Detecting of Barely Visible Impact Damage on Carbon Fiber Reinforced Polymer Using Diffusion Ultrasonic Improved by Time-Frequency Domain Disturbance Sensitive Zone.

Based on the decorrelation calculation of diffusion ultrasound in time-frequency domain, this paper discusses the repeatability and potential significance of Disturbance Sensitive Zone (DSZ) in time-frequency domain. The experimental study of Barely Visible Impact Damage (BVID) on Carbon Fiber Reinforced Polymer (CFRP) is carried out. The decorrelation coefficients of time, frequency, and time-frequency domains and DSZ are calculated and compared. It has been observed that the sensitivity of the scattered wave disturbance caused by impact damage is non-uniformly distributed in both the time and frequency domains. This is evident from the non-uniform distribution of the decorrelation coefficient in time-domain and frequency-domain decorrelation calculations. Further, the decorrelation calculation in the time-frequency domain can show the distribution of the sensitivity of the scattered wave disturbance in the time domain and frequency domain. The decorrelation coefficients in time, frequency, and time-frequency domains increase monotonically with the number of impacts. In addition, in the time-frequency domain decorrelation calculation results, stable and repetitive DSZ are observed, which means that the specific frequency component of the scattered wave is extremely sensitive to the damage evolution of the impact region at a specific time. Finally, the DSZ obtained from the first 15 impacts is used to improve the decorrelation calculation in the 16-th to 20-th impact. The results show that the increment rate of the improved decorrelation coefficient is 10.22%. This study reveals that the diffusion ultrasonic decorrelation calculation improved by DSZ makes it feasible to evaluate early-stage damage caused by BVID.

The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.

The inhibitory effect of IFN-γ on protease HTRA1 expression in rheumatoid arthritis.

The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ-deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ-deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.

Lipopolysaccharide increases the incidence of collagen-induced arthritis in mice through induction of protease HTRA-1 expression.

OBJECTIVE: The protease HTRA-1 is closely associated with rheumatoid arthritis (RA). The molecular mechanisms that control HTRA-1 expression are currently unknown. This study was undertaken to determine the regulatory role of Toll-like receptors (TLRs) on HTRA-1 expression in mice with collagen-induced arthritis (CIA) and in synovial cells from RA patients. METHODS: HTRA-1 messenger RNA and protein production in mouse fibroblasts, mouse macrophages, and freshly isolated RA patient synovial cells treated with TLR ligands were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Arthritis incidence and severity were determined using clinical scores and histopathologic analysis. Involvement of HTRA-1 in lipopolysaccharide (LPS)-increased arthritis incidence and severity in mice was determined using anti-HTRA-1 monoclonal antibody. The signal pathways involved in HTRA-1 expression were accessed by specific inhibitors, RNA interference, dual-luciferase reporter, and chromatin immunoprecipitation methods. RESULTS: LPS and tenascin-C, but not the other TLR ligands tested, strongly induced HTRA-1 expression. LPS significantly increased HTRA-1 expression in the joint tissue as well as arthritis incidence and severity in mice with CIA. Blocking HTRA-1 by antibody significantly decreased LPS-promoted CIA severity. Inhibiting NF-κB significantly decreased LPS-induced HTRA-1 expression in mouse and human cells. Dual-luciferase reporter assay and ChIP analysis showed that p65 directly binds to HTRA-1 promoter (amino acid 347). CONCLUSION: Our findings indicate that TLR-4 activation increases HTRA-1 expression through the NF-κB pathway in fibroblasts and macrophages. HTRA-1 expression is involved in the enhancing effects of LPS on CIA. This study offers new insights into the regulation of HTRA-1 expression via LPS/TLR-4 and the role of HTRA-1 in RA pathogenesis.

Cytoplasmatic Localization of Six1 in Male Testis and Spermatogonial Stem Cells.

