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Ye Tao,1,* Lei Geng,2,* Wei-Wei Xu,1 Li-Min Qin,1 Guang-Hua Peng,1 Yi-Fei Huang1 1Department of Ophthalmology, 2Department of Orthopaedics, Chinese People’s Liberation Army General Hospital, Ophthalmology & Visual Science Key Lab of PLA, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Hydrogen, one of the most well-known natural molecules, has been used in numerous medical applications owing to its ability to selectively neutralize cytotoxic reactive oxygen species and ameliorate hazardous inflammations. Hydrogen can exert protective effects on various reactive oxygen species-related diseases, including the transplantation-induced intestinal graft injury, chronic inflammation, ischemia–reperfusion injuries, and so on. Especially in the eye, hydrogen has been used to counteract multiple ocular pathologies in the ophthalmological models. Herein, the ophthalmological utilizations of hydrogen are systematically reviewed and the underlying mechanisms of hydrogen-induced beneficial effects are discussed. It is our hope that the protective effects of hydrogen, as evidenced by these pioneering studies, would enrich our pharmacological knowledge about this natural element and cast light into the discovery of a novel therapeutic strategy against ocular diseases. Keywords: hydrogen, therapeutic strategy, ocular diseases

A light beam can be spatially structured in the complex amplitude to possess orbital angular momentum (OAM), which introduces an extra degree of freedom alongside the intrinsic spin angular momentum (SAM) associated with circular polarization. Furthermore, superimposing two such twisted light (TL) beams with distinct SAM and OAM produces a vector vortex beam (VVB) in nonseparable states where not only complex amplitude but also polarization is spatially structured and entangled with each other. In addition to the nonseparability, the SAM and OAM in a VVB are intrinsically coupled by the optical spin-orbit interaction and constitute the profound spin-orbit physics in photonics. In this work, we present a comprehensive theoretical investigation, implemented on the first-principles base, of the intriguing light-matter interaction between VVBs and WSe 2 monolayers (WSe 2 -MLs), one of the best-known and promising two-dimensional (2D) materials in optoelectronics dictated by excitons, encompassing bright exciton (BX) as well as various dark excitons (DXs). One of the key findings of our study is that a substantial enhancement of the photoexcitation of gray excitons (GXs), a type of spin-forbidden DX, in a WSe 2 -ML can be achieved through the utilization of a 3D-structured TL with the optical spin-orbit interaction. Moreover, we show that a spin-orbit-coupled VVB surprisingly allows for the imprinting of the carried optical information onto GXs in 2D materials, which is robust against the decoherence mechanisms in the materials. This suggests a promising method for deciphering the transferred angular momentum from structured light to excitons.

Purpose: Retinal degeneration (RD) is a large cluster of retinopathies that is characterized by the progressive photoreceptor death and visual impairments. CX3CL1/CX3CR1 signaling has been documented to mediate the microglia activation and gliosis reaction during neurodegeneration. We intend to verify whether the CX3CL1/CX3CR1 signaling is involved in the RD pathology., Methods: A pharmacologically induced RD mice model was established. AZD8797, a CX3CR1 antagonist, was injected into the vitreous cavity of an RD model to modulate the neuroglia activation. Then, the experimental animals were subjected to functional, morphological, and behavioral analysis., Results: The CX3CL1/CX3CR1 signaling mediated neuroglia activation was implicated in the photoreceptor demise of an RD model. Intravitreal injection of AZD8797 preserved the retinal structure and enhanced the photoreceptor survival through inhibiting the CX3CL1/CX3CR1 expressions. Fundus photography showed that the distribution of retinal vessel was clear, and the severity of lesions was alleviated by AZD8797. In particular, these morphological benefits could be translated into remarkable functional improvements, as evidenced by the behavioral test and electroretinogram (mf-ERG) examination. A mechanism study showed that AZD8797 mitigated the microglia activation and migration in the degenerative retinas. The Müller cell hyper-reaction and secondary gliosis response were also suppressed by AZD8797., Conclusions: The neuroinflammation is implicated in the photoreceptor loss of RD pathology. Targeting the CX3CL1/CX3CR1 signaling may serve as an effective therapeutic strategy. Future refinements of these findings may cast light into the discovery of new medications for RD.

