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Actinomycin D (ActD) has been considered as one of the most effective and safe chemotherapeutic medications for treating a number of cancers. Although ActD has been used in the treatment of gynecological tumors and pediatric tumors for more than 50 years, the toxic effects of ActD on mammalian oocytes remain unknown. In this study, the influence of ActD on mouse and human oocyte maturation and the possible mechanisms were investigated. Notably, ActD inhibited oocyte maturation and arrested oocytes at the metaphase I (MI) stage in a dose-dependent manner. In addition, ActD arrested oocyte maturation when the oocytes were treated at different successive stages, including the germinal vesicle (GV), germinal vesicle breakdown, and MI stages. In ActD-treated oocytes, disordered chromosome condensation and irregular spindle assembly occurred, resulting in incomplete chromosome segregation and oocytes arresting at the MI phase; these results possibly occurred because ActD triggered the formation of reactive oxygen species, resulting in DNA damage and decreased ATP in mouse GV oocytes. Besides, in vivo treatment with ActD also inhibited mouse oocyte maturation. Similar effects were seen in human oocytes. Collectively, our results indicated that ActD exposure disrupted oocyte maturation by increasing DNA damage, which is a finding that might help with optimizing future methods for female fertility preservation before undergoing chemotherapy.Summary Sentence ActD blocks oocyte maturation at MI stage by inhibiting chromosome separation and spindle assembly, perhaps through inducing nuclear DNA damage and disruption of the mitochondrial function in oocytes.

Biomaterial surface chemistry engenders profound consequences on cell adhesion and the ultimate tissue response by adsorbing proteins from extracellular matrix, where vitronectin (Vn) is involved as one of the crucial mediator proteins. Deciphering the adsorption behaviors of Vn in molecular scale provides a useful account of how to design biomaterial surfaces. But the details of structural dynamics and consequential biological effect remain elusive. Herein, both experimental and computational approaches were applied to delineate the conformational and orientational evolution of Vn during adsorption onto self-assembled monolayers (SAMs) terminating with -COOH, -NH2, -CH3and -OH. To unravel the interplay between cell binding and the charge and wettability of material surface, somatomedin-B (SMB) domain of Vn holding the RGD cell-binding motif was employed in molecular dynamics (MD) simulations, with orientation initialized by Monte Carlo (MC) method. Experimental evidences including protein adsorption, cell adhesion and integrin gene expressions were thoroughly investigated. The adsorption of Vn on different surface chemistries showed very complex profiles. Cell adhesion was enabled on all Vn-adsorbed surfaces but with distinct mechanisms mostly determined by conformational change induced reorientation. Higher amount of Vn was observed on negatively charged surface (COOH) and hydrophobic surface (CH3). However, advantageous orientations defined by RGD loop conditions were only obtained on the charged surfaces (COOH and NH2). Specifically, COOH surface straightened up the Vn molecules and accumulated them into a higher density, whereas CH3surface squashed Vn and stacked them into higher density multilayer by tracking adsorption but with the RGD loops restrained. These findings may have a broad implication on the understanding of Vn functionality and would help develop new strategies for designing advanced biomaterials.

ABSTRACTDifferent strategies of ovarian stimulation are widely used in IVF to retrieve mature metaphase II (MII) oocytes for fertilization. On average, approximately 70% of recovered oocytes are mature, while personalized administration of hCG and/or GnRH agonist trigger and in vitromaturation (IVM) management can further improve the maturation rate. However, even under such conditions, a complete absence of oocyte maturation is still observed sporadically. The probable causes for such maturation-deficient (MD) oocytes – which arrest abnormally at metaphase I (MI) stage – are still under investigation. In the present study, using single-cell transcriptomic RNA sequencing (RNA-seq) and differential expression analysis, we showed that gene expression profiles were aberrant, and alternative splicing (AS) patterns were changed in MD oocytes when compared with normally mature (MN) oocytes. Gene ontology (GO) enrichment demonstrated that the differently expressed genes (DEGs) were mostly correlated with pre-mRNA splicing, RNA transportation, RNA processing, and mRNA regulation. Subsequently, analysis of AS events revealed that genes with altered AS patterns were primarily associated with metabolism and cell cycle. With these findings, we have demonstrated aberrant gene expression in complete maturation-deficient oocytes, and we propose that alterations in post-transcriptional regulation constitute a potential underlying mechanism governing oocyte maturation.

