Identification of small droplets of photosynthetic squalene in engineered Synechococcus elongatus PCC 7942 using TEM and selective fluorescent Nile red analysis.

To identify microbial squalene that has been widely used in various industrial applications, intracellular formation of photosynthetic squalene was investigated using the previously engineered Synechococcus elongatusPCC 7942 strain. Unlike the proposed localization of squalene in the membrane bilayer, small droplets were identified in the cytoplasm of S. elongatusPCC 7942 as squalene using transmission electron microscopy analysis. Determination of the diameters of the squalene droplets with manual examination of 1016 droplets in different squalene-producing strains indicated larger squalene droplets in larger cells. Based on the observation of a sole droplet of squalene in a cyanobacterium, fluorescent Nile red was used for the selective staining of squalene. The fluorescent intensities were correlated with squalene contents determined using gas chromatography-mass spectrometry. Photosynthetic squalene was identified as a small droplet in S. elongatusPCC 7942, and this noninvasive quantitative method could be useful to promote high-throughput strain development for squalene production. SIGNIFICANCE AND IMPACT OF THE STUDY: Engineering of Cyanobacteria has focused on sustainable production of squalene by converting CO2 . Before improving the photosynthetic squalene production, we characterized formation of squalene, showing small droplets in the cytoplasm instead of single granule. Based on the finding and the analysis, this study has provided valuable evidences how further metabolic engineering strategies should apply to enhance the production yield.

Application of whole-exome sequencing for detecting copy number variants in CMT1A/HNPP.

Large insertions and deletions (indels), including copy number variations (CNVs), are commonly seen in many diseases. Standard approaches for indel detection rely on well-established methods such as qPCR or short tandem repeat (STR) markers. Recently, a number of tools for CNV detection based on next-generation sequencing (NGS) data have also been developed; however, use of these methods is limited. Here, we used whole-exome sequencing (WES) in patients previously diagnosed with CMT1A or HNPP using STR markers to evaluate the ability of WES to improve the clinical diagnosis. Patients were evaluated utilizing three CNV detection tools including CONIFER, ExomeCNV and CEQer, and array comparative genomic hybridization (aCGH). We identified a breakpoint region at 17p11.2-p12 in patients with CMT1A and HNPP. CNV detection levels were similar in both 6 Gb (mean read depth = 80×) and 17 Gb (mean read depth = 190×) data. Taken together, these data suggest that 6 Gb WES data are sufficient to reveal the genetic causes of various diseases and can be used to estimate single mutations, indels, and CNVs simultaneously. Furthermore, our data strongly indicate that CNV detection by NGS is a rapid and cost-effective method for clinical diagnosis of genetically heterogeneous disorders such as CMT neuropathy.

Clinical and histopathological study of Charcot-Marie-Tooth neuropathy with a novel S90W mutation in BSCL2.

The objective of the study was to investigate the disease-causing mutation in an autosomal dominant Charcot-Marie-Tooth disease type 2 family and examine the clinical and histopathological evaluation. We enrolled a family of Korean origin with axonal Charcot-Marie-Tooth disease neuropathy (FC305; 13 males, six females) and applied genome-wide linkage analysis. Whole exome sequencing was performed for two patients. In addition, sural nerve biopsies were obtained from two patients. Through whole exome sequencing, we identified an average of 20,336 coding variants from two patients. We also found evidence of linkage mapped to chromosome 11p11-11q13.3 (LOD score of 3.6). Among these variants in the linkage region, we detected a novel p.S90W mutation in the Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) gene, after filtering 31 Korean control exomes. Our p.S90W patients had frequent sensory disturbances, pyramidal tract signs, and predominant right thenar muscle atrophy in comparison with reported p.S90L patients. The phenotypic spectra were wide and demonstrated intrafamilial variability. Two patients with different clinical features underwent sural nerve biopsies; the myelinated fiber densities were increased slightly in both patients, which differed from two previous case reports of BSCL2 mutations (p.S90L and p.N88S). This report expands the variability of the clinical spectrum associated with the BSCL2 gene and describes the first family with the p.S90W mutation.

Identifying the degree of major histocompatibility complex matching in genetically unrelated dogs with the use of microsatellite markers.

