Long-term engraftment and maturation of autologous iPSC-derived cardiomyocytes in two rhesus macaques.

Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.

Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium.

Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.

Predictive value of PIVKA-II and AFP for the non-objective response of HBV-associated hepatocellular carcinoma after transarterial chemoembolization: a prospective study.

BACKGROUND: To determine the predictive value of serum abnormal prothrombin (PIVKA-II) and alpha-fetoprotein (AFP) for the non-objective response of HBV-associated hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE). METHODS: This prospective study included HBV-associated HCC patients who underwent TACE at the Fourth People's Hospital of Qinghai Province between December 2021 and July 2022. According to contrast-enhanced ultrasound and upper abdomen contrast-enhanced MRI, the patients were divided into the objective response group and the non-objective response group 3 months after TACE. RESULTS: There were 54 patients, of whom 31 experienced non-objective responses. The PIVKA-II levels in the objective response group were significantly lower than in the non-objective response group at 1 month [352.00 (142.16-722.54) vs. 528.58(241.32-1681.23) mAU/ml, P = 0.005] and 3 months [28.96 (20.01-42.49) vs. 2082.55 (52.63-10 057.30) mAU/ml, P = 0.016] after TACE. The Spearman rank correlation analysis showed no significant correlation between PIVKA-II and AFP (r = 0.315, P > 0.05). The areas under the curve (AUCs) of AFP and PIVKA-II before TACE were 0.632 and 0.529. One month after TACE, the AUC of PIVKA-II combined with AFP (AUC = 0.787) was higher than for PIVKA-II (AUC = 0.658) and AFP (AUC = 0.749). CONCLUSION: PIVKA-II does not outperform AFP in predicting non-objective response after TACE in HCC patients. The combination of PIVKA-II and AFP might improve the diagnosis of HCC non-objective response after TACE.

Contrast Agents of Magnetic Resonance Imaging and Future Perspective.

In recent times, magnetic resonance imaging (MRI) has emerged as a highly promising modality for diagnosing severe diseases. Its exceptional spatiotemporal resolution and ease of use have established it as an indispensable clinical diagnostic tool. Nevertheless, there are instances where MRI encounters challenges related to low contrast, necessitating the use of contrast agents (CAs). Significant efforts have been made by scientists to enhance the precision of observing diseased body parts by leveraging the synergistic potential of MRI in conjunction with other imaging techniques and thereby modifying the CAs. In this work, our focus is on elucidating the rational designing approach of CAs and optimizing their compatibility for multimodal imaging and other intelligent applications. Additionally, we emphasize the importance of incorporating various artificial intelligence tools, such as machine learning and deep learning, to explore the future prospects of disease diagnosis using MRI. We also address the limitations associated with these techniques and propose reasonable remedies, with the aim of advancing MRI as a cutting-edge diagnostic tool for the future.

Cell based dATP delivery as a therapy for chronic heart failure.

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2'-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host contractility offers a novel approach to treating the chronically failing heart.

Graft-host coupling changes can lead to engraftment arrhythmia: a computational study.

After myocardial infarction (MI), a significant portion of heart muscle is replaced with scar tissue, progressively leading to heart failure. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) offer a promising option for improving cardiac function after MI. However, hPSC-CM transplantation can lead to engraftment arrhythmia (EA). EA is a transient phenomenon arising shortly after transplantation then spontaneously resolving after a few weeks. The underlying mechanism of EA is unknown. We hypothesize that EA may be explained partially by time-varying, spatially heterogeneous, graft-host electrical coupling. Here, we created computational slice models derived from histological images that reflect different configuration of grafts in the infarcted ventricle. We ran simulations with varying degrees of connection imposed upon the graft-host perimeter to assess how heterogeneous electrical coupling affected EA with non-conductive scar, slow-conducting scar and scar replaced by host myocardium. We also quantified the effect of variation in intrinsic graft conductivity. Susceptibility to EA initially increased and subsequently decreased with increasing graft-host coupling, suggesting the waxing and waning of EA is regulated by progressive increases in graft-host coupling. Different spatial distributions of graft, host and scar yielded markedly different susceptibility curves. Computationally replacing non-conductive scar with host myocardium or slow-conducting scar, and increasing intrinsic graft conductivity both demonstrated potential means to blunt EA vulnerability. These data show how graft location, especially relative to scar, along with its dynamic electrical coupling to host, can influence EA burden; moreover, they offer a rational base for further studies aimed to define the optimal delivery of hPSC-CM injection. KEY POINTS: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) hold great cardiac regenerative potential but can also cause engraftment arrhythmias (EA). Spatiotemporal evolution in the pattern of electrical coupling between injected hPSC-CMs and surrounding host myocardium may explain the dynamics of EA observed in large animal models. We conducted simulations in histology-derived 2D slice computational models to assess the effects of heterogeneous graft-host electrical coupling on EA propensity, with or without scar tissue. Our findings suggest spatiotemporally heterogeneous graft-host coupling can create an electrophysiological milieu that favours graft-initiated host excitation, a surrogate metric of EA susceptibility. Removing scar from our models reduced but did not abolish the propensity for this phenomenon. Conversely, reduced intra-graft electrical connectedness increased the incidence of graft-initiated host excitation. The computational framework created for this study can be used to generate new hypotheses, targeted delivery of hPSC-CMs.

Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

Dynamic chromatin organization and regulatory interactions in human endothelial cell differentiation.

Vascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. The gene-regulatory mechanisms underpinning endothelial specialization are largely unknown, as are the roles of chromatin organization in regulating endothelial cell transcription. To investigate the relationships between chromatin organization and gene expression, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-sequencing assays at specific time points. Long-range intrachromosomal contacts increase over the course of differentiation, accompanied by widespread heteroeuchromatic compartment transitions that are tightly associated with transcription. Dynamic topologically associating domain boundaries strengthen and converge on an endothelial cell state, and function to regulate gene expression. Chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes essential to vascular biology. Chromatin dynamics guide transcription in endothelial cell development and promote the divergence of endothelial cells from cardiomyocytes.

The appropriate expression and coordination of glycolate oxidase and catalase are vital to the successful construction of the photorespiratory metabolic pathway.

Photorespiration has emerged as a hotspot in the evolution of photosynthesis owing to the energy loss during the process. To ensure the physiological functions of photorespiration such as light protection, H2O2 signaling, and stress resistance, separate the photorespiration glycolic acid flow, and minimize photorespiration loss, a balance must be maintained during the construction of photorespiratory metabolic branch. In this study, glycolate oxidase (GLO) and catalase (CAT) were introduced into potato (Solanum tuberosum) chloroplasts through the expression of fusion protein. Through the examination of phenotypic characteristics, photosynthesis, anatomical structure, and enzyme activity, the efficiency of the photorespiration pathway was demonstrated. The results showed that certain transgenic lines plants had shorter plant height and deformed leaves and tubers in addition to the favorable photosynthetic phenotypes of thicker leaves and larger and denser mesophyll cells. By Diaminobenzidine (DAB) staining analysis of the leaves, the intermediate H2O2 could not be decomposed in time to cause biomass decline and malformation, and the excessive glycolate shunt formed by the overexpression of the fusion protein affected other important physiological activities. Hence, the appropriate and coordinated expression of glycolate oxidase and catalase is essential for the establishment of photorespiration pathways in chloroplasts.

The intervention effect of mental health knowledge integrated into ideological and political teaching on college students' employment and entrepreneurship mentality.

In order to analyze the intervention effect of integrating mental health knowledge into ideological and political teaching on college students' employment and entrepreneurship mentality, this paper proposes a study to predict the effect of integrated intervention. This research mainly investigates the ideological and psychological conditions of college students from divorced families through questionnaires, compares and analyzes the classification and statistical results of the survey data between groups and within groups, and analyzes the reasons for the ideological and psychological problems of college students. The experimental results show that 30% of college students from divorced families and college students from non-divorced families responded that they do not feel comfortable in places with many people, and the difference between the groups is not significant. Regarding the concept of entrepreneurship, 64.63% of college students from divorced families in urban areas believe that entrepreneurship is a form of learning and should be encouraged. 63.27% of college students from divorced families in rural areas believe that learning should be the first priority and that a business should not be started. 20.41% of college students from divorced families in rural areas and 25.61% of college students from divorced families in urban areas believe that because entrepreneurship provides economic income, it can reduce the burden on families, but the difference is not obvious. In short, this study can provide reference for the ideological and psychological status of college students from divorced families.

