A gene regulatory network combining Pax3/7, Sox10 and Mitf generates diverse pigment cell types in medaka and zebrafish.

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network for melanocytes (their only pigment cell type) contains three transcription factors, Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), gene regulatory networks governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasized, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification should be considered to result from the combinatorial activity of Mitf with other transcription factors.

Establishment of transgenic epithelium-specific Cre-recombinase driving medaka (Oryzias latipes) by homology repair mediated knock-in.

Deletion of gene expression in the target tissues and cells is an effective strategy for elucidating the physiological functions of the protein of interest. For tissue-specific and/or inducible gene deletion, the Cre-loxP system has been widely used in various model organisms including medaka (Oryzias latipes). The epithelium is the key tissue, locating at the outermost area and playing a role in barrier to external stimuli. Despite a large genetic toolbox developed in medaka, there is no available Cre-driver line that works in an epithelium-specific manner. Here, we established epithelium-specific Cre-driver lines in medaka using a homology-directed repair mediated knock-in approach with CRISPR/Cas9, targeting each of periplakin and keratin genes. We show that Cre-recombinase is expressed exclusively in the epithelium in the knock-in lines and that it efficiently and specifically induces recombination in the tissues. These Cre-driver lines are useful for studying the functions of proteins expressed in the epithelium.

Catalytic Substrate-Selective Silylation of Primary Alcohols via Remote Functional-Group Discrimination.

Silylation of alcohols has generally been known to take place at the sterically most accessible less-hindered hydroxy group. Herein, the catalyst-controlled substrate-selective silylation of primary alcohols, in which the selectivity was controlled independently of the innate reactivity of the hydroxy group, based on the steric environment, is reported. The chain-length-selective silylation of 1,n-amino alcohol derivatives was achieved and 1,5-amino alcohol derivatives showed outstandingly high reactivity in the presence of analogues with a shorter or longer chain length under catalyst-controlled conditions. A highly substrate-selective catalytic silylation of pentanol analogues was also developed, in which the remote functionality at C(5) from the reacting hydroxy groups was effectively discriminated on silylation.

Contribution of sox9b to pigment cell formation in medaka fish.

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a-/- ; sox10b-/- double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b-/- ; sox10a-/- and sox10a-/- ; sox10b-/- double mutants, but their numbers were significantly reduced in the sox9b-/- ; sox10a-/- mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish.

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

The Prevention of Carboxymethylcellulose on Bowel Adhesions Induced by Talc Peritonitis in Mice.

BACKGROUND: Postoperative bowel adhesions may lead to various disorders, including abdominal pain, bowel obstruction, ischemia, and necrosis. In previous reports, a dose-dependent increase in bowel adhesions was observed in talc-treated animals in comparison with control animals. Although various methods have been devised to prevent peritoneal adhesions, each of these methods has advantages and disadvantages. In this study, we have attempted to reassess the effect of a carboxymethylcellulose (CMC) solution in the reduction of peritoneal adhesions induced by an intraperitoneal injection of a talc suspension in mice. MATERIALS AND METHODS: Mice received an intraperitoneal injection of a talc suspension, followed by an injection of a CMC solution or vehicle. Two weeks after the injection, any adherent bowel mass was removed en bloc, weighed, and histologically observed. RESULTS: The administration of talc induced severe bowel adhesions. CMC treatment was unable to completely inhibit the development of bowel adhesions, but treatment did reduce their weight in a dose-dependent manner. According to a histopathologic analysis, the bowel adhesions were composed of a conglomerate of talc aggregate and granulation tissue. The conglomerate was divided into two zones: the cell-rich marginal zone and the cell-scarce central zone. The injection of CMC specifically reduced the width of the marginal zone and the number of infiltrated cells. CONCLUSIONS: This study demonstrated that CMC inhibited bowel adhesions induced by talc in mice. In addition, this is the first report on the effect of CMC on talc peritonitis accompanied by a detailed histologic examination. Our experimental model is very simple and easy to use. Therefore, it may help in the discovery of new antiadhesive agents and in the analysis of the kinetics of bowel adhesion.

Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements.

BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

Medaka and zebrafish contactin1 mutants as a model for understanding neural circuits for motor coordination.

A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)-superfamily domain-containing membrane-anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans-heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro-mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator-like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1-deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.