Plants are often simultaneously stressed by both UV radiation and phosphorus (P) deficiency in agricultural ecosystems. Coordinated responses and adaptations to these stressors are critical for plant growth, development, and survival. However, the underlying molecular response and adaptation mechanisms in plants are not fully understood. Here, we show that plants use a reciprocal antagonistic strategy in response to UV radiation and P deficiency. UV radiation inhibits P-starvation response processes and disrupts phosphate (Pi) homeostasis by suppressing the function of PHOSPHATE STARVATION RESPONSE PROTEINS (PHRs), the Pi central regulators. Conversely, P availability modulates plant UV tolerance and the expression of UV radiation response genes in a PHR-dependent manner. Therefore, reducing the P supply or increasing PHR activities can improve tolerance to UV stress in rice. Moreover, this antagonistic interaction is conserved across various plant species. Our meta-analysis showed that the increase in global UV radiation over the last 40 years may have reduced crop P-utilization efficiency worldwide. Our findings provide insights for optimizing P fertilizer management and breeding smart crops that are resilient to fluctuations in UV radiation and soil P levels.

Aromatic amino acid decarboxylases (AAADs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the decarboxylation of aromatic amino acid l-amino acids. In plants, apart from canonical AAADs that catalyze the straightforward decarboxylation reaction, other members of the AAAD family function as aromatic acetaldehyde synthases (AASs) and catalyze more complex decarboxylation-dependent oxidative deamination. The interconversion between a canonical AAAD and an AAS can be achieved by a single tyrosine-phenylalanine mutation in the large catalytic loop of the enzymes. In this work, we report implicit ligand sampling (ILS) calculations of the canonical l-tyrosine decarboxylase from Papaver somniferum(PsTyDC) that catalyzes l-tyrosine decarboxylation and its Y350F mutant that instead catalyzes the decarboxylation-dependent oxidative deamination of the same substrate. Through comparative analysis of the resulting three-dimensional (3D) O2free energy profiles, we evaluate the impact of the key tyrosine/phenylalanine mutation on oxygen accessibility to both the wild type and Y350F mutant of PsTyDC. Additionally, using molecular dynamics (MD) simulations of the l-tryptophan decarboxylase from Catharanthus roseus(CrTDC), we further investigate the dynamics of a large catalytic loop known to be indispensable to all AAADs. Results of our ILS and MD calculations shed new light on how key structural elements and loop conformational dynamics underlie the enzymatic functions of different members of the plant AAAD family.

The Daming Temple, built during 457–464 C.E., is one of the developing ancient temple heritage spaces located in Yangzhou city, P. R. China. Over the past 60 years, variation in visitors’ spatial perception along the tour routes in the temple has occurred. This research attempts to reveal the changes in visitors’ visual perception along the spatial sequences at 3 different times (i.e., 1962, 1973 and 2022). A quantitative GIS-based method, which includes analysing the distribution of visitors’ spatial preferences and spatial configuration, is proposed. Digital landscape tools and quantitative estimation methods are used, including mapping within Rhinoceros software, the kernel density estimation (KDE) method within ArcGIS software and spatial syntax analysis within DepthMap software. Extracted geodata from 500 photographs of the heritage space taken by volunteer visitors are analysed within the GIS environment. Values of the mean depth (MD) at both levels of visibility and accessibility are calculated within the visibility graph analysis (VGA) model. Comparisons between the visual preferences of the visitors and the spatial configuration along the spatial sequence are conducted. The results indicate that the spatial sequence has a significant impact on visitors’ visual preferences and tour routes. The phenomenon of spatial sequence among dynamic temporal variations and the effects of narrative spaces along the spatial sequence are highlighted and explained, which reveal the relationship between visitors’ geospatial preference and the spatial configuration of the temple. Some suggestions are put forwards for further studies on the revitalisation and management of East Asian ancient temple heritage spaces.

The use of antimicrobial peptides (AMPs)-functionalized titanium implants is an efficient method for preventing bacterial infection. However, the attachment of AMPs to the surface of titanium implants remains a challenge. In this study, a “clickable” titanium surface was developed by using a silane coupling agent with an alkynyl group. The antimicrobial titanium implant was then constructed through the reaction between the “clickable” surface and azido-AMPs (PEG-HHC36:N3-PEG12-KRWWKWWRR) via click chemistry of Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Such an antimicrobial titanium implant, with an AMP density of 897.4 ± 67.3 ng/cm2(2.5 ± 0.2 molecules per nm2) on the surface, exhibited good and stable antimicrobial activity, inhibited 90.2% of Staphylococcus aureusand 88.1% of Escherichia coliafter 2.5 h of incubation, and even inhibited 69.5% of Staphylococcus aureusafter 4 days of degradation. The CCK-8 assay indicated that the antimicrobial titanium implant exhibited negligible cytotoxicity to mouse bone mesenchymal stem cells. In vivoassay illustrated that this implant could kill 78.8% of Staphylococcus aureusafter 7 days. This method has great potential for the preparation of antimicrobial titanium implants and the prevention of infections in the clinic.

