation proceeded normally in Ras-NIH 3 T3 cells upon LPAR3 downregulation. Basal phosphorylation of MEK and ERK markedly increased in Ras-NIH 3 T3 cells, whereas being significantly lower in LPAR3 KO cells, suggesting that increased MEK signaling is involved in autophagosome-lysosome fusion in Ras-NIH 3 T3 cells. Conclusions Paradoxical downregulation of LPAR3 exerts cooperative tumor-promoting activity with MEK activation through autophagy induction in Ras-transformed cells. Our findings have implications for the development of cancer chemotherapeutic approaches. Keywords: LPAR3, Autophagy, Knockout, Cell transformation, Downregulation, Paradoxical, Methylation, Ras, Epigenetic
Ginseng contains three active components: ginsenosides, gintonin, and polysaccharides. After the separation of 1 of the 3 ingredient fractions, other fractions are usually discarded as waste. In this ...study, we developed a simple and effective method, called the ginpolin protocol, to separate gintonin-enriched fraction (GEF), ginseng polysaccharide fraction (GPF), and crude ginseng saponin fraction (cGSF).
Dried ginseng (1 kg) was extracted using 70% ethanol (EtOH). The extract was water fractionated to obtain a water-insoluble precipitate (GEF). The upper layer after GEF separation was precipitated with 80% EtOH for GPF preparation, and the remaining upper layer was vacuum dried to obtain cGSF.
The yields of GEF, GPF, and cGSF were 14.8, 54.2, and 185.3 g, respectively, from 333 g EtOH extract. We quantified the active ingredients of 3 fractions: L-arginine, galacturonic acid, ginsenosides, glucuronic acid, lysophosphatidic acid (LPA), phosphatidic acid (PA), and polyphenols. The order of the LPA, PA, and polyphenol content was GEF > cGSF > GPF. The order of L-arginine and galacturonic acid was GPF >> GEF = cGSF. Interestingly, GEF contained a high amount of ginsenoside Rb1, whereas cGSF contained more ginsenoside Rg1. GEF and cGSF, but not GPF, induced intracellular Ca2+i transient with antiplatelet activity. The order of antioxidant activity was GPF > GEF = cGSF. Immunological activities (related to nitric oxide production, phagocytosis, and IL-6 and TNF-α release) were, in order, GPF > GEF = cGSF. The neuroprotective ability (against reactive oxygen species) order was GEF > cGSP > GPF.
We developed a novel ginpolin protocol to isolate 3 fractions in batches and determined that each fraction has distinct biological effects.
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Although bisphenol F (BPF), the main replacement for bisphenol A, has been commonly used in polycarbonate production, its neurotoxicity and the underlying mechanisms remain poorly understood. To ...address this knowledge gap, this study aimed to assess the neurotoxicity caused by chronic exposure to BPF and to identify its underlying mechanisms. We exposed adult zebrafish chronically to BPF at environmentally relevant concentrations (0.001, 0.01, and 0.1 mg/L) for 4 weeks. The results revealed that with BPF crossing the blood–brain barrier and bioaccumulating in brain tissues, chronic exposure to BPF resulted in anxiety-like behaviors and disruptions in learning and memory function in adult zebrafish. Furthermore, BPF toxicity in the zebrafish brain involved the dysregulation of metabolic pathways for choline and kynurenine in neurotransmitter systems and for 17β-estradiol, cortisol, pregnenolone-sulfate, and Dehydroepiandrosterone (DHEA)-sulfate in neurosteroid systems. RNA-seq analysis revealed that BPF exposure affected metabolic pathways, calcium signaling pathways, neuroactive ligand–receptor interactions, tight junctions, gap junctions, and the gonadotropin-releasing hormone signaling pathway. Our results indicate that chronic exposure to BPF alters the neurochemical profile of the brain and causes neurobehavioral effects, such as anxiety and cognitive decline. Overall, the multimodal approach, including behavioral and neurochemical profiling technologies, has great potential for the comprehensive assessment of potential risks posed by environmental pollutants to human and ecosystem health.
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•BPF crosses the blood-brain barrier.•BPF alters neurochemical and transcriptomic profiles.•BPF causes neurobehavioral effects in zebrafish.•BPF induces anxiety-like behavior and disruption in learning and memory.
Numerous studies have revealed an association between particulate matter (PM) and emergency room (ER) visits, although few studies have investigated the association between health and PM components. ...The present study evaluated the associations of ER visits for cardiovascular and respiratory diseases with PM2.5 components, including organic carbon (OC), elemental carbon (EC), and ion species (SO42-, NO3-, and NH4+). Statistical analyses were performed using the time-series approach, and generalized linear models with natural spline functions were used to adjust for the non-linear relationship between the confounders and ER visits. Our single-pollutant models revealed that the greatest increase in cardiovascular ER visits was associated with NH4+ (relative risk: 1.05; 95% confidence interval: 1.01-1.09), which was followed by OC, SO42-, NO3-, and EC. The associations of cardiovascular ER visits with EC and OC varied according to age and sex, with elderly and female patients exhibiting stronger associations. Lagged SO42- was associated with respiratory ER visits. To the best of our knowledge, this is the first study to evaluate the associations between ER visits and PM components in South Korea. As PM components are related to traffic and industrial sources, and exhibited positive associations with ER visits, our results may help improve air pollution regulation and public health.
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