Noninvasive Evaluation of Breast Tumor Response to Combined Ultrasound-Stimulated Microbubbles and Hyperthermia Therapy Using Quantitative Ultrasound-Based Texture Analysis Method.

OBJECTIVES: Quantitative ultrasound (QUS) is a noninvasive imaging technique that can be used for assessing response to anticancer treatment. In the present study, tumor cell death response to the ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) treatment was monitored in vivo using QUS. METHODS: Human breast cancer cell lines (MDA-MB-231) were grown in mice and were treated with HT (10, 30, 50, and 60 minutes) alone, or in combination with USMB. Treatment effects were examined using QUS with a center frequency of 25 MHz (bandwidth range: 16 to 32 MHz). Backscattered radiofrequency (RF) data were acquired from tumors subjected to treatment. Ultrasound parameters such as average acoustic concentration (AAC) and average scatterer diameter (ASD), were estimated 24 hours prior and posttreatment. Additionally, texture features: contrast (CON), correlation (COR), energy (ENE), and homogeneity (HOM) were extracted from QUS parametric maps. All estimated parameters were compared with histopathological findings. RESULTS: The findings of our study demonstrated a significant increase in QUS parameters in both treatment conditions: HT alone (starting from 30 minutes of heat exposure) and combined treatment of HT plus USMB finally reaching a maximum at 50 minutes of heat exposure. Increase in AAC for 50 minutes HT alone and USMB +50 minutes was found to be 5.19 ± 0.417% and 5.91 ± 1.11%, respectively, compared to the control group with AAC value of 1.00 ± 0.44%. Furthermore, between the treatment groups, ΔASD-ENE values for USMB +30 minutes HT significantly reduced, depicting 0.00062 ± 0.00096% compared to 30 minutes HT only group, showing 0.0058 ± 0.0013%. Further, results obtained from the histological analysis indicated greater cell death and reduced nucleus size in both HT alone and HT combined with USMB. CONCLUSION: The texture-based QUS parameters indicated a correlation with microstructural changes obtained from histological data. This work demonstrated the use of QUS to detect HT treatment effects in breast cancer tumors in vivo.

Quantitative Ultrasound for Evaluation of Tumour Response to Ultrasound-Microbubbles and Hyperthermia.

Objectives: Prior study has demonstrated the implementation of quantitative ultrasound (QUS) for determining the therapy response in breast tumour patients. Several QUS parameters quantified from the tumour region showed a significant correlation with the patient's clinical and pathological response. In this study, we aim to identify if there exists such a link between QUS parameters and changes in tumour morphology due to combined ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) using the breast xenograft model (MDA-MB-231). Method: Tumours grown in the hind leg of severe combined immuno-deficient mice were treated with permutations of USMB and HT. Ultrasound radiofrequency data were collected using a 25 MHz array transducer, from breast tumour-bearing mice prior and post-24-hour treatment. Result: Our result demonstrated an increase in the QUS parameters the mid-band fit and spectral 0-MHz intercept with an increase in HT duration combined with USMB which was found to be reflective of tissue structural changes and cell death detected using haematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labelling stain. A significant decrease in QUS spectral parameters was observed at an HT duration of 60 minutes, which is possibly due to loss of nuclei by the majority of cells as confirmed using histology analysis. Morphological alterations within the tumour might have contributed to the decrease in backscatter parameters. Conclusion: The work here uses the QUS technique to assess the efficacy of cancer therapy and demonstrates that the changes in ultrasound backscatters mirrored changes in tissue morphology.

Effects of clonal integration and nutrient availability on the growth of Glechoma longituba under heterogenous light conditions.

