Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

STUDY QUESTION: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? SUMMARY ANSWER: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. WHAT IS KNOWN ALREADY: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-µM gallic acid on their maturation rate. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/ß-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. LARGE SCALE DATA: Raw data from this study can be accessed through GSE158539. LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Quantitative structure determination of adsorbed formate and surface hydroxyls on Fe3O4(001).

Using the chemically specific techniques of normal incidence X-ray standing waves and photoelectron diffraction, we have investigated the dissociative adsorption of formic acid on the Fe3O4(001) surface, specifically probing the local structures of both the adsorbed formate and resulting surface hydroxyl. Using model independent direct methods, we reinforce the observations of a previous surface X-ray diffraction study that the formate molecule adsorbs with both oxygens atop octahedrally coordinated surface Fe cations and that ∼60% of the formate is adsorbed in the so called tet site. We additionally determine, for the first time, that the surface hydroxyl species are found at the so called int site. This confirms previous DFT predictions and reinforces the pivotal role the surface hydroxyl plays in lifting the subsurface cation vacancy termination of the Fe3O4(001) surface.

Surfactant-Mediated Epitaxial Growth of Single-Layer Graphene in an Unconventional Orientation on SiC.

We report the use of a surfactant molecule during the epitaxy of graphene on SiC(0001) that leads to the growth in an unconventional orientation, namely R0° rotation with respect to the SiC lattice. It yields a very high-quality single-layer graphene with a uniform orientation with respect to the substrate, on the wafer scale. We find an increased quality and homogeneity compared to the approach based on the use of a preoriented template to induce the unconventional orientation. Using spot profile analysis low-energy electron diffraction, angle-resolved photoelectron spectroscopy, and the normal incidence x-ray standing wave technique, we assess the crystalline quality and coverage of the graphene layer. Combined with the presence of a covalently bound graphene layer in the conventional orientation underneath, our surfactant-mediated growth offers an ideal platform to prepare epitaxial twisted bilayer graphene via intercalation.

Probing structural changes upon carbon monoxide coordination to single metal adatoms.

In this work, the adsorption height of Ag adatoms on the Fe3O4(001) surface after exposure to CO was determined using normal incidence x-ray standing waves. The Ag adatoms bound to CO (Ag1 CO) are found to be pulled out of the surface to an adsorption height of 1.15 Å ± 0.08 Å, compared to the previously measured height of 0.96 Å ± 0.03 Å for bare Ag adatoms and clusters. Utilizing DFT+vdW+U calculations with the substrate unit cell dimension fixed to the experimental value, the predicted adsorption height for Ag1 CO was 1.16 Å, in remarkably good agreement with the experimental results.

Charge Transfer and Built-in Electric Fields between a Crystalline Oxide and Silicon.

We report charge transfer and built-in electric fields across the epitaxial SrNb_{x}Ti_{1-x}O_{3-δ}/Si(001) interface. Electrical transport measurements indicate the formation of a hole gas in the Si and the presence of built-in fields. Hard x-ray photoelectron measurements reveal pronounced asymmetries in core-level spectra that arise from these built-in fields. Theoretical analysis of core-level spectra enables built-in fields and the resulting band bending to be spatially mapped across the heterojunction. The demonstration of tunable charge transfer, built-in fields, and the spatial mapping of the latter, lays the groundwork for the development of electrically coupled, functional heterojunctions.

Direct measurement of Ni incorporation into Fe3O4(001).

The normal incidence X-ray standing wave (NIXSW) technique has been used to follow the evolution of the adsorption geometry of Ni adatoms on the Fe3O4(001)-(√2 × âˆš2)R45° surface as a function of temperature. Two primary surface region sites are identified: a bulk-continuation tetrahedral site and a sub-surface octahedral site, the latter site being preferred at higher annealing temperatures. The ease of incorporation is linked to the presence of subsurface cation vacancies in the (√2 × âˆš2)R45° reconstruction and is consistent with the preference for octahedral coordination observed in the spinel compound NiFe2O4.

Probing the interplay between geometric and electronic structure in a two-dimensional K-TCNQ charge transfer network.

