Construction of vector vortex beams on hybrid-order Poincaré sphere through highly scattering media.

Vector vortex beams (VVBs) have attracted extensive attention due to their unique properties and their wide applications in fields such as optical manipulation and optical imaging. However, the wavefronts of the vector vortex beams are highly scrambled when they encounter highly scattering media (HSM), such as thick biological tissues, which greatly prevents the applications of VVBs behind HSM. To address this issue, we propose a scheme to construct VVBs of freewill position on the surface of hybrid-order Poincaré sphere (HyOPS) through HSM. With the measurement of two orthogonal scalar transmission matrices, the conjugated wavefronts for constructing orbital angular momentum beams with arbitrary topological charge in right and left circularly polarized states through HSM can be calculated, respectively. When an input wavefront superimposed by the two conjugated wavefronts with an appropriate ratio and phase delay, impinges on the HSM, the desired VVB can be created through HSM. To demonstrate the viability of our scheme, a series of VVBs on different locations of various HyOPSs have been reconstructed through a ZnO scattering layer experimentally. Furthermore, to characterize the polarization distribution of the generated beams, the polarization maps of these beams are derived by measuring the four Stokes parameters, which agree well with the theoretical distributions. This work will promote the applications of VVBs in highly scattering environments.

CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis.

Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU), revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and results in dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.

Human retinal organoids with an OPA1 mutation are defective in retinal ganglion cell differentiation and function.

Autosomal dominant optic atrophy (ADOA), mostly caused by heterozygous OPA1 mutations and characterized by retinal ganglion cell (RGC) loss and optic nerve degeneration, is one of the most common types of inherited optic neuropathies. Previous work using a two-dimensional (2D) differentiation model of induced pluripotent stem cells (iPSCs) has investigated ADOA pathogenesis but failed to agree on the effect of OPA1 mutations on RGC differentiation. Here, we use 3D retinal organoids capable of mimicking in vivo retinal development to resolve the issue. We generated isogenic iPSCs carrying the hotspot OPA1 c.2708_2711delTTAG mutation and found that the mutant variant caused defective initial and terminal differentiation and abnormal electrophysiological properties of organoid-derived RGCs. Moreover, this variant inhibits progenitor proliferation and results in mitochondrial dysfunction. These data demonstrate that retinal organoids coupled with gene editing serve as a powerful tool to definitively identify disease-related phenotypes and provide valuable resources to further investigate ADOA pathogenesis and screen for ADOA therapeutics.

An optimized procedure to record visual evoked potential in mice.

Visual evoked potential (VEP) is commonly used to evaluate visual acuity in both clinical and basic studies. Subdermal needle electrodes or skull pre-implanted screw electrodes are usually used to record VEP in rodents. However, the VEP amplitudes recorded by the former are small while the latter may damage the brain. In this study, we established a new invasive procedure for VEP recording, and made a series of comparisons of VEP parameters recorded from different electrode locations, different times of day (day and night) and bilateral eyes, to evaluate the influence of these factors on VEP in mice. Our data reveal that our invasive method is reliable and can record VEP with good waveforms and large amplitudes. The comparison data show that VEP is greatly influenced by active electrode locations and difference between day and night. In C57 or CD1 ONC (optic nerve crush) models and Brn3bAP/AP mice, which are featured by loss of retinal ganglion cells (RGCs), amplitudes of VEP N1 and P1 waves are drastically reduced. The newly established VEP procedure is very reliable and stable, and is particularly useful for detecting losses of RGC quantities, functions or connections to the brain. Our analyses of various recording conditions also provide useful references for future studies.

In vivo Regeneration of Ganglion Cells for Vision Restoration in Mammalian Retinas.

Glaucoma and other optic neuropathies affect millions of people worldwide, ultimately causing progressive and irreversible degeneration of retinal ganglion cells (RGCs) and blindness. Previous research into cell replacement therapy of these neurodegenerative diseases has been stalled due to the incapability for grafted RGCs to integrate into the retina and project properly along the long visual pathway. In vivo RGC regeneration would be a promising alternative approach but mammalian retinas lack regenerative capacity. It therefore has long been a great challenge to regenerate functional and properly projecting RGCs for vision restoration in mammals. Here we show that the transcription factors (TFs) Math5 and Brn3b together are able to reprogram mature mouse Müller glia (MG) into RGCs. The reprogrammed RGCs extend long axons that make appropriate intra-retinal and extra-retinal projections through the entire visual pathway to innervate both image-forming and non-image-forming brain targets. They exhibit typical neuronal electrophysiological properties and improve visual responses in RGC loss mouse models. Together, our data provide evidence that mammalian MG can be reprogrammed by defined TFs to achieve in vivo regeneration of functional RGCs as well as a promising new therapeutic approach to restore vision to patients with glaucoma and other optic neuropathies.

Towards a better understanding of allograft-induced stress response in the pearl oyster Pinctada fucata martensii: Insights from iTRAQ-based comparative proteomic analysis.

