T-cell responses to SARS-CoV-2 Omicron spike epitopes with mutations after the third booster dose of an inactivated vaccine.

The rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains more than 30 mutations that mediate escape from antibody responses elicited by prior infection or current vaccines. Fortunately, T-cell responses are highly conserved in most individuals, but the impacts of mutations are not clear. Here, we showed that the T-cell responses of individuals who underwent booster vaccination with CoronaVac were largely protective against the SARS-CoV-2 Omicron spike protein. To specifically estimate the impact of Omicron mutations on vaccinated participants, 16 peptides derived from the spike protein of the ancestral virus or Omicron strain with mutations were used to stimulate peripheral blood mononuclear cells (PBMCs) from the volunteers. Compared with the administration of two doses of vaccine, booster vaccination substantially enhanced T-cell activation in response to both the ancestral and Omicron epitopes, although the enhancement was slightly weakened by the Omicron mutations. Then, the peptides derived from these spike proteins were used separately to stimulate PBMCs. Interestingly, compared with the ancestral peptides, only the peptides with the G339D or N440K mutation were detected to significantly destabilize the T-cell response. Although more participants need to be evaluated to confirm this conclusion, our study nonetheless estimates the impacts of mutations on T-cell responses to the SARS-CoV-2 Omicron variant.

Cell survival controlled by lens-derived Sema3A-Nrp1 is vital on caffeine-suppressed corneal innervation during chick organogenesis.

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho-Histone3 (pHIS3) and C-caspase3 revealed that cell survival was repressed by caffeine administration. Whole-mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin-1 (Nrp1) showed that both caffeine and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF-labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A-Nrp1 signaling exerts a key role on the caffeine-suppressed neuron survival. Knocking-down Sema3A through transfection with Sema3A-siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A-Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2-Robo1 and Ephb2. Taken together, we speculate here that caffeine-enhanced reactive oxygen species upregulates Sema3A-Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

Effects of oxidative stress on hyperglycaemia-induced brain malformations in a diabetes mouse model.

Pregestational diabetes mellitus (PGDM) enhances the risk of fetal neurodevelopmental defects. However, the mechanism of hyperglycaemia-induced neurodevelopmental defects is not fully understood. In this study, several typical neurodevelopmental defects were identified in the streptozotocin-induced diabetes mouse model. The neuron-specific class III beta-tubulin/forkhead box P1-labelled neuronal differentiation was suppressed and glial fibrillary acidic protein-labelled glial cell lineage differentiation was slightly promoted in pregestational diabetes mellitus (PGDM) mice. Various concentrations of glucose did not change the U87 cell viability, but glial cell line-derived neurotrophic factor expression was altered with varying glucose concentrations. Mouse maternal hyperglycaemia significantly increased Tunel(+) apoptosis but did not dramatically affect PCNA(+) cell proliferation in the process. To determine the cause of increased apoptosis, we determined the SOD activity, the expression of Nrf2 as well as its downstream anti-oxidative factors NQO1 and HO1, and found that all of them significantly increased in PGDM fetal brains compared with controls. However, Nrf2 expression in U87 cells was not significantly changed by different glucose concentrations. In mouse telencephalon, we observed the co-localization of Tuj-1 and Nrf2 expression in neurons, and down-regulating of Nrf2 in SH-SY5Y cells altered the viability of SH-SY5Y cells exposed to high glucose concentrations. Taken together, the data suggest that Nrf2-modulated antioxidant stress plays a crucial role in maternal hyperglycaemia-induced neurodevelopmental defects.

Investigating the effect of excess caffeine exposure on placental angiogenesis using chicken 'functional' placental blood vessel network.

It is now known that over-consumption of caffeine by pregnant mothers could have detrimental effects on normal fetal development. However, it remains obscure how caffeine's harmful effect impacts directly or indirectly on the developing embryo/fetus through damaging placenta development. In this study, we demonstrated the morphological similarities between the yolk sac and chorioallantoic membranes (CAM) of chick embryos and the villi of the mammalian placenta. Using the chick yolk sac and the CAM as a model, we found that 5-15 µmol per egg of caffeine exposure inhibited angiogenesis. Under the same condition, cell proliferation in extraembryonic mesoderm was reduced while apoptosis was enhanced. Semi-quantitative RT-PCR analysis revealed that caffeine treatment down-regulated VEGF, VEGFR2, PIGF, IGF2 and NRP1 expression, but up-regulated Ang1 and Ang2 expression. We performed in situ hybridization to show VE-cadherin expression and as to demonstrate the blood vessels in the CAM and yolk sac membranes. This distribution of the VE-cadherin(+) blood vessels was determined to be reduced after caffeine treatment. Furthermore, MDA activity was induced after caffeine exposure, but GSH-PX activity was inhibited after caffeine exposure; SOD activity was unchanged as compared with the control. In summary, our results suggest that caffeine exposure could negatively impact on angiogenesis in the chick yolk sac and CAM by targeting angiogenesis-related genes. Some of these genes are also involved in regulating excess ROS generation. The results implied that the negative impact of caffeine on fetal development was partly attributed to impaired placental angiogenesis.