Higher susceptibility to osmolality of the medaka (Oryzias latipes) mutants in orthologue genes of mammalian skin transglutaminases.

Transglutaminase (TG) is an essential enzyme to catalyze cross-linking reactions of epidermal proteins. Recently, we biochemically characterized human skin TG orthologues for medaka (Oryzias latipes), a model fish. By genome editing, gene-modified fishes for the two orthologues were obtained, both of which lack the ordinal enzymes. These fish appeared to exhibit higher susceptibility to osmolality at the period of larvae.

Biochemical characterization of the medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2).

Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (Oryzias latipes), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.

Leucophores are similar to xanthophores in their specification and differentiation processes in medaka.

Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.

Sox5 functions as a fate switch in medaka pigment cell development.

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).

A Novel Ataxic Mutant Mouse Line Having Sensory Neuropathy Shows Heavy Iron Deposition in Kidney.

BACKGROUND/AIMS: A novel ataxic mouse line was established from the offspring of a male mouse administered cyclophosphamide in a juvenile period. METHODS: We have attempted to examine the phenotype and histopathological changes of affected mice. Furthermore, linkage analysis and sequencing of the mutant was performed to reveal the causative gene locus. RESULTS AND CONCLUSION: The affected mouse was characterized by heavy hind limb ataxia with gait disorder, which was first recognized at about 4 weeks of age and slowly progressed with advancing age. The phenotype was inherited in an autosomal recessive pattern. The genetic locus associated with the phenotype was named hak and mapped to 107,305,356-108,637,615 on chromosome 2qE3, non-coding sequences in the vicinity of Bdnf gene. Many spheroids were noticed in the cerebellar medulla and the brain stem. In the peripheral nerves, some sensory ganglionic cells showed deposition of NF-200 in the perikaryon and NF-200-positive spheroids in nerve fibers. No inflammatory cell infiltration was observed. In addition, the adult affected mouse had distinct iron deposition in the kidney and the liver, but not in the heart, the skeletal muscle and the central nervous system. These results suggest that the hak mouse has a tissue-specific impairment in the expression of a type of Bdnf transcripts.

Microangiopathy triggers, and inducible nitric oxide synthase exacerbates dextran sulfate sodium-induced colitis.

Ulcerative colitis (UC) is a representative clinical manifestation of inflammatory bowel disease that causes chronic gastrointestinal tract inflammation. Dextran sulfate sodium (DSS)-induced colitis mice have been used to investigate UC pathogenesis, and in this UC model, disturbance and impairment of the mucosal epithelium have been reported to cause colitis. However, how DSS sporadically breaks down the epithelium remains unclear. In this study, we focused on the colonic microcirculation and myenteric neurons of DSS-induced colitis. Moreover, we examined the potential of myenteric neurons as a target to prevent exacerbation of colitis. Fluorescent angiographic and histopathological studies revealed that DSS administration elicited blood vessel disruption before epithelial disorders appeared. Ischemic conditions in the lamina propria induced inducible nitric oxide synthase (iNOS) expression in myenteric neurons as colitis aggravated. When neuronal activity was inhibited with butylscopolamine, neuronal iNOS expression decreased, and the exacerbation of colitis was prevented. These results suggested that DSS-induced colitis was triggered by microcirculatory disturbance in the mucosa, and that excessive neuronal excitation aggravated colitis. During remission periods of human UC, endoscopic inspection of the colonic microcirculation may enable the early detection of disease recurrence, and inhibition of neuronal iNOS expression may prevent the disease from worsening.

Stromal Cell-Derived Factor-1α Plays a Crucial Role Based on Neuroprotective Role in Neonatal Brain Injury in Rats.

Owing to progress in perinatal medicine, the survival of preterm newborns has markedly increased. However, the incidence of cerebral palsy has risen in association with increased preterm birth. Cerebral palsy is largely caused by cerebral hypoxic ischemia (HI), for which there are no effective medical treatments. We evaluated the effects of stromal cell-derived factor-1α (SDF-1α) on neonatal brain damage in rats. Left common carotid (LCC) arteries of seven-day-old Wistar rat pups were ligated, and animals were exposed to hypoxic gas to cause cerebral HI. Behavioral tests revealed that the memory and spatial perception abilities were disturbed in HI animals, and that SDF-1α treatment improved these cognitive functions. Motor coordination was also impaired after HI but was unimproved by SDF-1α treatment. SDF-1α reduced intracranial inflammation and induced cerebral remyelination, as indicated by the immunohistochemistry results. These data suggest that SDF-1α specifically influences spatial perception abilities in neonatal HI encephalopathy.

