Differential manifestation of ocular phenotypes in TALEN-mediated p19arf knockout FVB/N and C57BL/6J mouse lines.

BACKGROUND: p19arf, primarily known as a tumor suppressor, has also been reported to play an essential role in normal development of mouse eyes. Consistently, lack of p19arf has been associated with ocular defects, but the mixed background of the knockout (KO) mouse strain used raised a concern on the accuracy of the phenotypes observed in association with the targeted gene due to genetic heterogeneity. OBJECT: We carried out a study to investigate into the effect of genetic background on the manifestation of p19arf KO associated phenotypes. METHODS: We characterized the phenotypes of novel p19arf KO mouse lines generated in FVB/N and C57BL/6J using a transcription activator-like effector nuclease (TALEN) system in comparison to the reported phenotypes of three other p19arf-deficient mouse lines generated using homologous recombination. RESULTS: Ninety-five percent of FVB/N-p19arf KO mice showed ocular opacity from week 4 after birth which worsened rapidly until week 6, while such abnormality was absent in C57BL/6J-p19arf KO mice up to the age of 26 weeks. Histopathological analysis revealed retrolental masses and dysplasia in the retinal layer in FVB/N-p19arf KO mice from week 4. Besides these, both strains developed normally from birth to week 26 without increased tumorigenesis except for a subcutaneous tumor found in a C57BL/6J-p19arf KO mouse. CONCLUSION: Our findings demonstrated surprisingly variable manifestation of p19arf-linked phenotypes between FVB/N and C57BL/6J mice, and furthermore between our mouse lines and the established lines, indicating a critical impact of genetic background on functional study of genes using gene targeting strategies in mice.

Pediococcus spp.-fermented chicken meat for dogs.

An experiment was conducted to evaluate Pediococcus spp.-fermented chicken meat as a snack for dogs. The fermented or non-fermented snacks used in this study were prepared through the following process; meat mixtures containing 52.8% MDCM, 35.2% chicken breast meat (CBM) and 9.7% corn starch were inoculated with or without Pediococcus spp., incubated at 37°C for 24 h and then sterilized at 121°C for 20 min. During the 24-h fermentation, the pH of fermented chicken snack dropped rapidly with concomitant increase in number of lactic acid bacteria. The nutritional composition was not altered by fermentation. In vitro pepsin nitrogen digestibility was higher (p < 0.05) in the fermented snack compared with the non-fermented snack. Upon storage at room temperature for 14 days, bacteria grew slowly in fermented vs. non-fermented snack samples. In a palatability trial, dogs preferred non-fermented over fermented snack food. In 12-d-long feeding trial, fecal ammonia content was lowered, but fecal lactic acid content was increased in dogs fed the fermented vs. non-fermented snack food. Our study shows that the fermented MDCM-based snack exhibited good preservability upon storage, and improved in vitro nitrogen digestibility and fecal characteristics in dogs.

Lysates of a Probiotic, Lactobacillus rhamnosus, Can Improve Skin Barrier Function in a Reconstructed Human Epidermis Model.

The main function of the skin is to protect the body from the external environment. The barrier function of the skin is mainly provided by the stratum corneum, which consists of corneocytes bound with the corneodesmosomes and lamellar lipids. Skin barrier proteins like loricrin and filaggrin also contribute to the skin barrier function. In various skin diseases, skin barrier dysfunction is a common symptom, and skin irritants like detergents or surfactants could also perturb skin barrier function. Many efforts have been made to develop strategies to improve skin barrier function. Here, we investigated whether the microfluidized lysates of Lactobacillus rhamnosus (LR), one of the most widely used probiotic species for various health benefits, may improve the skin barrier function in a reconstructed human epidermis, Keraskin™. Application of LR lysate on Keraskin™ increased the expression of tight junction proteins; claudin 1 and occludin as determined by immunofluorescence analysis, and skin barrier proteins; loricrin and filaggrin as determined by immunohistochemistry and immunofluorescence analysis and qPCR. Also, the cytotoxicity of a skin irritant, sodium lauryl sulfate (SLS), was alleviated by the pretreatment of LR lysate. The skin barrier protective effects of LR lysate could be further demonstrated by the attenuation of SLS-enhanced dye-penetration. LR lysate also attenuated the destruction of desmosomes after SLS treatment. Collectively, we demonstrated that LR lysate has protective effects on the skin barrier, which could expand the utility of probiotics to skin-moisturization ingredients.

CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice.

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2 -/-) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2 -/- mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2 -/- mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2 -/- mice. These findings indicate that our RAG-2 -/- mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice.

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47 -/- hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47 -/- mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47 -/- mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47 -/- mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47 -/- mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47 -/- mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47 -/- mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1.

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21(Cip1) and p27(Kip1) expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G1 arrest, up-regulation of cell cycle-regulatory proteins p21(Cip1) and p27(Kip1) was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins.

Preparation, characterization, and cytotoxicity of CPT/Fe2O3-embedded PLGA ultrafine composite fibers: a synergistic approach to develop promising anticancer material.

The aim of this study was to fabricate camptothecin/iron(III) oxide (CPT/Fe2O3)-loaded poly(D,L-lactide-co-glycolide) (PLGA) composite mats to modulate the CPT release and to improve the structural integrity and antitumor activity of the released drug. The CPT/Fe2O3-loaded PLGA ultrafine fibers were prepared for the first time by electrospinning a composite solution of CPT/Fe2O3 and neat PLGA (4 weight percent). The physicochemical characterization of the electrospun composite mat was carried out by scanning electron microscopy, energy dispersive X-ray spectroscopy, electron probe microanalysis, thermogravimetry, transmission electron microscopy, ultraviolet-visible spectroscopy, and X-ray diffraction pattern. The medicated composite fibers were evaluated for their cytotoxicity on C2C12 cells using Cell Counting Kit-8 assay (Sigma-Aldrich Corporation, St Louis, MO). The in vitro studies indicated a slow and prolonged release over a period of 96 hours with mild initial burst. Scanning electron microscopy, thermogravimetry, and X-ray diffraction studies confirmed the interaction of CPT/Fe2O3 with the PLGA matrix and showed that the crystallinity of CPT decreased after loading. Incorporation of CPT in the polymer media affected both the morphology and the size of the CPT/Fe2O3-loaded PLGA composite fibers. Electron probe microanalysis and energy dispersive X-ray spectroscopy results confirmed well-oriented composite ultrafine fibers with good incorporation of CPT/Fe2O3. The cytotoxicity results illustrate that the pristine PLGA did not exhibit noteworthy cytotoxicity; conversely, the CPT/Fe2O3 composite fibers inhibited C2C12 cells significantly. Thus, the current work demonstrates that the CPT/Fe2O3-loaded PLGA composite fibers represent a promising chemotherapeutic system for enhancing anticancer drug efficacy and selectively targeting cancer cells in order to treat diverse cancers.

Vitamin D3 upregulated protein 1 deficiency promotes N-methyl-N-nitrosourea and Helicobacter pylori-induced gastric carcinogenesis in mice.

OBJECTIVE: Vitamin D(3) upregulated protein 1 (VDUP1) is a potent tumour suppressor whose expression is dramatically reduced in various types of human cancers, including gastric cancer. However, the precise mechanisms underlying tumour development remain unclear. In the present study, the authors examined the effect of VDUP1 on Helicobacter pylori-induced gastric carcinogenesis in mice. DESIGN: Gastric cancer was generated in VDUP1 knockout (KO) and wild-type mice using a combination of N-methyl-N-nitrosourea treatment and H pylori infection. Fifty weeks after treatment, gastric tissues from both types of mice were examined by histopathology, immunohistochemistry and immunoblotting. In vitro tests on the human gastric cancer cell line, AGS, were also performed to identify the underlying mechanisms of cancer development. RESULTS: The overall incidence of gastric cancer was significantly higher in VDUP1 KO mice than in wild-type mice. Similarly, VDUP1 KO mice showed more severe chronic gastritis, glandular atrophy, foveolar hyperplasia, metaplasia and dysplasia. Although no differences in the apoptotic index were apparent, lack of VDUP1 increased the rate of gastric epithelial cell proliferation in non-cancerous stomachs, with corresponding increases in tumour necrosis factor alpha (TNFα) level, nuclear transcription factor kappa B (NF-κB) activation and cyclooxygenase-2 (COX-2) expression. An in vitro study showed that H pylori-associated cell proliferation and induction of TNFα, NF-κB and COX-2 were inhibited in cells transfected with VDUP1. In addition, overexpression of VDUP1 in AGS cells suppressed TNFα-induced NF-κB activation and COX-2 expression. CONCLUSION: Our data show that VDUP1 negatively regulates H pylori-associated gastric carcinogenesis, in part by disrupting cell growth and inhibiting the induction of TNFα, NF-κB and COX-2. These findings provide important insights into the role of VDUP1 in H pylori-associated tumourigenesis.

