CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia.

Beta-thalassemia (ß-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of ß-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for ß-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including ß-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent ß-thalassemia (TDT). In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for ß-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of ß-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating ß-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.

Cytoplasmic SIRT1 promotes paclitaxel resistance in ovarian carcinoma through increased formation and survival of polyploid giant cancer cells.

Therapeutic resistance is a notable cause of death in patients with ovarian carcinoma. Polyploid giant cancer cells (PGCCs), commonly arising in tumor tissues following chemotherapy, have recently been considered to contribute to drug resistance. As a type III deacetylase, Sirtuin1 (SIRT1) plays essential roles in the cell cycle, cellular senescence, and drug resistance. Accumulating evidence has suggested that alteration in its subcellular localization via nucleocytoplasmic shuttling is a critical process influencing the functions of SIRT1. However, the roles of SIRT1 subcellular localization in PGCC formation and subsequent senescence escape remain unclear. In this study, we compared the differences in the polyploid cell population and senescence state of PGCCs following paclitaxel treatment between tumor cells overexpressing wild-type SIRT1 (WT SIRT1) and those expressing nuclear localization sequence (NLS)-mutated SIRT1 (SIRT1NLSmt ). We investigated the involvement of cytoplasmic SIRT1 in biological processes and signaling pathways, including the cell cycle and cellular senescence, in ovarian carcinoma cells' response to paclitaxel treatment. We found that the SIRT1NLSmt tumor cell population contained more polyploid cells and fewer senescent PGCCs than the SIRT1-overexpressing tumor cell population. Comparative proteomic analyses using co-immunoprecipitation (Co-IP) combined with liquid chromatography-mass spectrometry (LC-MS)/MS showed the differences in the differentially expressed proteins related to PGCC formation, cell growth, and death, including CDK1 and CDK2, between SIRT1NLSmt and SIRT1 cells or PGCCs. Our results suggested that ovarian carcinoma cells utilize polyploidy formation as a survival mechanism during exposure to paclitaxel-based treatment via the effect of cytoplasmic SIRT1 on PGCC formation and survival, thereby boosting paclitaxel resistance. © 2023 The Pathological Society of Great Britain and Ireland.

Anchor Association Learning for Unsupervised Video Person Re-Identification.

Video-based person re-identification (re-id) has attracted a significant attention in recent years due to the increasing demand of video surveillance. However, existing methods are usually based on the supervised learning, which requires vast labeled identities across cameras and is not suitable for real scenes. Although some unsupervised approaches have been proposed for video re-id, their performance is far from satisfactory. In this article, we propose an unsupervised anchor association learning (UAAL) framework to address the video-based person re-id task, in which the feature representation of each sampled tracklet is regarded as an anchor. Specifically, we first propose an intracamera anchor association learning (IAAL) term that learns the discriminative anchor by utilizing the affiliation relations between an image and the anchors in each camera. Then, the exponential moving average (EMA) strategy is employed to update the anchor and the updated anchors are stored into an anchor memory module. On top of that, a cross-camera anchor association learning (CAAL) term is introduced to mine potential positive anchor pairs across cameras by presenting a cyclic ranking anchor alignment and threshold filtering method. Extensive experiments conducted on two public datasets show the superiority of the proposed method; for example, our method achieves 73.2% for rank-1 accuracy and 60.1% for mean average precision (mAP) score, respectively, on MARS, similarly 89.7% and 87.0% on DukeMTMC-VideoReID.

Probing the therapeutic potential of TRPC6 for Alzheimer's disease in live neurons from patient-specific iPSCs.

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer's disease (AD) under patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher levels of AD pathological markers Aß and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neurons with TRPC6 protein fragment or agonist inhibited the elevation of Aß and phospho-tau. Our results in live AD neurons manifest that the compromised expression of TRPC6 substantially contributed to Aß pathology of sporadic AD, suggesting that targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.

Association of neck circumference and cognitive impairment among Chinese elderly.

Objectives: To investigate the association between neck circumference (NC) and cognitive impairment and interactions between relevant variables to the risk of cognitive impairment. Methods: A population-based survey was conducted among elderly inhabitants aged 60 years and over from a community in Shanghai suburb. Multivariate logistic regression analyses were performed to evaluate associations and log likelihood ratio tests to examine interactions. Results: Cognitive impairment was identified in 269 (10.8%) subjects from 2,500 participants. Higher BMI (OR = 1.55; 95% CI = 1.11-2.16), higher WHR (OR = 1.44; 95% CI = 1.07-1.95), and higher total cholesterol (TC) (OR = 1.52; 95% CI = 1.09-2.13) were significantly associated with the increased risk of cognitive impairment. Significant interactions were observed between TC and a few other relevant variables, respectively. Conclusions: NC was associated with the high risk of cognitive impairment. Additive effects of NC with TC on cognitive impairment were observed.