Ageing retina is prone to ferroptosis due to the iron accumulation and impaired efficiency of intracellular antioxidant defense system. Ferroptosis acts as a cell death modality that is characterized by the iron-dependent accumulation of lipid peroxidation. Ferroptosis is distinctively different from other types of regulated cell death (RCD) at the morphological, biochemical, and genetic levels. Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Its prevalence and severity increase progressively with age. Recent reports have shown that ferroptosis is implicated in the pathophysiology of DR. Under hyperglycemia condition, the endothelial cell and retinal pigment epithelium (RPE) cell will undergo ferroptosis, which contributes to the increased vascular permeability and the disrupted blood retinal barrier (BRB). The underlying etiology of DR can be attributed to the impaired BRB integrity and subsequent damages of the neurovascular units. In the absence of timely intervention, the compromised BRB can ultimately cause profound visual impairments. In particular, the ageing retina is vulnerable to ferroptosis, and hyperglycemia will accelerate the progression of this pathological process. In this article, we discuss the contributory role of ferroptosis in DR pathogenesis, and summarize recent therapeutic trials that targeting the ferroptosis. Further study on the ferroptosis mediated damage would enrich our knowledge of DR pathology, and promote the development of clinical treatment for this degenerative retinopathy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mitochondrial autophagy plays a contributary role in the pathogenesis of retina degeneration (RD). ZYAN1 is a novel proline hydroxylase domain (PHD) inhibitor that can enhance the expression of hypoxia-inducible factor 1-alpha (HIF-1α). This study investigated whether ZYAN1 could alleviate progressive photoreceptor loss and oxidative damage in a pharmacologically induced RD model via the modulation of mitophagy. ZYAN1 was injected into the vitreous body of the RD model, and the retinal autophagy level was analyzed. The therapeutic effects of ZYAN1 were evaluated via a function examination, a morphological assay, in situ reactive oxygen species (ROS) detection, and an immunofluorescence assay. It was shown that the thickness of the outer nuclear layer (ONL) increased significantly, and visual function was efficiently preserved via ZYAN1 treatment. The mitochondria structure of photoreceptors was more complete in the ZYAN1-treated mice, and the number of autophagosomes also increased significantly. Membrane disc shedding and ROS overproduction were alleviated after ZYAN1 treatment, and the axonal cilia were more structurally intact. A Western blot analysis showed that the expression levels of the autophagy-related proteins LC3-B, Beclin-1, and ATG5 increased significantly after ZYAN1 treatment, while the expression of P62 was down-regulated. Moreover, the expression levels of HIF-1α and BNIP3 were up-regulated after ZYAN1 treatment. Therefore, an intravitreal injection of ZYAN1 can act as part of the pharmacologic strategy to modulate mitophagy and alleviate oxidative stress in RD. These findings enrich our knowledge of RD pathology and provide insights for the discovery of a therapeutic molecule.

Background: Pepper (Capsicum annum L.) is the main crop in the vegetable industry. The growth and development of peppers are regulated by nitrate, but there is limited research on the molecular mechanisms of nitrate absorption and assimilation in peppers. A plant specific transcription factor NLP plays an important role in nitrate signal transduction., Methods and Results: In this study, a total of 7 NLP members were identified based on pepper genome data. Two nitrogen transport elements (GCN4) were found in the CaNLP5 promoter. In the phylogenetic tree, CaNLP members are divided into three branches, with pepper NLP and tomato NLP having the closest genetic relationship. The expression levels of CaNLP1, CaNLP3, and CaNLP4 are relatively high in the roots, stems, and leaves. The expression level of CaNLP7 gene is relatively high during the 5-7 days of pepper fruit color transformation. After various non-Biotic stress and hormone treatments, the expression of CaNLP1 was at a high level. The expression of CaNLP3 and CaNLP4 was down regulated in leaves, but up regulated in roots. Under conditions of nitrogen deficiency and sufficient nitrate, the expression patterns of NLP genes in pepper leaves and roots were determined., Conclusion: These results provide important insights into the multiple functions of CaNLPs in regulating nitrate absorption and transport., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Retinal degenerative diseases, characterized by retinal neuronal death and severe vision loss, affect millions of people worldwide. One of the most promising treatment methods for retinal degenerative diseases is to reprogram non-neuronal cells into stem or progenitor cells, which then have the potential to re-differentiate to replace the dead neurons, thereby promoting retinal regeneration. Müller glia are the major glial cell type and play an important regulatory role in retinal metabolism and retinal cell regeneration. Müller glia can serve as a source of neurogenic progenitor cells in organisms with the ability to regenerate the nervous system. Current evidence points toward the reprogramming process of Müller glia, involving changes in the expression of pluripotent factors and other key signaling molecules that may be regulated by epigenetic mechanisms. This review summarizes recent knowledge of epigenetic modifications involved in the reprogramming process of Müller glia and the subsequent changes to gene expression and the outcomes. In living organisms, epigenetic mechanisms mainly include DNA methylation, histone modification, and microRNA-mediated miRNA degradation, all of which play a crucial role in the reprogramming process of Müller glia. The information presented in this review will improve the understanding of the mechanisms underlying the Müller glial reprogramming process and provide a research basis for the development of Müller glial reprogramming therapy for retinal degenerative diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Si, Li, Zhang, Su, Liu, Chen, Peng, Cao and Zang.)

