Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF) gene in an Epstein-Barr virus (EBV)-based plasmid (pEBVHGF) showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF).ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures

Automating Rey Complex Figure Test scoring using a deep learning-based approach: a potential large-scale screening tool for cognitive decline

Background The Rey Complex Figure Test (RCFT) has been widely used to evaluate the neurocognitive functions in various clinical groups with a broad range of ages. However, despite its usefulness, the scoring method is as complex as the figure. Such a complicated scoring system can lead to the risk of reducing the extent of agreement among raters. Although several attempts have been made to use RCFT in clinical settings in a digitalized format, little attention has been given to develop direct automatic scoring that is comparable to experienced psychologists. Therefore, we aimed to develop an artificial intelligence (AI) scoring system for RCFT using a deep learning (DL) algorithm and confirmed its validity. Methods A total of 6680 subjects were enrolled in the Gwangju Alzheimers and Related Dementia cohort registry, Korea, from January 2015 to June 2021. We obtained 20,040 scanned images using three images per subject (copy, immediate recall, and delayed recall) and scores rated by 32 experienced psychologists. We trained the automated scoring system using the DenseNet architecture. To increase the model performance, we improved the quality of training data by re-examining some images with poor results (mean absolute error (MAE) ≥ 5 [points]) and re-trained our model. Finally, we conducted an external validation with 150 images scored by five experienced psychologists. Results For fivefold cross-validation, our first model obtained MAE = 1.24 [points] and R-squared (R2 ) = 0.977. However, after evaluating and updating the model, the performance of the final model was improved (MAE = 0.95 [points], R2 = 0.986). Predicted scores among cognitively normal, mild cognitive impairment, and dementia were significantly different. For the 150 independent test sets, the MAE and R2 between AI and average scores by five human experts were 0.64 [points] and 0.994, respectively. Conclusion We concluded that there was no fundamental difference between the rating scores of experienced psychologists and those of our AI scoring system. We expect that our AI psychologist will be able to contribute to screen the early stages of Alzheimers disease pathology in medical checkup centers or large-scale community-based research institutes in a faster and cost-effective way.This research was supported by the Technology Innovation Program (20022810, Development and Demonstration of a Digital System for the evaluation of geriatric Cognitive impairment) funded By the Ministry of Trade, Industry & Energy(MOTIE, Korea), by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1F1A1052932), by the Healthcare AI Convergence Research & Development Program through the National IT Industry Promotion Agency of Korea (NIPA) funded by the Ministry of Science and ICT(No.1711120216), by the KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science and ICT (22-BR-03–05), and by the Korea National Institute of Health research project (project No. 2021-ER1007-01)

Long-Term Glycaemic Durability of Early Combination Therapy Strategy versus Metformin Monotherapy in Korean Patients with Newly Diagnosed Type 2 Diabetes Mellitus

We assessed the glycaemic durability with early combination (EC; vildagliptin+metformin [MET], n=22) versus MET monotherapy (n=17), among newly-diagnosed type 2 diabetes mellitus (T2DM) enrolled (between 2012 and 2014) in the VERIFY study from Korea (n=39). Primary endpoint was time to initial treatment failure (TF) (glycosylated hemoglobin [HbA1c]>= 7.0% at two consecutive scheduled visits after randomization [end of period 1]). Time to second TF was assessed when both groups were receiving and failing on the combination (end of period 2). With EC the risk of initial TF significantly reduced by 78% compared to MET (n=3 [15%] vs. n=10 [58.7%], P=0.0228). No secondary TF occurred in EC group versus five patients (29.4%) in MET. Patients receiving EC treatment achieved consistently lower HbA1c levels. Both treatment approaches were well tolerated with no hypoglycaemic events. In Korean patients with newly diagnosed T2DM, EC treatment significantly and consistently improved the long-term glycaemic durability as compared with MET.Peer reviewe

Genome shotgun sequencing and development of microsatellite markers for gerbera (Gerbera hybrida H.) by 454 GS-FLX