Sine oculis homeobox 1 (Six1) is an important factor for embryonic development and carcinoma malignancy. However, the localization of Six1 varies due to protein size and cell types in different organs. In this study, we focus on the expression and localization of Six1 in male reproductive organ via bioinformatics analysis and immunofluorescent detection. The potential interacted proteins with Six1 were also predicted by protein-protein interactions (PPIs) and Enrichr analysis. Bioinformatic data from The Cancer Genome Atlas and Genotype-Tissue Expression project databases showed that SIX1 was highly expressed in normal human testis, but low expressed in the testicular germ cell tumor sample. Human Protein Atlas examination verified that SIX1 level was higher in normal than that in cancer samples. The sub-localization of SIX1 in different reproductive tissues varies but specifically in the cytoplasm and membrane in testicular cells. In mouse cells, single cell RNA-sequencing data analysis indicated that Six1 expression level was higher in mouse spermatogonial stem cells (mSSCs) and differentiating spermatogonial than in other somatic cells. Immunofluorescence staining showed the cytoplasmic localization of Six1 in mouse testis and mSSCs. Further PPIs and Enrichr examination showed the potential interaction of Six1 with bone morphogenetic protein 4 (Bmp4) and catenin Beta-1 (CtnnB1) and stem cell signal pathways. Cytoplasmic localization of Six1 in male testis and mSSCs was probably associated with stem cell related proteins Bmp4 and CtnnB1 for stem cell development.

Grhl2 deficiency impairs otic development and hearing ability in a zebrafish model of the progressive dominant hearing loss DFNA28.

Congenital and progressive hearing impairment is a common distressing disease. The progressive dominant hearing loss DFNA28 in human is associated with a frameshift mutation of Grainyhead-like 2 (GRHL2) but its etiology and mechanism remain unknown. Here we report a zebrafish grhl2b(T086) mutant line in which grhl2b expression is interrupted by an insertion of a Tol2 transposon element. The mutants exhibit enlarged otocysts, smaller or eliminated otoliths, malformed semicircular canals, insensitiveness to sound stimulation and imbalanced swimming motion. Since grainyhead-like family members can regulate epithelial adhesion, we examined the expression of some genes encoding junction proteins in mutants. We show that the expression of claudin b (cldnb) and epcam is abolished or dramatically reduced and apical junctional complexes are abnormal in otic epithelial cells of mutant embryos. Co-injection of cldnb and epcam mRNA could largely rescue the mutant phenotype. Injection of human wild-type GRHL2 mRNA but not the mutant GRHL2 mRNA derived from DFNA28 patients into grhl2b(T086) mutant embryos could rescue the inner-ear defects. Furthermore, we demonstrate that Grhl2b directly binds to the enhancers and promotes the expression of cldnb and epcam. Thus, this work reveals an evolutionarily conserved function of Grhl2 in otic development and provides a fish model for further studying mechanisms of Grhl2-related hearing loss.

Effect of High-Entropy Spinel Ferrite (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4 Doping Concentration on the Ferroelectric Properties of PVDF-Based Polymers.

Polyvinylidene fluoride (PVDF)-based dielectric energy storage materials have the advantages of environmental friendliness, high power density, high operating voltage, flexibility, and being light weight, and have enormous research value in the energy, aerospace, environmental protection, and medical fields. To investigate the magnetic field and the effect of high-entropy spinel ferrite (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4 nanofibers (NFs) on the structural, dielectric, and energy storage properties of PVDF-based polymers, (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4 NFs were prepared via the use of electrostatic spinning methods, and (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4/PVDF composite films were prepared via the use of the coating method. The effects of a 0.8 T parallel magnetic field, induced for 3 min, and the content of high-entropy spinel ferrite on the relevant electrical properties of the composite films are discussed. The experimental results show that, structurally, the magnetic field treatment causes the originally agglomerated nanofibers in the PVDF polymer matrix to form a linear fiber chain with different fiber chains parallel to each other along the magnetic field direction. Electrically, the introduction of the magnetic field enhanced the interfacial polarization, and the (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4/PVDF composite film with a doping concentration of 10 vol% had a maximum dielectric constant of 13.9, as well as a low energy loss of 0.068. The high-entropy spinel ferrite (Mn0.2Zr0.2Cu0.2Ca0.2Ni0.2)Fe2O4 NFs and the magnetic field influenced the phase composition of the PVDF-based polymer. The α-phase and γ-phase of the cohybrid-phase B1 vol% composite films had a maximum discharge energy density of 4.85 J/cm3 and a charge/discharge efficiency of 43%.