Photoreceptors are integral and crucial for the retina, as they convert light into electrical signals. Epigenetics plays a vital role in determining the precise expression of genetic information in space and time during the development and maturation of photoreceptors, cell differentiation, degeneration, death, and various pathological processes. Epigenetic regulation has three main manifestations: histone modification, DNA methylation, and RNA-based mechanisms, where methylation is involved in two regulatory mechanisms-histone methylation and DNA methylation. DNA methylation is the most studied form of epigenetic modification, while histone methylation is a relatively stable regulatory mechanism. Evidence suggests that normal methylation regulation is essential for the growth and development of photoreceptors and the maintenance of their functions, while abnormal methylation can lead to many pathological forms of photoreceptors. However, the role of methylation/demethylation in regulating retinal photoreceptors remains unclear. Therefore, this study aims to review the role of methylation/demethylation in regulating photoreceptors in various physiological and pathological situations and discuss the underlying mechanisms involved. Given the critical role of epigenetic regulation in gene expression and cellular differentiation, investigating the specific molecular mechanisms underlying these processes in photoreceptors may provide valuable insights into the pathogenesis of retinal diseases. Moreover, understanding these mechanisms could lead to the development of novel therapies that target the epigenetic machinery, thereby promoting the maintenance of retinal function throughout an individual's lifespan., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lu, Zhou, Zhang, Su, Liu, Peng, Zang and Cao.)

In this work, we present a comprehensive theoretical and computational investigation of exciton fine structures of WSe2-monolayers, one of the best-known two-dimensional (2D) transition-metal dichalcogenides (TMDs), in various dielectric-layered environments by solving the first-principles-based Bethe-Salpeter equation. While the physical and electronic properties of atomically thin nanomaterials are normally sensitive to the variation of the surrounding environment, our studies reveal that the influence of the dielectric environment on the exciton fine structures of TMD-MLs is surprisingly limited. We point out that the non-locality of Coulomb screening plays a key role in suppressing the dielectric environment factor and drastically shrinking the fine structure splittings between bright exciton (BX) states and various dark-exciton (DX) states of TMD-MLs. The intriguing non-locality of screening in 2D materials can be manifested by the measurable non-linear correlation between the BX-DX splittings and exciton-binding energies by varying the surrounding dielectric environments. The revealed environment-insensitive exciton fine structures of TMD-ML suggest the robustness of prospective dark-exciton-based optoelectronics against the inevitable variation of the inhomogeneous dielectric environment.

Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, play a critical role in metabolic regulation and intracellular communication. Stem cell-derived EVs are considered to have the potential for regeneration, like stem cells, while simultaneously avoiding the risk of immune rejection or tumour formation. The therapeutic effect of stem cell-derived EVs has been proven in many diseases. However, the molecular mechanism of stem cell-derived EVs in retinal repair and regeneration has not been fully clarified. In this review, we described the biological characteristics of stem cell-derived EVs, summarized the current research on stem cell-derived EV treatment in retinal repair and regeneration, and discussed the potential and challenges of stem cell-derived EVs in translational medicine., (© 2023. The Author(s).)

