Three-Dimensional Printing Quality Inspection Based on Transfer Learning with Convolutional Neural Networks.

Fused deposition modeling (FDM) is a form of additive manufacturing where three-dimensional (3D) models are created by depositing melted thermoplastic polymer filaments in layers. Although FDM is a mature process, defects can occur during printing. Therefore, an image-based quality inspection method for 3D-printed objects of varying geometries was developed in this study. Transfer learning with pretrained models, which were used as feature extractors, was combined with ensemble learning, and the resulting model combinations were used to inspect the quality of FDM-printed objects. Model combinations with VGG16 and VGG19 had the highest accuracy in most situations. Furthermore, the classification accuracies of these model combinations were not significantly affected by differences in color. In summary, the combination of transfer learning with ensemble learning is an effective method for inspecting the quality of 3D-printed objects. It reduces time and material wastage and improves 3D printing quality.

Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis Pathway.

Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, podocalyxin-like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self-renewal, protein expression of c-MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown-mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single-cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human-mouse chimeras, laminin and collagen signaling-related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.

Structural interaction between DISC1 and ATF4 underlying transcriptional and synaptic dysregulation in an iPSC model of mental disorders.

Psychiatric disorders are a collection of heterogeneous mental disorders arising from a contribution of genetic and environmental insults, many of which molecularly converge on transcriptional dysregulation, resulting in altered synaptic functions. The underlying mechanisms linking the genetic lesion and functional phenotypes remain largely unknown. Patient iPSC-derived neurons with a rare frameshift DISC1 (Disrupted-in-schizophrenia 1) mutation have previously been shown to exhibit aberrant gene expression and deficits in synaptic functions. How DISC1 regulates gene expression is largely unknown. Here we show that Activating Transcription Factor 4 (ATF4), a DISC1 binding partner, is more abundant in the nucleus of DISC1 mutant human neurons and exhibits enhanced binding to a collection of dysregulated genes. Functionally, overexpressing ATF4 in control neurons recapitulates deficits seen in DISC1 mutant neurons, whereas transcriptional and synaptic deficits are rescued in DISC1 mutant neurons with CRISPR-mediated heterozygous ATF4 knockout. By solving the high-resolution atomic structure of the DISC1-ATF4 complex, we show that mechanistically, the mutation of DISC1 disrupts normal DISC1-ATF4 interaction, and results in excessive ATF4 binding to DNA targets and deregulated gene expression. Together, our study identifies the molecular and structural basis of an DISC1-ATF4 interaction underlying transcriptional and synaptic dysregulation in an iPSC model of mental disorders.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Plasminogen/thrombomodulin signaling enhances VEGF expression to promote cutaneous wound healing.

Plasminogen (Plg) and thrombomodulin (TM) are glycoproteins well known for fibrinolytic and anticoagulant functions, respectively. Both Plg and TM are essential for wound healing. However, their significance during the reparative process was separately demonstrated in previous studies. Here, we investigate the interaction between Plg and epithelial TM and its effect on wound healing. Characterization of the wound margin revealed that Plg and TM were simultaneously upregulated at the early stage of wound healing and the two molecules were bound together. In vitro, TM silencing or knockout in keratinocytes inhibited Plg activation. Plg treatment enhanced keratinocyte proliferation and migration, and these actions were abolished by TM antibody. Keratinocyte-expressed vascular endothelial growth factor (VEGF), which presented a dose-response relationship with Plg treatment, can be suppressed by TM silencing. Moreover, treatment with VEGF antibody inhibited Plg-enhanced keratinocyte proliferation and wound recovery. In vivo, TM antibody treatment and keratinocyte-specific TM knockout can impede Plg-enhanced wound healing in mice. In high-glucose environments, Plg-enhanced VEGF expression and wound healing were suppressed due at least in part to downregulation of keratinocyte-expressed TM. Taken together, our findings suggest that activation of Plg/TM signaling may hold therapeutic potential for chronic wounds in diabetic or non-diabetic individuals. KEY MESSAGES: Plg binds to TM in cutaneous wound healing. TM facilitates the activation of Plg to Plm in keratinocytes. Epithelial TM regulates Plg-enhanced wound healing through VEGF expression.

Zika-Virus-Encoded NS2A Disrupts Mammalian Cortical Neurogenesis by Degrading Adherens Junction Proteins.

