Evaluation of Bis(2-ethylhexyl) phthalate toxicity on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

Bis(2-ethylhexyl) phthalate, generally known as DEHP is a synthetic compound mainly used as a plasticizer to make polyvinyl chloride products flexible and soft. The present work aimed to study the toxicity of Bis(2-ethylhexyl) phthalate on the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ) Bg9. The hsp70 gene is associated with the ß-galactosidase in our present transgenic strain therefore, the more activity of ß-galactosidase will indirectly correspond to hsp70 expression. The third instar larvae were allowed to feed on the diet for 24 h having 0.001, 0.005, 0.01, and 0.02 M of Bis(2-ethylhexyl) phthalate at the final concentration. After the exposure of 24hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers assays, and comet assay. A dose-dependent increase in hsp70 expression, tissue damage, Glutathione-S-transferase (GST) activity, lipid peroxidation, monoamine oxidase, caspase-9 & 3, protein carbonyl content (PCC), DNA damage and decrease in the glutathione (GSH) content, delta-aminolevulinic acid dehydrogenase (ẟ-ALD-D) and acetylcholinesterase activity were observed in the larvae exposed to 0.005, 0.01, 0.02 M of Bis-(2-ethylhexyl) phthalate. The dose of 0.001 M of Bis(2-ethylhexyl) phthalate did not showed any toxic effects and hence can be considered as No Observed Adverse Effect Level (NOAEL) for Bis(2-ethylhexyl) phthalate. The study supports the use of Drosophila for the evaluation of possible toxic effects associated with synthetic compounds.

Hydroxyapatite microspheres used as a drug delivery system for gliosarcoma strain 9l/Lacz treatment by photodynamic therapy protocols.

BACKGROUND: Hydroxyapatite (HAp) presents similarities with the human bone structure and presents properties such as biodegradability, biocompatibility, and osteoconductivity, which favors its use in prostheses implants and enables its use as a vehicle for the delivery of photosensitizers (PS) from systems of release (DDS) for photodynamic therapy applications Methods: In this work was to synthesized hydroxyapatite microspheres (meHAp), encapsulated with chloroaluminium phthalocyanine (ClAlPc), for DDS. meHAp was synthesized using vaterite as a template. The drug was encapsulated by mixing meHAp and a 50.0 mg.mL-1 ClAlPc solution. Photochemical, photophysical, and photobiological studies characterized the system. RESULTS: The images from the SEM analysis showed the spherical form of the particles. All spectroscopic results showed excellent photophysical parameters of the drug studied when served in the meHAp system. The incorporation efficiency was 57.8 %. The trypan blue exclusion test results showed a significant reduction (p < 0.05) in cell viability for the groups treated with PDT at all concentrations above 250 µg.mL-1. In 9 L/lacZ gliosarcoma cells, PDT mediated at concentrations from 250 to 62.5 µg.mL-1 reduced cell viability by more than 98 %. In the cell internalization study, it was possible to observe the internalization of phthalocyanines at 37 °C, with the accumulation of PS in the cytoplasm and inside the nucleus in the two tested concentrations. CONCLUSIONS: From all the results presented throughout the article, the meHAp system shows promise for use as a modified release system (DSD) in photodynamic therapy.

Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Optimized immunofluorescence staining protocol for identifying resident mesenchymal stem cells in bone using LacZ transgenic mice.

Glioma-associated oncogene homolog 1 (Gli1) marks a subpopulation of endogenous mesenchymal stem cells (MSCs) characterized by perivascular location. Here, we present an optimized immunofluorescence staining protocol to identify resident Gli1+ MSCs in fixed/frozen bone sections from LacZ transgenic mice. This protocol describes the preparation of fixed/frozen tissue sections and the use of LacZ immunofluorescent staining for the in vivo characterization of endogenous MSCs, regarding their specific identity and specialized niches, and is applicable to LacZ-expressing cells of diverse organs. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Photodynamic effect of protoporphyrin IX in gliosarcoma 9l/lacZ cell line.

Photodynamic Therapy (PDT) is an oncologic treatment, producing reactive oxygen species (ROS) to induce the death of cancer cells. This study aimed to evaluate the action of PDT on gliosarcoma cells, using protoporphyrin IX as PS by incubation with the precursor aminolevulinic acid (ALA). An LED device was used with a light dose of 10 J/cm². The success of the therapy proved to be dependent on the concentration of ALA, and an incubation time of 4 h required for an effective response. Cell death was prevalent due to necrosis when assessed 18 h post-PDT. ALA proved to be an option to PDT in cells of the 9 L/lacZ, with the protocol tested.

Zinc pthalocyanine loaded poly (lactic acid) nanoparticles by double emulsion methodology for photodynamic therapy against 9 L/LacZ gliosarcoma cells.