The objective of this research was to develop and characterize microsatellite markers for gerbera. We used shotgun sequencing with Roche 454 GS-FLX Titanium technology to identify microsatellite loci in gerbera genomic DNA (Gerbera hybrida). The total length of non-redundant sequences obtained was 22,527,019 bp, which consisted of 3,085 contigs and 28,249 singletons. We assembled 61,958 reads into 3,085 contigs, of which 114 (3.70%) contained microsatellite repeats. The average G+C content was 39.3%. Functional annotation to known sequences yielded 14.7% unigenes in the ‘Raon’ cultivar. Analysis of the gerbera genome DNA (‘Raon’) general library showed that sequences of (AT), (AG), (AAG) and (AAT) repeats appeared most often, whereas (AC), (AAC) and (ACC) were the least frequent. Primer pairs were designed for 80 loci. Only eight primer pairs produced reproducible polymorphic bands in the 28 gerbera accessions analyzed. A total of 30 alleles were identified from the eight polymorphic SSR loci, with two to eight alleles per locus (average level of 3.75). These markers will be useful for investigating genetic diversity and differentiation in gerbera. Keywords: Genetic diversity, genomics, microsatellite isolation, pyrosequencing, SSRs. African Journal of Biotechnology Vol. 11(29), pp. 7388-7396, 10 April, 201

Generation of Functional Insulin-Producing Cells from Neonatal Porcine Liver-Derived Cells by PDX1/VP16, BETA2/NeuroD and MafA

Abstract Surrogate b-cells derived from stem cells are needed to cure type 1 diabetes, and neonatal liver cells may be an attractive alternative to stem cells for the generation of b-cells. In this study, we attempted to generate insulin-producing cells from neonatal porcine liver-derived cells using adenoviruses carrying three genes: pancreatic and duodenal homeobox factor1 (PDX1)/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA), which are all known to play critical roles in pancreatic development. Isolated neonatal porcine liver-derived cells were sequentially transduced with triple adenoviruses and grown in induction medium containing a high concentration of glucose, epidermal growth factors, nicotinamide and a low concentration of serum following the induction of aggregation for further maturation. We noted that the cells displayed a number of molecular characteristics of pancreatic b-cells, including expressing several transcription factors necessary for b-cell development and function. In addition, these cells synthesized and physiologically secreted insulin. Transplanting these differentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the reversal of hyperglycemia, and more than 18% of the cells in the grafts expressed insulin at 6 weeks after transplantation. These data suggested that neonatal porcine liver-derived cells can be differentiated into functional insulin-producing cells under the culture conditions presented in this report and indicated that neonatal porcine liverderived cells (NPLCs) might be useful as a potential source of cells for b-cell replacement therapy in efforts to cure type I diabetes

Loss of Autophagy Diminishes Pancreatic β Cell Mass and Function with Resultant Hyperglycemia

SummaryAutophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic β cells and glucose homeostasis has not been described. We produced mice with β cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. β cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of β cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient β cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in β cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic β cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles

Early combination versus initial metformin monotherapy in the management of newly diagnosed type 2 diabetes : An East Asian perspective

Type 2 diabetes (T2D) in the East Asian population is characterized by phenotypes such as low body mass index, an index of beta-cell dysfunction, and higher percentage of body fat, an index of insulin resistance. These phenotypes/pathologies may predispose people to early onset of diabetes with increased risk of stroke and renal disease. Less than 50% of patients with T2D in East Asia achieve glycaemic targets recommended by national or regional guidelines, which may be attributable to knowledge and/or implementation gaps. Herein, we review the latest evidence with special reference to East Asian patients with T2D and present arguments for the need to use early combination therapy to intensify glycaemic control. This strategy is supported by the 5-year worldwide VERIFY study, which reported better glycaemic durability in newly diagnosed patients with T2D with a mean HbA1c of 6.9% treated with early combination therapy of vildagliptin plus metformin versus those treated with initial metformin monotherapy followed by addition of vildagliptin only with worsening glycaemic control. This paradigm shift of early intensified treatment is now recommended by the American Diabetes Association and the European Association for the Study of Diabetes. In order to translate these evidence to practice, increased awareness and strengthening of the healthcare system are needed to diagnose and manage patients with T2D early for combination therapy.Peer reviewe