Magnetic Field Effects on the Structure, Dielectric and Energy Storage Properties of High-Entropy Spinel Ferrite (La0.14Ce0.14Mn0.14Zr0.14Cu0.14Ca0.14Ni0.14)Fe2O4/PVDF Nanocomposites.

Energy depletion is one of the significant threats to global development. To increase the usability of clean energy, the energy storage performance of dielectric materials must be urgently enhanced. Semicrystalline ferroelectric polymer (PVDF) is the most promising candidate for the next generation of flexible dielectric materials thanks to its relatively high energy storage density. In this work, high-entropy spinel ferrite (La0.14Ce0.14Mn0.14Zr0.14Cu0.14Ca0.14Ni0.14Fe2O4) nanofibers (abbreviated 7FO NFs) were prepared by the sol-gel and electrostatic spinning methods, then blended with PVDF to prepare composite films using the coating method. A magnetic field was used to control the orientation distribution of the high-entropy spinel nanofibers in the PVDF matrix. We investigated the effects of the applied magnetic field and the content of high-entropy spinel ferrite on the structure, dielectric, and energy storage properties of the PVDF substrate films. The 3 vol% 7FO/PVDF film treated in a 0.8 T magnetic field for 3 min exhibited a good overall performance. The maximum discharge energy density was 6.23 J/cm3 at 275 kV/mm and the efficiency was 58% with 51% ß-phase content. In addition, the dielectric constant and dielectric loss were 13.3 and 0.035, respectively, at a frequency of 1 kHz.

Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta.

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition expected in reef ecosystems over the next several decades. Recognition of this reef degradation has prompted a range of novel research- and restoration-based active interventions. Ex situ aquaculture can play a supporting role through the establishment of robust coral culture protocols (e.g., to improve health and reproduction in long-term experiments) and through the provision of a consistent broodstock supply (e.g., for use in restoration projects). Here, simple techniques for the feeding and ex situ culture of brooding scleractinian corals are outlined using the common and well-studied coral, Pocillopora acuta, as an example. To demonstrate this approach, coral colonies were exposed to different temperatures (24 °C vs. 28 °C) and feeding treatments (fed vs. unfed) and the reproductive output and timing, as well as the feasibility of feeding Artemia nauplii to corals at both temperatures, was compared. Reproductive output showed high variation across colonies, with differing trends observed between the temperature treatments; at 24 °C, fed colonies produced more larvae than unfed colonies, but the opposite was found in colonies cultured at 28 °C. All colonies reproduced before the full moon, and differences in reproductive timing were only found between unfed colonies in the 28 °C treatment and fed colonies in the 24 °C treatment (mean lunar day of reproduction ± standard deviation: 6.5 ± 2.5 and 11.1 ± 2.6, respectively). The coral colonies fed efficiently on Artemia nauplii at both treatment temperatures. These proposed feeding and culture techniques focus on the reduction of coral stress and the promotion of reproductive longevity in a cost-effective and customizable manner, with versatile applicability in both flow-through and recirculating aquaculture systems.

Global identification of SMAD2 target genes reveals a role for multiple co-regulatory factors in zebrafish early gastrulas.

Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIP-chip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/ß-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos.

[The Nodal regulated dusp4 inhibits mesendoderm formation during zebrafish gastrulation].

