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Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell-based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow-derived mesenchymal stem cells (BMSCs) from 8- to 12-month-old heritage-breed turkeys. The isolated cells demonstrated fibroblast-like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow-derived MSCs from turkey would provide opportunities of studying cell-based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419-1428, 2019. more...

Objective Recombinant adeno-associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9- and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette. Results Our results showed comparable GFP knockout efficiencies (40–50%) upon a single low-dose infection. Three consecutive transductions of 25-fold higher doses of vectors showed 80% GFP knockout efficiency. To analyze the “AAV-SaCas9-resistant cell population”, we sorted the residual GFP-positive cells and assessed their permissiveness to super-infection with two AAV-Cas9-GFP vectors. We found the sorted cells were significantly more resistant to the GFP knockout mediated by the same AAV vector, but not by the other GFP-targeted AAV vector. Our data therefore demonstrate highly efficient genome-editing by the AAV-SaCas9-gRNA vector system. Differential susceptibilities of single cell-derived cells to the AAV-SaCas9-gRNA-mediated genome editing may represent a formidable barrier to achieve 100% genome editing efficiency by this vector system. more...

To explore the function of Dof transcription factors during kernel development in maize, we first identified Dof genes in the maize genome. We found that ZmDof3 was exclusively expressed in the endosperm of maize kernel and had the features of a Dof transcription factor. Suppression of ZmDof3 resulted in a defective kernel phenotype with reduced starch content and a partially patchy aleurone layer. The expression levels of starch synthesis-related genes and aleurone differentiation-associated genes were down-regulated in ZmDof3 knockdown kernels, indicating that ZmDof3 plays an important role in maize endosperm development. The maize endosperm, occupying a large proportion of the kernel, plays an important role in seed development and germination. Current knowledge regarding the regulation of endosperm development is limited. Dof proteins, a family of plant-specific transcription factors, play critical roles in diverse biological processes. In this study, an endosperm-specific Dof protein gene, ZmDof3, was identified in maize through genome-wide screening. Suppression of ZmDof3 resulted in a defective kernel phenotype. The endosperm of ZmDof3 knockdown kernels was loosely packed with irregular starch granules observed by electronic microscope. Through genome-wide expression profiling, we found that down-regulated genes were enriched in GO terms related to carbohydrate metabolism. Moreover, ZmDof3 could bind to the Dof core element in the promoter of starch biosynthesis genes Du1 and Su2 in vitro and in vivo. In addition, the aleurone at local position in mature ZmDof3 knockdown kernels varied from one to three layers, which consisted of smaller and irregular cells. Further analyses showed that knockdown of ZmDof3 reduced the expression of Nkd1, which is involved in aleurone cell differentiation, and that ZmDof3 could bind to the Dof core element in the Nkd1 promoter. Our study reveals that ZmDof3 functions in maize endosperm development as a positive regulator in the signaling system controlling starch accumulation and aleurone development. more...

Aims To investigate the prevalence of adult-onset autoimmune diabetes (ADM) and predisposition to autoimmune diseases by quantifying serum organ-specific autoantibodies in people with phenotype of type 2 diabetes (T2D). Materials and methods We included a nationally representative sample of 46 239 adults aged ≥20 years from 14 provinces, of whom 4671 had diabetes, plus 1000 control subjects with normal glucose tolerance (NGT). Participants were screened centrally for autoantibodies to glutamic acid decarboxylase (GAD), islet antigen 2 (IA2) and zinc transporter isoform-8 (Znt8) and were defined as having ADM where positive for these antibodies. We then assayed thyroid peroxidase (TPO), tissue transglutaminase (tTG) and 21-hydroxylase (21-OH) autoantibodies in randomly selected participants with ADM and in age-matched, sex-matched and non-ADM controls with T2D plus controls with NGT. Results Post-normalization, the standardized prevalence rate of ADM was 6.0% (95% confidence interval [CI] 5.3-6.8) in initially non-insulin-requiring participants with ADM, corresponding to six million adults in China, in whom adjusted antibody positivity was: TPO autoantibodies 16.3% (95% CI 10.8-21.8), tTG autoantibodies 2.1% (95% CI 0.0-4.2), and 21-OH autoantibodies 1.8% (95% CI -0.2 to 3.8). Those participants with ADM who were GAD autoantibody-positive had high risk of TPO autoantibody positivity (odds ratio [OR] 2.39, P = 0.0031) and tTG autoantibody positivity (OR 6.98, P = 0.027), while those positive for IA2 autoantibodies had a high risk of tTG autoantibody positivity (OR 19.05, P = 0.001). Conclusions A proportion of people with phenotype of T2D in China have ADM, with diabetes-associated autoantibodies, and may be at risk of developing other organ-specific autoimmune diseases; therefore, it may be clinically relevant to consider screening such Chinese populations. more...

