Jagged 2b induces intercellular signaling within somites to establish hematopoietic stem cell fate in zebrafish.

During development, the somites play a key role in the specification of hematopoietic stem cells (HSCs). In zebrafish, the somitic Notch ligands Delta-c (Dlc) and Dld, both of which are regulated by Wnt16, directly instruct HSC fate in a shared vascular precursor. However, it remains unclear how this signaling cascade is spatially and temporally regulated within somites. Here, we show in zebrafish that an additional somitic Notch ligand, Jagged 2b (Jag2b), induces intercellular signaling to drive wnt16 expression. Jag2b activated Notch signaling in segmented somites at the early stage of somitogenesis. Loss of jag2b led to a reduction in the expression of wnt16 in the somites and an HSC marker, runx1, in the dorsal aorta, whereas overexpression of jag2b increased both. However, Notch-activated cells were adjacent to, but did not overlap with, wnt16-expressing cells within the somites, suggesting that an additional signaling molecule mediates this intercellular signal transduction. We uncover that Jag2b-driven Notch signaling induces efna1b expression, which regulates wnt16 expression in neighboring somitic cells. Collectively, we provide evidence for previously unidentified spatiotemporal regulatory mechanisms of HSC specification by somites.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Formulation and Characterization of Soybean Oil-in-Water Emulsions Stabilized Using Gelatinized Starch Dispersions from Plant Sources.

Consumers are concerned about employing green processing technologies and natural ingredients in different manufacturing sectors to achieve a "clean label" standard for products and minimize the hazardous impact of chemical ingredients on human health and the environment. In this study, we investigated the effects of gelatinized starch dispersions (GSDs) prepared from six plant sources (indica and japonica rice, wheat, corn, potatoes, and sweet potatoes) on the formulation and stability of oil-in-water (O/W) emulsions. The effect of gelatinization temperature and time conditions of 85-90 °C for 20 min on the interfacial tension of the two phases was observed. Emulsification was performed using a primary homogenization condition of 10,000 rpm for 5 min, followed by high-pressure homogenization at 100 MPa for five cycles. The effects of higher oil weight fractions (15-25% w/w) and storage stability at different temperatures for four weeks were also evaluated. The interfacial tension of all starch GSDs with soybean oil decreased compared with the interfacial tension between soybean oil and water as a control. The largest interfacial tension reduction was observed for the GSD from indica rice. Microstructural analysis indicated that the GSDs stabilized the O/W emulsion by coating oil droplets. Emulsions formulated using a GSD from indica rice were stable during four weeks of storage with a volume mean diameter (d4,3) of ~1 µm, minimal viscosity change, and a negative ζ-potential.

Effect of marination in lemon juice on beef tenderization and in vitro gastric digestibility.

BACKGROUND: There is limited knowledge regarding digestion and absorption of nutrients after cooked marinated meat is ingested. Most of the previous studies on food gastric digestion have focused on chemical digestion and did not reflect upon physical digestion driven by peristalsis. In the present study, we examined the effects of marinating beef in lemon juice on gastric digestibility using a human gastric digestion simulator (GDS) that mimics peristaltic motion called antral contraction waves. RESULTS: Beef thigh slices were marinated in 100% lemon juice for 1 h and then grilled; an image of a stained tissue sample revealed that muscle tissue contraction (i.e. that usually occurs upon cooking) was suppressed. The measurement of physical properties using a rheometer and texture analyzer showed that the meat marinated in lemon juice had a soft texture. In vitro digestion experiments using the GDS revealed that the extent of both physical digestion driven by peristalsis and chemical digestion catalyzed by digestive enzymes was enhanced by the lemon juice marinade. CONCLUSION: The results of the present study suggest that marinating beef in lemon juice affects nutrient digestibility. An integrated evaluation of tissue structure, physical properties and GDS digestion to analyze meat digestion would enhance our understanding of the effects of seasoning and cooking methods on meat. © 2023 Society of Chemical Industry.