Scanning tunnelling microscopy (STM), low energy electron diffraction (LEED), ultraviolet and soft X-ray photoelectron spectroscopy (UPS and SXPS) have been used to characterise the formation of a coadsorption phase of TCNQ and K on Ag(111), while the normal incident X-ray standing waves (NIXSW) technique has been used to obtain quantitative structural information. STM and LEED show that an ordered incommensurate phase is formed in which the K atoms are surrounded by four TCNQ molecules in a 'windmill' motif, characteristic of other metal/TCNQ phases, in which the nominal TCNQ : K stoichiometry is 1 : 1. UPS and SXPS data indicate the TCNQ is in a negatively-charged state. NIXSW results show that the carbon core of the TCNQ is essentially planar at a height above the Ag(111) surface closely similar to that found without coadsorbed K. In the presence of TCNQ the height of the K ions above the surface is significantly larger than on clean Ag(111), and the ions occupy sites above 'holes' in the TCNQ network. NIXSW data also show that the N atoms in the molecules must occupy sites with at least two different heights above the surface, which can be reconciled by a tilt or twist of the TCNQ molecules, broadly similar to the geometry that occurs in bulk TCNQ/K crystals.

Structural and Electronic Properties of Nitrogen-Doped Graphene.

We investigate the structural and electronic properties of nitrogen-doped epitaxial monolayer graphene and quasifreestanding monolayer graphene on 6H-SiC(0001) by the normal incidence x-ray standing wave technique and by angle-resolved photoelectron spectroscopy supported by density functional theory simulations. With the location of various nitrogen species uniquely identified, we observe that for the same doping procedure, the graphene support, consisting of substrate and interface, strongly influences the structural as well as the electronic properties of the resulting doped graphene layer. Compared to epitaxial graphene, quasifreestanding graphene is found to contain fewer nitrogen dopants. However, this lack of dopants is compensated by the proximity of nitrogen atoms at the interface that yield a similar number of charge carriers in graphene.

Approaching truly freestanding graphene: the structure of hydrogen-intercalated graphene on 6H-SiC(0001).

We measure the adsorption height of hydrogen-intercalated quasifreestanding monolayer graphene on the (0001) face of 6H silicon carbide by the normal incidence x-ray standing wave technique. A density functional calculation for the full (6√3×6√3)-R30° unit cell, based on a van der Waals corrected exchange correlation functional, finds a purely physisorptive adsorption height in excellent agreement with experiments, a very low buckling of the graphene layer, a very homogeneous electron density at the interface, and the lowest known adsorption energy per atom for graphene on any substrate. A structural comparison to other graphenes suggests that hydrogen-intercalated graphene on 6H-SiC(0001) approaches ideal graphene.

Human Brain Organoid Research and Applications: Where and How to Meet Legal Challenges?

An ethical and legal framework is needed to regulate the rapidly developing human brain organoid research field properly. However, considering the legal issues involved in human brain organoid research remains underdeveloped and scattered. This article reviews the legal issues of human brain organoid research, grouping them into the following five broad themes: (1) consciousness, (2) legal status, (3) consent, (4) ownership, and (5) transplantation. The issues in each topic include both the urgent (e.g., appropriate forms of consent) and the speculative (e.g., protection of conscious human brain organoids). Therefore, we have attempted to be as explicit as possible about the timescale within which each issue will be realized and to prioritize each. Examining these issues has revealed legal issues specific to human brain organoid research and issues common to research in other fields. Further discussion of human brain organoid research from a legal perspective is needed in the future, considering discussions in related fields.

Donor-Acceptor Co-Adsorption Ratio Controls the Structure and Electronic Properties of Two-Dimensional Alkali-Organic Networks on Ag(100).

The results are presented of a detailed combined experimental and theoretical investigation of the influence of coadsorbed electron-donating alkali atoms and the prototypical electron acceptor molecule 7,7,8,8-tetracyanoquinodimethane (TCNQ) on the Ag(100) surface. Several coadsorption phases were characterized by scanning tunneling microscopy, low-energy electron diffraction, and soft X-ray photoelectron spectroscopy. Quantitative structural data were obtained using normal-incidence X-ray standing wave (NIXSW) measurements and compared with the results of density functional theory (DFT) calculations using several different methods of dispersion correction. Generally, good agreement between theory and experiment was achieved for the quantitative structures, albeit with the prediction of the alkali atom heights being challenging for some methods. The adsorption structures depend sensitively on the interplay of molecule-metal charge transfer and long-range dispersion forces, which are controlled by the composition ratio between alkali atoms and TCNQ. The large difference in atomic size between K and Cs has negligible effects on stability, whereas increasing the ratio of K/TCNQ from 1:4 to 1:1 leads to a weakening of molecule-metal interaction strength in favor of stronger ionic bonds within the two-dimensional alkali-organic network. A strong dependence of the work function on the alkali donor-TCNQ acceptor coadsorption ratio is predicted.

