Reproductive and developmental toxicity assessments of LPM3480392, a novel mu opioid receptor biased agonist in rats.

Opioids remain the most powerful analgesics for moderate to severe pain but their clinical use, misuse and abuse has been an alarming medical problem, especially for those users at child-bearing age. Mu-opioid receptor (MOR) biased agonists have been suggested as superior alternatives with better therapeutic ratios. We recently discovered and characterized a novel MOR biased agonist, LPM3480392, which demonstrates robust analgesic effect, favorable pharmacokinetic performance, and mild respiratory suppression in vivo. To understand the safety profile of LPM3480392 on the reproductive system and embryonic development, this study evaluated the effects of LPM3480392 on the fertility and early embryonic development, embryo-fetal development, and pre- and postnatal development in rats. Results showed that LPM3480392 had mild effects on parental male and female animals, accompanied by subtle early embryonic loss and delayed ossification of fetal development during organogenesis period. In addition, although minor effects were found on normal developmental milestones and behaviors in the pups, there was no evidence of malformed effect. In conclusion, these results suggest that LPM3480392 has a favorable safety profile with only minor effects on the reproductive and developmental outcomes in animals, which support the development of LPM3480392 as a novel analgesic.

Single-Cell Transcriptome Analysis Reveals Embryonic Endothelial Heterogeneity at Spatiotemporal Level and Multifunctions of MicroRNA-126 in Mice.

BACKGROUND: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. METHODS: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre-mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre-mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. RESULTS: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre-mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre-mediated ECs-specific miR-126 knockout mice. CONCLUSIONS: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.

ADP receptor P2y12 prevents excessive primitive hematopoiesis in zebrafish by inhibiting Gata1.

In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), ß-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.

Single-cell transcriptomics of murine mural cells reveals cellular heterogeneity.

Mural cells (MCs) wrap around the endothelium, and participate in the development and homeostasis of vasculature. MCs have been reported as heterogeneous population morphologically and functionally. However, the transcriptional heterogeneity of MCs was rarely studied. In this study, we illustrated the transcriptional heterogeneity of MCs with different perspectives by using publicly available single-cell dataset GSE109774. Specifically, MCs are transcriptionally different from other cell types, and ligand-receptor interactions of different cells with MCs vary. Re-clustering of MCs identified five distinct subclusters. The heterogeneity of MCs in tissues was reflected by MC coverage, various distribution of MC subclusters, and ligand-receptor interactions of MCs and parenchymal cells. The transcriptomic diversity of MCs revealed in this article will help facilitate further research into MCs.

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

BACKGROUND: Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. METHODS AND RESULTS: We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. CONCLUSIONS: We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.

Dynamic regulation of small RNAome during the early stage of cardiac differentiation from pluripotent embryonic stem cells.

Embryonic stem cells (mESCs), having potential to differentiate into three germ-layer cells including cardiomyocytes, shall be a perfect model to help understanding heart development. Here, using small RNA deep sequencing, we studied the small RNAome in the early stage of mouse cardiac differentiation. We found that the expression pattern of most microRNA (miRNA) were highly enriched at the beginning and declined thereafter, some were still insufficiently expressed on day 6, and most miRNAs recovered in the following days. When pluripotent embryonic stem cells are differentiating to cardiomyocytes, targeted genes are concentrated on TGF, WNT and cytoskeletal remodeling pathway. The pathway and network of dynamically changed target genes of the miRNAs at different time points were also investigated. Furthermore, we demonstrated that small rDNA-derived RNAs (srRNAs) were significantly up-regulated during differentiation, especially in stem cells. The pathways of srRNAs targeted genes were also presented. We described the existence and the differential expression of transfer RNA (tRNA), Piwi-interacting RNA (piRNA) and Endogenous siRNAs (endo-siRNAs) in this process. This study reports the genome-wide small RNAome profile, and provides a uniquely comprehensive view of the small RNA regulatory network that governs embryonic stem cell differentiation and cardiac development.

The DNMT3B -579 G>T promoter polymorphism and risk of lung cancer.

The present study aimed to investigate the association of the -579 G>T polymorphism in the DNMT3B promoter with susceptibility to lung cancer. A total of 174 lung cancer patients and 135 healthy controls from the northern part of China were enrolled, and were matched for gender and age. All subjects were genotyped by polymerase chain reaction-restriction-fragment length polymorphism analysis and confirmed by DNA sequencing. Stratification analyses were used to study the subgroups of subjects by age and gender, and evaluate the association between the -579 G>T polymorphism and the genetic susceptibility to lung cancer. The results revealed that individuals with the DNMT3B -579 GT genotype had a significantly decreased risk of lung cancer [odds ratio (OR), 0.517; 95% confidence interval (CI), 0.273-0.981] compared with those with a -579 TT genotype in the studied population. However, the deviation was significant (OR, 0.138, 95% CI, 0.034-0.549) between the risk of lung cancer and the GT and GG genotype, when the smoking factor was considered. The data from this study indicate that the DNMT3B genetic polymorphism varies among various races, ethnic groups and geographical areas. The DNMT3B -579 G>T polymorphism may contribute to the genetic susceptibility to lung cancer.

Sublingual vein extraction algorithm based on hyperspectral tongue imaging technology.

Among the parts of the human tongue surface, the sublingual vein is one of the most important ones which may have pathological relationship with some diseases. To analyze this information quantitatively, one primitive work is to extract sublingual veins accurately from tongue body. In this paper, a hyperspectral tongue imaging system instead of a digital camera is used to capture sublingual images. A hidden Markov model approach is presented to extract the sublingual veins from the hyperspectral sublingual images. This approach characterizes the spectral correlation and the band-to-band variability using a hidden Markov process, where the model parameters are estimated by the spectra of the pixel vectors forming the observation sequences. The proposed algorithm, the pixel-based sublingual vein segmentation algorithm, and the spectral angle mapper algorithm are tested on a total of 150 scenes of hyperspectral sublingual veins images to evaluate the performance of the new method. The experimental results demonstrate that the proposed algorithm can extract the sublingual veins more accurately than the traditional algorithms and can perform well even in a noisy environment.