High-intensity ultrasound modified the functional properties of Neosalanx taihuensis myofibrillar protein and improved its emulsion stability.

This study aimed to investigate the effects of high-intensity ultrasound treatment on the functional properties and emulsion stability of Neosalanx taihuensis myofibrillar protein (MP). The results showed that the carbonyl groups, emulsification properties, intrinsic fluorescence intensity, and surface hydrophobicity of the ultrasound treated MP solution were increased compared to the MP without ultrasound treatment. The results of secondary structure showed that the ultrasound treatment could cause a huge increase of ß-sheet and a decline of α-helix of MP, indicating that ultrasound induced molecular unfolding and stretching. Moreover, ultrasound reduced the content of total sulfhydryl and led to a certain degree of MP cross-linking. The microscopic morphology of MP emulsion indicated that the emulsion droplet decreased with the increase of ultrasound power. In addition, ultrasound could also increase the storage modulus of the MP emulsion. The results for the lipid oxidation products indicated that ultrasound significantly improved the oxidative stability of N. taihuensis MP emulsions. This study offers an important reference theoretically for the ultrasound modification of aquatic proteins and the future development of N. taihuensis deep-processed products represented by surimi.

Development of human retinal organoid models for bisphenol toxicity assessment.

Bisphenols, including Bisphenol A (BPA), Tetrabromobisphenol A (TBBPA), and Tetrabromobisphenol S (TBBPS), have been widely applied in the production of polycarbonate plastics and epoxy resins and have been detected in the environment worldwide. The frequent detection of bisphenols in maternal and fetal samples has raised concerns about their toxic effects on human embryonic development, especially on the development of the central nervous system. However, the effect of bisphenols on human retinal development is still unknown. In this study, to evaluate the toxicity of bisphenols on early retinal development, human embryonic stem cells were induced to differentiate into retinal organoids that responded to BPA, TBBPA, and TBBPS, at human exposure relevant concentrations. The global gene expression of retinal organoids was analyzed by RNA sequencing (RNA-seq). A set of retinal development-related biological processes, including neuron differentiation, phototransduction, axon guidance, and retina layer formation, were identified in retinal organoids corresponding to different developmental stages. The RNA-seq data also showed that BPA, TBBPA, and TBBPS influenced retinal development by interfering with the Cytokine-cytokine receptor interaction pathway. HSPA6, HIF1A-AS3, CDC20B, IL19, OAS1, HSPA7, and RN7SK were dysregulated by these chemicals. Additionally, BPA, TBBPA, and TBBPS exhibited different toxic effects on neural retina development, with TBBPA appearing to exert more toxicity than BPA and TBBPS. Furthermore, three bisphenols exhibited different effects at different stages of neural retina development. The sensitivity of retinal development to bisphenols depends on their developmental stage. This study provides new insights into the deep dissection of retinotoxicity after prenatal bisphenol exposure.

Disease-associated microglial activation prevents photoreceptor degeneration by suppressing the accumulation of cell debris and neutrophils in degenerating rat retinas.

Retinitis pigmentosa initially presents as night blindness owing to defects in rods, and the secondary degeneration of cones ultimately leads to blindness. Previous studies have identified active roles of microglia in the pathogenesis of photoreceptor degeneration in RP. However, the contribution of microglia to photoreceptor degeneration remains controversial, partly due to limited knowledge of microglial phenotypes during RP. Rationale: In this study, we investigated the pathways of microglial activation and its contribution to photoreceptor degeneration in RP. Methods: A classic RP model, Royal College of Surgeons rat, was used to explore the process of microglial activation during the development of RP. An inhibitor of colony-stimulating factor 1 receptor (PLX3397) was fed to RCS rats for sustained ablation of microglia. Immunohistochemistry, flow cytometry, RT-qPCR, electroretinography and RNA-Seq were used to investigate the mechanisms by which activated microglia influenced photoreceptor degeneration. Results: Microglia were gradually activated to disease-associated microglia in the photoreceptor layers of RCS rats. Sustained treatment with PLX3397 ablated most of the disease-associated microglia and aggravated photoreceptor degeneration, including the secondary degeneration of cones, by downregulating the expression of genes associated with photoreceptor function and components and exacerbating the impairment of photoreceptor cell function. Disease-associated microglial activation promoted microglia to engulf apoptotic photoreceptor cell debris and suppressed the increase of infiltrated neutrophils by increasing engulfment and inhibiting CXCL1 secretion by Müller cells, which provided a healthier microenvironment for photoreceptor survival. Conclusions: Our data highlight a key role of disease-associated microglia activation in the suppression of rod and cone degeneration, which reduces secondary damage caused by the accumulation of dead cells and infiltrated neutrophils in the degenerating retina.

