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关键词： non-viral delivery   Solupore   CAR T   CRISPR   CD19 CAR   TRAC   STRATEGIES  

摘要： Background aims: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. Methods: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a realtime cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. Results: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CART cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. Conclusions: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary huma

关键词： Autohaemotherapy   Gold therapy   Corneal wound healing   Human corneal epithelial cells   EPIDERMAL-GROWTH-FACTOR   INTERLEUKIN-6   EXPRESSION   MIGRATION   ALPHA   NANOPARTICLES   MMP-9   RNA  

摘要： Background: Wound healing disorders characterised by impaired or delayed reepithelialisation are a serious medical problem. In the present study, we show that gold-based blood serum therapy is a suitable therapeutic approach and shows a supportive effect in wound closure of human corneal epithelial cells (HCE) in primary in vitro experiments. Methods: For this purpose, blood from healthy individuals was incubated without (***) or with gold-microparticles (***) for 24 h. Prior to human epithelial cell stimulation (HCE), the gold particles were removed and the serum was diluted in DMEM (10 % or 30 %). Both groups of serum were compared after injury. HCE were cultured and injured (corneal in vitro wound model) and then stimulated with *** or ***. Results: Treatment with serum from a gold-based serum therapy (***) shows a supportive effect on wound healing in HCE cells in vitro. In addition, gold therapy supports the secretion of important cytokines normally associated with ocular surface wound healing (IL-1 beta, IL-6, TNF-alpha and TGF-beta) in HCE cells. Conclusions: Therapy with gold-based blood serum significantly promotes the secretion and expression of cytokines and growth factors in HCE cells in vitro. Further preclinical experiments are necessary to demonstrate the influence of this therapy on HCE cells for possible clinical application on the human ocular surface and to prove its function also in poorly healing corneal lesions. (C) 2021 Elsevier GmbH. All rights reserved.

关键词： COVID-19   SARS-CoV-2   Spike protein   Binding free energy   Molecular dynamics simulation  

摘要： SARS-CoV-2 is the causative agent of the ongoing viral pandemic of COVID-19. After the emergence of this virus, it became a global public health concern and quickly evolved into a pandemic. Mexico is currently in the third position in the number of deaths due to SARS-CoV-2. To date, there have been several lineages of SARS-CoV-2 worldwide; in the Mexican population, two variants of the spike protein (S-protein) are found, localized at H49Y and D614G, which have been related to increased infectivity with respect to the wild-type S-protein. To understand how these differences impact the structural behavior of the S-protein of SARS-CoV-2, as well as binding with ACE2, we performed MD simulations combined with the molecular mechanics generalized Born/PoissonBoltzmann surface area (MMGB(PB)SA) approach starting from X-ray crystallography data. Energetic and structural analysis showed that the differences in infectivity can be explained by differences in affinity of the protein-protein interface between the wild-type and mutant S-protein with ACE2. Conformational analysis showed that molecular recognition between the S-protein and ACE2 is linked to a decrease in the conformational flexibility of wild-type and mutant S-protein; however, an increase in the conformational mobility of ACE2 could also contribute to the binding affinity observed using the MMGB(PB)SA method.

关键词： Parkinson’s disease   Human amniotic epithelial cells   Neuroprotection   Dopaminergic neurons   Anti-inflammatory factors   Anti-oxidative stress   CILIARY NEUROTROPHIC FACTOR   STEM-CELLS   DOPAMINERGIC-NEURONS   RAT MODEL   GROWTH-FACTOR   OXIDATIVE STRESS   SUBSTANTIA-NIGRA   ANIMAL-MODELS   TRANSPLANTATION   BRAIN  

