Phenotypic features of genetically modified DMD-XKOXWT pigs.

Introduction: Duchenne muscular dystrophy (DMD) is a hereditary neuromuscular disorder caused by mutation in the dystrophin gene (DMD) on the X chromosome. Female DMD carriers occasionally exhibit symptoms such as muscle weakness and heart failure. Here, we investigated the characteristics and representativeness of female DMD carrier (DMD-XKOXWT) pigs as a suitable disease model. Methods: In vitro fertilization using sperm from a DMD-XKOY↔XWTXWT chimeric boar yielded DMD-XKOXWT females, which were used to generate F2 and F3 progeny, including DMD-XKOXWT females. F1-F3 piglets were genotyped and subjected to biochemical analysis for blood creatine kinase (CK), aspartate aminotransferase, and lactate dehydrogenase. Skeletal muscle and myocardial tissue were analyzed for the expression of dystrophin and utrophin, as well as for lymphocyte and macrophage infiltration. Results: DMD-XKOXWT pigs exhibited various characteristics common to human DMD carrier patients, namely, asymptomatic hyperCKemia, dystrophin expression patterns in the skeletal and cardiac muscles, histopathological features of skeletal muscle degeneration, myocardial lesions in adulthood, and sporadic death. Pathological abnormalities observed in the skeletal muscles in DMD-XKOXWT pigs point to a frequent incidence of pathological abnormalities in the musculoskeletal tissues of latent DMD carriers. Our findings suggest a higher risk of myocardial abnormalities in DMD carrier women than previously believed. Conclusions: We demonstrated that DMD-XKOXWT pigs could serve as a suitable large animal model for understanding the pathogenic mechanism in DMD carriers and developing therapies for female DMD carriers.

Phenotypic features of dystrophin gene knockout pigs harboring a human artificial chromosome containing the entire dystrophin gene.

Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via a somatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3 months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.

A Transgenic Pig Model With Human Mutant SOD1 Exhibits the Early Pathology of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) causes progressive degeneration of the motor neurons. In this study, we delivered the genetic construct including the whole locus of human mutant superoxide dismutase 1 (SOD1) with the promoter region of human SOD1 into porcine zygotes using intracytoplasmic sperm injection-mediated gene transfer, and we thereby generated a pig model of human mutant SOD1-mediated familial ALS. The established ALS pig model exhibited an initial abnormality of motor neurons with accumulated misfolded SOD1. The ALS pig model, with a body size similar to that of human beings, will provide opportunities for cell and gene therapy platforms in preclinical translational research.

Genome Editing of Pig.

Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.

Batteryless wireless magnetostrictive Fe30Co70/Ni clad plate for human coronavirus 229E detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been garnered increasing for its rapid worldwide spread. Each country had implemented city-wide lockdowns and immigration regulations to prevent the spread of the infection, resulting in severe economic consequences. Materials and technologies that monitor environmental conditions and wirelessly communicate such information to people are thus gaining considerable attention as a countermeasure. This study investigated the dynamic characteristics of batteryless magnetostrictive alloys for energy harvesting to detect human coronavirus 229E (HCoV-229E). Light and thin magnetostrictive Fe-Co/Ni clad plate with rectification, direct current (DC) voltage storage capacitor, and wireless information transmission circuits were developed for this purpose. The power consumption was reduced by improving the energy storage circuit, and the magnetostrictive clad plate under bending vibration stored a DC voltage of 1.9 V and wirelessly transmitted a signal to a personal computer once every 5 min and 10 s under bias magnetic fields of 0 and 10 mT, respectively. Then, on the clad plate surface, a novel CD13 biorecognition layer was immobilized using a self-assembled monolayer of -COOH groups, thus forming an amide bond with -NH2 groups for the detection of HCoV-229E. A bending vibration test demonstrated the resonance frequency changes because of HCoV-229E binding. The fluorescence signal demonstrated that HCoV-229E could be successfully detected. Thus, because HCoV-229E changed the dynamic characteristics of this plate, the CD13-modified magnetostrictive clad plate could detect HCoV-229E from the interval of wireless communication time. Therefore, a monitoring system that transmits/detects the presence of human coronavirus without batteries will be realized soon.

Moving towards a novel therapeutic strategy for hyperammonemia that targets glutamine metabolism.