Stability, Antioxidant Activity and Intestinal Permeation of Oleuropein Inclusion Complexes with Beta-Cyclodextrin and Hydroxypropyl-Beta-Cyclodextrin.

Compared to beta-cyclodextrins (beta-CD), hydroxypropyl-beta-cyclodextrins (HP-beta-CD) are a more popular material used to prepare inclusion complexes due to their superior solubility and intestinal absorption. In this study, oleuropein (OL) inclusion complexes with beta-CD (beta-CD:OL) and HP-beta-CD (HP-beta-CD:OL) were prepared and the formation of inclusion complexes was validated by IR, PXRD, and DSC. A phase solubility test showed that the lgK (25 °C) and binding energy of beta-CD:OL and HP-beta-CD:OL was 2.32 versus 1.98, and −6.1 versus −24.66 KJ/mol, respectively. Beta-CD:OL exhibited a more powerful effect than HP-beta-CD:OL in protecting OL from degradation upon exposure to light, high temperature and high humidity. Molecular docking, peak intensity of carbonyls in IR, and ferric reducing power revealed that beta-CD:OL formed more hydrogen bonds with the unstable groups of OL. Both inclusion complexes significantly enhanced the solubility, intestinal permeation and antioxidant activity of OL (p < 0.05). Though HP-beta-CD:OL had higher solubility and intestinal absorption over beta-CD:OL, the difference was not significant (p > 0.05). The study implies that lower binding energy is not always associated with the higher stability of a complex. Beta-CD can protect a multiple-hydroxyl compound more efficiently than HP-beta-CD with the intestinal permeation comparable to HP-beta-CD complex.

A cross-sectional study of the interaction between night shift frequency and age on hypertension prevalence among female nurses.

Night shift is a common work schedule. This study aimed to analyze the interaction between age and frequency of night shift on the hypertension prevalence. A census questionnaire was conducted in 512 medical institutions in 11 cities of Hebei Province. One lakh twenty-one thousand nine hundred three female nurses were included in this study. Binary Logistic regression analysis was done by SPSS Version 26.0. The youngest age group without night shift was used as the reference group. The odds ratio was calculated by different combinations of interaction items. Interaction coefficients were calculated by an Excel table designed by Andersson. Compared with the 18-25 year old ones without night shift, there existed an additive interaction between the age of 36-45 and more than 5-10 night shifts per month on hypertension prevalence. Odds ratio, the relative excess risk of interaction, the attributable proportion of interaction, and the synergy index and their 95% confidence intervals were 2.923(2.292-3.727), 0.631(0.309-0.954), 0.216(0.109-0.323), 1.488(1.158-1.913). Additive interaction was also found between the age of 36-45 and more than 10 night shifts per month. OR, RERI, API, SI, and their 95% confidence intervals were 3.430(2.273-5.175) 1.037(0.061-2.013), 0.303(0.089-0.516), and 1.746(1.093-2.788). There also existed an additive interaction between the age of 46-65 and more than 5-10 night shifts per month on hypertension prevalence. OR, RERI, API, SI, and their 95% confidence intervals were 7.398(5.595-9.781) 1.809(0.880-2.739), 0.245(0.148-0.341), and 1.394(1.199-1.622).There existed interaction between specific age groups and night shift frequency on the prevalence of hypertension among female nurses.

Photo-Embossed Surface Relief Structures with Improved Aspect Ratios and Their Applications in Liquid Crystal Devices.

Photo-embossing has been developed as a convenient and economical method for creating complex surface relief structures in polymer films. The pursuit for large aspect ratios of the photo-embossed structures has never stopped. Here, we demonstrate a simple strategy to obtain improved aspect ratios by adding a quick solvent developing step into the photo-embossing process. A good solvent for the monomer is used to remove unreacted monomers from the unexposed region, resulting in deepened valleys of the surface reliefs. In a polymer film as thin as 2.5 µm, the height of the surface reliefs can be increased by a factor of three to around 1.0 µm. This strategy is also shown to be compatible with other methods used to improve the aspect ratios of the photo-embossed structures. Lastly, we employ these surface relief structures in the fabrication of liquid crystal (LC) devices and investigate their performances for visible light regulation.