Evaluation of the influences of low dose polybrominated diphenyl ethers exposure on human early retinal development.

Increasing evidence in animal models has suggested that polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, can cause retinotoxicity. However, data on the influence of PBDE treatment on human retinal development are scarce due to the lack of appropriate models. In the present study, we report the utilization of human embryonic stem cell-derived retinal organoids (hESC-ROs) for toxicity assessment of the most common PBDE congener (BDE-47) during the early stages of retinal development. Exposure to BDE-47 decreased the thickness and area of the neural retina (NR) of hESC-ROs in a dose- and time-dependent manner. Abnormal retinal cell distributions, disordered NR structures, and neural rosette-like structures were found on hESC-ROs after low-level BDE-47 exposure. Moreover, BDE-47 exposure decreased cell proliferation, promoted cell apoptosis, and caused abnormal differentiation. Transcriptomic analysis demonstrated that differentially expressed genes, caused by BDE-47, were enriched in extracellular matrix organization. Metabolomic studies of hESC-ROs revealed significant changes in the metabolism of purine and glutathione after BDE-47 exposure for five weeks. This study clarifies the retinotoxicity of low-level BDE-47 treatment and highlights the powerfulness of the hESC-RO model, deepening our understanding of BDE-47-driven human early retina developmental toxicity.

Glucocorticoids Promote Extracellular Matrix Component Remodeling by Activating YAP in Human Retinal Capillary Endothelial Cells.

Remodeling of extracellular matrix (ECM) components of endothelial cells is the main cause of retinal vascular basement membrane (BM) thickening, which leads to the initiation and perpetuation of microvasculopathy of diabetic retinopathy (DR). Excessive amounts of glucocorticoids (GCs) are related to the presence and severity of DR, however transcriptional effects of GCs on the biology of human retinal capillary endothelial cells (HRCECs) and its impacts on DR are still unclear. Here, we showed that GC (hydrocortisone) treatment induced ECM component [fibronectin (FN) and type IV collagen (Col IV)] expression and morphological changes in HRCECs via the glucocorticoid receptor (GR), which depended on the nuclear translocation of YAP coactivator. Mechanistically, GCs induced stress fiber formation in HRCECs, while blocking stress fiber formation inhibited GC-induced YAP nuclear translocation. Overexpression of FN, but not Col IV, activated YAP through the promotion of stress fiber formation via ECM-integrin signaling. Thus, a feedforward loop is established to sustain YAP activity. Using mRNA sequencing of HRCECs with overexpressed YAP or GC treatment, we found a similarity in Gene Ontology (GO) terms, differentially expressed genes (DEGs) and transcription factors (TFs) between the two RNA-seq datasets. In vivo, YAP was activated in retina vascular ECs of STZ-induced diabetic mice, and TF prediction analysis of published RNA-seq data of dermal vascular ECs from T2DM patients showed that GR and TEAD (the main transcription factor for YAP) were enriched. Together, GCs activate YAP and promote ECM component (FN and Col IV) remodeling in retinal capillary endothelial cells, and the underlying regulatory mechanism may provide new insights into the vascular BM thickening of the retina in the early pathogenesis of DR.

