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

Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells

<p>Abstract</p> <p>Background</p> <p>The studies on cancer-stem-cells (CSCs) have attracted so much attention in recent years as possible therapeutic implications. This study was carried out to investigate the gene expression profile of CSCs in human lung adenocarcinoma A549 cells.</p> <p>Results</p> <p>We isolated CSCs from A549 cell line of which side population (SP) phenotype revealed several stem cell properties. After staining the cell line with Hoechst 33342 dye, the SP and non-side population (non-SP) cells were sorted using flow cytometric analysis. The mRNA expression profiles were measured using an Affymetrix GeneChip^®oligonucleotide array. Among the sixty one differentially expressed genes, the twelve genes inclusive three poor prognostic genes; Aldo-keto reductase family 1, member C1/C2 (AKR1C1/C2), Transmembrane 4 L six family member 1 nuclear receptor (TM4SF1), and Nuclear receptor subfamily 0, group B, member 1 (NR0B1) were significantly up-regulated in SP compared to non-SP cells.</p> <p>Conclusion</p> <p>This is the first report indicating the differences of gene expression pattern between SP and non-SP cells in A549 cells. We suggest that the up-regulations of the genes AKR1C1/C2, TM4SF1 and NR0B1 in SP of human adenocarcinoma A549 cells could be a target of poor prognosis in anti-cancer therapy.</p

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

Inter- and Intra-observer Reliability of MRI for Lumbar Lateral Disc Herniation

Background: The authors analyzed inter- and intra-observer agreement with respect to interpretation of simple magnetic resonance T1- and T2-weighted axial and sagittal images for the diagnosis of lumbar lateral disc herniation, including foraminal and extraforaminal disc herniations.Methods: Forty-two patients in whom lumbar lateral disc herniation was suspected or confirmed by simple magnetic resonance imaging at one institute between May 2003 and December 2004 were included. The magnetic resonance images consisting of T1- and T2-weighted axial and sagittal images, and these were reviewed blindly and independently by three orthopaedic spine surgeons in a random manner. The images were interpreted as positive or negative for lateral disc herniation on 2 different occasions 3 months apart. Results were analyzed using Cohens kappa statistic, and strengths of agreements were determined using the Landis and Koch criteria.Results: The kappa values for inter-observer agreement averaged 0.234 (0.282, 0.111, and 0.308 respectively) on the first occasion, and 0.166 (0.249, 0.111, and 0.137 respectively) on the second occasion, with an overall mean value of 0.200. Thus, the strength of agreement was only slight-to-fair according to the Landis and Koch criteria. Kappa values for intra-observer agreement averaged 0.479 (0.488, 0.491, and 0.459 respectively), indicating moderate agreement.Conclusions: The present study indicates that simple magnetic resonance imaging is not a reliable imaging modality for diagnosing lumbar lateral disc herniation. Another imaging study with improved diagnostic values should be developed to diagnose this pathologic finding.Keywords: Lumbar lateral disc herniation, Inter-observer reliability, Intra-observer reliability, Magnetic resonance imagingOAIID:oai:osos.snu.ac.kr:snu2009-01/102/0000004226/1SEQ:1PERF_CD:SNU2009-01EVAL_ITEM_CD:102USER_ID:0000004226EMP_ID:A076317DEPT_CD:801FILENAME:E019T_CiOS-2009_Kim_Inter-and Intra-observer Reliability of MRI for Lumbar.pdfDEPT_NM:의학과EMAIL:[email protected]_YN:NCONFIRM:YCONFIRM:

Visuospatial memory impairment as a potential neurocognitive marker to predict tau pathology in Alzheimers continuum

Background Given that tau accumulation, not amyloid-β (Aβ) burden, is more closely connected with cognitive impairment in Alzheimers disease (AD), a detailed understanding of the tau-related characteristics of cognitive function is critical in both clinical and research settings. We investigated the association between phosphorylated tau (p-Tau) level and cognitive impairment across the AD continuum and the mediating role of medial temporal lobe (MTL) atrophy. We also developed a prediction model for abnormal tau accumulation. Methods We included participants from the Gwangju Alzheimers Disease and Related Dementia Cohort in Korea, who completed cerebrospinal fluid analysis and clinical evaluation, and corresponded to one of three groups according to the biomarkers of A and T profiles based on the National Institute on Aging and Alzheimers Association research framework. Multiple linear and logistic regression analyses were performed to examine the association between p-Tau and cognition and to develop prediction models. Receiver operating characteristic curve analysis was performed to examine the discrimination ability of the models. Results Among 185 participants, 93 were classified as A-T-, 23 as A+T-, and 69 as A+T+. There was an association between decreased visuospatial delayed memory performance and p-Tau level (B = − 0.754, β = − 0.363, p < 0.001), independent of other relevant variables (e.g., Aβ). MTL neurodegeneration was found to mediate the association between the two. Prediction models with visuospatial delayed memory alone (area under the curve [AUC] = 0.872) and visuospatial delayed memory and entorhinal thickness (AUC = 0.921) for abnormal tau accumulation were suggested and they were validated in an independent sample (AUC = 0.879 and 0.891, respectively). Conclusion It is crucial to identify sensitive cognitive measures that capture subtle cognitive impairment associated with underlying pathological changes. Preliminary findings from the current study might suggest that abnormal tau accumulation underlies episodic memory impairment, particularly visuospatial modality, in the AD continuum. Suggested models are potentially useful in predicting tau pathology, and might be utilized practically in the field.This study was supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (21-BR-03-05), the Original Technology Research Program for Brain Science of the National Research Foundation (NRF) funded by the Korean government, MSIT (NRF-2014M3C7A1046041 and NRF-2016M3C7A1905469), the Brain Convergence Research Pro‑ gram of the NRF funded by the Ministry of Science and ICT (NRF2020M3E5D2A01084721) and a Basic Science Research Program through the NRF of Korea (NRF-2020R1F1A1052932

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method