摘要： Human amniotic epithelial cells (hAECs) have been reported to have neuroprotective roles in Parkinson's disease (PD) animal models. However, the molecular mechanism is not fully understood. The present study was designed to explore the possible mechanism by which hAECs ameliorate PD symptoms and the important paracrine factors produced by hAECs that attribute to the recovery of dopaminergic neurons. Thus, we performed in vivo and in vitro experiments with hAECs in PD models or lesioned dopaminergic neurons, respectively. First, hAECs were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and motor deficits were significantly attenuated. Second, the grafts prevented the loss of nigral dopaminergic neurons and promoted the outgrowth of neurites and striatal axon fibers in PD mice. In addition, decreased microglial activation, inflammatory factor levels and MPTP-induced excessive reactive oxygen species (ROS) levels were also observed in hAEC-treated PD mice. In vitro, we found that the conditioned medium (CM) from hAECs promoted the survival of mesencephalic dopaminergic neurons stimulated with 1-methyl-4-phenylpyridine (MPP+) and induced neurite outgrowth. Next, analysis of hAEC-CM with an antibody array of 507 soluble target proteins revealed that the levels of many neurotrophic factors, growth factors, neuronal cell adhesion molecule (NrCAM) and anti-inflammatory factors were evidently high. In addition, antibody neutralization experiments showed that many of these factors contributed to the survival and growth of dopaminergic neurons and neurite outgrowth. More importantly, we found that the anti-inflammatory factor interleukin-1 receptor antagonist (IL-1ra) also augmented the survival of dopaminergic neurons, demonstrating for the first time an anti-oxidative and anti-inflammatory role of hAECs in PD mice, which represents a novel molecular mechanism of hAECs in the treatment of *** molecular mechanism of hAE

关键词： Arsenic   In vitro   Genomics   Mode of action   Carcinogenicity   NF-KAPPA-B   GENE-EXPRESSION CHANGES   DOSE-RESPONSE   24-HR EXPOSURES   DRINKING-WATER   ACTIVATION   CANCER   MDM2   CARCINOGENICITY   PATHWAYS  

摘要： Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 mu M for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKK alpha). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-kappa B), leading to inhibition of P53 function.

关键词： Thyroid gland   T lymphocytes   Lymphoproliferative disorders   Hashimoto disease   Tertiary lymphoid structures   PRIMARY LYMPHOMA   GLAND  

摘要： Thyroid lymphoproliferative disorder is known to be associated with Hashimoto thyroiditis. The major histological types of thyroid lymphoproliferative disorders are malignant lymphoma, such as diffuse large B cell lymphoma, extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT), and follicular lymphoma. All of these are B cell lymphomas; however, T cell lymphoproliferative disorders represent only a minority of cases. Since T cell lymphoproliferative disorder rarely develops and T cell lymphoma is a heterogeneous entity, it is difficult to concisely summarize this complex topic. Here, we present a case of primary thyroid T cell lymphoproliferative disorder reminiscent of indolent T cell lymphoproliferative disorder of the gastrointestinal tract: the pathological findings from this case also were similar to those of MALT lymphoma such as small lymphoid cells, lymphoepithelial lesions, and a low Ki-67 labeling index. T cell lymphoproliferative disorders with these pathological features have been reported in the thyroid gland, lymph nodes, palate, spleen, and gastrointestinal tract. Furthermore, we reviewed and summarized the studies on thyroid T cell lymphoproliferative disorders and compared the cases and their clinicopathological characteristics.

关键词： HEREDITARY HEMORRHAGIC TELANGIECTASIA   FACTOR RECEPTOR-2 EXPRESSION   SP1-DEPENDENT DNA-BINDING   TGF-BETA RECEPTOR   SHEAR-STRESS   GENE   ANGIOGENESIS   MUTATIONS   BEVACIZUMAB   ANGIOPOIETIN-1  

摘要： Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor beta 1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphorAKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis. (Translational Research 2021; 235:129-143)

关键词： Terrain reconstruction   Remote sensing image   Multispectral image   Convolutional neural network   Material classification   EXTRACTION   AERIAL   RECONSTRUCTION   SEGMENTATION   REGISTRATION   MODELS   ROADS  

摘要： For real-world simulation, terrain models must combine various types of information on material and texture in terrain reconstruction for the three-dimensional numerical simulation of terrain. However, the construction of such models using the conventional method often involves high costs in both manpower and time. Therefore, this study used a convolutional neural network (CNN) architecture to classify material in multispectral remote sensing images to simplify the construction of future models. Visible light (i.e., RGB), near infrared (NIR), normalized difference vegetation index (NDVI), and digital surface model (DSM) images were examined. This paper proposes the use of the robust U-Net (RUNet) model, which integrates multiple CNN architectures, for material classification. This model, which is based on an improved U-Net architecture combined with the shortcut connections in the ResNet model, preserves the features of shallow network extraction. The architecture is divided into an encoding layer and a decoding layer. The encoding layer comprises 10 convolutional layers and 4 pooling layers. The decoding layer contains four upsampling layers, eight convolutional layers, and one classification convolutional layer. The material classification process in this study involved the training and testing of the RUNet model. Because of the large size of remote sensing images, the training process randomly cuts subimages of the same size from the training set and then inputs them into the RUNet model for training. To consider the spatial information of the material, the test process cuts multiple test subimages from the test set through mirror padding and overlapping cropping; RUNet then classifies the subimages. Finally, it merges the subimage classification results back into the original test image. The aerial image labeling dataset of the National Institute for Research in Digital Science and Technology (Inria, abbreviated from the French Institut national de recherche en