The orientation and conformation of adhesive proteins after adsorption play a central role in cell-binding bioactivity. Fibronectin (Fn) holds two peptide sequences that favor cell adhesion: the Arg–Gly–Asp (RGD) loop on the tenth type-III domain (Fn-III10) and the Pro–His–Ser–Arg–Asn (PHSRN) synergy site on the ninth type-III domain (Fn-III9). Herein, adsorption of Fn fragments (Fn-III10and Fn-III9–10) on self-assembled monolayers (SAMs) carrying various functional groups (−COOH, −NH2, −CH3, and −OH) was investigated by the Monte Carlo method and molecular dynamics simulation in order to understand its mediation effect on cell adhesion. The results demonstrated that Fn-III9could enhance the stiffness of the Fn molecule and further fix the adsorption orientation. The RGD site of the Fn fragment appeared to be deactivated on hydrophobic surfaces (CH3-SAM) because of the binding of adjacent nonpolar residues on surfaces, whereas charged surfaces (COOH-SAM and NH2-SAM) and hydrophilic surfaces (OH-SAM) were conducive to the formation of cell-binding-favorable orientation for Fn fragments. The cell adhesion capability of Fn fragments was highly improved on positively charged surfaces (NH2-SAM) and hydrophilic surfaces because of the advantageous steric structure and orientation of both RGD and PHSRN sites. This work provides an insight into the interplay at the atomic scale between protein adsorption and surface chemistry for designing biologically responsive substrate surfaces.

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.

Osteopontin (OPN) is a key mediator of cell interactions with biomaterials. However, few studies have been dedicated to studying cell adhesion on OPN-adsorbed substrates with controlled charge and wettability. Here, amino–carboxyl (NH2/COOH) and hydroxyl–methyl (OH/CH3) mixed self-assembled monolayers (SAMs) of varying charges and wettability, respectively, were used as controllable model surfaces to study OPN adsorption and subsequent mesenchymal stem cell (MSC) adhesion. The amount of OPN adsorbed onto the NH2/COOH mixed SAMs appeared to monotonically depend on the surface charge, whereas only a moderately hydrophilic surface was conducive to OPN adsorption on OH/CH3mixed SAMs. The results correlated well with cell spreading on OPN-coated surfaces in a serum-free medium culture. In addition, the OH/CH3mixed SAMs with moderate wettability tended to promote β1, β3, αvand α5integrins, indicating that wettability may guide cell adhesion by mediating the integrins signaling pathway. This work will have reference value for designing biologically responsive substrate surfaces.

Yeast cells have controllable biosorption on metallic ions during metabolism. However, few studies were dedicated to using yeast-regulated biomimetic mineralization process to control the strontium-doped positions in calcium phosphate microcapsules. In this study, the yeast cells were allowed to pre-adsorb strontium ions metabolically and then served as sacrificing template for the precipitation and calcination of mineral shell. The pre-adsorption enabled the microorganism to enrich of strontium ions into the inner part of the microcapsules, which ensured a slow-release profile of the trace element from the microcapsule. The co-culture with human marrow stromal cells showed that gene expressions of alkaline phosphatase and Collagen-I were promoted. The promotion of osteogenic differentiation was further confirmed in the 3D culture of cell-material complexes. The strategy using living microorganism as 'smart doping apparatus' to control incorporation of trace element into calcium phosphate paved a pathway to new functional materials for hard tissue regeneration.

Oligo(ethylene glycol) (-OEG) and amino (-NH 2 ) mixed self-assembled monolayers (SAMs) were employed as model substrates to investigate the effect of charge density on the fate of mesenchymal stem cells (MSCs) and osteopontin (OPN) adsorption. We found that all surfaces presenting -NH 2 groups favored cell responses regardless of the surface charge. Meanwhile, OPN adsorption could remain stable on the mixed SAMs over a certain range of charge densities. Our work provides some insights into cell responses and protein adsorption to surface charge. [ABSTRACT FROM AUTHOR]