BACKGROUND: The dog has served as an important experimental model for biomedical research such as transplantation and developing immunosuppressive agents. Although major histocompatibility complex (MHC) in dogs is a dominant factor of graft rejection, it has not been well investigated in dogs compared with human. For that reason, imprecise cross-matching or time-consuming sequence-based typing methods have generally been used to choose specific donor and recipient pairs. Investigation of matching distribution of MHC in dogs with the use of simple and accurate methods would be beneficial for biomedical researchers. The aim of this study was to identify the diversity of dog leukocyte antigen (DLA) types in genetically unrelated dogs by means of microsatellite markers. METHODS: Thirty-three Beagle and Shih-Tzu dogs, which were negative in cross-matching, were chosen. The genomic DNA was isolated from peripheral blood leukocytes, and highly polymorphic short tandem repeats located in MHC class I and II were amplified with the use of specific primers. RESULTS: Among all of the dogs, MHC matching groups, including class I full match-class II full match (M-M), class I full match-class II haplo match (M-H), class I haplo match-class II full match (H-M), class I haplo match-class II haplo match (H-H) groups, were ∼1.55%, 0.39%, 1.94%, and 6.59%, respectively. MHC class I nonmatch-class II nonmatch (U-U) groups were 58.14% of the total dogs. CONCLUSIONS: Because differences of histocompatibility between donor and recipient leads to various allograft rejections, knowledge of the distribution of MHC matching in unrelated dogs would be helpful in designing studies and to get more accurate results from experiments using dog transplantation models.

Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation.

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

Effects of amniotic membrane on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea.

AIMS: To investigate if the amniotic membrane (AM) promotes epithelial migration while inhibiting stromal remodelling associated with corneal haze after excimer laser keratectomy. METHODS: A wound 150 microm in depth and 6.0 mm in diameter was produced in 40 rabbits using an excimer laser. One eye was randomly chosen to be covered by the AM while the other eye served as a control. Epithelial wound healing was evaluated, together with any morphological changes of the anterior stroma connected with corneal haze. These morphological changes were histopathologically analysed using dichlortriazinyl aminofluorescein (DTAF), Masson trichrome staining, and an image analyser. RESULTS: The AM group had a short latent phase followed by fast epithelial healing (p<0.001) during the early wound healing period and a significant decrease in the inflammatory response, together with a smaller change in the number of keratocytes than the control group. The mean thickness of the regenerated stroma was significantly thinner in the AM group than in the control group at 8 weeks (p<0.0001). The AM group had a more regular architecture of regenerated stromal lamella at 8 weeks and significantly less haze after 4 weeks than the control group (p<0.05). CONCLUSION: Use of the AM as a dressing on a corneal wound created by excimer laser surgery, in which severe haze is expected, may induce rapid epithelial healing with less inflammatory response. The AM may inhibit the irregular synthesis of stromal collagen that is associated with corneal haze.

Multifocal phototherapeutic keratectomy for the treatment of persistent epithelial defect.

PURPOSE: To evaluate the use of multifocal phototherapeutic keratectomy (PTK) for the treatment of indolent and persistent epithelial defect (PED). SETTING: Department of Ophthalmology, Kangnam St. Mary's Hospital, The Catholic University of Korea, College of Medicine, Seoul, Korea. METHODS: Fifteen eyes diagnosed with PED were treated with Summit excimer laser PTK. In all patients (N = 15), the epithelium had previously failed to cover with at least 1 method of medical or surgical treatment. Multifocal ablations were delivered to the elevated margin surrounding the epithelial defect area using PTK with a diameter of 1.0 mm and depth of 150 to 200 pulse ablations. The mean follow-up was 17.7 months (range 9 to 37 months). RESULTS: Complete reepithelialization occurred within 7 days in 13 eyes and within 11 days and 12 days in 1 eye each. All corneas except 1 remained healed for at least 9 months of follow-up. One eye had a recurrence 3 months after PTK and was retreated. Postoperative visual acuity improved in 8 eyes and was unaltered in 7. CONCLUSION: Multifocal PTK with the 193 nm excimer laser is a safe, effective treatment for PED that is unresponsive to conventional therapy.

Γ-glutamyl transferase as an early and sensitive marker in ethanol-induced liver injury of rats.

γ-Glutamyl transferase (GGT) has been regarded as a biological marker of heavy alcohol consumption or hepatobiliary disease such as fatty liver. However, the role of GGT is unknown in the molecular pathway during alcohol-induced liver injury. To determine the role of GGT in alcohol-induced liver injury, Sprague-Dawley rats were administered 22% and 38% ethanol for 3 days as acute and 5 weeks as subchronic model. In serologic analysis, the level of GGT was significantly increased and the level of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were not changed at 3 days and 5 weeks. In histologic analysis, ethanol exposure induced granular deposit formation and sinusoidal dilation in the acute model for 3 days. In the subchronic model for 5 weeks, ethanol exposure further increased the granular deposit formation, sinusoidal congestion, and mild fatty liver change. To determine whether ethanol-exposed liver is associated with changes of antioxidants levels, we performed reverse-transcriptase polymerase chain reaction (RT-PCR) analysis on ethanol-exposed livers of rats. In RT-PCR analysis, the mRNA levels of GPX1 and SOD1 were significantly increased as well as up-regulation of CYP2E1. In the glutathione assay, the level of glutathione was significantly reduced in response to ethanol in rats. Therefore, in this study, ethanol increased the level of serum GGT but depleted the level of glutathione. Moreover, the CYP2E1 was rapidly reflected to ethanol in rats. Taken together, our findings suggest that the elevated GGT is associated with cellular antioxidant defense system, and the CYP2E1 can be used for early diagnosis in alcohol-related diseases.