Rationale: Müller glia (MG) play a key role in maintaining homeostasis of the retinal microenvironment. In zebrafish, MG reprogram into retinal progenitors and repair the injured retina, while this MG regenerative capability is suppressed in mammals. It has been revealed that microglia in zebrafish contribute to MG reprogramming, whereas those in mammals are over-activated during retinal injury or degeneration, causing chronic inflammation, acceleration of photoreceptor apoptosis, and gliosis of MG. Therefore, how to modulate the phenotype of microglia to enhance MG reprogramming rather than gliosis is critical. Methods: PLX3397, a colony-stimulating factor 1 receptor inhibitor, was applied to deplete microglia in the retinas of retinal degeneration 10 (rd10) mice, and withdrawal of PLX3397 was used to induce the repopulated microglia (Rep-MiG). The protective roles of the Rep-MiG on the degenerative retina were assessed using a light/dark transition test, and scotopic electroretinogram recordings. Immunofluorescence, western blot, transcriptomic sequencing, and bioinformatics analysis were performed to investigate the effects and mechanisms of microglia on MG reprogramming. Results: Following PLX3397 withdrawal, Rep-MiG replenished the entire retina with a ramified morphology and significantly improved the retinal outer nuclear layer structure, the electroretinography response, and the visual behavior of rd10 mice. Coincidentally, MG were activated, de-differentiated, and showed properties of retina progenitors in a spatial correlation with Rep-MiG. Morphological and transcriptomic analyses revealed Rep-MiG significantly enhanced protease inhibitor activity and suppressed extracellular matrix (ECM) levels during retinal degeneration. Conclusions: It suggested that Rep-MiG with the homeostasis characteristic stimulated the progenitor cell-like properties of MG, probably through regulating ECM remodeling, which protected photoreceptors and improved visual function of rd10 mice. It might be a potential protocol to reprogram MG and delay mammal retinal degeneration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: Retinitis pigmentosa (RP) is a group of neurodegenerative retinopathies causing blindness due to progressive and irreversible photoreceptor cell death. The alkylating agent methyl methanesulfonate (MMS) can induce selective photoreceptor cell death, which is used to establish RP animal models. MMS induces DNA base damage by adding alkyl groups to DNA, and epigenetic modifications influence DNA damage response. Here, we aimed to explore the relationship between DNA methylation and DNA damage response in dying photoreceptors of RP., Methods: The mouse RP model was established by a single intraperitoneal injection of MMS. The retinal structure and function were assessed by H&E, OCT, TUNEL, and ERG at several time points. The expression of DNA methylation regulators was assessed by qPCR and Western blot. DNMT inhibitor 5-aza-dC was applied to inhibit the activity of DNA methyltransferases and improve the retinal photoreceptor damage., Results: The outer nuclear layer (ONL) and IS/OS layer were significantly thinner and the retinal function was impaired after MMS treatment. The cell death was mainly located in the ONL. The retinal damage induced by MMS was accompanied by hyperexpression of DNMT3A/3B. The application of DNMT inhibitor 5-aza-dC could suppress the expression level of DNMT3A/3B, resulting in the remission of MMS-induced photoreceptor cell damage. The ONL and IS/OS layers were thicker than that of the control group, and the retinal function was partially restored. This protective effect of 5-aza-dC was associated with the down-regulated expression of DNMT3A/3B., Conclusion: These findings identified a functional role of DNMT3A/3B in MMS-induced photoreceptor cell damage and provided novel evidence to support DNMTs as potential therapeutic targets in retinal degenerative diseases.Graphical Abstract., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ji, Zhao, Qiao and Peng.)

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Du, Yuan and Peng.)

Background: Acute kidney injury (AKI) is a common and severe complication in patients with cirrhosis, and is associated with poor prognosis. Therefore, identifying cirrhotic patients with AKI who are at high risk of mortality is very important and may be helpful for providing timely medical interventions to improve the prognosis of these patients. However, studies focused on investigating the risk factors for the mortality of cirrhotic patients with AKI were scarce., Aim: To identify risk factors for mortality and establish a nomogram for predicting the prognosis of these patients., Methods: Two hundred fifty consecutive patients with cirrhosis and AKI were recruited and randomly divided into training cohort ( n = 173) and validation cohort ( n = 77). In the training cohort, potential risk factors for death were identified by performing a Cox regression analysis, and a nomogram was established. The predictive performance of the nomogram was internally and externally validated by calculating the area under the receiver operating characteristic curve (AUROC), constructing a calibration curve and performing decision curve analysis., Results: The serum sodium level, international normalized ratio, peak serum creatinine level > 1.5 mg/dL, the presence of hepatic encephalopathy and diabetes were potential risk factors for mortality of cirrhotic patients with AKI in the training dataset. A prognostic nomogram incorporating these variables was established for predicting the overall survival of these patients. Compared with Child-Turcotte-Pugh, the model for end-stage liver disease (MELD) and the MELD-Na scores, the nomogram in predicting 90- and 180-d mortality exhibited better discriminatory power with AUROCs of 0.792 and 0.801 for the training dataset and 0.817 and 0.862 for the validation dataset, respectively. With a nomogram score of 98, patients were divided into low- and high-risk groups, and high-risk patients had a higher mortality rate., Conclusion: A prognostic nomogram displayed good performance for predicting the overall survival of cirrhotic patients with AKI, and will assist clinicians in evaluating the prognosis of these patients., Competing Interests: Conflict-of-interest statement: We have no financial relationships to disclose., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: It has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear., Methods: Following treatment with the alkylating agent methyl methane sulfonate (MMS), the C3 mRNA and protein level was measured, DNA damage and photoreceptor cell death were assessed in both wild-type (WT) C57BL/6J and C3 knockout (KO) mice., Results: We determined that complement pathway is activated following MMS treatment, and C3 knockout (KO) increased the rate of photoreceptor cell survival and preserved visual function. The mRNA levels of nuclear erythroid-related factor 2 (Nrf2) and related genes were higher after MMS application in C3 KO mice., Conclusion: In summary, our study found that C3 KO promotes photoreceptor cell survival and activates the Nrf2 signaling pathway in the context of alkylation-induced retinal degeneration., (© 2022. The Author(s).)

