Dissecting Somatic Cell Reprogramming by MicroRNAs and Small Molecules: A Dissertation

Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood. This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could achieve fully reprogrammed state. We demonstrate that loss of microRNA biogenesis pathway abolished the potential of mouse embryonic fibroblasts (MEFs) to be reprogrammed and revealed that several clusters of mES-specific microRNAs were highly induced by four factors during early stage of reprogramming. Among them, miR-93 and 106b were further confirmed to enhance iPSC generation by promoting mesenchymal-to-epithelial transition (MET) and targeting key p53 and TGFβ pathway components: p21 and Tgfbr2, which are important barrier genes to the process. To expand our view of microRNAs function during reprogramming, a systematic approach was used to analyze microRNA expression profile in iPSC-enriched early cell population. From a list of candiate microRNAs, miR-135b was found to be most highly induced and promoted reprogramming. Subsequent analysis revealed that it targeted an extracellular matrix network by directly modulating key regulator Wisp1. By regulating several downstream ECM genes including Tgfbi, Nov, Dkk2 and Igfbp5, Wisp1 coordinated IGF, TGFβ and Wnt signaling pathways, all of which were strongly involved in the reprogramming process. Therefore, we have identified a microRNA-regulated network that modulates somatic cell reprogramming, involving both intracellular and extracellular networks. In addition to microRNAs, in order to identify new regulators and signaling pathways of reprogramming, we utilized small molecule kinase inhibitors. A collection of 244 kinase inhibitors were screened for both enhancers and inhibitors of the process. We identified that inhibition of several novel kinases including p38, IP3K and Aurora kinase could significantly enhance iPSC generation, the effects of which were also confirmed by RNAi of specific target genes. Further characterization revealed that inhibition of Aurora A kinase enhanced phosphorylation and inactivation of GSK3β, a process mediated by Akt kinase. All together, in this dissertation, we have identified novel role of both small non-coding RNAs and kinases in regulating the reprogramming of MEFs to iPSCs

Feasibility and Initial Efficacy of Home-Based Cardiac Telerehabilitation— A Pilot Study

Background: Cardiovascular Disease (CVD) is the top health problem all over the world, including China. Home-based rehabilitation after cardiac surgery has been shown to be beneficial. In our study, a clinical study has been carried out to investigate the feasibility and effectiveness of using emergent mobile and information technology to deliver monitoring and feedback function in Home-Based Cardiac Telerehabilitation program (HBCTR). The main purpose of this study is to assess the feasibility and acceptance of the HBCTR program in low risk patients post Percutaneous Coronary Intervention (PCI). The secondary purpose is to assess the initial efficacy of the HBCTR program. Method: A single-blinded parallel two-arm Randomized Controlled Trial (RCT) has been conducted at the First Affiliated Hospital of Shantou University Medical College, China. A total of 24 post PCI patients were recruited and randomly divided into two equal groups. The control group (Usual Care (UC) program) received paper-based CVD educational booklets and biweekly outpatient review. The experiment group (HBCTR program) carried out outdoor walking/jogging exercise with real time physiological monitoring along with CVD education materials. Feasibility and acceptance of the HBCTR program were evaluated by using an acceptance questionnaire, a satisfaction questionnaire, patients’ adherence evaluation, system abnormalities analysis, and safety evaluation. The effectiveness of this program was measured by using 6 Minutes Walking Test (6MWT), Fagerstrom Test for Nicotine Dependence (FTND), Cardiac Depression Scale (CDS)), and SF-36 Health Survey. Results: A total of 53 respondents completed the HBCTR patient acceptance questionnaire, and 22 participants completed the RCT. One experiment group participant withdrew, and one control group participant lost contact during the RCT. 67.9% of participants deemed the HBCTR program acceptable due to real time exercise monitoring and emergency alert function. Features including real time exercise monitoring and emergency alerts are attributed to the high acceptance of the HBCTR program. The HBCTR program is perceived to allow patients to exercise in a safer and more independent manner compared to the UC method, and 81.8% of participants (n=9) felt satisfied with the HBCTR program. The average adherence rates of HBCTR in terms of exercise trainings, self-reporting, and medication intake are 92.9%, 88.4%, and 90.0% respectively. No serious adverse event was reported in the study; Out of the 184 exercise trainings the HBCTR remote monitoring system had only 7(3.8%) temporary system failures. After the six- week intervention, both groups resulted in statistically significant improvements in SF-36 physical component summary (PCS), SF-36 mental component summary (MCS), 6MWT, FTND, DASI, and CDS. Furthermore, patients in the experimental group had better improvements compared to patients in the control group in PCS scores in SF-36 (HBCTR: Δ12.5±7.8 vs UC: Δ 4.6±5.7), DASI (HBCTR: Δ0.7±0.5 vs UC: Δ 0.3±0.4), and 6 MWT (HBCTR: Δ45.5±17.4 vs UC: Δ27.6±14.7). Conclusion: The proposed HBCTR program is feasible and safe for low-risk post PCI patients. Although improvements were observed in both groups, the physical indicators of PCS and 6MWT of HBCTR patients exceeded those of the UC patients. The patients in the HBCTR program showed high satisfaction and decreased fears in performing rehabilitation exercise “at home” with remote monitoring. With this decreased fears in exercise, the adherence among the HBCTR patients was high which resulted in their benefit physical outcomes. Future study with multiple centers and a large-scale randomized controlled trial can be carried out to further assess the efficiency of the HBCTR program in long term. Cost analysis also can be added into the further study to compare the cost effective between HBCTR and traditional center-based CR