Patients with urea cycle disorders intermittently develop episodes of decompensation with hyperammonemia. Although such an episode is often associated with starvation and catabolism, its molecular basis is not fully understood. First, we attempted to elucidate the mechanism of such starvation-associated hyperammonemia. Using a mouse embryonic fibroblast (MEF) culture system, we found that glucose starvation increases ammonia production, and that this increase is associated with enhanced glutaminolysis. These results led us to focus on α-ketoglutarate (AKG), a glutamate dehydrogenase inhibitor, and a major anaplerotic metabolite. Hence, we sought to determine the effect of dimethyl α-ketoglutarate (DKG), a cell-permeable AKG analog, on MEFs and found that DKG mitigates ammonia production primarily by reducing flux through glutamate dehydrogenase. We also verified that DKG reduces ammonia in an NH4 Cl-challenged hyperammonemia mouse model and observed that DKG administration reduces plasma ammonia concentration to 22.8% of the mean value for control mice that received only NH4 Cl. In addition, we detected increases in ornithine concentration and in the ratio of ornithine to arginine following DKG treatment. We subsequently administered DKG intravenously to a newborn pig with hyperammonemia due to ornithine transcarbamylase deficiency and found that blood ammonia concentration declined significantly over time. We determined that this effect is associated with facilitated reductive amination and glutamine synthesis. Our present data indicate that energy starvation triggers hyperammonemia through enhanced glutaminolysis and that DKG reduces ammonia accumulation via pleiotropic mechanisms both in vitro and in vivo. Thus, cell-permeable forms of AKG are feasible candidates for a novel hyperammonemia treatment.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

Introduction: Previously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model. Methods: The IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity. Results: Tumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed. Conclusions: IL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Asymmetric Transfer Hydrogenative Amination of Benzylic Ketones Catalyzed by Cp*Ir(III) Complexes Bearing a Chiral N-(2-Picolyl)sulfonamidato Ligand.

A convenient asymmetric reductive amination of benzylic ketones (α-arylated ketones) catalyzed by newly designed Cp*Ir complexes bearing a chiral N-(2-picolyl)sulfonamidato ligand was developed. Using readily available ß-amino alcohols as chiral aminating agents, a range of benzo-fused and acyclic ketones were successfully reduced with formic acid in methanol at 40 °C to afford amines with favorable chemo- and diastereoselectivities. The amino alcohol-derived chiral auxiliary was easily removed by mild periodic oxidants, leading to optically active primary ß-arylamines without erosion of the optical purity (up to 97% ee). The excellent catalytic performance was retained even upon lowering the amount of catalyst to a substrate/catalyst (S/C) ratio of 20,000, and the amination could be performed on a large scale exceeding 100 g. The precise hydride transfer to iminium species generated from the ketonic substrate and the chiral amine counterpart was suggested by the mechanistic studies on stoichiometric reactions of isolable hydridoiridium complexes and model intermediates such as N,O-acetal, enamine, and iminium compounds.

Aspects of the Complement System in New Era of Xenotransplantation.

After producing triple (Gal, H-D and Sda)-KO pigs, hyperacute rejection appeared to no longer be a problem. However, the origin of xeno-rejection continues to be a controversial topic, including small amounts of antibodies and subsequent activation of the graft endothelium, the complement recognition system and the coagulation systems. The complement is activated via the classical pathway by non-Gal/H-D/Sda antigens and by ischemia-reperfusion injury (IRI), via the alternative pathway, especially on islets, and via the lectin pathway. The complement system therefore is still an important recognition and effector mechanism in xeno-rejection. All complement regulatory proteins (CRPs) regulate complement activation in different manners. Therefore, to effectively protect xenografts against xeno-rejection, it would appear reasonable to employ not only one but several CRPs including anti-complement drugs. The further assessment of antigens continues to be an important issue in the area of clinical xenotransplantation. The above conclusions suggest that the expression of sufficient levels of human CRPs on Triple-KO grafts is necessary. Moreover, multilateral inhibition on local complement activation in the graft, together with the control of signals between macrophages and lymphocytes is required.

The Innate Cellular Immune Response in Xenotransplantation.