Latent reservoir persistence remains a major obstacle for curing human immunodeficiency virus type 1 (HIV-1) infection. Thus, strategies for the elimination of latent HIV-1 are urgently needed. As a bromodomain and extra-terminal (BET) inhibitor, BMS-986158 has been used in clinical trials for advanced solid tumors and hematological malignancies. Here, we found that BMS-986158 reactivated latent HIV-1 in three types of HIV-1 latency cells in vitro, and in combination antiretroviral therapy (cART)-treated patient-derived peripheral blood mononuclear cells ex vivo, without influencing global immune cell activation. BMS-986158 reactivated latent HIV-1 by increasing phosphorylation of CDK9 at Thr186 and promoting recruitment of CDK9 and RNA polymerase II to the HIV-1 long terminal repeat in J-Lat cells. Furthermore, BMS-986158 exerted strong synergism in reactivating latent HIV-1 when combined with prostratin and vorinostat and enhanced the antiviral activity of anti-HIV-1 drugs. Finally, BMS-986158 showed antiviral activity in an HIV-1 acute infection model, possibly by arresting the cell cycle in infected cells. Thus, these results suggest that BMS-986158 is a potential candidate for AIDS/HIV-1 therapy.

The retina is an important visual organ, which is responsible for receiving light signals and transmitting them to the optic nerve center step by step. The retina contains a variety of cells, among which photoreceptor cells receive light signals and convert them into nerve signals, and are mainly responsible for light and dark vision. Retinal degeneration is mainly the degeneration of photoreceptor cells, and retinitis pigmentosa (RP) is characterized by rod degeneration followed by cone degeneration. So far, there is still a lack of effective drugs to treat RP. Here, we established a stable RP model by tail vein injection of methyl methanesulfonate to study the mechanism of retinal photoreceptor degeneration. Mechanistic target of rapamycin (mTOR) is located in the central pathway of growth and energy metabolism and changes in a variety of diseases in response to pathological changes. We found that the mTOR was activated in this model. Therefore, the inhibitor of mTOR, rapamycin was used to suppress the expression of mTOR and interfere with photoreceptor degeneration. Electroretinogram assay showed that the function of mice retina was improved. Hematoxylin and eosin staining results displayed that retinal photoreceptor thickness and morphology were improved. Also, the autophagy in rapamycin group was activated, which revealed that rapamycin may protect the retinal photoreceptor by inhibiting mTOR and then activating autophagy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Lv, Yang and Peng.)

Human retina development involves multiple well-studied signaling pathways that promote the genesis of a wide arrange of different cell types in a complex architectural structure. Human embryonic stem cells (hESCs)-derived retinal organoids could recapitulate the human retinal development. We performed single-cell RNA-seq of retinal organoids from 5 time points (D36, D66, D96, D126, D186) and identified 9 distinct populations of cells. In addition, we analyzed the molecular characteristics of each main population and followed them from genesis to maturity by pseudotime analysis and characterized the cell-cell interactions between different cell types. Interestingly, we identified insulin receptor (INSR) as a specifically expressed receptor involved in the genesis of photoreceptors, and pleiothropin (PTN)-protein tyrosine phosphatase receptor type Z1 (PTPRZ1) as a mediator of a previously unknown interaction between Müller and retinal progenitor cells. Taken together, these findings provide a rich transcriptome-based lineage map for studying human retinal development and modeling developmental disorders in retinal organoids., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.

The current dogma in ophthalmology and vision research presumes the intraocular environment to be sterile. However, recent evidence of intestinal bacterial translocation into the bloodstream and many other internal organs including the eyes, found in healthy and diseased animal models, suggests that the intraocular cavity may also be inhabited by a microbial community. Here, we tested intraocular samples from over 1000 human eyes. Using quantitative PCR, negative staining transmission electron microscopy, direct culture, and high-throughput sequencing technologies, we demonstrated the presence of intraocular bacteria. The possibility that the microbiome from these low-biomass communities could be a contamination from other tissues and reagents was carefully evaluated and excluded. We also provide preliminary evidence that a disease-specific microbial signature characterized the intraocular environment of patients with age-related macular degeneration and glaucoma, suggesting that either spontaneous or pathogenic bacterial translocation may be associated with these common sight-threatening conditions. Furthermore, we revealed the presence of an intraocular microbiome in normal eyes from non-human mammals and demonstrated that this varied across species (rat, rabbit, pig, and macaque) and was established after birth. These findings represent the first-ever evidence of intraocular microbiota in humans.