Introduction: Clonal integration of connected ramets within clones is an important ecological advantage. In this study, we tested the hypothesis that the effects of clonal integration on performance of donor and recipient ramets when one resource is heterogeneous can be influenced by the availability of another resource of donor ramets. Methods: We conducted a greenhouse experiment on the widespread, perennial herb Glechoma longituba. Clonal fragments consisting of pairs of connected ramets were grown for seven weeks. The younger, apical ramets were exposed under 30% or 100% light condition and the older, basal ramets were treated with three levels of nutrients. The connections between ramets were either severed or left intact. 30% light condition negatively affected the growth of apical ramets, basal ramets and the whole fragments. Results: Clonal integration significantly increased the growth of apical ramets, but decreased the growth of the basal ramets. Medium and high level nutrient availability of basal ramets significantly increased the growth of apical ramets, basal ramets and the whole fragments. At the high nutrient level, the reduction in growth of basal ramets from clonal integration was decreased, but the growth responses of apical ramets and the whole fragments to clonal integration were not influenced by nutrient availability. Conclusion: The results suggested that clonal integration was benefit to the growth of apical ramets of Glechoma longituba but at the cost of reducing the growth of basal ramets. Although the high nutrient level could reduce the cost that clonal integration brought to the unshaded basal ramets, but could not increase the benefit that clonal integration brought to the shaded apical ramets and whole fragment.

Cadmium stress interacts with nutrient availability and light condition to affect the growth of Hydrocotyle vulgaris.

Heavy metal pollution is becoming a serious problem in wetland and often co-occurs with nutrient availability and light conditions variation. We hypothesized that nutrient availability and light condition can affect the growth of wetland plants under heavy metal stress. To test this hypothesis, single ramets of a common, clonal wetland plant Hydrocotyle vulgaris were grown for four weeks at three levels of cadmium with three levels of nutrient availability under 30% or 100% light conditions. High level of nutrient availability and high light condition overall promoted growth of H. vulgaris under Cd stress. Under the two light conditions, responses of H. vulgaris to Cd treatments differed among three nutrient levels. Under 30% light condition, 2 mg L-1 Cd2+ treatment decreased total mass at the low nutrient level and decreased ramet number at the medium nutrient level; 0.5 and 2 mg L-1 Cd2+ treatments decreased leaf mass ratio at the low and the medium nutrient levels. Under 100% light condition, 2 mg L-1 Cd2+ treatments significantly decreased total mass at the high level of nutrients; 2 mg L-1 Cd2+ treatment decreased ramet number at the medium and the high nutrient levels and decreased leaf mass ratio at the medium nutrient levels. Our results suggested that Cd stress can interact with nutrient availability and light condition to affect the performance of wetland plants such as H. vulgaris.

MiR-208b Regulates the Conversion of Skeletal Muscle Fiber Types by Inhibiting Mettl8 Expression.

Skeletal muscle, the main source of animal meat products, contains muscle fiber as a key unit. It is well known that transformation takes place between different types of muscle fibers, however, the conversion mechanism is not clear. In a previous study, our lab has demonstrated that there is a decrease in type I muscle fibers and an increase in type IIB muscle fibers in skeletal muscle of myostatin gene-edited Meishan pigs. Very interestingly, we observed the down regulation of miR-208b expression and an increase in expression the predicted target gene Mettl8 (Methyltransferase like 8) in skeletal muscle of MSTN gene-edited Meishan pigs. These results reveal that there is a potential connection between the conversion of skeletal muscle fiber types and miR-208b and Mettl8 expression. In this study, we first explored the expression patterns of miR-208b and Mettl8 in skeletal muscle in Meishan pigs; and then C2C12 cells were used to simulate the development and maturation of muscle fibers. Our results indicated that Myh4 expression level decreased and Myh7 expression level increased following overexpression of miR-208b in C2C12 cells. We therefore speculate that miR-208b can promote the conversion of fast-twitch fibers to slow-twitch fibers. The targeting relationship between Mettl8 and miR-208b was confirmed by results obtained using dual luciferase assay, RT-qPCR, and WB analysis. Following the transfection of Mettl8 siRNA into C2C12 cells, we observed that Mettl8 expression decreased significantly while Myh7 expression increased and Myh4 expression decreased, indicating that Mettl8 promotes the conversion of slow muscle fibers to fast muscle fibers. Additionally, changes in skeletal muscle fiber types are observed in those mice where miR-208b and Mettl8 genes are knocked out. The miR-208b knockout inhibits the formation of slow muscle fibers, and the Mettl8 knockout inhibits the formation of fast muscle fibers. In conclusion, our research results show that miR-208b regulates the conversion of different muscle fiber types by inhibiting Mettl8 expression.

Fusion of 3D lung CT and serum biomarkers for diagnosis of multiple pathological types on pulmonary nodules.