Human Retinal Progenitor Cells Derived Small Extracellular Vesicles Delay Retinal Degeneration: A Paradigm for Cell-free Therapy.

Retinal degeneration is a leading cause of irreversible vision impairment and blindness worldwide. Previous studies indicate that subretinal injection of human retinal progenitor cells (hRPCs) can delay the progression of retinal degeneration, preserve retinal function, and protect photoreceptor cells from death, albeit the mechanism is not well understood. In this study, small extracellular vesicles derived from hRPCs (hRPC-sEVs) were injected into the subretinal space of retinal dystrophic RCS rats. We find that hRPC-sEVs significantly preserve the function of retina and thickness of the outer nuclear layer (ONL), reduce the apoptosis of photoreceptors in the ONL, and suppress the inflammatory response in the retina of RCS rats. In vitro, we have shown that hRPC-sEV treatment could significantly reserve the low-glucose preconditioned apoptosis of photoreceptors and reduce the expression of pro-inflammatory cytokines in microglia. Pathway analysis predicted the target genes of hRPC-sEV microRNAs involved in inflammation related biological processes and significantly enriched in processes autophagy, signal release, regulation of neuron death, and cell cycle. Collectively, our study suggests that hRPC-sEVs might be a favorable agent to delay retinal degeneration and highlights as a new paradigm for cell-free therapy.

Improving cell survival and engraftment in vivo via layer-by-layer nanocoating of hESC-derived RPE cells.

BACKGROUND: Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of hESC-RPE cells is a great challenge. METHODS: Here, we report hESC-RPE cells that are embedded with polyelectrolytes gelatin and alginate by layer-by-layer (LbL) self-assembly technique, based on the opposite charge of alternate layers. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo. RESULTS: This strategy obviously decreased the immunogenicity of hESC-RPE cells without affecting its activity. LbL-RPE cell transplants into the subretinal space of Royal College of Surgeons (RCS) rats optimized cell engraftment and decreased immunogenicity compared to untreated RPE cell transplants (immunosuppression was not used during the 21-week study). Visual-functional assay with electroretinogram recordings (ERGs) also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants. CONCLUSIONS: We demonstrate that transplanted LbL-RPE cells have better viability and grafting efficiency, optimized immunogenicity, and visual function. Therefore, LbL engineering is a promising method to increase the efficacy of hESC-RPE cell transplantation.

Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia.

Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1ß, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

Microglia Mediate Synaptic Material Clearance at the Early Stage of Rats With Retinitis Pigmentosa.

Resident microglia are the main immune cells in the retina and play a key role in the pathogenesis of retinitis pigmentosa (RP). Many previous studies on the roles of microglia mainly focused on the neurotoxicity or neuroprotection of photoreceptors, while their contributions to synaptic remodeling of neuronal circuits in the retina of early RP remained unclarified. In the present study, we used Royal College of Surgeons (RCS) rats, a classic RP model characterized by progressive microglia activation and synapse loss, to investigate the constitutive effects of microglia on the synaptic lesions and ectopic neuritogenesis. Rod degeneration resulted in synapse disruption and loss in the outer plexiform layer (OPL) at the early stage of RP. Coincidentally, the resident microglia in the OPL increased phagocytosis and mainly engaged in phagocytic engulfment of postsynaptic mGluR6 of rod bipolar cells (RBCs). Complement pathway might be involved in clearance of postsynaptic elements of RBCs by microglia. We pharmacologically deleted microglia using a CSF1 receptor (CSF1R) inhibitor to confirm this finding, and found that it caused the accumulation of postsynaptic mGluR6 levels and increased the number and length of ectopic dendrites in the RBCs. Interestingly, the numbers of presynaptic sites expressing CtBP2 and colocalized puncta in the OPL of RCS rats were not affected by microglia elimination. However, sustained microglial depletion led to progressive functional deterioration in the retinal responses to light in RCS rats. Based on our results, microglia mediated the remodeling of RBCs by phagocytosing postsynaptic materials and inhibiting ectopic neuritogenesis, contributing to delay the deterioration of vision at the early stage of RP.