Summary Generation of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However, it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors, critical for β-cell development and function, in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1, NEUROG3, and MAFA triad (PNM) enabled in vitro generation of psBCs with glucose and GLP-1 responsiveness within 3 weeks. PNM transduction upregulated genes associated with glucose sensing, insulin secretion, and β-cell maturation. In recipient diabetic mice, PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1 week post transplantation. Thus, enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in vitro, offering a competent tissue-primed biotherapy., Highlights • PNM transduction facilitates generation of glucose- and GLP1-responsive β cells • Stepwise PNM improves glucose sensing, insulin secretion, and β-cell maturation • PNM-transduced psBCs showed glucose-responsive insulin secretion in diabetic mice, In this article, Ikeda Yasuhiro and colleagues show that stepwise transduction of the triad of transcription factors PDX1, NEUROG3, and MAFA (PNM) enabled in vitro generation of glucose- and GLP-1-responsive β cells from iPSCs within 3 weeks. PNM transduction improves glucose sensing, insulin secretion, and β-cell maturation of generated cells. PNM-transduced β cells showed glucose-responsive insulin secretion as early as 1 week post transplantation in diabetic mice. more...

Tendon injuries are among the most common and severe hand injuries with a high demand for functional recovery. Stem cells have been identified and isolated from different species and a variety of tissues for the sake of regenerative medicine. Recently, turkey has been suggested as a potential new large animal model for flexor tendon-related research. However, turkey tissue-specific stem cells have not been investigated. Here, we presented the isolation and verification of tendon-derived stem cells (TDSCs) from 6- to 8-month-old heritage-breed turkey. TDSCs were isolated from turkey flexor tendon by plating nucleated cells at the determined optimal density. Approximately 4% of the nucleated cells demonstrated clonogenicity, high proliferation rate, and trilineage differentiation potential after induction culturing. These cells expressed surface antigens CD90, CD105, and CD44, but did not express CD45. There was a high level of gene expression of tenogenic markers in TDSCs, including mohawk, collagen type I, tenascin C, and elastin. Turkey TDSCs also expressed transcription factorsPouV,Nanog, andSox2, which are critically involved in the regulation of stemness. The successful isolation of tendon-derived stem cells from turkey was beneficial for future studies in tendon tissue engineering and would help in the development of new treatment for tendon diseases using this novel animal model. more...

Transcription factors regulate gene expression through binding to specific enhancer sequences. Pancreas/duodenum homeobox protein 1 (PDX1), Neurogenin-3 (NEUROG3), and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) are transcription factors critical for beta cell development and maturation. NEUROG3 is expressed in endocrine progenitor cells and controls islet differentiation and regeneration. PDX1 is essential for the development of pancreatic exocrine and endocrine cells including beta cells. PDX1 also binds to the regulatory elements and increases insulin gene transcription. Likewise, MAFA binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. In addition to those natural roles in beta cell development and maturation, ectopic expression of PDX1, NEUROG3, and/or MAFA has been successfully used to reprogram various cell types into insulin-producing cells in vitro and in vivo, such as pancreatic exocrine cells, hepatocytes, and pluripotent stem cells. Here, we review biological properties of PDX1, NEUROG3, and MAFA, and their applications and limitations for beta cell regenerative approaches. The primary source literature for this review was acquired using a PubMed search for articles published between 1990 and 2017. Search terms include diabetes, insulin, trans-differentiation, stem cells, and regenerative medicine. more...