Disparate hypervariable region-1 of mitochondrial DNA did not induce skin allograft rejection in cloned porcine models.

INTRODUCTION: Alloantigen recognition in skin transplantation is the bane for surgeons. Several studies have mainly focused on the immunogenicity of major histocompatibility (MHC) antigens and H-Y minor histocompatibility antigens. However, the roles of the mitochondrial DNA (mtDNA) encorded miHA have not been identified. Therefore, we sought to address the antigenicity of the hypervariable region 1 (HV-1) of mtDNA in skin transplantation using cloned pig models. METHODS: Swine leukocyte antigen and HV-1 of mtDNA were analyzed using sequencing methods. Skin transplantation was performed between MHC-matched, mtDNA-mismatched cloned miniature pigs. Full-thickness skin was grafted between cloned pigs without any immunosuppressants. The grafted tissues were observed for 3 months and evaluated histologically. RESULTS: The cloned pigs shared identical MHC but mtDNA mismatched at 9 positions. Skin grafts between the cloned pigs were accepted and hair growth maintained, whereas MHC-mismatched grafts showed acute rejection within 7 days after transplantation and were replaced by hairless scar tissue. CONCLUSIONS: HV-1 disparate skin grafts were not recognized as alloantigenic by MHC-matched cloned pigs.

Fabrication of a biodegradable xenoantigen-free rat liver scaffold for potential drug screening applications.

BACKGROUND: The increasing market in biological pharmaceuticals raises the demand for human test systems. Although 2-dimensional (2D) models are mostly used for these purposes, these models not mimic responses of 3-dimensional (3D) native tissue. METHODS: After generation of a rat liver scaffold using 0.1% sodium dodecyl sulfate, we characterized the histology, blood vessel integrity, and residual DNA as well as retained amounts of collagen and glycosaminoglycan (GAG). Then, we examined the susceptibility of extracellular matrix (ECM) to enzymatic remodeling. Finally, a mixed lymphocyte reaction (MLR) was performed to evaluate the in vitro immunogenicity of the ECM against human peripheral blood mononuclear cells (PBMCs). RESULTS: Histologic examination of decellularized liver revealed the removal of nuclear and cytoplasmic materials with preservation of architecture. The vascular network was intact after decellularization. Biochemical analysis of ECM components revealed that only a negligible amount of DNA was retained compared with the native liver with preservation of large amounts of GAG and collagen. Scaffolds were degraded in response to collagenase treatment. MLR demonstrated that decellularized matrices did not exert any xenostimulatory response against human PBMCs. CONCLUSION: Our findings suggested that naturally derived rat liver scaffolds show natural biocompatibility besides the ability to preserve the intact 3D structure and components. Because of these characteristics, the whole decellularized rat liver can retain many aspects of native tissue structure and function upon recellularization enabling it to be used for drug screening.

Mouse adipose tissue-derived adult stem cells expressed osteogenic specific transcripts of osteocalcin and parathyroid hormone receptor during osteogenesis.

INTRODUCTION: Adult mesenchymal stem cells (MSCs) have potential to differentiate into various lineages, replacing cells during normal turnover and tissue regeneration to replace damaged or lost adult tissues during osteoporosis and arthritis, or traumatic injuries. We investigated the osteogenic signature in mouse adipose tissue (AD)- and bone marrow (BM)-derived MSCs. MATERIALS AND METHODS: MSCs from adipose tissue and bone marrow were compared for osteogenic endogenous mRNA markers by reverse-transcription polymerase chain reaction (RT-PCR). Cellular proliferation and immunophenotype analyzed by flow cytometry revealed that mouse AD-MSCs and BM-MSCs shared similar characteristics. RESULT: Isolated AD-MSC and BM-MSC showed high proliferation rates and fibroblast morphology. Flow cytometry revealed positive markers for mesenchyme, but negative for primitive hematopoietic and endothelial cells. At day 21, Alizarin red S and Von-kossa staining of differentiated cells showed high calcium deposits compared with undifferentiated cells. After 21 days of osteogenic differentiation, AD-MSCs expressed osteocalcin and parathyroid hormone (PTH) compared with undifferentiated cells. Osteogenic-specific transcript of osteocalcin (OC), bone gamma carboxyglutamate protein, and PTH receptor (PTHr) were detected only in differentiated not undifferentiated cells. Undifferentiated BM-MSCs, expressed all markers at low intensity, which amplified during differentiation. CONCLUSION: Our findings suggest that the OC and PTHr can be used as differentiation markers for osteogenesis of mouse AD-MSC.