Effect of polymer molecular weight on the fiber morphology of electrospun mats.

In this work, different fractions of solvent-induced polymer degraded solution were mixed with freshly prepared solution of same polymer, and its effect on fiber morphology of electrospun mats was investigated. Nylon-6 solution in formic acid was allowed to degrade for 3 weeks and different fractions of it were mixed with freshly prepared nylon-6 solution to get the electrospun mats. FE-SEM images of the mats indicated that the a large amount of sub-nanofibers (<50 nm in diameter) in the form of spider-net like structures were achieved by tailoring the amount of solvent degraded polymer solution in the freshly prepared nylon-6 solution. Large quantity of these ultrafine sub-nanofibers present in electrospun nylon-6 mats could increase its hydrophilicity and mechanical strength. The decreased average pore diameter and increased BET surface area of the mat, caused by spider-net like structure, can make it as a potential candidate for air/water filtration.

Effect of adhesive on the morphology and mechanical properties of electrospun fibrous mat of cellulose acetate.

Ultrafine fibers of cellulose acetate/poly(butyl acrylate) (CA/PBA) composite in which PBA acted as an adhesive and CA acted as a matrix, were successfully prepared as fibrous mat via electrospinning. The morphology observation from the electrospun CA/PBA composite fibers, after treatment with heat hardener, revealed that the fibers were cylindrical and had point-bonded structures. SEM, FT-IR spectra, Raman spectra, TGA analysis, and mechanical properties measurement were used to study the different properties of hybrid mats. The tensile strength of blend fibrous electrospun mats was found to be effectively increased. This resultant enhancement of the mechanical properties of polymer fibrous mats, caused by generating the point-bonded structures (due to adhesive), could increase the number of potential applications of mechanically weak electrospun CA fibers.

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm.

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2(EGFP), appear normal. However, further reductions in Sox2, using Sox2(LP) and Sox2(COND) hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2(EGFP/COND) embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63(+) basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2(EGFP/COND) embryos in which Sox2 levels are approximately 18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63(+) cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.

Health surveillance of specific pathogen-free and conventionally-housed mice and rats in Korea.

The present study contains information about proper microbiological monitoring of laboratory animals' health and the standardization of microbiological monitoring methods in Korea. Microbiological quality control for laboratory animals, composed of biosecurity and health surveillance, is essential to guard against research complications and public health dangers that have been associated with adventitious infections. In this study, one hundred and twenty-two mice and ninety rats from laboratory animal breeding companies and one animal facility of the national universities in Korea were monitored in 2000-2003. Histopathologically, thickening of the alveolar walls and lymphocytic infiltration around the bronchioles were observed in mice and rats from microbiologically contaminated facilities. Cryptosporidial oocysts were observed in the gastric pits of only conventionally-housed mice and rats. Helicobacter spp. infection was also detected in 1 of 24 feces DNA samples in mice and 9 of 40 feces DNA samples in rats by PCR in 2003, but they were not Helicobacter hepaticus. This paper describes bacteriological, parasitological, and virological examinations of the animals.

Phenotype of peroxisome proliferator-activated receptor-alpha(PPARalpha)deficient mice on mixed background fed high fat diet.

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-alpha PPARalpha) on obesity. Previous reports demonstrated that PPARalpha is a critical modulator of lipid homeostasis, but the overt, obese phenotypic characterization in the strain of PPAR deficient (PPARalpha-/-) mice is influenced by other factors, including diet and genetics. Therefore, it is necessary to establish the phenotypic characterization of PPARalpha-/- mice prior to the obesity-related study. In this study, we observed phenotype of PPARalpha-/- mice on mixed genetic background (C57BL/6Nx129/Sv) fed a high fat diet for 16 weeks. PPARalpha-/- mice, regardless of sex, raised body growth rate significantly comparing with wild type and showed male-specific fatty change in the liver. They were shown to lack hepatic induction of PPARalpha target genes encoding enzymes for fatty acid beta-oxidation.