Zika virus (ZIKV) directly infects neural progenitors and impairs their proliferation. How ZIKV interacts with the host molecular machinery to impact neurogenesis in vivo is not well understood. Here, by systematically introducing individual proteins encoded by ZIKV into the embryonic mouse cortex, we show that expression of ZIKV-NS2A, but not Dengue virus (DENV)-NS2A, leads to reduced proliferation and premature differentiation of radial glial cells and aberrant positioning of newborn neurons. Mechanistically, in vitro mapping of protein-interactomes and biochemical analysis suggest interactions between ZIKA-NS2A and multiple adherens junction complex (AJ) components. Functionally, ZIKV-NS2A, but not DENV-NS2A, destabilizes the AJ complex, resulting in impaired AJ formation and aberrant radial glial fiber scaffolding in the embryonic mouse cortex. Similarly, ZIKA-NS2A, but not DENV-NS2A, reduces radial glial cell proliferation and causes AJ deficits in human forebrain organoids. Together, our results reveal pathogenic mechanisms underlying ZIKV infection in the developing mammalian brain.

Molecular signatures associated with ZIKV exposure in human cortical neural progenitors.

Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.

Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen.

In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.

CCL5/RANTES is important for inducing osteogenesis of human mesenchymal stem cells and is regulated by dexamethasone.

In this study, we examine the effect of chemokine (C-C motif) ligand 5 (CCL5)/Regulated on Activation Normal T cell Expressed and Secreted (RANTES), a pro-inflammatory cytokine on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We found CCL5 expression was increased during osteogenic differentiation of hMSCs and CCL5 expression is dependent on the presence of dexamethasone. Knocking down endogenous CCL5 expression blocked osteogenesis, as revealed by decreasing alkaline phosphatase (ALP) activity and a reduction in the expression levels of ALP, bone sialoprotein (BSP), and osteopontin (OPN). Of note, the overexpression of CCL5 was sufficient to increase ALP expression and activity. Moreover, the down-regulation of chemokine (C-C motif) receptor 1 (CCR1), one of the CCL5 receptors, significantly decreased the osteogenesis of hMSCs. Interestingly, the down-regulation of CCR1, but not CCL5, was sufficient to affect the cell numbers during the process of osteogenesis. Our findings reveal that both CCL5 and CCR1 are required for osteogenesis of human MSCs, CCL5 is sufficient for the osteogenesis, and provide a novel link between dexamethasone and CCL5 in human osteogenesis.

Chicken essence improves exercise performance and ameliorates physical fatigue.

Chicken essence (CE) is a liquid nutritional supplement made from cooking whole chickens. In traditional Chinese medicine, CE is used to support health, promote healing, increase metabolism, and relieve fatigue. However, few studies have examined the effect of CE on exercise performance and physical fatigue. We aimed to evaluate the potential beneficial effects of CE on fatigue and ergogenic functions following physical challenge in mice. Male ICR mice were divided into four groups to receive vehicle or CE by oral gavage at 0, 845, 1690, or 4225 mg/kg/day for 4 weeks. Exercise performance and anti-fatigue function were evaluated by forelimb grip strength, exhaustive swimming time, and levels of physical fatigue-related biomarkers serum lactate, ammonia, glucose, and creatine kinase (CK) after physical challenge. CE supplementation dose-dependently elevated endurance and grip strength. CE supplementation significantly decreased lactate, ammonia, and CK levels after physical challenge. Tissue glycogen content, an important energy source for exercise, was significantly increased with CE supplementation. In addition, CE supplementation had few subchronic toxic effects. The supplementation with CE can have a wide spectrum of bioactivities on health promotion, performance improvement and anti-fatigue.

High-performance large-scale flexible dye-sensitized solar cells based on anodic TiO2 nanotube arrays.

A simple strategy to fabricate flexible dye-sensitized solar cells involves the use of photoanodes based on TiO2 nanotube (TNT) arrays with rear illumination. The TNT films (tube length ∼35 µm) were produced via anodization, and sensitized with N719 dye for photovoltaic characterization. Pt counter electrodes of two types were used: a conventional FTO/glass substrate for a device of rigid type and an ITO/PEN substrate for a device of flexible type. These DSSC devices were fabricated into either a single-cell structure (active area 3.6×0.5 cm2) or a parallel module containing three single cells (total active area 5.4 cm2). The flexible devices exhibit remarkable performance with efficiencies η=5.40% (single cell) and 4.77% (parallel module) of power conversion, which outperformed their rigid counterparts with η=4.87% (single cell) and 4.50% (parallel model) under standard one-sun irradiation. The flexible device had a greater efficiency of conversion of incident photons to current and a broader spectral range than the rigid device; a thinner electrolyte layer for the flexible device than for the rigid device is a key factor to improve the light-harvesting ability for the TNT-DSSC device with rear illumination. Measurements of electrochemical impedance spectra show excellent catalytic activity and superior diffusion characteristics for the flexible device. This technique thus provides a new option to construct flexible photovoltaic devices with large-scale, light-weight, and cost-effective advantages for imminent applications in consumer electronics.

Enhanced light harvesting with a reflective luminescent down-shifting layer for dye-sensitized solar cells.