MAP kinase phosphatase-2 (MKP-2/DUSP4), a dual specificity protein phosphatase with tyrosine/serine/ threonine phosphatase activity, is associated with cellular proliferation and differentiation, but its functions during embryo development are unclear. To study the developmental function of dusp4, we first examined the spatiotemporal expression pattern of this gene during zebrafish embryonic development by whole mount in situ hybridization. We found that dusp4 was maternally expressed since its transcripts were present from the one-cell to the 256-cell stages. At early gastrulation stages, dusp4 transcripts specifically distributed at margin region, where the mesendodermal cells were located. Further-more, Nodal signal was crucial for dusp4 expression. The expression of dusp4 was obviously increased in Nodal ligand overexpressed embryos, while its expression was almost lost in the Nodal signal-deficient MZoep mutants. In addition, dusp4 MO was also designed to knock down its expression in embryos. The mesendoderm formation was significantly in-creased in dusp4 morphants, but not obviously changed in dusp4 overexpressed embryos, suggesting that dusp4 is necessary, but not sufficient for the inhibitory of mesendoderm induction. Thus, our results indicate that Nodal regulated dusp4 plays a repressive role in mesendoderm induction.

Effects of Cosmetic Emulsions on the Surface Properties of Mongolian Hair.

In the light of clean beauty and sustainability requirements emerging in the personal care market, the urgent need for the replacement of silicones in hair conditioners-with comparable performance and customer experience-has been highlighted in the industry. In this context, the goal of the present study was to investigate the physical effects of different silicone-free conditioner formulations on Mongolian hair after damage due to bleaching and compare the results to property changes induced by a classical silicone-containing formulation. To that end, the morphology, structure, and composition of strands and individual fibers of this hair type were characterized before and after bleaching by means of optical microscopy, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). It is shown that oxidative bleaching causes significant damage to the native hair surface, leading to local depletion or even large-area removal of the outer hydrophobic lipid layer. This results in enhanced wettability of the bleached hair by water (as confirmed by contact angle measurements) and is accompanied by an undesired loss of hair gloss and softness. Upon treatment with suitable cosmetic emulsions, the natural hydrophobicity of intact Mongolian hair can be partially or fully restored, with silicone-free formulations having effects similar to those of established silicone-containing products. The successive influence of bleaching and conditioning was further monitored using inverse gas chromatography (iGC), a technique that probes changes in surface energetics and polarity over an ensemble of an entire hair strand through interactions with specific molecules at the solid/gas interface. The resulting data mirror the macroscopic behavior of the bleached/conditioned hair and provide a quantitative scale for measuring damage and repair effects. Most notably, the effect of bleaching and subsequent conditioning on the haptic perception of hair strands could also be quantified with the aid of a biomimetic measurement system, which identifies increased friction (both tactile and sliding) as the major cause for the strawy feel of bleached hair and indicates successful relubrication after treatment with suitable conditioner formulations. Finally, the different physical properties determined for native, bleached, and reconditioned Mongolian hair are found to be reflected in application-oriented tests, namely in vitro measurements of wet and dry combing work. Overall, the data collected in this work shed novel light on the surface properties of Mongolian hair and highlight that effective hair conditioning after damage can be achieved without silicones in advanced cosmetic emulsions based on octyldodecyl myristate and glyceryl oleate.

Ni2P nanowire arrays grown on Ni foam as an efficient monolithic cocatalyst for visible light dye-sensitized H2 evolution.

Nanostructured H2 evolution cocatalysts are able to promote charge separation and thus enhance the efficiency of the photocatalytic H2 evolution reaction (HER). However, the nanosized cocatalyst particles are easily detached from the surfaces of semiconductors or severely aggregated in reaction systems, which not only greatly reduces the photocatalytic HER efficiency during long-term use but also greatly increases the difficulty of recovery. Moreover, powdery cocatalysts have poor compatibility with the scale-up photoelectrochemical devices. In this paper, a monolithic cocatalyst is developed by controllably growing Ni2P nanowire arrays on Ni foam substrate (Ni2P NWAs/NF) via a direct vapor-phase phosphorization method. The grown Ni2P NWAs with high specific surface areas can not only offer ample active sites for the HER, but also serve as scaffolds for anchoring dye molecules to maximize the light utilization efficiency, which endows the Ni2P NWAs/NF monolithic cocatalyst with excellent HER activity. When sensitized with Erythrosin B (ErB) in triethanolamine (TEOA) solution, the turnover number (TON) of H2 evolution based on ErB reaches 9.7 in 5 h under visible light. Notably, the good structural integrity and inherent magnetism enable the Ni2P NWAs/NF to be easily separated from the reaction solution and excellent catalytic H2 evolution stability over a 45 h cycling reaction. This work presents a new strategy of fabricating monolithic cocatalysts with controllable microstructure and functionalities as well as high activity, durability, and device-compatibility for large-scale solar energy conversion applications.

