Direct identification of A-to-I editing sites with nanopore native RNA sequencing.

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.

Cardiomyocyte differentiation of pluripotent stem cells with SB203580 analogues correlates with Wnt pathway CK1 inhibition independent of p38 MAPK signaling.

Differentiation of human pluripotent stem cells as embryoid bodies (EBs) has been achieved previously with p38alfa MAPK inhibitors such as SB203580 with moderate efficiency of 10-15%. We synthesized and screened 42 compounds that are 2,4,5-trisubstituted azole analogues of SB203580 for efficient cardiomyocyte differentiation. Our screen identified novel compounds that have similar cardiac differentiation activity as SB203580. However, the cardiac differentiation did not correlate with p38alfa MAPK inhibition, indicating an alternative mechanism in cardiac differentiation. Upon profiling several 2,4,5-trisubstituted azole compounds against a panel of 97 kinases we identified several off targets, among them casein kinases 1 (CK1). The cardiomyogenic activities of SB203580 and its analogues showed a correlation with post mesoderm Wnt/beta-catenin pathway inhibition of CK1 epsilon and delta. These findings united the mechanism of 2,4,5-trisubstituted azole with the current theory of Wnt/beta-catenin regulated pathway of cardiac differentiation. Consequently an efficient cardiomyocyte protocol was developed with Wnt activator CHIR99021 and 2,4,5-trisubstituted azoles to give high yields of 50-70% cardiomyocytes and a 2-fold increase in growth.

Semaglutide ameliorates Alzheimer's disease and restores oxytocin in APP/PS1 mice and human brain organoid models.

AIMS: To investigate the therapeutic effects and mechanisms of Semaglutide in Alzheimer's disease (AD), and identify its potential targets. METHODS: We systematically evaluated the effect of Semaglutide on Alzheimer's disease (AD), using both mice and human organoid models. RESULTS: Behavioral analyses on APP/PS1 mice demonstrated that Semaglutide improved the cognitive capabilities, particularly in the learning and memory domains. Biochemical investigations further highlighted its role in reducing amyloid plaque deposition and down-regulating the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) expression in the mouse brain tissues. Meanwhile, oxytocin (OXT) was up-regulated after Semaglutide treatment. Subsequent studies using human AD-brain organoids (BOs) models revealed that, upon Semaglutide treatment, these AD-BO models also exhibited reduced levels of amyloid-beta (Aß), phosphorylated Tau (p-Tau) and GFAP expression as well as increased OXT level. CONCLUSIONS: Semaglutide can ameliorate Alzheimer's disease in pre-clinical models, suggesting the promising therapeutic potential in AD patients.

Identification of a novel prognostic model for gastric cancer utilizing glutamine-related genes.

Background: Glutamine metabolism presents a promising avenue for cancer prevention and treatment, but the underlying mechanisms in gastric cancer (GC) progression remain elusive. Methods: The TCGA-STAD and GEO GSE62254 datasets, containing gene expression, clinical information, and survival outcomes of GC, were meticulously examined. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were employed to excavate a key module (MEturquoise), which was used to intersect with glutamine metabolism-related genes (GMRGs) and differentially expressed genes (DEGs) to identify differentially expressed GMRGs (DE-GMRGs). LASSO and Cox Univariate analyses were implemented to determine risk model genes. Correlation of the risk model with clinical parameters, pathways, and tumor immune microenvironments, was analyzed, and its prognostic independence was validated by Cox analyses. Finally, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the expression levels of MYB, LRFN4, LMNB2, and SLC1A5 in GC and para-carcinoma tissue. Results: The excavation of 4521 DEGs led to the discovery of the key MEturquoise module, which exhibited robust correlations with GC traits. The intersection analysis identified 42 DE-GMRGs, among which six genes showed consistency. Further LASSO analysis established MYB, LRFN4, LMNB2, and SLC1A5 as pivotal risk model genes. The risk model demonstrated associations with oncogenic and metabolism-related pathways, inversely correlating with responses to immune checkpoint blockade therapies. This risk model, together with "age", was validated to be an independent prognostic factor for GC. RT-qPCR result indicated that MYB, LRFN4, LMNB2, and SLC1A5 expressions were remarkably up-regulated in GC tissues comparison with para-carcinoma tissue. Conclusion: The present study has generated a novel risk module containing four DE-GMRGs for predicting the prognosis and the response to immune checkpoint blockade treatments for GC. This risk model provides new insights into the involvement of glutamine metabolism in GC, warranting further investigation.

Combination of Walnut Peptide and Casein Peptide alleviates anxiety and improves memory in anxiety mices.