Precise control of stem cells, such as human bone marrow-derived mesenchymal stem cells (hMSCs), is critical for the development of effective cellular therapies for tissue engineering and regeneration medicine. Emerging evidence suggests that several miRNAs act as key regulators of diverse biological processes, including differentiation of various stem cells. In this study, we have described a delivery system for miR-29b using PEI-capped gold nanoparticles (AuNPs) to synergistically promote osteoblastic differentiation. The cell proliferation assay revealed that AuNPs and AuNPs/miR-29b exert negligible cytotoxicity to hMSCs and MC3T3-E1 cells. With the assistance of AuNPs as a delivery vector, miR-29b could efficiently enter the cytoplasm and regulate osteogenesis. AuNPs/miR-29b more effectively promoted osteoblast differentiation and mineralization through induced the expression of osteogenesis genes (RUNX2, OPN, OCN, ALP) for the long-term, compared to the widely used commercial transfection reagent, Lipofectamine. With no obvious cytotoxicity, PEI-capped AuNPs showed great potential as an adequate miRNA vector for osteogenesis differentiation. Interestingly, we observed loading of AuNPs as well as AuNPs/miR-29b into the lumen of the endoplasmic reticulum (ER). Our findings collectively suggest that AuNPs, together with miR-29b, exert a synergistic promotory effect on osteogenic differentiation of hMSCs and MC3T3-E1 cells.

Fibronectin (Fn) is significant to the performance of biomaterials, and the chemistry of biomaterial surface play important roles in Fn adsorption and subsequent cell behavior. However, the “molecular scale” mechanism is still unclear. Herein, we combined experimental strategies with molecular simulations to solve this problem. We prepared self-assembled monolayers with varying chemistries, i.e., SAMs-CH3, SAMs-NH2, SAMs-COOH and SAMs-OH, and characterized Fn adsorption and cell behaviors on them. Next, Monte Carlo method and all-atom molecular dynamics simulations were employed to reveal the orientation/conformation of Fn on surfaces. We found that SAMs-CH3strongly adsorbed Fn via hydrophobic interactions, but show poor bioactivity as the low exposure of RGD/PHSRN motifs and the deformation of Fn. SAMs-NH2and SAMs-COOH could adsorb Fn efficiently via vdW interactions, electrostatic interactions, hydrogen bonds and salt bridges. Fn exhibited excellent bioactivity for cell adhesion, proliferation and osteogenic differentiation as high exposure of bioactive motifs on SAMs-NH2, or as the activation of other inferior cell-binding motifs on SAMs-COOH. SAMs-OH showed poor Fn adsorption as the water film. However, the adsorbed Fn displayed non-negligible bioactivity due to high exposure of PHSRN motif and large degree of protein flexibility. We believe that the revealed mechanism presents great potential to rationally design Fn-activating biomaterials.

Chemokine receptor type 4 (CXCR4) has been suggested to regulate cell migration and invasion in human somatic cells. However, its role in human oocytes and embryos has not been investigated directly. Here we show that CXCR4mRNA was initially expressed at the 4-cell stage, and its expression gradually increased until the blastocyst stage, whereas its protein was detectable only after the 8-cell stage. In addition, CXCR4 mRNA and protein were expressed in the inner cell mass (ICM) and trophectoderm (TE) cell of the blastocyst. Furthermore, we collected embryos from women whose embryos had undergone successful implantation (SI) and those whose embryos had failed implantation (FI) in their fresh cycles. TE cells from the FI group had reduced CXCR4mRNA expression relative to those from the SI group but not in the ICM. Through ICM replacement, we constructed mouse blastocysts in which Cxcr4was specifically knocked down in TE cells to simulate the CXCR4expression profile of human blastocysts from the FI group. In this case, we found that the implantation rate significantly decreased after transfer of reconstructed embryos. Bioinformatic analysis indicated that CXCR4can induce cell apoptosis and migration mediated by Rho signaling. This hypothesis was confirmed by invasion and migration experiments, using a human trophoblast cell line. The present study is the first to explore the characteristics of CXCR4expression using human oocytes and embryos and suggests that CXCR4is required upstream of TE cell apoptosis and migration. CXCR4expression is a potential biomarker to predict implantation competence during assisted reproductive technologies.

Trophoblast stem cells differentiate into different trophoblast cell populations that are indispensable for successful pregnancy through interactions with the maternal uterine decidua. Rho GTPases play an important role in the regulation of trophoblast stem cell (TSC) self-renewal and differentiation; however, the role of Rho GDP-dissociation inhibitors (Rho GDIs) remains unclear. Here we report that overexpression of Rho GDIalpha resulted in rapid apoptosis of TSCs, while its knockdown promoted proliferation. Moreover, Rho GDIalpha knockdown also enhanced TSC invasion. Collectively, these results establish a potential mechanism whereby TSCs can balance growth and apoptosis, and thus ensure normal fetal development.

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