BACKGROUND AND OBJECTIVE: Current researches on pulmonary nodules mainly focused on the binary-classification of benign and malignant pulmonary nodules. However, in clinical applications, it is not enough to judge whether pulmonary nodules are benign or malignant. In this paper, we proposed a fusion model based on the Lung Information Dataset Containing 3D CT Images and Serum Biomarkers (LIDCCISB) we constructed to accurately diagnose the types of pulmonary nodules in squamous cell carcinoma, adenocarcinoma, inflammation and other benign diseases. METHODS: Using single modal information of lung 3D CT images and single modal information of Lung Tumor Biomarkers (LTBs) in LIDCCISB, a Multi-resolution 3D Multi-classification deep learning model (Mr-Mc) and a Multi-Layer Perceptron machine learning model (MLP) were constructed for diagnosing multiple pathological types of pulmonary nodules, respectively. To comprehensively use the double modal information of CT images and LTBs, we used transfer learning to fuse Mr-Mc and MLP, and constructed a multimodal information fusion model that could classify multiple pathological types of benign and malignant pulmonary nodules. RESULTS: Experiments showed that the constructed Mr-Mc model can achieve an average accuracy of 0.805 and MLP model can achieve an average accuracy of 0.887. The fusion model was verified on a dataset containing 64 samples, and achieved an average accuracy of 0.906. CONCLUSIONS: This is the first study to simultaneously use CT images and LTBs to diagnose multiple pathological types of benign and malignant pulmonary nodules, and experiments showed that our research was more advanced and more suitable for practical clinical applications.

Swallowtail-type diffraction catastrophe beams.

We demonstrate a universal approach for generating high-order diffraction catastrophe beams, specifically for Swallowtail-type beams (abbreviated as Swallowtail beams), using diffraction catastrophe theory that was defined by potential functions depending on the control and state parameters. The three-dimensional curved caustic surfaces of these Swallowtail catastrophe beams are derived by the potential functions. Such beams are generated by mapping the cross sections of the high-order control parameter space to the corresponding transverse plane. Owing to the flexibility of the high-order diffraction catastrophe, these Swallowtail beams can be tuned to a diverse range of optical light structures. Owing to the similarity in their frequency spectra, we found that the Swallowtail beams change into low-order Pearcey beams under given conditions during propagation. Our experimental results are in close agreement with our simulated results. Such fantastic catastrophe beams that can propagate along curved trajectories are likely to give rise to new applications in micromachining and optical manipulation, furthermore, these diverse caustic beams will pave the way for the tailoring of arbitrarily accelerating caustic beams.

Lung Respiratory Motion Estimation Based on Fast Kalman Filtering and 4D CT Image Registration.

Respiratory motion estimation is an important part in image-guided radiation therapy and clinical diagnosis. However, most of the respiratory motion estimation methods rely on indirect measurements of external breathing indicators, which will not only introduce great estimation errors, but also bring invasive injury for patients. In this paper, we propose a method of lung respiratory motion estimation based on fast Kalman filtering and 4D CT image registration (LRME-4DCT). In order to perform dynamic motion estimation for continuous phases, a motion estimation model is constructed by combining two kinds of GPU-accelerated 4D CT image registration methods with fast Kalman filtering method. To address the high computational requirements of 4D CT image sequences, a multi-level processing strategy is adopted in the 4D CT image registration methods, and respiratory motion states are predicted from three independent directions. In the DIR-lab dataset and POPI dataset with 4D CT images, the average target registration error (TRE) of the LRME-4DCT method can reach 0.91 mm and 0.85 mm respectively. Compared with traditional estimation methods based on pair-wise image registration, the proposed LRME-4DCT method can estimate the physiological respiratory motion more accurately and quickly. Our proposed LRME-4DCT method fully meets the practical clinical requirements for rapid dynamic estimation of lung respiratory motion.

Deep model with Siamese network for viable and necrotic tumor regions assessment in osteosarcoma.