Introduction: Anxiety disorders continue to prevail as the most prevalent cluster of mental disorders following the COVID-19 pandemic, exhibiting substantial detrimental effects on individuals' overall well-being and functioning. Even after a search spanning over a decade for novel anxiolytic compounds, none have been approved, resulting in the current anxiolytic medications being effective only for a specific subset of patients. Consequently, researchers are investigating everyday nutrients as potential alternatives to conventional medicines. Our prior study analyzed the antianxiety and memory-enhancing properties of the combination of Walnut Peptide (WP) and Casein Peptide (CP) in zebrafish. Methods and Results: Based on this work, our current research further validates their effects in mice models exhibiting elevated anxiety levels through a combination of gavage oral administration. Our results demonstrated that at 170 + 300 mg human dose, the WP + CP combination significantly improved performances in relevant behavioral assessments related to anxiety and memory. Furthermore, our analysis revealed that the combination restores neurotransmitter dysfunction observed while monitoring Serotonin, gamma-aminobutyric acid (GABA), dopamine (DA), and acetylcholine (ACh) levels. This supplementation also elevated the expression of brain-derived neurotrophic factor mRNA, indicating protective effects against the neurological stresses of anxiety. Additionally, there were strong correlations among behavioral indicators, BDNF (brain-derived neurotrophic factor), and numerous neurotransmitters. Conclusion: Hence, our findings propose that the WP + CP combination holds promise as a treatment for anxiety disorder. Besides, supplementary applications are feasible when produced as powdered dietary supplements or added to common foods like powder, yogurt, or milk.

Nutrient combinations exhibit universal antianxiety, antioxidant, neuro-protecting, and memory-improving activities.

Anxiety disorders are the most common mental disorders and, without proper treatment, may lead to severe conditions: e.g., somatic disorders or permanent damage to central nervous system. Although there are drugs in clinical trials, this study focuses on exploring the efficacy of nutrients in treating these diseases. We built different zebrafish models and screened several nutrient combinations for their antianxiety, antioxidant, neuro-protecting, and memory-improving activities. Our results showed that the combinations of nutrients (e.g., Walnut Peptides + Theanine at 14.2 + 33.3 µg/ml) have similar or better activities than the positive control drugs. In addition, we discovered that the effects of the nutrients in the above four aspects were universal and highly related. This study is noteworthy as it suggested that nutrients could be healthier and greener drug alternatives and provide similar or better universal treatments for anxiety and related conditions.

[Synthesis and antibacterial activity of 7 beta-[2-(2-substituted aminothiazol-4-yl)-(Z)-2-methoxyiminoacetylamido]-3-quaternaryammoniummethyl-cephalosporins].

In order to find new cephalosporin with more and more potent antibacterial activity, nine new fourth-generation cephalosporins (N1-N9) were synthesized from ethyl 2-(2-aminothiazol-4-yl)-(Z)-2-methoxyiminoacetate (1) via acylation, substitution, hydrolysis, active esterification, condensation and salt formation. The structures of compounds (N1-N9) were confirmed by IR, MS, 1H NMR and elemental analysis. The target compounds possess different antimicrobial activities against Gram-positive and Gram-negative bacteria. The preliminary results of antibacterial activities revealed that they showed better antibacterial activities against Gram-positive bacteria than cefpirome sulfate. In particular, their activities against Staphylococcus aureus and Streptococcus albus are better.

Biological macromolecule delivery system fabricated using zein and gum arabic to control the release rate of encapsulated tocopherol during in vitro digestion.

Nanoparticles were fabricated by adsorbing gum arabic (GA) on zein nanoparticles by antisolvent precipitation. The most stable mass ratio of zein:GA was 1:1.5 with a stable zeta-potential (-32.8 mV) in a pH range of 3.0-9.0. The surface hydrophobicity of zein-GA nanoparticles indicated formation of a stable structure through electrostatic attraction at a pH range of 3.0-6.0 and hydrophobic interaction at pH 7.0-9.0. The FTIR spectrogram showed an additional role of hydrogen bonds to promote the adsorption of GA on zein nanoparticles. Tocopherol (TOC) was encapsulated within the prepared zein-GA nanoparticles with a high loading capacity. The presence of GA not only prevented the precipitation of zein nanoparticles but also controlled the release of TOC from zein-GA nanoparticles during in vitro gastrointestinal digestion. Zein-GA biopolymer nanoparticles can be stably fabricated in a wide pH range for applications in the food and pharmacy industries.

Influencing the Fate of Cardiac and Neural Stem Cell Differentiation Using Small Molecule Inhibitors of ALK5.

In this study, 50 tri-substituted imidazoles (TIs), which are analogs of the small molecules TA-01 and SB203580, were synthesized and screened for cardiomyogenic activities. Several TIs displayed cardiomyogenic activities when applied during the differentiation from days 3-5. The TIs did not affect the Wnt/ß-catenin pathway during cardiomyogenesis and the likely mechanism of action is through the inhibition of ALK5 of the TGFß pathway. Interestingly, these TIs promoted the neural differentiation of human pluripotent stem cells (hPSCs) with a similar potency to that of the dual SMAD inhibitors SB431542/LDN-193189 when dosed from days 1 to 9. The neural induction activities of the TIs correlated with their ALK5 inhibitory activities. This study reports the discovery of small molecule inhibitors of ALK5, which can promote the differentiation of hPSCs into cardiomyocytes or neural cells depending on the time of dosing, showing potential for the production of clinical-grade cardiac/neural cells for regenerative therapy. Stem Cells Translational Medicine 2018;7:709-720.

Unraveling the Inconsistencies of Cardiac Differentiation Efficiency Induced by the GSK3ß Inhibitor CHIR99021 in Human Pluripotent Stem Cells.

Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3ß inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3ß inhibitors to induce cardiac differentiation. GSK3ß inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes.

Ex Vivo Expansion of CD34+ CD90+ CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds.

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.