A bibliometric profile of optogenetics: quantitative and qualitative analyses

IntroductionOptogenetics is a rapidly developing field combining optics and genetics, with promising applications in neuroscience and beyond. However, there is currently a lack of bibliometric analyses examining publications in this area.MethodPublications on optogenetics were gathered from the Web of Science Core Collection Database. A quantitative analysis was conducted to gain insights into the annual scientific output, and distribution of authors, journals, subject categories, countries, and institutions. Additionally, qualitative analysis, such as co-occurrence network analysis, thematic analysis, and theme evolution, were performed to identify the main areas and trends of optogenetics articles.ResultsA total of 6,824 publications were included for analysis. The number of articles has rapidly grown since 2010, with an annual growth rate of 52.82%. Deisseroth K, Boyden ES, and Hegemann P were the most prolific contributors to the field. The United States contributed the most articles (3,051 articles), followed by China (623 articles). A majority of optogenetics-related articles are published in high-quality journals, including NATURE, SCIENCE, and CELL. These articles mainly belong to four subjects: neurosciences, biochemistry and molecular biology, neuroimaging, and materials science. Co-occurrence keyword network analysis identified three clusters: optogenetic components and techniques, optogenetics and neural circuitry, optogenetics and disease.ConclusionThe results suggest that optogenetics research is flourishing, focusing on optogenetic techniques and their applications in neural circuitry exploration and disease intervention. Optogenetics is expected to remain a hot topic in various fields in the future

The Long Noncoding RNA HEAL Regulates HIV-1 Replication through Epigenetic Regulation of the HIV-1 Promoter.

A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named HIV-1-enhanced lncRNA (HEAL), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of HEAL Importantly, HEAL is a broad enhancer of multiple HIV-1 strains because depletion of HEAL inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by HEAL overexpression. HEAL forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) HEAL-FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) HEAL-FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, CDK2, to enhance CDK2 expression. Notably, HEAL knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment in vitro Our results suggest that silencing of HEAL or perturbation of the HEAL-FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS.IMPORTANCE Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named HIV-1-enhanced lncRNA (HEAL) is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. HEAL forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, HEAL knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting HEAL as a potential therapeutic target to cure HIV-1/AIDS

Upper urinary dilatation and treatment of 26 patients with diabetes insipidus: A single-center retrospective study

ObjectiveTo describe the urinary tract characteristics of diabetes insipidus (DI) patients with upper urinary tract dilatation (UUTD) using the video-urodynamic recordings (VUDS), UUTD and all urinary tract dysfunction (AUTD) systems, and to summarize the experience in the treatment of DI with UUTD.MethodsThis retrospective study analyzed clinical data from 26 patients with DI, including micturition diary, water deprivation tests, imaging data and management. The UUTD and AUTD systems were used to evaluate the urinary tract characteristics. All patients were required to undergo VUDS, neurophysiologic tests to confirm the presence of neurogenic bladder (NB).ResultsVUDS showed that the mean values for bladder capacity and bladder compliance were 575.0 ± 135.1 ml and 51.5 ± 33.6 cmH2O in DI patients, and 42.3% (11/26) had a post-void residual >100 ml. NB was present in 6 (23.1%) of 26 DI patients with UUTD, and enterocystoplasty was recommended for two patients with poor bladder capacity, compliance and renal impairment. For the 24 remaining patients, medication combined with individualized and appropriate bladder management, including intermittent catheterization, indwelling catheter and regular voiding, achieved satisfactory results. High serum creatinine decreased from 248.0 ± 115.8 μmoI/L to 177.4 ± 92.8 μmoI/L in 12 patients from a population with a median of 108.1 μmoI/L (IQR: 79.9-206.5 μmoI/L). Forty-four dilated ureters showed significant improvement in the UUTD grade, and the median grade of 52 UUTD ureters decreased from 3 to 2.ConclusionBladder distension, trabeculation and decreased or absent sensations were common features for DI patients with UUTD. Individualized therapy by medication combined with appropriate bladder management can improve UUTD and renal function in DI patients

Effect of urban innovation on land finance dependence: evidence from 233 Chinese cities

Unlike most studies on urban innovation, which focus on the effects of land-use policies, this study investigates the effect of urban innovation (UI) on land finance dependence (LFD). In China, innovation-related policies are intended to create a shared business environment where new entrepreneurs can contribute to achieving sustainable economic growth. This study analyzes the spatiotemporal dynamics of LFD and UI from 2005 to 2019 using the spatial autocorrelation model and dynamic spatial Durbin model. It finds a positive spatial association between LFD and UI throughout the studied regions during the study period. It also reveals a significant inhibitory effect of UI on LFD and a negative impact on the LFD of neighbouring cities. Moreover, the inhibitory effects will increase over time. Lastly, this study shows varied impacts of UI on LFD in different regions, especially in eastern China. Important policy recommendations for high-quality development in China are provided

Surface Immobilization of Redox-Labile Fluorescent Probes: Enabling Single-Cell Co-Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities.

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single-cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition

The long noncoding RNA THRIL regulates TNFalpha expression through its interaction with hnRNPL

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFalpha expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992-hnRNPL complex that regulated transcription of the TNFalpha gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFalpha expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFalpha and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFalpha expression and may play important roles in the innate immune response and inflammatory diseases in humans