Xenotransplantation is very attractive strategy for addressing the shortage of donors. While hyper acute rejection (HAR) caused by natural antibodies and complement has been well defined, this is not the case for innate cellular xenogeneic rejection. An increasing body of evidence suggests that innate cellular immune responses contribute to xenogeneic rejection. Various molecular incompatibilities between receptors and their ligands across different species typically have an impact on graft outcome. NK cells are activated by direct interaction as well as by antigen dependent cellular cytotoxicity (ADCC) mechanisms. Macrophages are activated through various mechanisms in xenogeneic conditions. Macrophages recognize CD47 as a "marker of self" through binding to SIRPα. A number of studies have shown that incompatibility of porcine CD47 against human SIRPα contributes to the rejection of xenogeneic target cells by macrophages. Neutrophils are an early responder cell that infiltrates xenogeneic grafts. It has also been reported that neutrophil extracellular traps (NETs) activate macrophages as damage-associated pattern molecules (DAMPs). In this review, we summarize recent insights into innate cellular xenogeneic rejection.

Additive Manufacturing of Magnetostrictive Fe-Co Alloys.

Fe-Co alloys are attracting attention as magnetostrictive materials for energy harvesting and sensor applications. This work investigated the magnetostriction characteristics and crystal structure of additive-manufactured Fe-Co alloys using directed energy deposition. The additive-manufactured Fe-Co parts tended to exhibit better magnetostrictive performance than the hot-rolled Fe-Co alloy. The anisotropy energy ΔK1 for the Fe-Co bulk, prepared under a power of 300 W (referred to as bulk-300 W), was larger than for the rolled sample. For the bulk-300 W sample in a particular plane, the piezomagnetic constant d was large, irrespective of the direction of the magnetic field. Elongated voids that formed during additive manufacturing changed the magnetostrictive behavior in a direction perpendicular to these voids. Magnetic property measurements showed that the coercivity decreased. Since sensors should be highly responsive, Fe-Co three-dimensional parts produced via additive manufacturing can be applied as force sensors.

Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

Study of the CRISPR/Cas3 System for Xenotransplantation.

The CRISPR/Cas3 system, classified in class I system, was recently focused as a new technology. For application of this system to porcine cells, the plasmids of bpNLS-Cascade, BPNLS-hCas3, and pBS-U6icrRNA were prepared. Initially, 2 crRNAs were established in the exon 9 of pig Gal-T (GGTA1) as #45 and #86. Next, hCas3 + #45 + #86 (group 1, control), Cascade + hCas3 + #45 (group 2), Cascade + hCas3 + #86 (group 3), and Cascade + hCas3 + #45 + #86 (group 4) were set and transfected into pig fibroblasts. Transfected cells were analyzed for bulk expression of α1,3Gal epitope by fluorescence-activated cell sorting (FACS), using a GSI-B4 lectin 2 days after the transfection. As the results, changes of expression are observed in order of G4>G2>G3, indicating the effect of the Cas3 system. Therefore, the nested polymerase chain reaction (PCR) for target region of GGTA1 was performed. Next, the PCR products from each group were checked in blotting, and the products were placed into the cloning sit of TOPO vector and transformed into Escherichia coli. Sixteen colonies of each group were checked by PCR, and clones containing PCR product with slightly varying length were evaluated. The direct sequence of these PCR changes were demonstrated as 294 to 754 bp deletions. In conclusion, we confirmed the effect of the CRISPR/Cas3 system on pig cell, especially in xenotransplantation.

Subjective and Objective Outcomes of Surgery for Rheumatoid Forefoot Deformities Under the Current Treatment Paradigm.

We investigated the clinical outcomes of surgical procedures for the treatment of forefoot deformities in patients with rheumatoid arthritis. Twenty feet in 16 women (mean age 62.1 years) underwent corrective osteotomy of the first metatarsal bone with shortening oblique osteotomy of the lesser metatarsophalangeal joints (joint-preservation group), while 13 feet in 12 women (mean age 67.4 years) underwent arthrodesis of the first metatarsophalangeal joint with resection arthroplasty of the lesser metatarsophalangeal joints (joint-sacrifice group); mean follow-up for each group was 25.8 and 23.8 months, respectively. The mean total Japanese Society for Surgery of the Foot (JSSF) scale improved significantly from 64.2 to 89.2 in the joint-preservation group (p < .001), and from 54.2 to 74.2 in the joint-sacrifice group (p = .003). In the joint-preservation group, the postoperative range of motion (ROM) of the joint, walking ability, and activities of daily living scores of the JSSF scale were significantly higher than those in the joint-sacrifice group (p = .001, p = .001, and p = .019, respectively). There were no differences in the subscale scores of the self-administered foot evaluation questionnaire between 2 groups either pre- or postoperatively. No differences in the postoperative complications were found between 2 groups. Although the joint-sacrificing procedure resulted in lower objective outcomes than the joint-preserving procedure regarding the ROM of the joint, the walking ability, and the level of activities of daily living, both procedures resulted in similar treatment outcomes when evaluated by the subjective measures.