To confirm the effect of melatonin on 3-nitropropionic acid (3-NP)-induced striatal interneuron injury in rats, behavioral test, histology, immunohistochemistry and Western blotting were respectively used to characterize the behavioral changes of experimental animals in motor and cognition, the morphological changes of striatal interneurons and the expression level of protein markers induced by 3-NP. The results showed that (1) 3-NP induced dysfunction of experimental animals in movement, motor coordination and cognition could be relieved by melatonin treatment; (2) The 3-NP-induced lesion area was unvaryingly in dorsolateral striatum, with almost all neuronal loss in the lesion core, however, lots of neurons survived after melatonin treatment; (3) Immunohistochemical staining of the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y-neuronal nitric oxide synthase co-containing) showed that, in the lesion core of 3-NP group, loss of the four interneuron types was obvious, but in transition zone, the processes and varicosities of calretinergic, and neuropeptide Y-neuronal nitric oxide synthase co-containing interneurons increased significantly. Melatonin treatment reduced the loss of the four interneuron types in the lesion core, and inhibited the increase of processes and varicosities in the transition zone; (4) Consistent with above results, the expression level of five interneuron protein markers were significantly increased in the striatum after melatonin treatment. Notably, in both the transition zone and the lesion core induced by 3-NP, TUNEL-positive cells were detected, but decreased significantly after melatonin treatment. The present results indicate that melatonin effectively protects the striatal neurons against the injury induced by 3-NP in rats. more...

Oxidative stress is closely involved in neurodegenerative diseases. The present study aimed to examine the effect of anti-oxidant DHM (dihydromyricetin) on 3NP (3-nitropropionic acid) -induced behavioral deficits of experimental rats and striatal histopathological injury by using behavioral, imaging, biochemistry, histochemistry and molecular biology technologies. The experimental results showed that both motor dysfunctions and learning and memory impairments induced by 3NP were significantly reduced after DHM treatment. 3NP-induced striatal metabolic abnormality was also remarkably improved by DHM treatment, showed as the increased glucose metabolism in PET/CT scan, decreased MDA (malondialdehyde) and increased SOD (superoxide dismutase) activity in enzyme histochemical staining. In addition, the cell apoptosis was evidently detected in the striatum of the 3NP group, while in the 3NP + DHM group, the number of apoptotic cells was remarkably reduced. 3NP treatment obviously induced down-regulation of Bcl-2, and up-regulations of Bax and Cleaved Caspase-3, while these changes were significantly reversed by DHM treatment. The present results suggested that DHM showed its protective effect by anti-oxidant and anti-apoptosis mechanisms. more...

Background In the China National Diabetes and Metabolic Disorders Study, age-standardised prevalence of total diabetes was 9·7%, accounting for 92·4 million adults, in China in 2010. Using clinical data and serum samples from this study, we aimed to investigate the prevalence of several organ-specific autoantibodies in patients with phenotypic type 2 diabetes. Methods This study included a nationally representative sample of 46 239 adults aged 20 years or older from 14 provinces and municipalities. After an overnight fast, participants had an oral glucose tolerance test. Previously diagnosed diabetes was self-reported. We randomly selected 4671 patients with diabetes and 1002 individuals with normal glucose tolerance as controls. Participants were screened centrally for glutamic acid decarboxylase autoantibodies (GAD-Ab), protein tyrosine phosphatase-like protein autoantibodies (IA2-Ab), and zinc transporter isoform 8 autoantibodies (ZnT8-Ab) using validated methods of the Islets Autoantibody Standardization Program 2012. We then assayed for thyroid peroxidase autoantibodies (TPO-Ab), tissue transglutaminase autoantibodies (tTG-Ab), and 21-hydroxylase autoantibodies (21-OH-Ab) in patients with islet autoantibody-positive diabetes (n=175), in randomly selected age-matched and sex-matched patients with islet autoantibody-negative diabetes (n=3052 for TPO-Ab, n=525 for tTG-Ab, and n=525 for 21-OH-Ab), and in individuals with normal glucose tolerance (n=954 for TPO-Ab, n=200 for tTG-Ab, and n=200 for 21-OH-Ab). We used multiple logistic regression analysis to determine the risk factor of each autoantibody. Ethics approval was granted by the Second Xiangya Hospital of Central South University, and all participants provided written informed consent. Findings After adjustment for age and sex using results from the national population census, the standardised prevalence of autoimmune diabetes was 6·0% in all patients with diabetes, which corresponded to about 6 million adults in China. In patients with diabetes, the adjusted antibody positivity was 3·6% for GAD-Ab, 1·2% for IA2-Ab, and 1·5% for ZnT8-Ab. In those with autoimmune diabetes, the adjusted antibody positivity was 16·3% for TPO-Ab, 2·1% for tTG-Ab, and 1·8% for 21-OH-Ab. In patients with islet autoantibody-negative diabetes, the adjusted positivity was 10·2% for TPO-Ab, 0·3% for tTG-Ab, and 0·5% for 21-OH-Ab. In Chinese adults with autoimmune diabetes, those positive for GAD-Ab had a high risk for TPO-Ab positivity (odds ratio 2·39, 95% CI 1·35–4·24; p=0·0031) and tTG-Ab positivity (6·983, 1·24–39·22; p=0·027), and those positive for IA2-Ab had a high risk for tTG-Ab positivity (19·05, 3·14–115·61; p=0·0010). Patients positive for TPO-Ab were at high risk of being positive for 21-OHAb (7·48, 1·44–38·74; p=0·016) and vice versa (7·21, 1·35–38·45; p=0·021). Of note, women had a high risk for TPO-Ab positivity compared with men (3·83, 2·20–6·68; p Interpretation Patients positive for islet autoantibodies have a high risk for other organ-specific autoimmune diseases. Screening of TPO-Ab and tTG-Ab in Chinese individuals with autoimmune diabetes should be considered a priority. Funding National Key Technology R&D Program (2012BAI02B04), the Grant of National Clinical Research Center for Metabolic Diseases (2013BAI09B12), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1195), and the Key Project of Chinese Ministry of Education (113050A). more...