关键词： Ectopic   Primary hyperparathyroidism   Giant parathyroid adenoma   Water clear cell adenoma   Case report   PATIENT   LOCALIZATION   SCINTIGRAPHY   TUMOR   GLAND  

摘要： Background: Ectopic parathyroid gland is not uncommon, and is associated with primary hyperparathymidism. Giant parathyroid adenoma (PA) and concurrent presence of enlarged thyroid nodule increases the probability of ectopic location. The combination of a giant PA that is ectopic (within the strap muscle) in the neck is very rare, especially in cases with no previous surgery. The rare histopathological findings of the current case, water clear cell parathyroid adenoma (WCCPA), could explain the patient's presentation, since it has low endocrine function. Case presentation: A 56-year-old Qatari female on routine visit to primary health care physician for hypertension, was incidentally discovered to be hypercalcemic and was referred to the emergency department of our institution. Neck ultrasound showed a thyroid nodule on the left side, but Tc-99m-sestamibi scintigraphy identified a left PA. FNAC of the thyroid nodule showed that it was a colloid nodule. She underwent left hemithyroidectomy and excision of left PA. Intraoperatively, the PA was giant and in the sternohyoid muscle. Intraoperative monitoring of intact PTH (IOiPTH) confirmed successful excision. Discussion: Ectopic giant parathyroid adenoma is rare especially with the intramuscular location in sternohyoid muscle in the neck without previous neck surgeries. The presence of thyroid nodule could be a precipitating factor for migration of the PA. Preoperative assessment with the radiological image is crucial for diagnosis but sometimes fail to localized the PA. Conclusion: Giant asymptomatic PA with long standing low function before hyperfunctioning should raise the suspicion of WCCPA. If diagnosis is confirmed, metastasis from a clear cell renal cell carcinoma should be ruled out.

关键词： Glaucoma   Optineurin   RGCs   Human primary retinal cells   TBK1   TBK1 inhibitor   Amlexanox   OPEN-ANGLE GLAUCOMA   GANGLION-CELLS   STEM-CELLS   OPTINEURIN   EXPRESSION   PROTEIN   MUTATIONS   AUTOPHAGY   DEATH   TBK1  

摘要： Studies carried out on the pathogenesis of glaucoma using murine cell lines and animal models require to be validated in human cells. Therefore, we explored the possibility of using human primary retinal cells (hPRCs) in culture as a model for molecular studies and testing of potential therapeutic drugs. For this purpose, central retinal tissue, obtained from the enucleated eyes of patients with anterior staphyloma, was digested with trypsin and grown in a medium containing supplements (basic fibroblast growth factor and fetal bovine serum). hPRCs at passage 1 and 2, show expression of either GFAP, a glial cell marker, or beta-III tubulin, a retinal ganglion cell (RGC)-specific marker. But at passages 3-5 nearly all of hPRCs express several RGC-specific markers (Brn3 proteins, Thy-1, beta-III tubulin, RBPMS and NeuN) but not GFAP. Expression of these markers indicated that these cells may have functional properties of RGCs. As RGCs are sensitive to glaucoma-associated mutants of OPTN, we analysed the survival of hPRCs upon overexpression of OPTN mutants. Glaucoma-associated mutants, E50KOPTN and M98K-OPTN, induced significantly higher cell death in hPRCs compared to WT-OPTN, whereas an amyotrophic lateral sclerosis-associated mutant, E478G-OPTN, did not. TBK1 inhibitor Amlexanox protected hPRCs from E50K-OPTN and M98K-OPTN induced cell death. M98K-OPTN induced cell death was suppressed by inhibitors of CaMKK beta and AMPK in hPRCs as well as in 661W, a mouse cell line that expresses several markers of RGCs and RGC precursor cells. Our results suggest that hPRCs under appropriate culture condition show RGClike properties. These cells can be used to explore the molecular mechanisms of cell death relevant for glaucoma pathogenesis and for testing of cytoprotective compounds.