For a dye-sensitized solar cell with a near-infrared squaraine (SQ1) sensitizer, the photovoltaic performance was enhanced remarkably with a reflective luminescent down-shifting (R-LDS) layer to increase the light-harvesting efficiency at the wavelength region 400-550 nm where the SQ1 dye has weak absorption. Relative enhancements greater than 200% in IPCE near 500 nm and 40-54% in JSC were achieved with red phosphor CaAlSiN3:Eu(2+) as the LDS material, attaining 5.0 and 4.8% overall efficiencies of power conversion for the R-LDS layer coated on the counter electrode (front illumination) and working electrode (back illumination), respectively.

Hybrid Titania Photoanodes with a Nanostructured Multi-Layer Configuration for Highly Efficient Dye-Sensitized Solar Cells.

To construct a hybrid titania photoanode containing nanoparticles and nanorods of varied size in a multilayer (ML) configuration for dye-sensitized solar cells, the essence of our ML design is a bilayer system with additional layers of nanorods of well-controlled size inserted between the transparent and the scattering layers to enhance the light-harvesting capability for photosensitizers with small absorptivity, such as Z907. We measured charge-extraction and intensity-modulated photoelectric spectra to show the advantages of one-dimensional nanorods with an improved electron-transport property and an upward shift of the potential band edge; a favorable ML configuration was constructed to have a cascade potential feature for feasible electron transport from long nanorods, to normal nanorods, to small nanoparticles. On the basis of the ML system reported herein, we demonstrate how the performance of a Z907 device is improved to attain η ∼10%, which is a milestone for its future commercialization.

Size-controlled anatase titania single crystals with octahedron-like morphology for dye-sensitized solar cells.

A simple hydrothermal method with titanium tetraisopropoxide (TTIP) as a precursor and triethanolamine (TEOA) as a chelating agent enabled growth in the presence of a base (diethylamine, DEA) of anatase titania nanocrystals (HD1-HD5) of controlled size. DEA played a key role to expedite this growth, for which a biphasic crystal growth mechanism is proposed. The produced single crystals of titania show octahedron-like morphology with sizes in a broad range of 30-400 nm; a typical, extra large, octahedral single crystal (HD5) of length 410 nm and width 260 nm was obtained after repeating a sequential hydrothermal treatment using HD3 and then HD4 as a seed crystal. The nanocrystals of size ~30 nm (HD1) and ~300 nm (HD5) served as active layer and scattering layer, respectively, to fabricate N719-sensitized solar cells. These HD devices showed greater V(OC) than devices of conventional nanoparticle (NP) type; the overall device performance of HD attained an efficiency of 10.2% power conversion at a total film thickness of 28 µm, which is superior to that of a NP-based reference device (η = 9.6%) optimized at a total film thickness of 18-20 µm. According to results obtained from transient photoelectric and charge extraction measurements, this superior performance of HD devices relative to their NP counterparts is due to the more rapid electron transport and greater TiO(2) potential.

Spirally configured cis-stilbene/fluorene hybrids as bipolar, organic sensitizers for solar cell applications.

Hybrids based on a dibenzosuberene core bearing a spiro-fluorene junction at the C-5 position and with amino donor and ß-thiophenyl-α-cyanoacrylic acid acceptor groups at C-3 and C-7, respectively, serve as new organic sensitizer materials for solar cell applications. Solar cell devices based on these materials show a conversion efficiency (η) of up to 6.1% (V(oc) = 697 mV, J(sc) = 12.2 mA cm(-2), FF = 0.72) under AM 1.5 G conditions. The best IPCE values exceed 75% within the 450-550 nm absorption range.

Design and Characterization of Heteroleptic Ruthenium Complexes Containing Benzimidazole Ligands for Dye-Sensitized Solar Cells: The Effect of Fluorine Substituents on Photovoltaic Performance.

We designed heteroleptic ruthenium complexes (RD12-RD15) containing fluoro-substituted benzimidazole ligands for dye-sensitized solar cells (DSSCs). These dyes were synthesized according to a typical one-pot procedure with the corresponding ancillary ligands produced in two simple steps; they were prepared into DSSC devices according to the same conditions of fabrication. The eventual devices show a systematic trend of increasing VOC and decreasing JSC with fluorine atoms of increasing number substituted on the ligand. The charge-extraction results show that upward shifts of the TiO2 potential occurred when the fluoro-substituted dyes were sensitized on TiO2 with a systematic trend of shift N719 > RD15 (with 5 F) > RD12 (with 2 F) >RD5 (no F); the intensity-modulated photovoltage spectra indicate that those fluoro substituents retard charge recombination with the electron lifetimes (τR) in the order RD15 > RD12 > RD5 > N719, consistent with the variation of VOC for the systems.