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.
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.
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.
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•Biological macromolecule delivery system were fabricated by zein and GA.•Zein-AG nanoparticles have a good storage stability in a wide pH range from 3 to 9.•Electrostatic interaction and hydrophobic interaction existed in GA and zein.•GA controlled the release of TOC from zein-GA nanoparticles in simulate digestion.
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.
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 GSK3β 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 GSK3β inhibitors to induce cardiac differentiation. GSK3β 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.
•Lineage variety and cell culture density affect the cell cycle in hPSCs•CHIR99021 is cytotoxic to hPSCs with reduced S/G2/M cell-cycle phases•Cardiac differentiation reproducibility depends on cell-cycle consistency in hPSCs•Cell cycle and TCF protein levels modulate CHIR99021-induced differentiation
In this article, Laco and colleagues show that human pluripotent stem cells are characterized by individual cell-cycle profiles. Changes in cell-cycle profiles, mainly due to cell culture density, affect Wnt pathway-induced cardiac differentiation and cell death when GSK3β inhibitors are applied. We show that cell-cycle validation is crucial for stem cell quality and experimental reproducibility.
Umbilical cord blood (UCB) transplantation in adults have slow hematopoietic recovery compared to bone marrow (BM) or peripheral blood grafts mainly due to low number of total nucleated cells (TNC) ...and hematopoietic stem & progenitor cells (HSPC). Current investigational clinical strategies focus on increasing HSPC dosage by expanding CD34 enriched grafts that have resulted in early neutrophil recovery followed by long term hematopoietic reconstitution.
In an effort to expand HSPC, specifically those expressing primitive phenotype (CD34+CD90+CD49f+) from non-enriched UCB, we developed a proprietary library of 50 small molecules using structure-activity-relationship studies. Freshly-thawed UCB-mononucleated cells (MNC), were cultured in serum or animal component free expansion medium supplemented with optimal concentration of respective compound. The effects of the expansion protocol were measured based on phenotypic and functional assays. Cell cultures with basal cytokines served as control.
Screening of the small molecule library showed negligible acute adverse effects on CD45+ leukocyte population and its viability within 72 hours compared to cytokine control. In long term expansion cultures lasting up to 11 days, one specific structural analog, C7, resulted in 1195.8±71.7-folds increase of absolute CD45+CD34+CD38-CD45RA- progenitors which was at least 9.2-folds higher than control cultures (P<0.01; n=4). Colony forming unit assay showed significant increase of granulocyte-macrophage colonies from C7 treated cells compared to cytokine control (P<0.01; n=6) although TNC expansion was comparable between the culture conditions. It was necessary for the cytokine cocktail to comprise of at least stem cell factor, thrombopoietin and Fms-related tyrosine kinase 3 ligand for mediating HSPC expansion in presence of C7, although further addition of insulin like growth factor binding protein 2 marginally boosted expansion (P<0.001; n=3). Majority of HSPC expansion occurred between the seventh and tenth day of the culture period. In MNC initiated cultures, addition of C7 boosted primitive HSPC (CD45+CD34+CD38-CD45RA-CD90+CD49f+) by 633.3±8.5-folds over 10 days which was at least 7.4-folds higher than control cultures (P<0.001; n=3). In cultures initiated with purified CD34+CD38- cells, there was at least 15.9-folds higher expansion of HSPC defined by CD45+CD34+CD38-CD45RA-CD90+ in presence of C7 compared to cytokine cultures (P<0.05; n=3). Expansion of HSPC by C7 was at least 2-folds higher in comparison to mesenchymal stromal co-culture system which is the only known clinical protocol that allows for UCB expansion without prior CD34/CD133 selection (P<0.001; n=6). Transplantation of C7 expanded UCB grafts (n=11) at equivalent dosage of 2.5x107 cells/kg to sub-lethally irradiated NOD SCID Gamma (NSG) mice resulted in 3.21- and 2.09-folds higher engraftment of human CD45+ cells in the peripheral blood by day 21 compared to non-expanded (P=0.0030; n=6) and cytokine expanded grafts (P=0.0005; n=12) respectively. The frequency of SCID repopulating cells contributing to early peripheral blood engraftment was 2.48-folds higher in C7 expanded graft compared to unmanipulated graft. C7 expanded graft sustained human cell engraftment over 19 weeks which were primarily myeloid cells (CD33+/CD15+) as opposed to non-expanded graft which consisted of CD3+ T cells. Analysis of NSG BM at week 19 post-transplantation, showed significantly better (P<0.0001) chimerism of human CD45 cells in female (n=15) recipient compared to male (n=14) irrespective of graft type (transplantation dosage: 2.5x107 cells/kg). C7 expanded graft gave comparable level of human CD45 and CD34 progenitor cell engraftment as that of the non-expanded grafts. Multi-lineage reconstitution of NSG BM comprising of both myeloid and lymphoid human cells could be achieved with the C7 expanded graft. At higher transplantation dosage of 5.0x107 cells/kg, the expanded grafts had a higher survival rate of 75% compared to 50% for non-expanded graft mainly due to lower incidence of graft-versus-host-disease.
In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment or stromal cell co-culture. The expanded UCB consists of phenotypically defined primitive HSPC that maintains in vitro and in vivo functionality.
Hwang:Celgene Corporation: Honoraria, Other: Travel Support; Roche Singapore: Honoraria, Other: Travel Support; Pfizer Singapore: Honoraria, Other: Travel Support; Novartis International AG: Honoraria, Other: Travel Support; Bristol-Myers Squibb Pte Ltd: Honoraria, Other: Travel Support; MSD Pharma (Singapore): Honoraria, Other: Travel Support; Sanofi Aventis Singapore: Honoraria, Other: Travel Support; Janssen-Cilag Singapore: Honoraria, Other: Travel Support.
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
This study identified a novel structural analog of SB203580 (p38‐MAPK inhibitor) that expands hematopoietic stem and progenitor cells (HSPC) from non‐enriched, frozen‐thawed umbilical cord blood (UCB)–mononucleated cells. The UCB graft expanded with the lead small molecule C7 consists of primitive HSPC phenotype (CD34+CD90+CD49f+), maintains enhanced in vitro colony formation capacity and engrafts both primary and secondary immunodeficient mice. If CD34 selection is further used, this molecule is also able to attain superior HSPC expansion compared to current technologies.
Abstract 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.
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