Twisted light carries a well-defined orbital angular momentum (OAM) of l ℏ per photon. The quantum number l of its OAM can be arbitrarily set, making it an excellent light source to realize high-dimensional quantum entanglement and ultrawide bandwidth optical communication structures. In spite of its interesting properties, twisted light interaction with solid state materials, particularly two-dimensional materials, is yet to be extensively studied via experiments. In this work, photoluminescence (PL) spectroscopy studies of monolayer molybdenum disulfide (MoS 2 ), a material with ultrastrong light-matter interaction due to reduced dimensionality, are carried out under photoexcitation of twisted light. It is observed that the measured spectral peak energy increases for every increment of l of the incident light. The nonlinear l -dependence of the spectral blue shifts is well accounted for by the analysis and computational simulation of this work. More excitingly, the twisted light excitation revealed the unusual lightlike exciton band dispersion of valley excitons in monolayer transition metal dichalcogenides. This linear exciton band dispersion is predicted by previous theoretical studies and evidenced via this work's experimental setup.

Objective: To investigate the prevalence and risk factors of Blastocystis hominis infections among AIDS patients in Nanchang City., Methods: A cross-sectional questionnaire survey was conducted among AIDS patients in Nanchang City during the period between May and September, 2016. B. hominis infection was detected in patients'stool samples using a PCR assay, and the CD4 + T cell count was measured in subjects'blood samples. In addition, the risk factors of B. hominis infection in AIDS patients were identified using univariate and multivariate logistic regression analyses., Results: A survey was conducted in Nanchang City from May to September 2016. A total of 505 AIDS patients were investigated, and the prevalence of B. hominis infection was 4.16%. Univariate analysis revealed that B. hominis infection correlated with the occupation ( χ 2 = 8.595, P = 0.049), education level ( χ 2 = 14.494, P = 0.001), type of daily drinking water ( χ 2 = 10.750, P = 0.020), root of HIV infections ( χ 2 = 8.755, P = 0.026) and receiving anti-HIV therapy ( χ 2 = 23.083, P = 0.001) among AIDS patients, and multivariate logistic regression analysis identified daily direct drinking of tap water as a risk factor of B. hominis infections [odds ratio ( OR ) = 7.988, 95% confidential interval ( CI ): (1.160, 55.004)] and anti-HIV therapy as a protective factor of B. hominis infection [ OR = 0.183, 95% CI : (0.049, 0.685)]., Conclusions: The prevalence of B. hominis is 4.16% among AIDS patients in Nanchang City. Daily direct drinking of tap water is a risk factor, and anti-HIV therapy is a protective factor of B. hominis infection among AIDS patients living in Nanchang City.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electrical signals. The differentiation and degeneration of photoreceptors are regulated by specific genes and proteins. With the development of epigenetic approaches, scientists have discovered that histone modifications, such as acetylation, methylation, ubiquitylation, and phosphorylation, may modulate the processes of photoreceptor differentiation and degeneration. Histone acetylation is regulated by two opposing classes of enzymes, namely, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which add and remove acetyl groups to and from target histones, respectively, causing changes in transcriptional activity. Herein, we review the effects of HATs and HDACs on the differentiation and degeneration of photoreceptors and discuss the underlying mechanisms of these effects., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Modulation of the embryonic stem cell state is beneficial for elucidating the innate mechanisms of development and regenerative medicine. Ion flux plays important roles in modulating the transition between stemness and differentiation in mouse embryonic stem cells (mESCs). Optogenetics is a novel tool for manipulating ion flux. To investigate the impact of optical stimulation on embryonic stem cells, optogenetically engineered V6.5 mESCs were used to measure the depolarization mediated by ChR2 on the proliferation, self-renewal, and differentiation of mESCs. Blue light stimulation significantly inhibited ChR2-GFP-V6.5 ESC proliferation and disrupted the cell cycle progression, reducing the proportion of cells in the S phase. Interestingly, optical stimulation could inhibit ChR2-GFP-V6.5 ESC self-renewal and trigger differentiation by activating the extracellular regulated protein kinase (ERK) signaling pathway. Our data suggest that membrane potential changes play pivotal roles in regulating the proliferation, self-renewal and initiation of differentiation of mESCs., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