A Review of Radio Frequency Identification Sensing Systems for Structural Health Monitoring.

Structural health monitoring (SHM) plays a critical role in ensuring the safety of large-scale structures during their operational lifespan, such as pipelines, railways and buildings. In the last few years, radio frequency identification (RFID) combined with sensors has attracted increasing interest in SHM for the advantages of being low cost, passive and maintenance-free. Numerous scientific papers have demonstrated the great potential of RFID sensing technology in SHM, e.g., RFID vibration and crack sensing systems. Although considerable progress has been made in RFID-based SHM, there are still numerous scientific challenges to be addressed, for example, multi-parameters detection and the low sampling rate of RFID sensing systems. This paper aims to promote the application of SHM based on RFID from laboratory testing or modelling to large-scale realistic structures. First, based on the analysis of the fundamentals of the RFID sensing system, various topologies that transform RFID into passive wireless sensors are analyzed with their working mechanism and novel applications in SHM. Then, the technical challenges and solutions are summarized based on the in-depth analysis. Lastly, future directions about printable flexible sensor tags and structural health prognostics are suggested. The detailed discussion will be instructive to promote the application of RFID in SHM.

Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models.

Type 2 diabetes mellitus is one of the most prevalent metabolic diseases presenting with systemic pathologies, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contributing to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we perform STRT-seq to examine the transcriptome of diabetic patients' testes at single-cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure are dramatically impaired. Among these deregulate pathways, the Apelin (APLN) peptide/Apelin-receptor (APJ) axis is hyper-activated in diabetic patients' testes. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppress the production of carnitine and repress the expression of cell adhesion genes in Sertoli cells. Together, these effects culminate in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorate the BTB damage and, importantly, improve functional spermatogenesis in diabetic db/db mice. We also translate and validate these findings in cultured human testes. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.

Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis.

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

Cell-fate transition and determination analysis of mouse male germ cells throughout development.

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells' identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

The chromatin accessibility landscape reveals distinct transcriptional regulation in the induction of human primordial germ cell-like cells from pluripotent stem cells.

In vitro induction of human primordial germ cell-like cells (hPGCLCs) provides an ideal platform to recapitulate hPGC development. However, the detailed molecular mechanisms regulating the induction of hPGCLCs remain largely uncharacterized. Here, we profiled the chromatin accessibility and transcriptome dynamics throughout the process of hPGCLC induction. Genetic ablation of SOX15 indicated the crucial roles of SOX15 in the maintenance of hPGCLCs. Mechanistically, SOX15 exerted its roles via suppressing somatic gene expression and sustaining latent pluripotency. Notably, ETV5, a downstream regulator of SOX15, was also uncovered to be essential for hPGCLC maintenance. Finally, a stepwise switch of OCT4/SOX2, OCT4/SOX17, and OCT4/SOX15 binding motifs were found to be enriched in closed-to-open regions of human embryonic stem cells, and early- and late-stage hPGCLCs, respectively. Collectively, our data characterized the chromatin accessibility and transcriptome landscapes throughout hPGCLC induction and defined the SOX15-mediated regulatory networks underlying this process.

Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale.

BACKGROUND: The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. METHODS: The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. RESULTS: In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. DISCUSSION: The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.

Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis.

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.