PURPOSE: To achieve automatic classification of viable and necrotic tumor regions in osteosarcoma, most of the existing deep learning methods can only design a simple model to prevent overfitting on small datasets, which leads to the weak ability of extracting image features and low accuracy of the models. In order to solve the above problem, a deep model with Siamese network (DS-Net) was designed in this paper. METHODS: The DS-Net constructed on the basis of full convolutional networks is composed of an auxiliary supervision network (ASN) and a classification network. The construction of the ASN based on the Siamese network aims to solve the problem of a small training set (the main bottleneck of deep learning in medical images). It uses paired data as the input and updates the network through combined labels. The classification network uses the features extracted by the ASN to perform accurate classification. RESULTS: Pathological diagnosis is the most accurate method to identify osteosarcoma. However, due to intraclass variation and interclass similarity, it is challenging for pathologists to accurately identify osteosarcoma. Through the experiments on hematoxylin and eosin (H&E)-stained osteosarcoma histology slides, the DS-Net we constructed can achieve an average accuracy of 95.1%. Compared with existing methods, the DS-Net performs best in the test dataset. CONCLUSIONS: The DS-Net we constructed can not only effectively realize the histological classification of osteosarcoma, but also be applicable to many other medical image classification tasks affected by small datasets.

Enhanced skeletal muscle growth in myostatin-deficient transgenic pigs had improved glucose uptake in stretozotocin-induced diabetes.

The size of skeletal muscle mass plays a significant role in glucose uptake in healthy and diabetic human subjects. Previously, we have generated myostatin-deficient (MSTN-/-) transgenic pigs via animal cloning technology. MSTN-/- pigs had dramatic phenotype with individual muscle mass increase by 100% over their wild-type controls, which provides a unique large animal model to investigate how enhanced skeletal muscles are beneficial to glucose update in diabetes. We employed intravenous administration of stretozotocin (STZ) to male MSTN-/- and wild-type pigs (100 mg/kg body weight). One month later, blood glucose and insulin concentrations and pancreas histology were examined, STZ-induced diabetes occurred in both MSTN transgenic and wild-type pigs. Histology of pancreas, analysis of pAKT and Glut4 transporter proteins by Western blotting, and real-time qPCR for MSTN gene expression were used in the study. The STZ-treated pigs had increased levels of fasting plasma glucose and insulin levels in comparison with animals receiving sodium citrate buffer, their pancreas also had reduced beta cells and slight increases in lymphocyte. There are significant lower concentrations of fasting plasma glucose and insulin in MSTN-/- pigs than that of wild-type pigs after STZ administration. Detections of pAKT and Glut4 transporter proteins by Western blotting in muscle tissue indicates significant elevations of both proteins in MSTN-/- pigs compared with the wild-type pigs. The results from this pig model suggest that enhanced skeletal muscle by manipulation of myostatin function can improve glucose uptake even in the status of diabetes.

Identification of genes related to skeletal muscle growth and development by integrated analysis of transcriptome and proteome in myostatin-edited Meishan pigs.

Embryonic development of skeletal muscle is a complex process that is important to the growth of skeletal muscle after birth. However, the mechanisms by which skeletal muscle growth and development in embryonic phase remain unclear. We have previously produced myostatin-knockout (MKO) Meishan pigs with double-muscle (DM) phenotype via zinc finger nucleases (ZFN) technology. To further investigate the molecular mechanisms involved in skeletal muscle growth and development, in this study, we performed an integrated analysis of transcriptome and proteome in longissimus dorsi muscle from myostatin wild type (MWT) and MKO pigs on 65 days post coitus. Results showed that, compared with MWT group, there were 438 differentially expressed genes (DEGs) and 66 differentially expressed proteins (DEPs) in MKO group. These DEGs and DEPs were mainly enriched in signaling pathways that are involved in skeletal muscle growth and development, glucose metabolism and apoptosis. Furthermore, we identified two key genes, Troponin T 1 (TNNT1) and Myosin regulatory light chain 9 (MYL9), which showed significant changes in both mRNA and protein levels with the similar changing trends in MKO group. It is thus speculated that TNNT1 and MYL9 may play an important role in skeletal muscle growth and development. SIGNIFICANCE: Our study analyzed some important regulatory genes and proteins during skeletal muscle growth and development, our results provided (1) a new insight to further understanding of the molecular mechanisms by which growth and development are regulated in porcine skeletal muscle, and (2) some possible molecular makers for improvement of meat quality in the animal husbandry and diagnosis of human muscle diseases in medicine.

An integrated analysis of mRNA and miRNA in skeletal muscle from myostatin-edited Meishan pigs.