With both spin and valley degrees of freedom, the low-lying excitonic spectra of photoexcited transition-metal dichalcogenide monolayers (TMDC-MLs) are featured by rich fine structures, comprising the intravalley bright exciton states as well as various intra- and intervalley dark ones. The latter states can be classified as those of the spin- and momentum-forbidden dark excitons according to the violated optical selection rules. Because of their optical invisibility, these two types of the dark states are in principle hardly observed and even distinguished in conventional spectroscopies although their impacts on the optical and dynamical properties of TMDC-MLs have been well noticed. In this Letter, we present a theoretical and computational investigation of the exciton fine structures and the temperature-dependent photoluminescence spectra of strained tungsten diselenide monolayers (WSe 2 -MLs) where the intravalley spin-forbidden dark exciton lies in the lowest exciton states and other momentum-forbidden states are in the higher energies that are tunable by external stress. The numerical computations are carried out by solving the Bethe-Salpeter equation for an exciton in a WSe 2 -ML under the stress-control in the tight-binding scheme established from the first principle computation in the density functional theory. According to the numerical computation and supportive model analysis, we reveal the distinctive signatures of the spin- and momentum-forbidden exciton states of strained WSe 2 -MLs in the temperature-dependent photoluminescences and present the guiding principle to infer the relative energetic locations of the two types of dark excitons.

Purpose: A growing body of evidence suggests that the microbiome of the ocular surface confers potent immunoregulatory functions and has a key role in the physiologic maintenance of healthy eyes and in the pathogenesis of ocular diseases. Although the microbiome is known to be affected by age and sex, the influence of these factors on ocular surface microbiota in healthy adults remains largely unknown., Methods: Ocular surface microbiome samples were obtained from the inferior bulbar conjunctiva of 48 young and 42 old adults at Zhongshan Ophthalmic Center. Using metagenomic shotgun sequencing, we characterized the sex- and age-differences in conjunctival microbiome profiles of healthy adults., Results: Male and female groups differed only in the β diversity of bacterial communities, while there were significant differences in bacterial composition, metabolic functions, and the abundance of antibiotic resistance genes between young and old adult groups., Conclusions: Our findings suggest that age and sex collectively shape the conjunctival microbiome, and may change the immune homeostasis of the ocular surface through alterations of its commensal microbiome.

Retinal degenerative diseases are among the leading causes of blindness worldwide, and cell replacement is considered as a promising therapeutic. However, the resources of seed cells are scarce. To further explore this type of therapy, we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore, we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks, resulting in beneficial effects on the morphology of the host retina, and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function.

Purpose: To quantify the transcorneal electrical stimulation (TES)-induced effects on regional photoreceptors and visual signal pathway of N-methyl-N-nitrosourea (MNU)-treated retinas via topographic measurements., Methods: N-methyl-N-nitrosourea-administered mice received TES or sham stimulations and were subsequently subjected to electroretinography (ERG), multielectrode array (MEA), and histologic and immunohistochemistry examinations. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were also performed to determine the mRNA levels of Bax, Bcl-2, Calpain-2, Caspase-3, brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF)., Results: Amplitudes of ERG b-wave in the TES-treated mice were significantly larger than those in the sham controls (P < 0.01). Microelectrode array examination revealed that the photoreceptors in TES-treated retina were efficiently preserved (P < 0.01). Morphologic measurements showed that the central retina region was more consolidated than the other areas in the TES-treated mice. Together with the disproportionate distribution of immunostaining in retinal flat mounts, these findings indicated that different rescuing kinetics existed among regional photoreceptors. Compared with the sham controls, a significantly increased signal-to-noise ratio was also found in the TES-treated mice (TES100: 2.02 ± 1.12; TES200: 4.42 ± 1.51; sham: 0.25 ± 0.13; P < 0.01). Moreover, qRT-PCR measurements suggested that the altered expression of several apoptotic factors and neurotrophic cytokines was correlated with TES-induced protection., Conclusions: Regional photoreceptors in the MNU-administered retinas exhibit different sensitivities to TES. Transcorneal electrical stimulation is capable of ameliorating MNU-induced photoreceptor degeneration and rectifying abnormalities in the inner visual signal pathways.

Transcorneal electrical stimulation (TES) is a novel therapeutic approach to activate the retina and related downstream structures. TES has multiple advantages over traditional treatments, such as being minimally invasive and readily applicable in a routine manner. Series of animal experiments have shown that TES protects the retinal neuron from traumatic or genetic induced degeneration. These laboratory evidences support its utilization in ophthalmological therapies against various retinal and optical diseases including retinitis pigmentosa (RP), traumatic optic neuropathy, anterior ischemic optic neuropathy (AION), and retinal artery occlusions (RAOs). Several pioneering explorations sought to clarify the functional mechanism underlying the neuroprotective effects of TES. It seems that the neuroprotective effects should not be attributed to a solitary pathway, on the contrary, multiple mechanisms might contribute collectively to maintain cellular homeostasis and promote cell survival in the retina. More precise evaluations via functional and morphological techniques would determine the exact mechanism underlying the remarkable neuroprotective effect of TES. Further studies to determine the optimal parameters and the long-term stability of TES are crucial to justify the clinical significance and to establish TES as a popularized therapeutic modality against retinal and optic neuropathy.