Effect of mechanical properties on in vitro dynamic digestion of starch contained in hydrogels.

BACKGROUND: This study evaluates the effect of mechanical properties on the in vitro dynamic gastrointestinal digestion of hydrogels containing starch (HCSs) as a model for studying the nutrient digestibility of solid foods. It provides a useful theoretical basis for the processing of specific foods. RESULT: Four types of HCSs with two levels of fracture stress (17.4-20.9 kPa and 55.5-57.6 kPa) and two levels of fracture strain (25.4-28.5% and 53.7-57.4%) were prepared. For these HCSs, the degree of gastric disintegration of hydrogels reduced significantly when fracture strain exceeded 30% (P < 0.05). The gastric emptying of HCS particles was also affected by mechanical properties. For example, even at the same level of fracture stress (ca. 20 kPa), the dry solids retention ratio decreased markedly from 0.90 to 0.43 with a decrease in fracture strain from 53.7% to 25.4% (P < 0.05). For the starch hydrolysis of HCSs after gastric digestion, more than 70% of starch in the particles of all types of HCSs emptied did not undergo digestion. The starch hydrolysis of HCSs during small intestinal digestion was also influenced by their mechanical properties. Fracture strains of HCSs, rather than their fracture stress, affected starch digestibility in hydrogels. CONCLUSION: The gastric disintegration, the gastric emptying, and the starch hydrolysis of HCSs are suppressed when fracture strain exceeded 30%. Even with the amount of nutritional components contained in hydrogels being the same, the in vitro gastrointestinal digestion behavior of HCSs depends on their mechanical properties. This behavior has the potential to be used in the design of processed foods with controlled bioaccessibility. © 2023 Society of Chemical Industry.

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants.

Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.

Dual role of Jam3b in early hematopoietic and vascular development.

In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

Revisiting lycopene extraction: Caprylic acid-based emulsion for the highest recovery.

In this study, we evaluated the caprylic acid-based oil-in-water (O/W) emulsion-assisted extraction of lycopene from tomatoes. Emulsion-assisted extraction was performed using two types of micron-sized O/W emulsions: (a) O/W emulsion with absence or (b) presence of 0.1% (w/w) of Tween 20 emulsifier. This green extraction technique was compared with the conventional method using soybean oil, tributyrin, and caprylic acid. The results show that caprylic acid, a green solvent, is significantly more effective for lycopene recovery than soybean oil and tributyrin. In the absence of an emulsifier, caprylic acid-based O/W emulsion significantly improved the lycopene content by 14.69 mg/g, corresponding to a 98.59% extraction efficiency at 50 ˚C. The capability of the proposed approach to lycopene recovery was explained in terms of lycopene affinity, the ability to swell the tomato cell, and some other standard parameters. In addition, caprylic acid has the significant advantage that once developed with the extracted lycopene can be used directly as a food additive.

Melatonin suppresses both osteoblast and osteoclast differentiation through repression of epidermal Erk signaling in the zebrafish scale.

Melatonin has been implicated in the regulation of bone metabolism; however, the molecular mechanisms underlying its involvement in fracture healing are still obscure. We previously developed an in vivo fracture healing model using the scale of a double-transgenic zebrafish, trap:GFP; osterix:mCherry, which labels osteoclasts and osteoblasts with GFP and mCherry, respectively. Here we show using this model that melatonin inhibits both osteoblast and osteoclast differentiation under fracture stress through the repression of Erk signaling in epidermal cells of the scale. Melatonin treatment resulted in reduced numbers of both osteoblasts and osteoclasts in the fractured scale. Immunochemistry analysis revealed that Erk signals in epidermal cells, which express melatonin receptors, were greatly enhanced in response to fracture stress, but this enhancement was blocked by melatonin treatment. Moreover, inhibition of Erk signaling phenocopied the effects of melatonin treatment in the fractured scale. Collectively, these data suggest that the activation of epidermal Erk signaling is required for both osteoblast and osteoclast differentiation in the early stage of fracture healing, and melatonin suppresses epidermal Erk signaling, leading to impaired fracture healing.

Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

Oil-in-water emulsions containing tamarind seed gum during in vitro gastrointestinal digestion: rheological properties, stability, and lipid digestibility.

BACKGROUND: Polysaccharides may enhance/inhibit lipid digestibility of oil-in-water (O/W) emulsions because of their emulsifying and/or stabilizing ability and can also affect the formation, stability, and viscosity of emulsions. Tamarind seed gum (TSG) was used as the sole emulsifier/stabilizer to stabilize an O/W emulsion prepared using high-speed homogenization. We investigated the effects of various TSG concentrations (50-150 g kg-1 ) on the lipid digestibility, rheological properties, and stability of O/W emulsions during in vitro gastrointestinal digestion. RESULTS: A low concentration (50 g kg-1 ) and a high concentration (150 g kg-1 ) of TSG reduced lipid digestibility by about 33% and 45%, respectively, compared to the control sample (without TSG). However, the emulsion containing the intermediate TSG concentration at 100 g kg-1 was the most efficient in the inhibition of lipid digestion, reducing lipid digestibility by about 70% compared to that of the control sample. The stability of emulsion tended to enhance as the concentration of TSG increased. The size of oil droplets before passing through the intestinal phase and the viscosity of the intestinal digested system may be important factors for enhancing/inhibiting lipid digestibility of emulsions. The destabilization of the emulsion during digestion was not clearly detected by rheological analysis because rheological characteristics (e.g. flow behavior index) were mainly driven by TSG. CONCLUSIONS: The addition of TSG in O/W emulsions inhibited lipid digestibility. TSG at a concentration of 100 g kg-1 was the most efficient in the inhibition of lipid digestibility, suggesting that TSG is an attractive alternative ingredient for control of lipid digestibility of emulsion foods. © 2020 Society of Chemical Industry.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Microchannel emulsification: A promising technique towards encapsulation of functional compounds.

This review provides an overview of microchannel emulsification (MCE) for production of functional monodispersed emulsion droplets. The main emphasis has been put on functional bioactives encapsulation using grooved-type and straight-through microchannel array plates. MCE successfully encapsulates the bioactives like ß-carotene, oleuropein, γ-oryzanol, ß-sitosterol, L-ascorbic acid and ascorbic acid derivatives, vitamin D and quercetin. These bioactives were encapsulated in a variety of delivery systems like simple and multiple emulsions, polymeric particles, microgels, solid lipid particles and functional vesicles. The droplet generation process in MCE is based upon spontaneous transformation of interfaces rather than high energy shear stress systems. The scale-up of MCE can increase the productivity of monodispersed droplets >100 L h-1 and makes it a promising tool at industrial level.

RANKL, Ephrin-Eph and Wnt10b are key intercellular communication molecules regulating bone remodeling in autologous transplanted goldfish scales.

This study aimed to investigate the precise data of gene expression, functions, and chronological relationships amongst communication molecules involved in the bone remodeling process with an in vivo model using autologous transplanted scales of goldfish. Autotransplantation of methanol-fixed cell-free scales triggers scale resorption and regeneration, as well as helps elucidate the process of bone remodeling. We investigated osteoclastic markers, osteoblastic markers, and gene expressions of communicating molecules (RANKL, ephrinB2, EphB4, EphA4, Wnt10b) by qPCR, in situ hybridization for Wnt10b, and immunohistochemistry for EphrinB2 and EphA4 proteins to elucidate the bone remodeling process. Furthermore, functional inhibition experiments for the signaling of ephrinB2/Eph, ephrin/EphA4, and Wnt10b using specific antibodies, revealed that these proteins are involved in key signaling pathways promoting normal bone remodeling. Our data suggests that the remodeling process comprises of two successive phases. In the first absorption phase, differentiation of osteoclast progenitors by RANKL is followed by the bone absorption by mature, active osteoclasts, with the simultaneous induction of osteoblast progenitors by multinucleated osteoclast-derived Wnt10b, and proliferation of osteoblast precursors by ehprinB2/EphB4 signaling. Subsequently, during the second formation phase, termination of bone resorption by synergistic cooperation occurs, with downregulation of RANKL expression in activated osteoblasts and Ephrin/EphA4-mediated mutual inhibition between neighboring multinucleated osteoclasts, along with simultaneous activation of osteoblasts via forward and reverse EphrinB2/EphB4 signaling between neighboring osteoblasts. In addition, the present study shows that autologous transplantation of methanol-fixed cell-free scale is an ideal in vivo model to study bone remodeling.