ROCK Inhibitor Y27632 Induced Morphological Shift and Enhanced Neurite Outgrowth-Promoting Property of Olfactory Ensheathing Cells via YAP-Dependent Up-Regulation of L1-CAM.

Olfactory ensheathing cells (OECs) are heterogeneous in morphology, antigenic profiles and functions, and these OEC subpopulations have shown different outcomes following OEC transplantation for central nervous system (CNS) injuries. Morphologically, OECs are divided into two subpopulations, process-bearing (Schwann cells-like) and flattened (astrocytes-like) OECs, which could switch between each other and are affected by extracellular and intracellular factors. However, neither the relationship between the morphology and function of OECs nor their molecular mechanisms have been clarified. In the present study, we first investigated morphological and functional differences of OECs under different cytokine exposure conditions. It demonstrated that OECs mainly displayed a process-bearing shape under pro-inflammatory conditions (lipopolysaccharide, LPS), while they displayed a flattened shape under anti-inflammatory conditions [interleukin-4 (IL-4) and transforming growth factor-ß1 (TGF-ß1)]. The morphological changes were partially reversible and the Rho-associated coiled-coil-containing protein kinase (ROCK)/F-actin pathway was involved. Functionally, process-bearing OECs under pro-inflammatory conditions showed increased cellular metabolic activity and a higher migratory rate when compared with flattened OECs under anti-inflammatory conditions and significantly promoted neurite outgrowth and extension. Remarkably, the morphological shift towards process-bearing OECs induced by ROCK inhibitor Y27632 enhanced the neurite outgrowth-promoting property of OECs. Furthermore, as the downstream of the ROCK pathway, transcriptional co-activator Yes-associated protein (YAP) mediated morphological shift and enhanced the neurite outgrowth-promoting property of OECs through upregulating the expression of the neural adhesion molecule L1-CAM. Our data provided evidence that OECs with specific shapes correspond to specific functional phenotypes and opened new insights into the potential combination of OECs and small-molecule ROCK inhibitors for the regeneration of CNS injuries.

Sialotranscriptome sequencing and analysis of Anopheles sinensis and comparison with Psorophora albipes sialotranscriptome (Diptera: Culicidae).

Most of adult female mosquitoes secrete saliva to facilitate blood sucking, digestion and nutrition, and mosquito-borne disease prevention. The knowledge of classification and characteristics of sialotranscriptome genes are still quite limited. Anopheles sinensis is a major malaria vector in China and southeast Asian countries. In this study, the An. sinensis sialotranscriptome was sequenced using Illumina sequencing technique with a total of 10 907 unigenes to be obtained and annotated in biological functions and pathways, and 10 470 unigenes were mapped to An. sinensis reference genome with 70.46% of genes having 90%-100% genome mapping through bioinformatics analysis. These mapped genes were classified into four categories: housekeeping (6632 genes), secreted (1177), protein-coding genes with function-unknown (2646) and transposable element (15). The housekeeping genes were divided into 27 classes, and the secreted genes were divided into 11 classes and 96 families. The classification, characteristics and evolution of these classes/families of secreted genes are further described and discussed. The comparison of the 1177 secreted genes in An. sinensis in the Anophelinae subfamily with 811 in Psorophora albipes in the Culicinae subfamily show that six classes/subclasses have the gene number more than twice and two classes (uniquely found in anophelines, and Orphan proteins of unique standing) are unique in the former compared with the latter, whereas four classes/subclasses are much expanded and uniquely found in the Aedes class and is unique in the later. The An. sinensis sialotranscriptome sequence data is the most complete in mosquitoes to date, and the analyses provide a comprehensive information frame for further research of mosquito sialotranscriptome.