Angiogenesis is repressed by ethanol exposure during chick embryonic development.

It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development.

The impact of high-salt exposure on cardiovascular development in the early chick embryo.

In this study, we show that high-salt exposure dramatically increases chick mortality during embryo development. As embryonic mortality at early stages mainly results from defects in cardiovascular development, we focused on heart formation and angiogenesis. We found that high-salt exposure enhanced the risk of abnormal heart tube looping and blood congestion in the heart chamber. In the presence of high salt, both ventricular cell proliferation and apoptosis increased. The high osmolarity induced by high salt in the ventricular cardiomyocytes resulted in incomplete differentiation, which might be due to reduced expression of Nkx2.5 and GATA4. Blood vessel density and diameter were suppressed by exposure to high salt in both the yolk sac membrane (YSM) and chorioallantoic membrane models. In addition, high-salt-induced suppression of angiogenesis occurred even at the vasculogenesis stage, as blood island formation was also inhibited by high-salt exposure. At the same time, cell proliferation was repressed and cell apoptosis was enhanced by high-salt exposure in YSM tissue. Moreover, the reduction in expression of HIF2 and FGF2 genes might cause high-salt-suppressed angiogenesis. Interestingly, we show that high-salt exposure causes excess generation of reactive oxygen species (ROS) in the heart and YSM tissues, which could be partially rescued through the addition of antioxidants. In total, our study suggests that excess generation of ROS might play an important role in high-salt-induced defects in heart and angiogenesis.

Excess caffeine exposure impairs eye development during chick embryogenesis.

Caffeine has been an integral component of our diet and medicines for centuries. It is now known that over consumption of caffeine has detrimental effects on our health, and also disrupts normal foetal development in pregnant mothers. In this study, we investigated the potential teratogenic effect of caffeine over-exposure on eye development in the early chick embryo. Firstly, we demonstrated that caffeine exposure caused chick embryos to develop asymmetrical microphthalmia and induced the orbital bone to develop abnormally. Secondly, caffeine exposure perturbed Pax6 expression in the retina of the developing eye. In addition, it perturbed the migration of HNK-1(+) cranial neural crest cells. Pax6 is an important gene that regulates eye development, so altering the expression of this gene might be the cause for the abnormal eye development. Thirdly, we found that reactive oxygen species (ROS) production was significantly increased in eye tissues following caffeine treatment, and that the addition of anti-oxidant vitamin C could rescue the eyes from developing abnormally in the presence of caffeine. This suggests that excess ROS induced by caffeine is one of the mechanisms involved in the teratogenic alterations observed in the eye during embryogenesis. In sum, our experiments in the chick embryo demonstrated that caffeine is a potential teratogen. It causes asymmetrical microphthalmia to develop by increasing ROS production and perturbs Pax6 expression.

Effects of high salt-exposure on the development of retina and lens in 5.5-day chick embryo.

BACKGROUND/AIMS: Excess maternal salt intake during pregnancy may alter fetal development. However, our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high-salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. METHODS: 5.5-day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole-mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high-salt treatment. RT-PCR analyses were conducted from chick retina tissues. RESULTS: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high-salt treatment. High-salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt-treatment was detrimental to the migration of neural crest cells. CONCLUSION: Taken together, our study demonstrated that high-salt exposure of 5.5-day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development.

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10(-8)-10(-6)µmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly, histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

BACKGROUND: Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. METHODS: The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. RESULTS: The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. CONCLUSIONS: This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Enhanced beta-catenin expression and inflammation are associated with human ectopic tubal pregnancy.