Development of a radio frequency heating system for sterilization of vacuum-packed fish in water.

We developed equipment that quickly and uniformly heats packed whole fish in circulating tap water using radio frequency (RF) heating. Four vacuumed plastic-packed Pacific sauries in tap water were set in a radial arrangement between coaxial cylindrical electrodes in a closed vessel. For sterilization testing, Bacillus subtilis spores added in the center of the sauries were counted after treatment. For quality assurance, meat color and backbone hardness were measured after treatment. The temperature at the center of the sauries was increased up to 130 °C for 19 min using 9 kW RF heating, and up to 119 °C for 45 min using conventional heating (CH) at 120 °C. B. subtilis spores were decreased by five logarithmic orders using RF heating and by four logarithmic orders using CH. The RF-treated meat was brighter than the CH-treated meat, and the RF-treated backbone was softer than CH-treated one.

Effects of hyperglycemia on bone metabolism and bone matrix in goldfish scales.

Increased risk of fracture associated with type 2 diabetes has been a topic of recent concern. Fracture risk is related to a decrease in bone strength, which can be affected by bone metabolism and the quality of the bone. To investigate the cause of the increased fracture rate in patients with diabetes through analyses of bone metabolism and bone matrix protein properties, we used goldfish scales as a bone model for hyperglycemia. Using the scales of seven alloxan-treated and seven vehicle-treated control goldfish, we assessed bone metabolism by analyzing the activity of marker enzymes and mRNA expression of marker genes, and we measured the change in molecular weight of scale matrix proteins with SDS-PAGE. After only a 2-week exposure to hyperglycemia, the molecular weight of α- and ß-fractions of bone matrix collagen proteins changed incrementally in the regenerating scales of hyperglycemic goldfish compared with those of euglycemic goldfish. In addition, the relative ratio of the γ-fraction significantly increased, and a δ-fraction appeared after adding glyceraldehyde-a candidate for the formation of advanced glycation end products in diabetes-to isolated type 1 collagen in vitro. The enzymatic activity and mRNA expression of osteoblast and osteoclast markers were not significantly different between hyperglycemic and euglycemic goldfish scales. These results indicate that hyperglycemia is likely to affect bone quality through glycation of matrix collagen from an early stage of hyperglycemia. Therefore, non-enzymatic glycation of collagen fibers in bone matrix may lead to the deterioration of bone quality from the onset of diabetes.

Identification of genes for sulfolipid synthesis in primitive red alga Cyanidioschyzon merolae.

Sulfoquinovosyl diacylglycerol is one of the lipids that construct thylakoid membranes, and is distributed from cyanobacteria to plastids in plants including a red lineage. One of the most primitive red algae, Cyanidioschyzon melorae, similar to cyanobacteria and green plants, possesses homologs of the SQD1 and SQD2 genes that code for UDP-sulfoquinovose and sulfoquinovosyl diacylglycerol synthases, respectively, for the synthesis of sulfoquinovosyl diacylglycerol. We here revealed the structural properties of SQD1 and SQD2 homologs in C. melorae intrinsic to those of the authentic proteins, and verified their enzymatic functions through heterologous expression in cyanobacterial disruptants as to the corresponding genes. The results demonstrated that the system of sulfoquinovosyl diacylglycerol synthesis could have been conserved through evolution of cyanobacteria to plastids in a red lineage, which is compatible with the monophyletic origin of plastids.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14-17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.