On the Possibility of Developing Magnetostrictive Fe-Co/Ni Clad Plate with Both Vibration Energy Harvesting and Mass Sensing Elements.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has spread rapidly around the world. In order to prevent the spread of infection, city blockades and immigration restrictions have been introduced in each country, but these measures have a severe serious impact on the economy. This paper examines the possibility of both harvesting vibration energy and detecting mass by using a magnetostrictive alloy. Few efforts have been made to develop new magnetostrictive biosensor materials. Therefore, we propose magnetostrictive Fe-Co/Ni clad steel vibration energy harvesters with mass detection, and we numerically and experimentally discuss the effect of the proof mass weight on the frequency shift and output voltage induced by bending vibration. The results reveal that the frequency and output voltage decrease significantly as the mass increases, indicating that the energy harvesting device is capable of mass detection. In the future, device miniaturization and the possibility of virus detection will be considered.

Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes.

Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.

Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes.

INTRODUCTION: Pancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by maintaining undifferentiated cells. Hes1 encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering Pdx1 and Hes1 genes. RESEARCH DESIGN AND METHODS: A transgenic (Tg) chimera pig with germ cells carrying a construct expressing Hes1 under the control of the Pdx1 promoter was used to mate with wild-type gilts to obtain Tg piglets. RESULTS: The Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. ß cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage. CONCLUSION: Pdx1-Hes1 Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.

Dorsal Vein Complex Preserving Technique During Robot-Assisted Radical Prostatectomy.

Background: Recently, two techniques of robot-assisted radical prostatectomy (RARP), which preserve dorsal vein complex (DVC), endopelvic fascia, and full neurovascular bundle (NVB), through anterior approach were reported. The techniques in a relatively large workspace seem less technically demanding than Retzius-sparing RARP. In this case report, we present a further modified technique of transperitoneal-anterior-antegrade approach with a division of the endopelvic fascia to reduce the technical demands. Case Presentation: In a routine evaluation, a 65-year-old man was shown to have a prostate-specific antigen level of 5.07 ng/mL. Prostatic biopsy revealed a Gleason score of 6 (3 + 3) adenocarcinoma in 2 of the 12 specimens, and the clinical stage was diagnosed as cT2aN0M0. RARP was performed including transperitoneal full NVB sparing, antegrade preservation of DVC, and division of endopelvic fascia to increase the prostate mobility and reduce technical demands. The patient completely gained continence on the day after removal of the catheter, and potency was recovered 30 days after surgery. Conclusion: Our DVC preservation technique in the transperitoneal-anterior-antegrade approach with a division of the endopelvic fascia during RARP may be safe, reduce technical demands, and facilitate early recovery of continence and sexual function after surgery.

Prognostic significance of p16 expression in high-grade prostate adenocarcinoma.

Management of advanced hormone-naïve prostate cancer (HNPC) is a critical public health issue. Useful prognostic markers are thus needed to select patients who will benefit from recently introduced upfront therapies. p16 expression is an adverse prognostic marker in prostate cancer. The present study aimed to determine whether p16 expression would serve as an adverse prognostic marker in advanced HNPC. A total of 79 patients diagnosed by needle biopsy with adenocarcinoma Gleason score ≥8 between 2010 and 2013 at Aichi Medical University were included in this study. The median patient age was 73 (range 52-87) years. The median follow-up was 62 months (range 2-98). Fourteen patients had p16-positive samples. Fifteen patients died from prostate cancer, 10 of whom were in the p16-positive group. p16 positivity was associated with clinical T stage (P < 0.001), presence of IDC-P (P < 0.001), distant metastasis (P < 0.001) and lymph node metastasis (P < 0.001). These results indicate that p16 expression is associated with adverse prognostic factor of prostate cancer and suggest that p16 expression may provide useful information for treatment planning and identifying suitable candidates for upfront chemotherapy or androgen receptor axis-targeted therapy.

Correction: Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy.

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