An updated review on the potential antineoplastic actions of oleuropein.

Oleuropein is an ester of elenolic acid and hydroxytyrosol (3, 4-dihydroxyphenylethanol). It is a phenolic compound and the most luxuriant in olives. The detailed information related to the anticancer effects of oleuropein was collected from the internet database PubMed/Medline, ResearchGate, Web of Science, Wiley Online Library, and Cnki using appropriate keywords until the end of October 2021. Oleuropein has been shown to have antioxidant, anticancer, antiinflammatory, cardioprotective, neuroprotective, and hepatoprotective effects. Previous studies also revealed that oleuropein could effectively inhibit the malignant progression of esophageal cancer, gastric cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, and cervical cancer. Recently, the role of oleuropein in inhibiting tumor cell proliferation, invasion, and migration and inducing tumor cell apoptosis has gained extensive attention. In this review, we have summarized the latest research progress related to the antioncogenic mechanisms and the potential role of oleuropein in targeting different human malignancies. Based on these findings, it can be concluded that oleuropein can function as a promising chemopreventive and chemotherapeutic agent against cancer, but its more detailed anticancer effects and underlying mechanisms need to be further validated in future preclinical as well as clinical studies.

Cholesterol metabolism is decreased in patients with diminished ovarian reserve.

RESEARCH QUESTION: Does cholesterol metabolism differ in patients with diminished ovarian reserve (DOR) compared to patients with normal ovarian reserve (NOR)? DESIGN: The current research included 72 women with NOR and 86 women with DOR. Data on the cholesterol metabolism in granulosa cells of these women were analysed. RESULTS: On the day of human chorionic gonadotrophin injection, serum oestradiol and progesterone in the DOR group were significantly lower than in the control group (P < 0.001). There were no significant differences in serum concentrations of total cholesterol, triglyceride, high-density lipoprotein and low-density lipoprotein between the NOR and DOR groups. The cholesterol-regulated gene SCAP in granulosa cells from women with DOR was down-regulated (P = 0.024). Cholesterol synthesis and transport genes (e.g. IDI1, FDFT1, CYP51A1, SRB1 and STARD1) were also significantly decreased (P = 0.026, P = 0.044, P = 0.049, P = 0.004 and P < 0.001, respectively). In granulosa cells of patients with DOR, cholesterol-related substances such as coprostanone, 11A-acetoxyprogesterone and 17α-hydroxyprogesterone were significantly reduced (P = 0.0008, P = 0.0269, P = 0.0337, respectively). CYP19A1, a key steroidogenesis gene, was significantly reduced (P = 0.009). 17α-hydroxyprogesterone and oestradiol decreased (P = 0.004 and P = 0.039, respectively). CONCLUSION: Decreased cholesterol metabolism affecting steroid hormone synthesis in granulosa cells might be a possible mechanism for DOR.

Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes.

Heart failure remains a significant cause of morbidity and mortality following myocardial infarction. Cardiac remuscularization with transplantation of human pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). Although transient, the risk posed by EA presents a barrier to clinical translation. We hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. This study uses a porcine model to provide proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively suppress EA. None of the nine treated subjects experienced the primary endpoint of cardiac death, unstable EA, or heart failure compared with five out of eight (62.5%) in the control cohort (hazard ratio = 0.00; 95% confidence interval: 0-0.297; p = 0.002). Pharmacologic treatment of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.

Progress on the study of the anticancer effects of artesunate.

Artesunate (ART) is a derivative of artemisinin that is extracted from the wormwood plant Artemisia annua. ART is an antimalarial drug that has been shown to be safe and effective for clinical use. In addition to its antimalarial properties, ART has been attracting attention over recent years due to its reported inhibitory effects on cancer cell proliferation, invasion and migration. Therefore, ART has a wider range of potential clinical applications than first hypothesized. The aim of the present review was to summarize the latest research progress on the possible anticancer effects of ART, in order to lay a theoretical foundation for the further development of ART as a therapeutic option for cancer.

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.

SARS-CoV-2 Infects Human Pluripotent Stem Cell-Derived Cardiomyocytes, Impairing Electrical and Mechanical Function.

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2.