Development delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (NP) were prepared using a double-emulsion method to load zinc(II) phthalocyanine (ZnPc). NP were obtained using poly (lactic acid) (PLA). ZnPc is a second generation of photosensitizer used in photodynamic therapy (PDT). ZnPc loaded PLA nanoparticles (NPLA-ZnPc) were prepared by double-emulsion method, characterized and available in cellular culture. The mean nanoparticle size presented particle size was 384.7 ± 84.2 nm with polydispersity index (PDI) of 0.150 ± 0.015, and the encapsulation efficiency was of 83%. The nanoparticle formulations presented negative zeta potential values (-27.5 ± 1.0 mV), explaining their colloidal stability. ZnPc loaded nanoparticles maintain its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained over 168 h with a biphasic ZnPc release profile. An in vitro phototoxic effect in range of 80% was observed in 9 L/LacZ gliosarcoma cells at laser light doses (10 J cm-2) with 3.0 µg mL-1 of NPLA-ZnPc. All the physical-chemical, photophysical and photobiological measurements performed allow us to conclude that ZnPc loaded PLGA nanoparticles is a promising drug delivery system for PDT.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Transcription Factor ß-Catenin Plays a Key Role in Fluid Flow Shear Stress-Mediated Glomerular Injury in Solitary Kidney.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3ß-ß-catenin signaling. The present study was undertaken to confirm (i) the activation of ß-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the ß-galactosidase reporter gene for ß-catenin activation, (ii) ß-catenin translocation in FFSS-treated mouse podocytes, and (iii) ß-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased ß-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-ß-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-ß-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-ß-catenin (Ser552) but not phospho-ß-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified ß-catenin as a key upstream regulator. We conclude that transcription factor ß-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

Folate Intake Alters Mutation Frequency and Profiles in a Tissue- and Dose-Specific Manner in MutaMouse Male Mice.

BACKGROUND: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk. OBJECTIVE: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice. METHODS: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test. RESULTS: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific. CONCLUSIONS: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.

Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice.

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system.

Analysis of the effects of Photodynamic therapy with Photodithazine on the treatment of 9l/lacZ cells, in vitro study.

Gliosarcoma is an aggressive brain tumor. Photodynamic Therapy (PDT) is a treatment that can be used for various cancers of the CNS. The aim of this study was to analyze the effects of PDT with Photodithazine (PDZ) in the treatment of gliosarcoma, using 9 L/lacZ cells and serial concentrations of 200 µg/mL to 3.1 µg/mL of PDZ. The samples were divided into two groups: dark and light (10 J/cm²). The PDZ was internalized along all the cytoplasmic extension. Viability tests demonstrated a reduction in viable cells after PDT. The production of ROS was concentration-dependent and PDZ was found in mitochondria and lysosomes, presenting a discrete connection with α-tubulin. However, this structure is likely damaged, evidenced by changes in the morphological analysis. Thus, according to the parameters of this study, PDZ proved to be an interesting PS in PDT for the treatment of gliosarcoma, with the inherent limitations of an in vitro study.

Single-stranded DNA versus tailed duplex in sequence conversion of lacZα DNA.

Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence.

Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions.

BACKGROUND: The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure. RESULTS: The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3' non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing. CONCLUSION: The ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.

Solithromycin Can Specifically Induce Macrolide-Lincosamide-Streptogramin B Resistance.

Objectives: Solithromycin is a fluoroketolide that is considered to be a noninducing antibiotic for macrolide-lincosamide-streptogramin B resistance mediated by erm genes. The exact activity of solithromycin to induce erm gene expression remains to be determined. Materials and Methods: The potential of solithromycin to induce erm(A), erm(C), and erm(B) gene expression was examined using a lacZ reporter assay, double-disk diffusion test, and determination of the minimal inhibitory concentration after incubation with subinhibitory concentration of different antibiotics. Results: Neither solithromycin nor the ketolides telithromycin and cethromycin induced erm(A) or erm(C) gene expression. However, solithromycin could significantly induce erm(B) gene expression at levels greater than that seen for cethromycin and clindamycin, but less than that for erythromycin, rokitamycin, and telithromycin. Conclusion: Solithromycin does not induce erm(A) and erm(C) gene expression, but does induce erm(B) gene expression, although to a weaker extent than that seen for macrolides.

Adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced fibrillin-2 cleavage.

Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10-/- mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10-/- mice, similar to Fbn1-/- mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10-/- zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10-/- eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10-/- mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.

In Utero Exposure to Benzo[a]pyrene Induces Ovarian Mutations at Doses That Deplete Ovarian Follicles in Mice.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA-reactive hematopoietic carcinogen with potent and well-characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig-a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose-related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig-a assays even at the lowest dose tested. PCH-induced lacZ and Pig-a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505-512, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model.

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018.

Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.

VemP ( Vibrio protein export monitoring polypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156 codon of vemP encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met120-Phe137) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.

Characterization of a new, inducible transgenic mouse model with GFP expression in melanocytes and their precursors.

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties.