IGF2BP2 Maintains Retinal Pigment Epithelium Homeostasis by Stabilizing PAX6 and OTX2.

Purpose: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE. Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, ß-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography. Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo. Conclusions: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.

Initiating PeriCBD to probe perinatal influences on neurodevelopment during 3-10 years in China.

Adverse perinatal factors can interfere with the normal development of the brain, potentially resulting in long-term effects on the comprehensive development of children. Presently, the understanding of cognitive and neurodevelopmental processes under conditions of adverse perinatal factors is substantially limited. There is a critical need for an open resource that integrates various perinatal factors with the development of the brain and mental health to facilitate a deeper understanding of these developmental trajectories. In this Data Descriptor, we introduce a multicenter database containing information on perinatal factors that can potentially influence children's brain-mind development, namely, periCBD, that combines neuroimaging and behavioural phenotypes with perinatal factors at county/region/central district hospitals. PeriCBD was designed to establish a platform for the investigation of individual differences in brain-mind development associated with perinatal factors among children aged 3-10 years. Ultimately, our goal is to help understand how different adverse perinatal factors specifically impact cognitive development and neurodevelopment. Herein, we provide a systematic overview of the data acquisition/cleaning/quality control/sharing, processes of periCBD.

Estimation of surface free energy at microstructured surface to investigate intermediate wetting state for partial wetting model.

While partial wetting at nano-/microstructured surfaces can be described using the intermediate wetting state between the Cassie-Baxter and Wenzel states, the limitations of the partial wetting model remain unclear. In this study, we performed surface free energy analysis at a microstructured Si-water interface from both theoretical and experimental viewpoints. We experimentally measured the water contact angle on microstructured Si surfaces with square holes and compared the measured values with theoretical predictions. Furthermore, the surface free energy was analyzed using the effective wetting area estimated from the measured contact angle and electrochemical impedance spectroscopy results. We verified the validity of the partial wetting model for fabricated Si surfaces with a hole aperture a less than 230 µm and a hole height h of 12 µm, and for a < 400 µm, h = 40 µm. The model was found to be applicable to microstructured Si surfaces with a/h < 10.

Cis-regulatory chromatin loops analysis identifies GRHL3 as a master regulator of surface epithelium commitment.

Understanding the regulatory network of cell fate acquisition remains a major challenge. Using the induction of surface epithelium (SE) from human embryonic stem cells as a paradigm, we show that the dynamic changes in morphology-related genes (MRGs) closely correspond to SE fate transitions. The marked remodeling of cytoskeleton indicates the initiation of SE differentiation. By integrating promoter interactions, epigenomic features, and transcriptome, we delineate an SE-specific cis-regulatory network and identify grainyhead-like 3 (GRHL3) as an initiation factor sufficient to drive SE commitment. Mechanically, GRHL3 primes the SE chromatin accessibility landscape and activates SE-initiating gene expression. In addition, the evaluation of GRHL3-mediated promoter interactions unveils a positive feedback loop of GRHL3 and bone morphogenetic protein 4 on SE fate decisions. Our work proposes a concept that MRGs could be used to identify cell fate transitions and provides insights into regulatory principles of SE lineage development and stem cell-based regenerative medicine.

Physiological and comparative transcriptome analyses reveal the mechanisms underlying waterlogging tolerance in a rapeseed anthocyanin-more mutant.

BACKGROUND: Rapeseed (Brassica napus) is the second largest oil crop worldwide. It is widely used in food, energy production and the chemical industry, as well as being an ornamental. Consequently, it has a large economic value and developmental potential. Waterlogging is an important abiotic stress that restricts plant growth and development. However, little is known about the molecular mechanisms underlying waterlogging tolerance in B. napus. RESULTS: In the present study, the physiological changes and transcriptomes of germination-stage rapeseed in response to waterlogging stress were investigated in the B. napus cultivar 'Zhongshuang 11' (ZS11) and its anthocyanin-more (am) mutant, which was identified in our previous study. The mutant showed stronger waterlogging tolerance compared with ZS11, and waterlogging stress significantly increased anthocyanin, soluble sugar and malondialdehyde contents and decreased chlorophyll contents in the mutant after 12 days of waterlogging. An RNA-seq analysis identified 1370 and 2336 differently expressed genes (DEGs) responding to waterlogging stress in ZS11 and am, respectively. An enrichment analysis revealed that the DEGs in ZS11 were predominately involved in carbohydrate metabolism, whereas those in the am mutant were particularly enriched in plant hormone signal transduction and response to endogenous stimulation. In total, 299 DEGs were identified as anthocyanin biosynthesis-related structural genes (24) and regulatory genes encoding transcription factors (275), which may explain the increased anthocyanin content in the am mutant. A total of 110 genes clustered in the plant hormone signal transduction pathway were also identified as DEGs, including 70 involved in auxin and ethylene signal transduction that were significantly changed in the mutant. Furthermore, the expression levels of 16 DEGs with putative roles in anthocyanin accumulation and biotic/abiotic stress responses were validated by quantitative real-time PCR as being consistent with the transcriptome profiles. CONCLUSION: This study provides new insights into the molecular mechanisms of increased anthocyanin contents in rapeseed in response to waterlogging stress, which should be useful for reducing the damage caused by waterlogging stress and for further breeding new rapeseed varieties with high waterlogging tolerance.

Wolfberry-derived zeaxanthin dipalmitate delays retinal degeneration in a mouse model of retinitis pigmentosa through modulating STAT3, CCL2 and MAPK pathways.

Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry-derived carotenoid, has anti-inflammatory and anti-oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi-electrode-array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the visual behavior of rd10 mice and delayed the degeneration of retinal photoreceptors. It also improved the light responses of photoreceptors, bipolar cells and retinal ganglion cells. The expression of genes that are involved in inflammation, apoptosis and oxidative stress were up-regulated in rd10 mice, and were reduced by ZD. ZD further reduced the activation of two key factors, signal transducer and activator of transcription 3 and chemokine (C-C motif) ligand 2, down-regulated the expression of the inflammatory factor GFAP, and inhibited extracellular signal regulated protein kinases and P38, but not the JNK pathways. In conclusion, ZD delays the degeneration of the rd10 retina both morphologically and functionally. Its anti-inflammatory function is mediated primarily through the signal transducer and activator of transcription 3, chemokine (C-C motif) ligand 2 and MAPK pathways. Thus, ZD may serve as a potential clinical candidate to treat RP.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Generating an MEIS1 homozygous knockout human embryonic stem cell line using the CRISPR/Cas9 system.

Myeloid ecotropic viral integration site 1 (MEIS1) plays an essential role in the development of several embryonic organs, such as the central nervous system and eyes. To further investigate the role of MEIS1 in embryonic development, herein, we generated a MEIS1 homozygous knockout human embryonic stem cell (hESC) line using the CRISPR/Cas9 genome-editing technology. We believe that this cell line will be a good resource for exploring the function of the MEIS1 gene in embryonic development in vitro. Furthermore, the gene-knockout method reported in this study is efficient and labor-saving, which may provide an effective strategy for hESC gene deletion.

Mechanism and Regulation of Silique Dehiscence, Which Affects Oil Seed Production.

Silique dehiscence is an important physiological process during natural growth that enables mature seeds to be released from plants, which then undergo reproduction and ensure the survival of future generations. In agricultural production, the time and degree of silique dehiscence affect the harvest time and processing of crops. Premature silique dehiscence leads to seeds being shed before harvest, resulting in substantial reductions to yields. Conversely, late silique dehiscence is not conducive to harvesting, and grain weight and oil content will be reduced due to the respiratory needs of seeds. In this paper, the mechanisms and regulation of silique dehiscence, and its application in agricultural production is reviewed.

D609 protects retinal pigmented epithelium as a potential therapy for age-related macular degeneration.