Acute rejection after swine leukocyte antigen-matched kidney allo-transplantation in cloned miniature pigs with different mitochondrial DNA-encoded minor histocompatibility antigen.

INTRODUCTION: Graft rejection remains a major cause of morbidity and mortality following renal transplantation. One of the main determinants of success after renal transplantation is histocompatibility between donor and recipient. Most of the research on this topic has addressed human leukocyte antigen (HLA), but the roles played by minor histocompatibility antigens (mHAgs), such as mitochondrially transmitted antigens, are poorly understood. In this study, we evaluated immune responses induced by minor antigens originating from mitochondrial DNA (mtDNA) in a large animal model. METHODS: To characterize whole swine leukocyte antigen (SLA) allele in 8 cloned pigs, we performed SLA genotyping for SLA-1, SLA-2, SLA-3, SLA-DQB1, and SLA-DRB1 as well as the hypervariable region 1 (HV1) of mtDNA. Renal transplantation was performed using SLA-matched pigs with different mtDNA as well as SLA-mismatched cloned animals. Cytokine profiling was performed by incubating peripheral leukocytes with cellular components from SLA-matched different mtDNA and SLA-mismatched cells to evaluate mtDNA-mediated immune response. RESULTS: SLA types were confirmed to be identical, but mtDNA sequences of HV1 varied among cloned pigs. Rejection episodes in the SLA-matched group with different mtDNA were similar to those in the SLA-mismatched group; that is, plasma creatinine and BUN levels were increased and mononuclear cell infiltration was observed in perivascular regions in the matched and SLA-mismatched groups. Furthermore, in vitro studies showed interleukin (IL)-1ß expression to be elevated in SLA-matched and SLA-mismatched groups. CONCLUSION: Cloned pigs are a useful preclinical model to evaluate the immunogenicity of mtDNA encoding minor antigens. The mtDNA originating from nongenomic DNA induced cell-mediated immune rejection after kidney transplantation.

Porcine bioengineered scaffolds as new frontiers in regenerative medicine.

Porcine organs are attractive for xenotransplantation, if severe immunologic concerns can be overcome. Recently, reengineered organs, with heterologous cellular materials removed but preserved organ architecture and vasculature have been created using small rodents in an effort to produce customized bioengineered organs. However, few studies have been performed to generate bioengineered organs from porcine sources. The aim of this work was to produce 3-D bioengineered scaffolds from major porcine organs, preserving the native morphology and vascular structures with complete removal of cellular and nuclear materials. We decellularized porcine heart, liver, and kidney using a peristaltic pump system with 1% sodium dodecyl sulfate. The preservation of major architecture and vasculature was confirmed by gross findings, ultrasonography, and angiography. Hematoxylin and eosin staining revealed no evidence of nuclear or cytoplasmic residues. Quantitative DNA analysis demonstrated a substantial reduction (0%-8%) of porcine DNA in the scaffolds. These results suggested that 3-D bioengineered scaffolds of porcine organs may have tremendous potential to produce non-immunogenic transplantable organs as well as beneficial tools for biomedical studies on organ re-engineering and repair.

Systemic decellularization for multi-organ scaffolds in rats.

INTRODUCTION: Bioscaffolds derived from animal organs are promising materials for xenotransplantation and regenerative medicine. For effective generation of biological scaffolds from diverse organs, there have been many technical challenges. In this study, we introduced a novel approach to create multiorgan bioscaffolds through systemic decellularization. METHODS AND MATERIALS: To obtain acellular bioscaffolds, the healthy adult rats were systemically perfused with ionic detergent through the carotid artery. Additional liver perfusion was set up to prevent potential obstruction from the influx of the decellularized debris via the portal vein. The perfusion system was controlled to maintain a constant physiological cardiac output of approximately 50 mL/min and was designed to minimize air entrapment. After decellularization, every organ designated for bioscaffold was harvested for evaluation of vascular structure and histology. RESULTS: The perfusion times were different for each organ. In our histological analysis, the decellularized bioscaffolds harvested from most organs including major solid organs (ie, heart, liver, and kidney) as well as the others (such as stomach, intestines, spleen, etc) represented no evidence of residual cellular materials. Furthermore, the well-preserved collagen materials and intact vascular structures were also confirmed. CONCLUSION: The results from this study suggested that this systemic decellularization has the advantages to obtain a variety of bioscaffolds from single donor, and we can even decellularize organs with complex influx vascular structures. This method may also be used to study organ bioengineering for patients who need simultaneous combined organ transplantation.