[A case of cerebellar hemangioblastoma complicated by pregnancy and concerns about the surgical period].

We report herein a case of cerebellar hemangioblastoma complicated by pregnancy and concerns about the period in which surgery could be performed successfully. A 19-year-old woman, who was also 35 weeks pregnant, was admitted to our hospital with headache, nausea, and general fatigue. Neurological examination on admission revealed disturbed consciousness, and the patient's general condition was poor. Computed tomography and magnetic resonance imaging showed a large tumor in the cerebellar vermis along with an obstructive hydrocephalus. Computed tomographic angiography with three-dimensional reconstruction revealed feeding arteries and a draining vein in this tumor. Based on the clinical features, hemangioblastoma was suspected, and surgical excision and extraction of the fetus were scheduled. However, because of rapid neurological deterioration due to tumor progression, an emergency cesarean section was performed under general anesthesia. After extracting the fetus, the level of consciousness improved, so a tumor resection was planned after the patient's general condition improved. However, the neurological state deteriorated again due to the worsening hydrocephalus, which was suspected to be caused by the increased cerebral blood flow following uterine contraction. Emergency surgery for the brain tumor was performed two days after delivery. The tumor was resected completely and histopathological examination confirmed a diagnosis of hemangioblastoma. The postoperative course was uneventful, and the patient and newborn were discharged with no neurological deficits three weeks after the operation. This case suggested that if we encounter patients with brain tumors complicated by pregnancy, not only is earlier diagnosis from clinical features important, but also persistent additional treatment should be carried out without delay to effectively control intracranial pressure.

Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein.

The Bone morphogenetic protein (Bmp) signalling gradient has a major function in the formation of the dorso-ventral axis. The zebrafish ventralized mutant, ogon, encodes Secreted Frizzled (Sizzled). sizzled is ventrally expressed in a Bmp-dependent manner and is required for the suppression of Bmp signalling on the ventral side of zebrafish embryos. However, it remains unclear how Sizzled inhibits Bmp signalling and controls ventro-lateral cell fate. We found that Sizzled stabilizes Chordin, a Bmp antagonist, by binding and inhibiting the Tolloid-family metalloproteinase, Bmp1a, which cleaves and inactivates Chordin. The cysteine-rich domain of Sizzled is required for inhibition of Bmp1a activity. Loss of both Bmp1a and Tolloid-like1 (Tll1; another Tolloid-family metalloproteinase) function leads to a complete suppression and reversal of the ogon mutant phenotype. These results indicate that Sizzled represses the activities of Tolloid-family proteins, thereby creating the Chordin-Bmp activity gradient along the dorso-ventral axis. Here, we describe a previously unrecognized role for a secreted Frizzled-related protein.

Physical properties of single-wall carbon nanotubes in cell culture and their dispersal due to alveolar epithelial cell response.

Concern over the influence of carbon nanotubes (CNTs) on human health has arisen due to advances; however, little is known about the potential toxicity of CNTs. In this study, impurity-free single-wall carbon nanotubes (SWCNTs), with different physical properties in cell culture medium, were prepared by a novel dispersion procedure. SWCNTs with small bundles (short linear shape) and SWCNTs with large bundles (long linear shape) did not cause a significant inhibition of cell proliferation, induction of apoptosis or arrest of cell cycle progression in A549 alveolar epithelial cells. Expression of many genes involved in the inflammatory response, apoptosis, response to oxidative stress and degradation of the extracellular matrix were not markedly upregulated or downregulated. However, SWCNTs with relatively large bundles significantly increased the level of intracellular reactive oxygen species (ROS) in a dose-dependent manner, and the levels of these ROS were higher than those of SWCNTs with relatively small bundles or commercial SWCNTs with residual metals. Transmission electron microscopy (TEM) revealed that impurity-free SWCNTs were observed in the cytoplasm and vacuoles of cells after 24 h. These results suggested that the physical properties, especially the size and length of the bundles of the SWCNTs dispersed in cell culture medium, contributed to a change in intracellular ROS generation, even for the same bulk SWCNTs. Additionally, the residual metals associated with the manufacturing of SWCNTs may not be a definitive parameter for intracellular ROS generation in A549 cells.