Molecular Investigation and Preliminary Validation of Candidate Genes Associated with Neurological Damage in Heat Stroke.

Heat stroke (HS) is a severe medical condition characterized by a systemic inflammatory response that may precipitate multi-organ dysfunction, with a particular predilection for inducing profound central nervous system impairments. We aim to employ bioinformatics techniques for the retrieval and analysis of genes associated with heat stroke-induced neurological damage. We performed a comprehensive analysis of the GSE64778 dataset from the Sequence Read Archive, resulting in the identification of 1178 significantly differentially expressed genes (DEGs). We retrieved 2914 genes associated with heat stroke from the GeneCards database and 2377 genes associated with heat stroke from the Comparative Toxicogenomics Database (CTD). The intersection of the top 300 DEGs in the GSE64778 dataset intersected with the search results of GeneCards and CTD, yielding 25 final candidates for DEGs associated with heat stroke. Gene Ontology functional annotation results indicated that the target genes were mainly involved in apoptosis, stress response, and negative regulation of cellular processes and function in processes such as protein dimerization and protein binding. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed a predominant enrichment of candidate target genes within the PI3K-AKT signaling pathway. Subsequent protein-protein interaction network analysis highlighted HSP90aa1 as a central gene, indicating its pivotal role by possessing the highest number of edges among the genes enriched in the PI3K-AKT signaling pathway. Quantitative reverse transcription-polymerase chain reaction analysis performed on blood samples from patients validated the expression of Hsp90aa1 in individuals exhibiting early neurological damage in HS, consistent with the findings from the mRNA bioinformatics analysis. Additionally, the bioinformatics analysis of the upstream microRNAs (miRNAs) regulating HSP90aa1 and the target miRNAs associated with candidate long non-coding RNAs (lncRNAs) identified three lncRNAs, eight miRNAs, and one mRNA in the regulatory network. The DIANA Tools database and algorithms were employed for pathway enrichment and correlation analysis, revealing a significant association between LOC102547734 and MIR-206-3p, with the latter being identified as a target binding site Moreover, the analysis unveiled a correlation between MIR-206-3p and HSP90aa1, implicating the latter as a potential target binding site within the regulatory network.

[Rapid determination of 13 ß-blockers in health foods by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry].

A method based on ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry was developed for the rapid determination of 13 ß-blockers in health foods. The MS fragmentation pathways of the analytes were subsequently investigated. The optimal MS conditions, extraction solvents, mobile phases, and matrix effects were evaluated in detail. The samples were extracted with methanol, filtered by high-speed centrifugation and ultrasonic treatment, and then separated on an Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm) with gradient elution using acetonitrile and 0.1% (v/v) formic acid aqueous solution as mobile phases. MS analysis was conducted in positive-ion mode, and the data were collected using full mass and data-dependent MS2 scans (Full MS/dd-MS2). The efficient separation and high-precision primary and secondary scanning of the 13 ß-blockers in health foods were realized within 10 min, and accurate mass numbers and fragment-ion information were obtained. The methodological validation showed good linear relationships in the range of 0.5-100 µg/L, with correlation coefficients (r)≥0.9912. The limits of detection ranged from 1 to 10 µg/kg. When the standard substances were added to the blank sample in the amount of 10-200 µg/kg, the recoveries were in the range of 75.3%-108.4%, and the relative standard deviations ranged from 0.9% to 10.0% (n=6). The method was used to screen 30 batches of commercially available health foods, and none of the 13 ß-blockers was detected. The proposed method is fast, accurate, and sensitive, and can be used for the rapid determination of ß-blockers in health foods.

Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction.

Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases, such as myocardial infarction (MI). We evaluated the cardio-protective effects of the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) stably expressing lymphoid enhancer-binding factor 1 (LEF1; LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell-proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proven by echocardiogram parameters, including greatly improved left-ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that these overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy, conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease.

Targeted Genome Engineering to Control VEGF Expression in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells: Potential Implications for the Treatment of Myocardial Infarction.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell-based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF-secreting stem cell (VEGF/hUCB-MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB-MSC chromosome, the transcription activator-like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB-MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB-MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB-MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB-MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB-MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF-secreting stem cell system is an effective therapeutic approach for the treatment of MI. Stem Cells Translational Medicine 2017;6:1040-1051.

[Mobilization of peripheral blood stem cells with mitoxantrone and high-dose cytarabine chemotherapy and rhG-CSF in patients with hematopoietic malignancies].

OBJECTIVE: To evaluate the efficacy of mitoxantrone combined high dose of cytarabine and recombinant human granulocyte colony-stimulating factor (MAG) regimen for mobilizing autologous peripheral blood stem cells (APBSC) in patients with hematopoietic malignancies. METHODS: From December 1995 to April 2003, 14 lymphoma and 29 acute leukemia patients were treated with high-dose cytarabine (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3), followed by 300 microgram recombinant human granulocyte colony-stimulating factor per day (rhG-CSF 300 microg/d) i.e, the MAG regimen as mobilization regimen of peripheral blood stem cells. rhG-CSF was given subcutaneously when the white blood cell (WBC) count below 1.0 x 10(9)/L following the MA chemotherapy, APBSC were harvested when WBC count increased using Baxter CS3000plus or Cobe Spectra. RESULTS: Mobilization was successful in 13 of 14 lymphoma patients with MNC (3.91 +/- 2.70) x 10(8)/kg, CD34+ cells (17.79 +/- 12.90) x 10(6)/kg. Meanwhile, mobilization was successful in 24 of 29 acute leukemia patients with average of 2.13 times for apheresis. The median MNC and CD34+ cells yielded were 3.62 x 10(8)/kg and 7.37 x 10(6)/kg respectively, rhG-CSF was used for a median time of 7 days. Excepting for grade I-II gastrointestinal toxicity in 8 and infection in 14 cases, no major side effects were observed. There was no mobilization-related mortality. Minimal residual diseases became undetectable after mobilization in some patients. CONCLUSION: MAG is a safe and highly effective mobilization regimen in patients with lymphoma and acute leukemia.