Accumulated oxidative damage may lead to irreversible retinal pigmented epithelium (RPE) cell death, which is considered to be the primary cause of dry age-related macular degeneration (AMD), leading to blindness in the elderly. However, an effective therapy for this disease is lacking. Here, we described a robust high-content screening procedure with a library of 814 protective compounds and found that D609 strongly protected RPE cells from sodium iodate (SI)-induced oxidative cell death and prolonged their healthy survival. D609 effectively attenuated excessive reactive oxygen species (ROS) and prevented severe mitochondrial loss due to oxidative stress in the RPE cells. Surprisingly, the potent antioxidative effects of D609 were not achieved through its own reducibility but were primarily dependent on its ability to increase the expression of metallothionein. The injection of this small water-soluble molecule also showed an explicit protective effect of the RPE layer in an SI-induced AMD mouse model. These findings suggested that D609 could serve as a novel antioxidative protector of RPE cells both in vitro and in vivo and unveiled a novel antioxidative mechanism of D609, which may ultimately have clinical applications for the treatment of AMD.

The influence of relative humidity and ground material on indoor walking-induced particle resuspension.

The resuspension of indoor particulate matters caused by people indoor walking could affect indoor air quality and human health. Therefore, it is particularly important to study the resuspension rules of the particulate matters in different indoor environments. The influence of the ground material and the relative humidity on resuspension of the particulate matters were investigated under three kinds of ground materials and three different relative humidity. Results showed that different relative humidity and different ground materials had different effects on the mass concentration of the particulate matters. In addition, different particle sizes had diverse influence on the mass concentration. Compared with low-level loop pile carpet and shaggy carpet, hardwood floor was more conductive to human health which was less likely to cause the resuspension of the particulate matters. At the same time, relative humidity had a great influence on the resuspension of the particulate matters. With the increase of relative humidity, the resuspension rate of fine particulate matters decreased.

Action potential response of human induced-pluripotent stem cell derived cardiomyocytes to the 28 CiPA compounds: A non-core site data report of the CiPA study.

We used the whole-cell current clamp technique to examine the response of our in-house hiPSC-CMs to the 28 CiPA-selected compounds, aiming to compare field potential via MEA from core-sites and action potential via current clamp measurement. Our blinded study showed that all seven high-risk test compounds, including bepridil, caused early afterdepolarizations (EADs) at mid-high and/or high concentration(s). All hERG channel blockers in the mid-risk category prolonged APD30 and APD90 at mid-high, and then led to EADs at their respective high concentrations; while chlorpromazine, clarithromycin and risperidone showed little effects. In addition, ranolazine was the only low-risk test compound to prolong APD30 and APD90 at mid-high, and then produce EADs at high concentration. In conclusion, our results generally agreed with data from all core-sites of the CiPA consortium using the MEA method. Moreover, our assay can successfully detect pro-arrhythmic risk of drug candidates such as bepridil with superior sensitivity.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Bradycardiac Effects Caused by Co-Administration of Sofosbuvir and Amiodarone.

Co-administration of sofosbuvir, an anti-hepatitis C virus medication, and antiarrhythmic amiodarone causes symptomatic severe bradycardia in patients and animal models. However, in a few in vitro studies, the combination of sofosbuvir and amiodarone resulted in tachycardiac effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This discrepancy may be attributable to the use of immature hiPSC-CMs in the in vitro studies. To address this, we evaluated the ability of our in-house hiPSC-CMs to assess the interactions between sofosbuvir and amiodarone in vitro. We performed whole-cell patch recordings on hiPSC-CMs to examine the cardiac effect of sofosbuvir and amiodarone, alone or in combination. We found that sofosbuvir and amiodarone caused bradycardiac effects (the beating rate decreased to 75% of the vehicle control, P < 0.001) on our hiPSC-CMs when applied in combination, but they had no significant effect when applied alone. Furthermore, the bradycardiac effect was membrane potential dependent: it increased with depolarization. This raised the possibility that the bradycardiac effects in vivo may originate in nodal cells, which have a more depolarized resting membrane potential compared with ventricular cells. The bradycardiac effects of sofosbuvir plus amiodarone in vitro are consistent with the clinical phenotype and suggest that our hiPSC-CMs may serve as a useful tool in assessing cardiac safety during drug discovery and development process.