Interneurons are involved in the physiological function and the pathomechanism of the spinal cord. Present study aimed to examine and compare the characteristics of Cr+, Calb+ and Parv+ interneurons in morphology and distribution by using immunhistochemical and Western blot techniques. Results showed that 1) Cr-Calb presented a higher co-existence rate than that of Cr-Parv, and both of them were higher in the ventral horn than in the dosal horn; 2) Cr+, Calb+ and Parv+ neurons distributing zonally in the superficial dosal horn were small-sized. Parv+ neuronswere the largest, and Cr+ and Calb+ neurons were higher density among them. In the deep dorsal horn, Parv+ neurons were mainly located in nucleus thoracicus and the remaining scatteredly distributed. Cr+ neuronal size was the largest, and Calb+ neurons were the least among three interneuron types; 3) Cr+, Calb+ and Parv+ neurons of ventral horns displayed polygonal, round and fusiform, and Cr+ and Parv+ neurons were mainly distributed in the deep layer, but Calb+ neurons mainly in the superficial layer. Cr+ neurons were the largest, and distributed more in ventral horns than in dorsal horns; 4) in the dorsal horn of lumbar cords, Calb protein levels was the highest, but Parv protein level in ventral horns was the highest among the three protein types. Present results suggested that the morphological characteristics of three interneuron types imply their physiological function and pathomechanism relevance. more...

The CRISPR/Cas9 and guide RNA system has emerged as a promising genome editing platform, due to its simplicity, versatility and efficiency, compared to prior techniques for genome modification. Although the original Cas9 gene is too long to be packaged by AAV vectors, Dr. Zhang and colleagues have identified a shorter Cas9 protein from S. aureus (saCas9), which allows generation of a single AAV vector carrying both saCas9 and a guide RNA. Previously, we and others have demonstrated that AAV serotypes 8 and 9-based vectors are highly pancreas-tropic. Our long-term goal is to develop efficient beta-cell- and acinar-cell-targeted gene editing systems based on the AAV-saCas9 system. We first assessed on-target effects of the AAV-saCas-gRNA vector system in vitro. We generated two GFP sequence-targeted gRNA vectors, and tested their genome editing effects using two HT1080 cell lines carrying a single copy of GFP. Infection of HT1080 lines with an multiplicity of infection (MOI) of 2×104 resulted in 40-55% GFP knockout by two AAV-gRNA constructs. Gene editing efficiency was comparable in two HT1080 lines, and dose-dependent. Consecutive, multiple high-dose infections for 3 days achieved up to 80% GFP knockdown. To test whether a subset of cells are more resistant to the AAV-saCas9-mediated genome editing, we sorted the remaining 20% of GFP-positive cells and re-infected recovered GFP-positive cells with the two saCas9 vectors. Our data showed those cells were equally permissive to the saCas9 vectors, when compared to their parental line. To assess the off-target effects of the AAV-saCas9-gRNA system, we compared the exome sequences of the vector-treated cells. Our preliminary result identified multiple off-target deletions upon AAV-saCas9 treatments. We are currently analyzing whether those off-target effects were due to their similarity to the GFP targeting gRNA sequences, or independent from the gRNA sequences. We then tested the feasibility of the AAV-saCas9-gRNA vector system for beta-cell-targeted gene editing in vivo. We generated AAV8 vectors carrying Pdx1-targeting gRNA, with saCas9 driven by CMV promoter. Single intraperitoneal injection of AAV saCas9 vectors led to efficient knockout of Pdx1 expression in the pancreatic beta-cells. To limit potential off-target effects, we need to restrict saCas9 expression in beta-cells. However, our original beta-cell targeted vectors carry a relatively long, 1.1kb insulin promoter sequence. We therefore optimized the insulin promoter sequence and determined the minimum, essential promoter sequence, which facilitates robust transgene expression with high tissue-specificity. We are in the process of testing the AAV-saCas9-gRNA vectors with the optimized insulin promoter sequence for in vivo beta-cell gene editing. more...