STUDY QUESTION: Is there a molecular link between Wnt signaling in fallopian tube inflammation and ectopic tubal implantation? SUMMARY ANSWER: Enhanced beta-catenin expression, reduced E-cadherin expression and glycogen accumulation in the tubal epithelia and hyperplasia in tubal arteries were found in ectopic tubal pregnancy, consistent with the effects induced by Wnt signaling and inflammation. WHAT IS KNOWN ALREADY: Chronic inflammation caused by infection can alter gene expression in the fallopian tube cells possibly leading to the development of ectopic pregnancy. Knockout mouse models have shown a relationship between Wnt/beta-catenin signaling and predisposition to tubal ectopic pregnancy. STUDY DESIGN, SIZE, DURATION: Women with ectopic tubal pregnancy (n = 18) were included in the case group, while women with chronic salpingitis (n = 13) and non-pregnant women undergoing sterilization procedures or salpingectomy for benign uterine disease (n = 10) were set as the controls. This study was performed between January 2012 and November 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ampullary segments of fallopian tubes were collected from patients. Tissues of tubal pregnancy were separated into implantation sites and non-implantation sites. Beta-catenin and E-cadherin expression were determined using immunohistological and immunofluorescence staining. Glycogen production was measured with periodic acid Schiff by staining. The diameter and wall thickness of tubal arteries were evaluated by histological analysis method. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistological staining revealed that beta-catenin protein expression was 100% positive in the ectopic pregnant and inflamed tubal tissues, and the staining intensity was significantly higher than in non-pregnant tubal tissues. In contrast, E-cadherin expression was reduced in ectopic pregnant fallopian tubes, possibly as a consequence of increased Wnt signaling. Moreover, glycogen accumulated in the tubal cells, and hyperplasia was observed in the tubal arteries with ectopic pregnancy, which is consistent with the effects induced by Wnt signaling and inflammation. All these changes could create the permissive environment that promotes embryos to ectopically implant into the fallopian tube. LIMITATIONS, REASONS FOR CAUTION: This finding requires a further confirmation about what activates Wnt signaling in ectopic tubal pregnancies. Also, it is generally recognized that Chlamydia infection is associated with ectopic pregnancy, and disturbs tubal epithelia via the Wnt signaling. However, the infection type in the samples used was salpingitis. WIDER IMPLICATIONS OF THE FINDINGS: A better understanding of the underlying mechanisms leading to ectopic pregnancies may contribute to our knowledge of the pathogenesis of tubal disorders and infertility and to the prevention of tubal ectopic pregnancy.

Changing roles of CD3 + CD8 low T cells in combating HIV-1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8 T) cells play critical roles in eradicating human immunodeficiency virus (HIV)-1 infection, but little is known about the effects of T cells expressing CD8 at low levels (CD8 low ) or high levels (CD8 high ) on HIV-1 replication inhibition after HIV-1 invasion into individual. METHODS: Nineteen patients who had been acutely infected with HIV-1 (AHI) and 20 patients with chronic infection (CHI) for ≥2 years were enrolled in this study to investigate the dynamics of the quantity, activation, and immune responses of CD3 + CD8 low T cells and their counterpart CD3 + CD8 high T cells at different stages of HIV-1 infection. RESULTS: Compared with healthy donors, CD3 + CD8 low T cells expanded in HIV-1-infected individuals at different stages of infection. As HIV-1 infection progressed, CD3 + CD8 low T cells gradually decreased. Simultaneously, CD3 + CD8 high T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed. The classical activation of CD3 + CD8 low T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage. Meanwhile, activated CD38 - HLA-DR + CD8 low T cells did not increase in the first month of AHI, and the number of these cells was inversely associated with viral load ( r = -0.664, P = 0.004) but positively associated with the CD4 T-cell count ( r = 0.586, P = 0.014). Increased programmed cell death protein 1 (PD-1) abundance on CD3 + CD8 low T cells was observed from the 1st month of AHI but did not continue to be enhanced, while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) abundance increase was observed in the 12th month of infection. Furthermore, increased PD-1 and TIGIT abundance on CD3 + CD8 low T cells was associated with a low CD4 T-cell count (PD-1: r = -0.456, P = 0.043; TIGIT: r = -0.488, P = 0.029) in CHI. Nonetheless, the nonincrease in PD-1 expression on classically activated CD3 + CD8 low T cells was inversely associated with HIV-1 viremia in the first month of AHI ( r = -0.578, P = 0.015). Notably, in the first month of AHI, few CD3 + CD8 low T cells, but comparable amounts of CD3 + CD8 high T cells, responded to Gag peptides. Then, weaker HIV-1-specific T-cell responses were induced in CD3 + CD8 low T cells than CD3 + CD8 high T cells at the 3rd and 12th months of AHI and in CHI. CONCLUSIONS: Our findings suggest that CD3 + CD8 low T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response. Subsequently, CD3 + CD8 low T-cell number decreased gradually as infection persisted, and their anti-HIV functions were inferior to those of CD3 + CD8 high T cells.

Weak SARS-CoV-2-specific responses of TIGIT-expressing CD8 + T cells in people living with HIV after a third dose of a SARS-CoV-2 inactivated vaccine.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS: TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.

Generation of human blastocyst-like structures from pluripotent stem cells.

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

A lncRNA-miRNA-mRNA network for human primed, naive and extended pluripotent stem cells.

Human pluripotent stem cells (hPSCs) represent a promising platform for studying embryonic development, and different states of pluripotency reflect the different stages of embryo development. Here, we successfully converted three in-house-derived primed hPSC lines (H10, H24, and iPS) to a naive state and an expanded pluripotent stem cell (EPS) state. Primed, naive and EPS cells displayed state-specific morphologies and expressed pluripotent markers. The expression of SSEA4 and TRA-1-60 was downregulated in the conversion process. The H3K27me3 expression level also decreased, indicating that global methylation was reduced and that the X chromosome started to reactivate. RNA-sequencing analysis results revealed that differentially expressed genes (DEGs) were significantly enriched in both naive hPSCs and EPS cells when compared to the primed state. However, imprinted gene expression barely changed before and after state reversion. Gene ontology (GO) analyses showed that the upregulated DEGs were mostly enriched in RNA processing, DNA replication and repair, and regulation of cell cycle process, while downregulated DEGs were related to extracellular adhesion and various tissue developmental processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that EPS cells were enriched in the PI3K-Akt and Wnt signaling pathways. Analysis of the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network between primed, naive hPSCs and EPS cells revealed that hsa-miR-424-5p, has-miR-16-5p, has-miR-27b-3p, has-miR-29c-3p, and KCNQ1OT1 were crucial nodes with high degrees of connectivity. Our work may represent new insight into the intrinsic molecular features of different hPSC states.

Transplanted mouse liver stem cells at different stages of differentiation ameliorate concanavalin A-induced acute liver injury by modulating Tregs and Th17 cells in mice.

Acute liver failure (ALF) is a disease with a considerably high mortality rate that still lacks a safe and effective treatment. Transplantation of liver stem cells (LSCs) has been considered to be a promising therapeutic alternative for ALF since LSCs have been shown to be involved in immunomodulation and functional reconstruction of the liver. Our present study evaluated and compared the protective effects of the two mouse LSC lines, YE and R5, as well as those of adult mouse hepatocyte (HC), on concanavalin A (ConA)-induced acute liver injury. YE and R5 cells were analyzed by microscopy, functional assays, and gene expression. We confirmed that YE and R5 cells were undifferentiated cells that had partial hepatocytic functions and a potential to differentiate into hepatocytes. YE cells has characteristics of LSCs at the early stage of differentiation, whereas the differentiation stage of R5 cells was later than that of YE cells. Subsequently, YE, R5, and HC cells were intraperitoneally transplanted into three groups of mice, followed by injection of ConA through the tail vein of each mouse at 12 h later. Blood tests, histology, flow cytometry, and quantitative PCR were then used to evaluate the therapeutic effects of the cell transplantations at 24 h after ConA injections. Compared with that of the ConA control group, YE, R5, and HC cells reduced the expression of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and alleviated the degree of hepatic necrosis. Moreover, transplantation of these cells induced more regulatory T cells (Tregs) and less T-helper 17 (Th17) cells in the liver and spleen, and also promoted the expression of forkhead box protein 3 (Foxp3) and interleukin (IL)-10; in contrast, these transplantations induced various degrees of inhibition in the expression of retinoic acid-related orphan receptor γt (RORγt), IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α). The protective effects of YE and R5 cells were significantly stronger than those of HC cells, and YE cells at the earlier differentiation stage than that of R5 cells exhibited the strongest protective effects. These results demonstrate that mouse LSCs at different stages of differentiation alleviate ConA-induced acute liver injury in mice by modulating Tregs, Th17 cells, and cytokine secretion.

Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs.

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Acute and sub-chronic toxicity of tetrandrine in intravenously exposed female BALB/c mice.

OBJECTIVE: To evaluate the acute and sub-chronic toxicity of intravenously administered tetrandrine (TET) in female BALB/c mice. METHODS: The median lethal dose (LD50) of intravenously administered TET was calculated in mice using Dixon's up-and-down method. In the acute toxicity study, mice were intravenously administered with TET at a single dose of 20, 100, 180, 260 and 340 mg/kg, respectively and were evaluated at 14 days after administration. In the sub-acute toxicity study, mice were intravenously administered various doses of TET (30, 90 and 150 mg/kg) each day for 14 consecutive days. Clinical symptoms, mortality, body weight, serum biochemistry, organ weight and histopathology were examined at the end of the experiment, as well as after a 1-week recovery period. RESULT: LD50 was found to be 444.67±35.76 mg/kg. In the acute toxicity study, no statistically signifificant differences in body weight, blood biochemistry, or organ histology were observed between the administration and control groups when mice were intravenously administered with single dose at 20, 100, 180, 260 and 340 mg/kg of TET (P >0.05). In the sub-acute toxicity study, no signifificant changes in body weight, biochemistry and organ histology were observed with up to 90 mg/kg of TET compared with the control group (P >0.05), however, in the 150 mg/kg administered group, TET induced transient toxicity to liver, lungs and kidneys, but withdrawal of TET can lead to reversal of the pathological conditions. CONCLUSIONS: The overall fifindings of this study indicate that TET is relatively non-toxic from a single dose of 20, 100, 180, 260 or 340 mg/kg, and that up to 90 mg/kg daily for 14 consecutive days can be considered a safe application dose.

Dexamethasone Exposure Accelerates Endochondral Ossification of Chick Embryos Via Angiogenesis.

Dexamethasone (Dex) is widely used to treat chronic inflammatory diseases in the clinic. Increasingly, there is more attention being paid to the side effect of Dex. In this study, we investigated the involvement and mechanism of Dex exposure in accelerating mineralization during long bone formation. We first determined that Dex exposure could accelerate long bone mineralization in vivo, but there was no apparent difference between control and Dex-treated in the phalanges model in vitro. Next, we established that Dex exposure promoted angiogenesis in the chick yolk sac membrane model. In addition, it increased human umbilical vein endothelial cell proliferation and migration in culture. We found that Dex could enhance angiogenesis when phalanges were cultured on chick chorioallantoic membrane and correspondingly increased the expression of angiogenesis-related genes in the phalanges. Furthermore, we also revealed that Dex exposure reduced the number of osteoblasts and simultaneously increased the number of osteocytes in ex vivo-cultured phalanges. Runx-2 and Col10α1 expressions were up-regulated by Dex exposure, indicating that Dex exposure accelerated the terminal differentiation of osteoblasts. Lastly, we demonstrated that MC3T3-E1 cells cultured in the presence of Dex accelerated their mineralization. In summary, we have shown that the ability of Dex to initiate angiogenesis is the mechanism that allows it to accelerate mineralization during long bone formation.

Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta.

It is widely accepted that diabetes mellitus impairs placental development, but the mechanism by which the disease operates to impair development remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in placental development in mice are mainly characterized by the changes of morphological structure of placenta. The alteration of differentiation-related gene expressions in trophoblast cells rather than cell proliferation/apoptosis is responsible for the phenotypes found in mouse placenta. Meanwhile, excess reactive oxygen species (ROS) production and activated nuclear factor erythroid2-related factor 2 (Nrf2) signalling were observed in the placenta of mice suffering from PGDM. Using BeWo cells, we also demonstrated that excess ROS was produced and Nrf2 signalling molecules were activated in settings characterized by a high concentration of glucose. More interestingly, differentiation-related gene expressions in trophoblast cells were altered when endogenous Nrf2 expression is manipulated by transfecting Nrf2-wt or Nrf2-shRNA. In addition, PGDM interferes with autophagy in both mouse placenta and BeWo cells, implying that autophagy is also involved, directly or indirectly, in PGDM-induced placental phenotypes. Therefore, we revealed that dysfunctional oxidative stress-activated Nrf2 signalling and autophagy are probably responsible for PGDM-induced defects in the placental development of mice. The mechanism was through the interference with differentiation-related gene expression in trophoblast cells.