Tetramethylpyrazine nitrone protects retinal ganglion cells against N-methyl-d-aspartate-induced excitotoxicity.

Adding a free radical-scavenging nitrone moiety on tetramethylpyrazine, we have previously synthesized a chemical named 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (tetramethylpyrazine nitrone, or TBN) and proved its neuroprotective effect but with limited understanding of its mechanism. Here we ask if TBN protects retinal ganglion cells (RGCs) against excitotoxicity induced by NMDA and explore the underlying mechanism. NMDA was intravitreally injected to induce RGC injury in rats, followed by daily intraperitoneal administrations of TBN. Measurements of TBN concentration at different times after intraperitoneal administration showed that more than 200 µM TBN reached the aqueous humor quickly. Then RGCs' survival was evaluated by quantifying Brn3-positive cells, and retinal functions were examined by electroretinogram and visual behaviors. TBN significantly increased the survival of RGCs after NMDA insult, recovered the amplitude of photopic negative responses to flash, and restored the visual behavior. Furthermore, TBN inhibited the apoptotic process, as indicated by the elevated ratios of cleaved caspase-3/caspase-3 and of Bax/Bcl-2, and decreased the level of reactive oxygen species. Moreover, TBN reduced RGC's calcium overload induced by NMDA or by KCl. Whole-cell patch recording from RGCs further showed that TBN slightly but significantly inhibited L-type calcium channels, but had little effect on T-type calcium channel or NMDA-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)-induced current. Thus our data indicate that TBN alleviates NMDA-elicited injury of rat RGCs both morphologically and functionally, possibly by inhibiting the L-type calcium channel thus reducing Ca2+ overload and by directly scavenging free radicals. Therefore, TBN may be a novel candidate for treating excitotoxicity-related visual disorders such as glaucoma.

Frizzled3 Shapes the Development of Retinal Rod Bipolar Cells.

PURPOSE: Frizzled3 (Fzd3), a member of the core planar cell polarity (PCP) family in mammals, contributes to visual development by guiding axonal projections of some retinal ganglion cells. However, its other functions in the maturation of the visual system, especially the retina, remain elusive. The present study explores the role of Fzd3 in retinal development by focusing on rod bipolar cells (RBCs). METHODS: Frizzled3 was conditionally removed from the retina of Isl1-Cre;Fzd3f/- mice using the Cre-loxP system. Electroretinograms (ERGs) were performed to measure the light response of retinas. Frizzled3 expression was monitored by ß-galactosidase (ß-gal) staining and anti-ß-gal immunostaining. Immunofluorescence was used to examine cellular distribution and morphology during development, and electron microscopy was applied to visualize the dendritic invaginations of RBCs. RESULTS: Electroretinograms showed decreased b-wave amplitudes, and lower b- to a-wave ratios in Isl1-Cre;Fzd3f/- than in control (Isl1-Cre;Fzd3f/+) mice. In RBCs, where Fzd3 was expressed and inactivated, the planar organization, shape, and orientation of somas were disrupted. From P10, dendrites of these RBCs displayed reduced arborization with mistargeting. Furthermore, their dendritic invaginations into rod terminals were suppressed, and the density of rod ribbons in the OPL was reduced. CONCLUSIONS: Frizzled3 is required to shape the pattern of RBC somas and dendrites, and the structural and functional connectivity between rods and RBCs. Our results highlight novel functions for Fzd3 in regulating retinal development.

PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. However, little is known about its presence or effects in human adipose-derived stem cells (hADSCs). In this study, the expression of PACAP type I receptor (PAC1R) was first confirmed in hADSCs. Maxadilan, a specific agonist of PAC1R, could increase hADSC proliferation as determined by Cell Counting Kit-8 and cell cycle analysis and promote migration as shown in wound-healing assays. Maxadilan also showed anti-apoptotic activity in hADSCs against serum withdrawal-induced apoptosis based on Annexin V/propidium iodide analysis and mitochondrial membrane potential assays. The anti-apoptotic effects of maxadilan correlated with the down-regulation of Cleaved Caspase 3 and Caspase 9 as well as up-regulation of Bcl-2. The chemical neural differentiation potential could be enhanced by maxadilan as indicated through quantitative PCR, Western blot and cell morphology analysis. Moreover, cytokine neural redifferentiation of hADSCs treated with maxadilan acquired stronger neuron-like functions with higher voltage-dependent tetrodotoxin-sensitive sodium currents, higher outward potassium currents and partial electrical impulses as determined using whole-cell patch clamp recordings. Maxadilan up-regulated the Wnt/ß-catenin signalling pathway associated with dimer-dependent activity of PAC1R, promoting cell viability that was inhibited by XAV939, and it also activated the protein kinase A (PKA) signalling pathway associated with ligand-dependent activity of PAC1R, enhancing cell viability and neural differentiation potential that was inhibited by H-89. In summary, these results demonstrated that PAC1R is present in hADSCs, and maxadilan could enhance hADSC viability and neural differentiation potential in neural differentiation medium.

[Effect of Staphylococcus aureus on the expressions of TLR2, IL-1ß, TNF-α and NF-κB in Bcap-37 cells].

OBJECTIVE: To observe the effects of Staphylococcus aureus (S.aureus) on the expressions of Toll-like receptor 2 (TLR2), proinflammatory cytokines (IL-1ß, TNF-α) and nuclear factor κB(NF-κB) in human breast cancer Bcap-37 cells in vitro. METHODS: At the different time points after Bcap-37 cells were challenged with S.aureus, the expressions of TLR2 mRNA and protein were respectively detected using RT-PCR and flow cytometry, the concentrations of IL-1ß and TNF-α were examined by ELISA and NF-κB level by Western blotting. RESULTS: The mRNA and protein levels of TLR2 gradually increased in Bcap-37 cells since the stimulations of S.aureus and then dropped after the levels peaked at 4 and 6 hours, respectively (P<0.05). Meanwhile, the expressions of IL-1ß and TNF-α was significantly elevated when Bcap-37 cells were exposed to S.aureus, and reached the maximal levels at 12 and 16 hours, respectively (P<0.05), and then IL-1ß went down. Expression of NF-κB rose in a time-dependent manner after stimulated by S.aureus up to the maximum at 12 hours, and declined after 14 hours. CONCLUSION: In Bcap-37 cells, S.aureus exposure could up-regulate the expression of TLR2 at both mRNA and protein levels, increase the production of IL-1ß and TNF-α, and stimulate the expression of NF-κB.

[The efficacy of enhancing murine zona pellucid 3 DNA vaccine immunized through intranasal vaccination by IL-15].

AIM: To observe the efficacy of enhancing murine zona pellucid 3(mZP3) DNA vaccine immunized through intranasal vaccination by IL-15 as adjuvant and chitosan as delivery vactor. METHODS: C57BL/6 mice were delivered with chitosan-loaded mZP3 DNA vaccine or IL-15+pcD-mZP3 vaccine by intranasal route, and to detect specific IgG in serum and sIgA in vaginal fluid by indirect ELISA, and to perform fertility experiment that all female C57BL/6 mice were mated(one male for each female) with normal males of similar age. To do histology analysis of immunized mice ovary and lungs for morphology of ovary. RESULTS: The results of ELISA showed IL-15 can promote IgG antibody level of immunized mice, and the fertility rate was decreased to some degrees. CONCLUSION: IL-15 and chitosan can enhance the antibody levels induced by mZP3 DNA vaccine, and reduce murine fertility rate to a certain extent.