Maize is not only a staple food crop but also an important raw material for feed and industry; however, the threat of weeds leads to a serious decline in its output and quality. The G2-aroA gene confers glyphosate herbicide tolerance to crops. In this study, the food safety of genetically modified (GM), glyphosate-tolerant maize with the G2-aroA gene was evaluated in a 90-day feeding study in Sprague-Dawley (SD) rats. Maize grain from GM or non-GM isogenic control lines were separately formulated into rodent diets at concentrations of 12.5% (low level), 25% (middle level), and 50% (high level). An additional group of rats were fed a commercialized diet as a control. The toxicological response variables, including body weights, food consumption, serum biochemistry, hematology, and absolute and relative organ weights, were compared between rats fed GM maize and those fed non-GM maize after consumption of test diets for 90days. In addition, gross and microscopic pathology were conducted among treatment groups. No adverse effects related to the consumption of GM maize were detected in the subchronic feeding study. These results indicated that the GM glyphosate-tolerant maize was as safe and nutritious as conventional maize. more...

Histology, immunohistochemistry, and Western blotting were used to characterize the changes in morphology, distribution pattern, and marker protein expression of striatal interneurons in the transition zone of striatal injury induced by 3-NP. The 3-NP treatment in rats yielded movement, motor coordination, and cognitive dysfunction. The 3-NP-induced lesion core was unvaryingly in the dorsolateral striatum, with a transition zone of lesser damage around the lesion core, in which medium-sized neurons were significantly decreased in abundance, but larger neurons survived. In both the transition zone and the lesion core, many TUNELpositive cells negative for the interneuron markers were detected, indicating widespread projection neuron death. Immunohistochemical staining for the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y–neuronal nitric oxide synthase cocontaining) showed that few immunolabeled interneurons were observed in the lesion core, but interneuron perikarya showed no evident loss in the transition zone. Consistently with this, Western blotting showed that the five interneuron protein markers were significantly decreased in the striatum after 3-NP treatment. Transition-zone calretinergic and neuropeptide Y– neuronal nitric oxide synthase-cocontaining interneurons, however, possessed more processes and varicosities than normal. These results show that, although striatal interneurons survive in the transition zone after 3-NP-mediated striatal injury, they have enhanced marker protein levels in their processes. V C 2011 Wiley more...

Inflammatory response after middle cerebral artery occlusion (MCAO) has been a focus of research recently, but the effect of inflammatory cells on ischemic neurons remains unclear. In order to study the effect of the inflammatory reaction on brain ischemic injury, we observed the morphology, number and distribution of CD3-, CD8-, ED1- and ED2-positive cells systematically in the caudate-putamen of rats in a MCAO model. The present results show that all four types of inflammatory cells first infiltrated the ischemic penumbra and then migrated into the center of the ischemic area, but the morphological changes and infiltration processes differed significantly; the infiltration of CD3- and CD8-positive cells into the ischemic area started at 3 days postischemia, and their number peaked at 1 week; however, although ED1- and ED2-positive cells were also observed at 3 days after ischemia, they reached their maximum number at 2 and 4 weeks, respectively. Moreover, ED1-and ED2-positive cells showed evident hyperplasia and hypertrophy in morphology. Our results also showed that the response of CD3-, CD8-, ED1- and ED2-positive cells in the ischemic area and the pathological changes in ischemic brain tissue could be inhibited by cyclosporine A. The results suggest that the infiltration and reaction of inflammatory cells are involved in the pathological process of ischemic brain injury. more...