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Abstract Background OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14–18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency. Methods In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them. Results OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group. Conclusion The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.

Objective: To analyze the frequency and spectrum of thalassemia mutations in amniotic fluid samples collected from Han and Li people in Hainan province of China. Methods: We carried out a retrospective analysis on prenatal diagnosis of amniotic fluid samples collected from pregnant women who may have next generation with high risks of medium or severe thalassemia between 2005 and 2016. Diverse fetal thalassemia genotypes and mutated alleles in Han and Li people were analyzed and cmpared. Results: We examined 536 amniotic fluid samples from Han people and 588 from Li people, among which 406 Han and 500 Li samples were found to carry at least one thalassemia gene mutation, with a detection rate of 75.75% and 85.03%, respectively. Among all α- and β-thalassemia mutant alleles detected, the most frequently found mutations in Han and Li samples were SEA-type of α-thalassemia and 41/42 (–CTTT) of β-thalassemia, respectively. A total of 75 severe thalassemia cases were identified in Han samples and 53 in Li samples. In most of these severe cases, parents chose to terminate pregnancy after being informed of thalassemia-related risks. Conclusions: The thalassemia mutations shows ethnic and area specificity, and that prenatal diagnosis for high-risk thalassemia carrier pregnant women is an efficient approach to prevent and control the occurrence of severe thalassemia in the high-prevalence areas.

Purpose: Aim of the study is to investigate whether Wnt/β-catenin pathway plays a correlating effect in the damages of central nervous system (CNS) induced by chronic fluorosis and SKL2001, an agonist of Wnt pathway, attenuates the neurotoxicity resulted from high dose of fluoride. Methods: Sprague-Dawley rats were divided randomly into the control group, in which the rats were fed with normal diet and drinking water, and the fluoride exposure group, treated with 50 ppm fluoride in their drinking water for 6 months; primary rat hippocampal neurons were divided into three groups, i.e., the control (untreated), the fluoride exposure (1 mM fluoride in cultured medium), and the fluoride plus SKL2001 (30 M) treatments. The cultured neurons were incubated for 24, 48 and 72 hrs. Expressions of Wnt/β-catenin pathway and apoptosis-related proteins were detected using Western blotting and immunofluorescence assays. Apoptosis levels were detected by TUNEL staining and Flow cytometry. Results: The results showed that as compared to controls, the protein levels of Wnt1, β-catenin, cyclin D1 and c-Myc were lower in the hippocampus of rat brains with fluoride exposure and while the levels of phospho-β-catenin, glycogen synthase kinase-3β and apoptosis higher. Moreover, the similar biochemical changes as the rat brans with chronic fluorosis were found in primary neurons exposed to fluoride. In addition, the B-cell lymphoma-2 (Bcl2) level was decreased, and Bcl2 associated X protein (Bax) and caspase-3 were increased in the primary neurons with fluoride exposure. Interestingly, SKL2001 treatment attenuated the changes of Wit/β-catenin pathway and the relating proteins with apoptosis induced by fluorosis. Conclusion: The results indicate that the expression of Wnt/β-catenin pathway was inhibited by fluorosis in CNS, which was correlated with the enhanced apoptosis. Importantly, the agonist of Wnt pathway, SKL2001, can attenuate the neurotoxicity of fluoride. [ABSTRACT FROM AUTHOR]

Excessive fluoride is known to induce oxidative stress and cell damage, particularly in the nervous system. We aimed to investigate changes in the expression of the receptor for advanced glycation end products (RAGE), p47phox, phosphop47phox, gp91phox, p22phox, and nuclear factor kappa B (NF-κB) in SH-SY5Y cells exposed to different levels of sodium fluoride and explore the molecular mechanisms underlying cell injury caused by excessive fluoride exposure. The cells were divided into the following groups: control, low-dose fluorinated (NaF 0.4 mmol/L), high-dose fluorinated (NaF 4 mmol/L), and FPS-ZM1 (1 µM). The protein levels of RAGE, p47phox, phospho-p47phox, gp91phox, p22phox, and NF-κB were detected using western blotting. Additionally, levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using respective biochemical methods. Compared with those in the control group, the protein levels of RAGE, p47phox, phospho-p47phox, gp91phox, p22phox, and NF-κB were significantly upregulated in the high-dose fluorinated group (p<0.05). Moreover, compared with those in the high-dose fluorinated group, the protein levels of p47phox, phospho-p47phox, gp91phox, p22phox, and NF-κB were downregulated in the FPS-ZM1 group (p<0.05). Furthermore, compared with that in the control and high-dose fluorinated groups (p<0.05), the expression of SOD decreased in the high-dose fluorinated group and increased in the FPS-ZM1 group, respectively. Compared with that in the control group and high-dose fluorinated groups (p<0.05), MDA expression increased in the high-dose fluorinated group and reduced in the FPSZM1 group, respectively. Thus, excessive fluoride can upregulate the RAGE/NADPH oxidase signaling pathway in SH-SY5Y cells, and these changes may be related to the nerve cell injury caused by fluoride. [ABSTRACT FROM AUTHOR]

Abstract Hyoscyamine and scopolamine (HS), two valuable tropane alkaloids of significant medicinal importance, are found in multiple distantly related lineages within the Solanaceae family. Here we sequence the genomes of three representative species that produce HS from these lineages, and one species that does not produce HS. Our analysis reveals a shared biosynthetic pathway responsible for HS production in the three HS-producing species. We observe a high level of gene collinearity related to HS synthesis across the family in both types of species. By introducing gain-of-function and loss-of-function mutations at key sites, we confirm the reduced/lost or re-activated functions of critical genes involved in HS synthesis in both types of species, respectively. These findings indicate independent and repeated losses of the HS biosynthesis pathway since its origin in the ancestral lineage. Our results hold promise for potential future applications in the artificial engineering of HS biosynthesis in Solanaceae crops.