Does F4TCNQ Adsorption on Cu(111) Form a 2D-MOF?

The results of a quantitative experimental structural investigation of the adsorption phases formed by 2,3,5,6-tetrafluoro-7,7',8,8'-tetracyanoquinodimethane (F4TCNQ) on Cu(111) are reported. A particular objective was to establish whether Cu adatoms are incorporated into the molecular overlayer. A combination of normal incidence X-ray standing waves, low-energy electron diffraction, scanning tunneling microscopy, and X-ray photoelectron spectroscopy measurements, complemented by dispersion-inclusive density functional theory calculations, demonstrates that F4TCNQ on Cu(111) does cause Cu adatoms to be incorporated into the overlayer to form a two-dimensional metal-organic framework (2D-MOF). This conclusion is shown to be consistent with the behavior of F4TCNQ adsorption on other coinage metal surfaces, despite an earlier report concluding that the adsorption structure on Cu(111) is consistent with the absence of any substrate reconstruction.

Exome sequencing identifies novel compound heterozygous mutations of IL-10 receptor 1 in neonatal-onset Crohn's disease.

Inflammatory bowel disease is well recognized for a strong genetic involvement in its pathogenesis. Homozygous mutations in interleukin-10 receptor 1 (IL-10R1) identified by linkage analysis were shown to be involved in this disorder. However, the underlying molecular mechanism and the causal nature of the mutations in the disease process remain to be clarified. In this study, using whole exome sequencing, we identified novel compound heterozygous missense mutations in the extracellular domain of IL-10R1 in a Crohn's disease patient from a non-consanguineous family. These mutations did not affect IL-10R1 expression, nor IL-10 binding. However, they abrogated IL-10R1 phosphorylation induced by IL-10, therefore leading to impaired STAT3 activation and suppression of inflammatory responses. After reconstitution with wild-type IL-10R1, the patient cells showed fully restored IL-10R function including IL-10-induced STAT3 activation and expression of suppressor of cytokine signaling 3. Thus, our results demonstrated that the mutations in IL-10R1 extracellular domain impair IL-10R1 activation rather than IL-10 binding, indicating these residues are important in IL-10 signal transduction through IL-10R1. The reconstitution data also confirmed the causality of the IL-10R1 mutations.

Association of CD247 with systemic lupus erythematosus in Asian populations.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. CD247 (CD3Z, TCRZ) plays a vital role in antigen recognition and signal transduction in antigen-specific immune responses, and is known to be involved in SLE pathogenesis. Weak disease association was reported for genetic variants in this gene in Caucasian studies for SLE, Crohn's disease and systemic sclerosis, but its role as a genetic risk factor was never firmly established. METHODS: In this study, using a collection of 612 SLE patients and 2193 controls of Chinese ethnicity living in Hong Kong in a genome-wide study, single nucleotide polymorphisms (SNPs) in and around CD247 were identified as being associated with SLE. The two most significant SNPs in this locus were selected for further replication using TaqMan genotyping assay in 3339 Asian patients from Hong Kong, Mainland China, and Thailand, as well as 4737 ethnically and geographically matched controls. RESULTS: The association of CD247 with SLE in Asian populations was confirmed (rs704853: odds ratio [OR] = 0. 81, p = 2.47 × 10(-7); rs858543: OR = 1.10, p = 0.0048). Patient-only analysis suggested that rs704853 is also linked to oral ulcers, hematologic disorders and anti-double-stranded DNA (dsDNA) antibody production. CONCLUSION: A significant association between variants in CD247 and SLE was demonstrated in Asian populations. Understanding the involvement of CD247 in SLE may shed new light on disease mechanisms and development of new treatment paradigms.

Initial corrosion observed on the atomic scale.