Myostatin (MSTN) is a key muscle factor that negatively regulates skeletal muscle growth and development. Our laboratory recently produced genetically engineered Meishan pigs containing a ZFN-edited MSTN loss-of-function mutation (MSTN-/-, MKO) that led to the hypertrophy of skeletal muscles. In this study, we performed transcriptome sequencing and miRNA sequencing in skeletal muscle samples from MKO and wildtype Meishan (MWT) pigs to investigate the effect of MSTN-/- on expression of mRNA and miRNA. Our results indicated that, compared to MWT pigs, there were 200 genes and 4 miRNAs being significantly up-regulated, and 238 genes and 5 miRNAs being significantly down-regulated in MKO pigs. Analysis by GO and KEGG pathways revealed that differentially expressed miRNAs and their target genes of those differentially expressed miRNAs were involved in the signal pathways of skeletal muscle growth and development such as AMPK, mTOR, and TGF-beta. An integrated analysis of the correlation between miRNA-mRNA and transcriptome predicated that XK and METTL8 were target genes for miR-499-5p, while LRP4 was a target gene for miR-490-3p. Our results provide important clues to help us further investigate MSTN's regulatory mechanisms during skeletal muscle growth and development.

Insertional mutagenesis in ChordinA induced by endogenous ΔTgf2 transposon leads to bifurcation of axial skeletal systems in grass goldfish.

The grass goldfish appeared early in the evolutionary history of goldfish, and shows heritable stability in the development of the caudal fin. The twin-tail phenotype is extremely rare, however, some twin-tail individuals were produced in the process of breeding for ornamental value. From mutations in the twin-tail goldfish genome, we identified two kinds of Tgf2 transposons. One type was completely sequenced Tgf2 and the other type was ΔTgf2, which had 858 bp missing. We speculate that the bifurcation of the axial skeletal system in goldfish may be caused by an endogenous ΔTgf2 insertion mutation in Chordin A, as ΔTgf2 has no transposition activity and blocks the expression of Chordin A. The twin-tail showed doubled caudal fin and accumulation of red blood cells in the tail. In addition, in situ hybridization revealed that ventral embryonic tissue markers (eve1, sizzled, and bmp4) were more widely and strongly expressed in the twin-tail than in the wild-type embryos during the gastrula stage, and bmp4 showed bifurcated expression patterns in the posterior region of the twin-tail embryos. These results provide new insights into the artificial breeding of genetically stable twin-tail grass goldfish families.

Effect of ZFN-edited myostatin loss-of-function mutation on gut microbiota in Meishan pigs.

Intestine contains the body's second largest genetic information, so a relatively stable microbiota ecosystems and interactions between intestinal micro-organisms play a pivotal role in the normal growth and development in animals. The establishment of intestinal microflora is affected by a variety of factors such as species, environmental factors, developmental stage, organizational structure and physiological characteristics of various parts of the digestive tract. Gene editing technology such as ZFN has recently been used as a new approach to replace the traditional transgenic technology and to make genetic modifications in animals. However, it is not known if genetic modification by gene editing technology will have any impact on gut microbiota. In this study, by sequencing 16S rRNA collected from rectum, we investigated the effects of ZFN-mediated myostatin (MSTN) loss-of-function mutation (MSTN-/-) on gut microbiota in Meishan pigs. Our results showed that the fecal microbial composition is very similar between MSTN-/- Meishan pigs and wild type Meishan pigs. Although significant differences in certain individual strains were observed, all the dominant microorganism species are basically the same between MSTN-/- and wild type pigs. However, these differences do not adversely affect MSTN-/- Meishan pigs. Thus, it is concluded that ZFN-mediated MSTN loss-of-function mutation did not have any adverse effect on the gut microbiota in Meishan pigs.

Gene Location, Expression, and Function of FNDC5 in Meishan Pigs.

Irisin is a new muscular regulatory factor that is generated by the cleavage of its precursor protein fibronectin type III domain-containing protein 5 (FNDC5). Irisin promotes fat consumption due to its stimulatory role in the browning of the adipocytes in mice. Currently, there is no report on FNDC5 functions in pigs as model animals. In this study, we investigated the expression patterns and functions of FNDC5 in Meishan pigs. Our results showed that FNDC5 gene in Meishan pigs contains five transcripts, all of which can be translated into functional intact irisin proteins. Porcine FNDC5 is mainly expressed in skeletal muscle, with the expression level being significantly higher during the embryonic and juvenile periods than in the adulthood stage. In vitro study showed that FNDC5 stimulates the proliferation and adipogenic differentiation of primary adipocytes isolated from Meishan pigs, and FNDC5 enhances the expression of browning marker genes during adipogenic differentiation. Our study was the first report on FNDC5 expression patterns and functions in pigs. Data from this study provide valuable information related to the study on FNDC5 functions and future development of novel treatment for obesity.

Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs.

Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn -/- ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn -/- pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue. Additionally, the serum insulin level decreased and the insulin sensitivity increased significantly in Mstn -/- pigs. Moreover, we found a significant increase in levels of insulin receptor and insulin receptor substrate proteins in skeletal muscle of Mstn -/- pigs, which then activating the insulin signaling pathway. Irisin-mediated regulation is not the only pathway for the activation of insulin signal in Mstn -/- skeletal muscle. This study provides valuable insight for the treatment of human obesity and diabetes mellitus.

MicroRNA-95 promotes myogenic differentiation by down-regulation of aminoacyl-tRNA synthase complex-interacting multifunctional protein 2.

MicroRNA-95 (miR-95) is well known for its ability to promote the proliferation of a variety of cancer cells, but its function in skeletal muscle development has not been reported so far. Our laboratory has recently generated genetically engineered Meishan pigs containing a loss-of-function myostatin (MSTN) mutant (MSTN-/-). These MSTN-/- pigs grow and develop normally but show clear double muscle phenotype as observed in Belgian cattle. We observed that the expression of miR-95 was up-regulated in the longissimus dorsi from MSTN-/- Meishan pigs at day 65 during embryo development. In this study, we investigated the role of miR-95 in the myogenic differentiation using a murine myoblast cell line C2C12. Our results revealed that miR-95 may play a very important role in regulating the expression of myogenic differentiation marker genes myosin heavy chain (MHC) and myogenin. By use of bioinformatical analysis and luciferase reporter gene assay, aminoacyl-tRNA synthase complex-interacting multifunctional protein 2 (AIMP2) gene was identified as a miR-95 target gene involved in myogenic differentiation. Our results indicated that higher miR-95 expression level leads to lower level of AIMP2 protein expression. When the endogenous expression of AIMP2 is inhibited by siRNA, the expression levels of myogenic differentiation marker genes MHC and myogenin increased, implying that AIMP2 negatively regulates myogenic differentiation. Taken together, it is likely that miR-95 promotes myogenic differentiation in C2C12 myoblasts and may play a positive functional role in skeletal muscle development by down regulating the expression of AIMP2 at protein level.

A 90-Day Feeding Study in Rats to Assess the Safety of Genetically Engineered Pork.

Our laboratory recently produced genetically engineered (GE) Meishan pigs containing a ZFN-edited myostatin loss-of-function mutant. These GE pigs develop and grow as normal as wild type pigs but produce pork with greater lean yield and lower fat mass. To assess any potential subchronic toxicity risks of this GE pork, a 90-day feeding study was conducted in Sprague-Dawley rats. Rats were randomly divided into five groups, and fed for 90 days with basic diet and basic diets formulated with low dose and high dose pork prepared from wild type pigs and GE pigs, respectively. Animal behaviors and clinical signs were monitored twice daily, and body weight and food consumption were measured and recorded weekly. At days 45 and 90, blood tests (lipid panel, electrolytes, parameters related to liver and kidney functions, and complete blood counts) were performed. Additionally, gross pathology and histopathological analyses were performed for major organs in each group. Data analysis shows that there were no significant differences in growth rate, food consumption, and blood test parameters between rat groups fed with GE pork and wild type pork. Although differences in some liver function parameters (such as aspartate aminotransferase, total proteins, albumin, and alkaline phosphatase) and white blood cell counts (such as lymphocyte percentage and monocyte percentage) were observed between rats fed with high dose GE pork and basic diet, all test results in rats fed with GE pork are in the normal range. Additionally, there are no apparent lesions noted in all organs isolated from rats in all five feeding groups on days 45 and 90. Overall, our results clearly indicate that food consumption of GE pork produced by ZFN-edited myostatin loss-of-function mutant pigs did not have any long-term adverse effects on the health status in rats.

Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology.

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.

Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.