[Gene expression of apolipoprotein and lipids synthesis and secretion in RPE-J cells].

PURPOSE: To determine whether the temperature-sensitive rat retinal pigment epithelial cell line (RPE-J) express mRNA transcripts for apolipoprotein(apo) and microsomal triglyceride transfer protein (MTP) and whether it can synthesize and secrete neutral lipids. METHODS: RPE-J cells were cultured in plastic tissue culture flasks and on Transwell filters. RT-PCR was used to check mRNA expression. Phagocytosis was checked by adding FITC-labeled beads and measuring the intensity of the fluorescence. Lipids were extracted from cultured cells that were fed photoreceptor outer segments (OS) for 24h; thin layer chromatography and enzymatic fluorimetry were used to assay lipid content. Cells were supplemented with [3H]-oleate (5000 dpm/nmole) bound to 1.5% fatty acid free bovine serum albumin in order to permit detection of the new synthesis and secretion of neutral lipids. RESULTS: RPE-J cells express mRNA for apoA-I, apoA-II, apoB, apoC-I, II, III, and apo E, as well as MTP. RPE-J cells can phagocytose OS and beads. OS-fed RPE-J cells, relative to untreated cells, have 5.8 times more triglyceride, 2.6 times more phospholipid, 1.5 times more unesterified cholesterol, and 0.3 times more esterified cholesterol. Radiolabeled neutral lipid is detected in cells and medium. All lipids increase in cells with time but only phospholipid increases in the medium with time. CONCLUSIONS: RPE cells express genes encoding apolipoproteins and MTP. Following the ingestion of OS, RPE-J cells contain abundant lipid. Following the ingestion of long-chain fatty acids, RPE-J cells can synthesize neutral lipids. Future experiments will determine if RPE-J cells secrete lipoprotein particles containing these proteins.

Molecular mechanism of bovine trabecular meshwork cells apoptosis induced by dexamethasone and protection by pilocarpine.

PURPOSE: To study the molecular mechanism of trabecular meshwork cells apoptosis induced by dexamethasone and the protection of pilocarpine. METHODS: Determining mRNA expression with reverse transcription-polymerase chain protein expression with Western blots and the percentage of reaction (RT-PCR), apoptotic cells with fluorescent microscopy. RESULTS: Dexamethasone up-regulated Fas proteins and affected Bax, caspase-8 and caspase-9 proteins in an action of first decrease then increase. Pre -treatment with pilocarpine decreased the four proteins expression, which were increased by dexamethasone. Pilocarpine self could decrease pro-apoptotic factors Bax, caspase-8 and caspase-9 proteins expression. CONCLUSION: Fas/FasL pathway participated in apoptotic process induced by dexamethasone in trabecular meshwork cells and the process was probably related with both caspase-8 and caspase-9 pathways. Pilocarpine protected the cells against apoptosis through down-regulating Fas, Bax, caspase-8 and caspase-9 proteins expression.

Muscarinic receptor agonists protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone.

PURPOSE: To study whether muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone. METHODS: The third to fifth passages of bovine trabecular meshwork cells were grown to confluence and incubated for 1-14 days in growth media with dexamethasone or pretreatment of pilocarpine or carbachol. The cultures were evaluated for apoptosis by phase-contrast microscopy, fluorescence microscopy, DNA laddering and flow cytometric analysis. RESULTS: Dexamethasone (0.24-0.96 mmol.L-1) induced apoptosis of trabecular meshwork cells in a dose and time-dependent manner. Before 0.48 mmol.L-1 dexamethasone-treatment, 1.84 mmol.L-1 of pilocarpine or 2.74 mmol.L-1 of carbachol added could significantly reduce apoptotic percentage. CONCLUSION: Muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone.

[Apoptosis of bovine trabecular meshwork cells induced by dexamethasone].

OBJECTIVE: To investigate apoptosis of bovine trabecular meshwork cells induced by dexamethasone. METHODS: In the incubation method of system in vitro, dexamethasone (1 - 500 mg/L) was added into the syncretic culture solution of the third to fifth generation of bovine trabecular meshwork cells. After 1 - 14 days, the culture solution was observed by phase-contrast microscopy, fluorescence microscopy and transmission electron microscopy, and was studied by DNA laddering and flow cytometric analysis. RESULTS: Dexamethasone (125 - 500 mg/L) induced apoptosis of trabecular meshwork cells in a dose-time-dependent manner. CONCLUSIONS: Apoptosis of cultured bovine trabecular meshwork cells can be induced by dexamethasone, which may be one of the pathogenic mechanisms of steroid-induced glaucoma.