Corrosion destroys more than three per cent of the world's GDP. Recently, the electrochemical decomposition of metal alloys has been more productively harnessed to produce porous materials with diverse technological potential. High-resolution insight into structure formation during electrocorrosion is a prerequisite for an atomistic understanding and control of such electrochemical surface processes. Here we report atomic-scale observations of the initial stages of corrosion of a Cu3Au111 single crystal alloy within a sulphuric acid solution. We monitor, by in situ X-ray diffraction with picometre-scale resolution, the structure and chemical composition of the electrolyte/alloy interface as the material decomposes. We reveal the microscopic structural changes associated with a general passivation phenomenon of which the origin has been hitherto unclear. We observe the formation of a gold-enriched single-crystal layer that is two to three monolayers thick, and has an unexpected inverted (CBA-) stacking sequence. At higher potentials, we find that this protective passivation layer dewets and pure gold islands are formed; such structures form the templates for the growth of nanoporous metals. Our experiments are carried out on a model single-crystal system. However, the insights should equally apply within a crystalline grain of an associated polycrystalline electrode fabricated from many other alloys exhibiting a large difference in the standard potential of their constituents, such as stainless steel (see ref. 5 for example) or alloys used for marine applications, such as CuZn or CuAl.

Two missense variants in UHRF1BP1 are independently associated with systemic lupus erythematosus in Hong Kong Chinese.

UHRF1BP1 encodes a highly conserved protein with unknown function. Previously, a coding variant in this gene was found to be associated with systemic lupus erythematosus (SLE) in populations of European ancestry (rs11755393, R454Q, P=2.22 x 10⁻8, odds ratio=1.17). In this study, by a combination of genome-wide study and replication involving a total of 1230 patients and 3144 controls, we confirmed the association of this coding variant to SLE in Hong Kong Chinese. We also identified another coding variant in this gene that independently contributes to SLE susceptibility (rs13205210, M1098T, P=4.44 x 10⁻9, odds ratio=1.49). Cross-population confirmation establishes the involvement of this locus in SLE and indicates that distinct alleles are contributing to disease susceptibility.

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer.

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples. METHODS: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs). RESULTS: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system. CONCLUSION: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

Structural investigation of Au(111)/butylthiolate adsorption phases.

The structures of the high-coverage ('standing-up') and low-coverage ('lying-down') phases of butylthiolate on Au(111) have been investigated by a range of experimental methods. Normal incidence X-ray standing waves, photoelectron diffraction and near-edge X-ray absorption fine structure results all identify the local S headgroup site as atop a surface Au atom in a bulk continuation site for both high- and low-coverage phases. Low energy electron diffraction shows the low-coverage phase to have a (12 x radical 3)rect. surface mesh with glide-line symmetry (pmg space group), the long dimension of this mesh being approximately four times the length of the butylthiolate molecule. A structural model is proposed for this phase based on two different enantiomers of an Au-adatom-dithiolate species that is consistent with these results and with recent finding for propylthiolate on this surface using low-temperature scanning tunnelling microscopy (O. Voznyy, J. J. Dubowski, J. T. Yates Jr. and P. Maksymovych, J. Am Chem. Soc., 2009, 131, 12989).

Microalgae for "Healthy" Foods-Possibilities and Challenges.

Microalgae have the potential to become a novel source of bioactive molecules, especially for those who might wish to enhance the nutritional and functional quality of foods. Spirulina, one of the most popular microalgae, has been described by the World Health Organization as one of the greatest superfoods on earth serving as an example of the potential of microalgae. This review provides background on current and future uses of microalgae in the human diet, lists the most common species of microalgae used to this end, and describes some production methods used in research and industrial production and recovery. The review also discusses some of the difficulties so far encountered such as low productivities and recovery rates, as well as challenges in the production of compounds of interest. Many scientists and engineers in research centers around the globe are currently dedicated to solve these problems as the various capabilities of microalgae have caught the attention of the energy, environmental, and agricultural industries, we propose that the food industry should as well evaluate the potential of microalgae as a novel source of "health promoting" compounds.

Validation of the inverted adsorption structure for free-base tetraphenyl porphyrin on Cu(111).

Utilising normal incidence X-ray standing waves we rigourously scrutinise the "inverted model" as the adsorption structure of free-base tetraphenyl porphyrin on Cu(111). We demonstrate that the iminic N atoms are anchored at near-bridge adsorption sites on the surface displaced laterally by 1.1 ± 0.2 Å in excellent agreement with previously published calculations.