Implantation of a spherical nucleus into a recipient oyster is a critical step in artificial pearl production. The implanted nucleus is known to trigger cellular stress responses at several levels, yet the molecular mechanism underpinning physiological adaptation of the pearl oysters to nucleus implantation is still poorly understood. In this study, we took advantage of the iTRAQ-based proteomics and LC-MS/MS approach to look into allograft induced gene regulation at the protein expression level in the pearl oyster Pinctada fucata martensii, across a period of 30 days following nucleus implantation. A wide variety of proteins, including a group of immune-related proteins such as E3 ubiquitin-ligase and heat shock proteins, exhibited differential expression in response to the surgical operation. Further comparisons between different sampling points revealed that GO terms including "translation" and "oxidation-reduction process" and KEGG pathways including "glycolysis/gluconeogenesis" and "pyruvate metabolism" were significantly enriched at several time points, indicating the important roles of these molecular events in the stress response of pearl oysters to nucleus implantation. In addition, considerable discrepancy between protein expression level and gene transcript abundancy was identified, as only a few genes showed at least 2-fold expression changes at both proteomic and transcriptomic levels. The result implies that post-transcriptional gene regulation for the key proteins may represent an important aspect of allograft-induced stress response in the pearl oysters. Taken together, the data obtained would contribute to a deeper understanding of the molecular mechanisms enabling stress adaptation of the pearl oysters in response to nucleus implantation.

Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol.

Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency. Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4-7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm. Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2. Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.

A novel toll-like receptor from the pearl oyster Pinctada fucata martensii is induced in response to stress.

Graft rejection due to immune incompatibility is a common occurrence in pearl culture, which often cause death to the host oyster. To improve cultured pearl production, host mortality and bead rejection rates must be reduced. Toll-like receptor 4 (TLR4) plays an important role in innate immunity, and may be related to allograft rejection. Here, we cloned the TLR4 cDNA from the pearl oyster Pinctada fucata martensii (PmTLR4). PmTLR4 cDNA was 3138bp, including a 2625bp open reading frame encoding 874 amino acids. The predicted PmTLR4 protein was structurally typical of the TLR family. PmTLR4 had relatively high sequence similarity and identity to the TLR4 of the Cyclina sinensis (48.1% and 27.6%, respectively). Multiple alignment of TLR4 sequences across species indicated that the Toll/interleukin-1 (IL-1) receptor domain was conserved among species. PmTLR4 mRNA was expressed in all tissues tested, with the most abundant mRNA expression in hepatopancreas and gill in P. fucata martensii. After being stressed by either lipopolysaccharide (LPS) exposure or the nucleus insertion operation, PmTLR4 mRNA expression increased significantly in the hemocytes as compared to controls. Peak level of PmTLR4 mRNA was observed 6h after the LPS injection, and 2d after the nucleus insertion operation. These data suggest that PmTLR4 may play a vital role in the induction of innate immunity and is therefore associated with allograft immunity in the pearl oyster P. fucata martensii.

Deep transcriptome profiling sheds light on key players in nucleus implantation induced immune response in the pearl oyster Pinctada martensii.

Immunological rejection of the pearl oysters following nucleus implantation is a major issue limiting the successful rate of cultured pearls. To date, the molecular mechanism of immune tolerance during pearl formation in the pearl oysters is still largely unknown. Through the RNA sequencing platform and comparative transcriptomic analysis, we investigated the chronic gene expression changes at seven time points (0, 5, 10, 15, 20, 30, 60 days post implantation or dpi) over a period of 60 days following nucleus implantation in the pearl oyster Pinctada martensii. A total of 81,390 unique transcripts (or unigenes) with a combined length of 96.8 million bp and a N50 value of 2227 bp were obtained. When compared with sequences in the nr, nt, Swiss-Prot, KEGG, COG and GO databases, 36,380 unigenes can find homologous genes. Pairwise comparison of gene expression among all the samples showed that the largest number (or 6846) of differentially expressed genes was observed at 10 dpi. The number then decreased to below 5000 at 15, 20 and 30 dpi and increased again to 6679 at 60 dpi. PCA analysis further showed that the seven time points can be roughly divided into four groups. Comparative transcriptomic analysis between the four groups identified a variety of genes showing differential expression at different time points, including many immune-related genes such as those encoding for toll-like receptor, lectin, scavenger receptor, and peroxidase. In addition, GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly associated with metabolism, ribosome function, immune response, signaling transduction, and cytoskeleton organization. Notably, two KEGG pathways, namely "cell adhesion molecules" and "primary immunodeficiency" were significantly enriched during the whole process. This finding indicates that genes in these pathways are likely to play critical roles in the immune tolerance of the pearl oysters. To conclude, the data obtained contribute to a better understanding of the molecular mechanisms of nucleus implantation induced immune response in the pearl oysters, and will facilitate the development of effective measures to improve the performance of pearl culture.

De novo assembly, gene annotation, and simple sequence repeat marker development using Illumina paired-end transcriptome sequences in the pearl oyster Pinctada maxima.

We analyzed the mantle transcriptome of pearl oyster Pinctada maxima and developed EST-SSR markers using Illumina HiSeq 2000 paired-end sequencing technology. A total of 49,500,748 raw reads were generated. De novo assembly generated 108,704 unigenes with an average length of 407 bp. Sequence similarity search with known proteins or nucleotides revealed that 30,200 (27.78%) and 25,824 (23.76%) consensus sequences were homologous with the sequences in the non-redundant protein and Swiss-Prot databases, respectively, and that 19,701 (18.12%) of these unigenes were possibly involved in approximately 234 known signaling pathways in the Kyoto Encyclopedia of Genes and Genomes database. Ninety one biomineralization-related unigenes were detected. In a cultured stock, 1764 simple sequence repeats were identified and 56 primer pairs were randomly selected and tested. The rate of successful amplification was 68.3%. The developed molecular markers are helpful for further studies on genetic linkage analysis, gene localization, and quantitative trait loci mapping.