Retinitis pigmentosa (RP) is a group of heterogeneous inherited retinal diseases that is characterized by primary death rod photoreceptors and the secondary loss of cones. The degeneration of cones causes gradual constriction of visual fields, leaving the central islands that are eventually snuffed out. Studies indicate that the hyperoxia causes oxidative damage in the retina and contributes to the cone death of RP. Moreover, abundant reactive oxidative species (ROS) which are generated in cones may result in mitochondria membrane depolarization, which has been ascribed a central role in the apoptotic process and has been proposed to act as a forward feeding loop for the activation of downstream cascades. Anthocyanin is a potent antioxidant which has been evidenced to be able to counteract oxidative damages, scavenge surplus ROS, and rectify abnormities in the apoptotic cascade. Taken together with its ability to attenuate inflammation which also contributes to the etiology of RP, it is reasonable to hypothesize that the anthocyanin could act as a novel therapeutic strategy to retard or prevent cone degeneration in RP retinas, particularly if the treatment is timed appropriately and delivered efficiently. Future pharmacological investigations will identify the anthocyanin as an effective candidate for PR therapy and refinements of that knowledge would ignite the hope of restoring the visual function in RP patients.

Objective: To evaluate the effect of schistosomiasis control in towns that had reached the criteria of infection controlled in Nanchang City., Methods: The residents and livestock of 14 towns that had reached the criteria of infection controlled in Nanchang, Xinjian and Jinxian counties of Nanchang City were sampled to investigate the schistosomiasis endemic situation, Oncomelania hupensis snail status, and data of schistosomiasis control work., Results: The positive rate of serum tests of the residents was 2.58%, and no positive stool samples were found. No cattle or sheep were positive in stool tests. Totally 1 635 snails were caught, and 1278 were alive with an average density of living snails of 0.051/0.1 M2, while no schistosome infected snails were found. All the surveyed administrative villages had the completed files of schistosomiasis control. Conclusion All the 14 towns that had reached the criteria of infection controlled of schistosomiasis have already reached the criteria of transmission controlled in Nanchang City.

Definitive surface markers for retinal progenitor cells (RPCs) are still lacking. Therefore, we sorted c-Kit(+) and stage-specific embryonic antigen-4(-) (SSEA4(-)) retinal cells for further biological characterization. RPCs were isolated from human fetal retinas (gestational age of 12-14 weeks). c-Kit(+)/SSEA4(-) RPCs were sorted by fluorescence-activated cell sorting, and their proliferation and differentiation capabilities were evaluated by using immunocytochemistry and flow cytometry. The effectiveness and safety were assessed following injection of c-Kit(+)/SSEA4(-) cells into the subretina of Royal College of Surgeons (RCS) rats. c-Kit(+) cells were found in the inner part of the fetal retina. Sorted c-Kit(+)/SSEA4(-) cells expressed retinal stem cell markers. Our results clearly demonstrate the proliferative potential of these cells. Moreover, c-Kit(+)/SSEA4(-) cells differentiated into retinal cells that expressed markers of photoreceptor cells, ganglion cells and glial cells. These cells survived for at least 3 months after transplantation into the host subretinal space. Teratomas were not observed in the c-Kit(+)/SSEA4(-)-cell group. Thus, c-Kit can be used as a surface marker for RPCs, and c-Kit(+)/SSEA4(-) RPCs exhibit the ability to self-renew and differentiate into retinal cells., (© 2015. Published by The Company of Biologists Ltd.)

Aim: To complete the data of ocular trauma in central China, as a well-known tertiary referral center for ocular trauma, we documented the epidemiological characteristics and visual outcomes of patients hospitalized for ocular trauma in this region., Methods: A retrospective study of patients hospitalized for ocular trauma in central China from 2006 to 2011 was performed., Results: This study included 5964 eyes of 5799 patients. The average age was 35.5±21.8y with a male-to-female ratio of 2.8:1. The most common age was 45-59y age group. Most patients were farmers and workers (51.9%). The most common injuries were firework related (24.5%), road traffic related (24.2%), and work related (15.0%). Among the most common causative agents were firecrackers (24.5%), followed by metal/knife/scissors (21.4%). Most injuries occurred in January (14.2%), February (27.0%), and August (10.0%). There were 8.5% patients with ocular injuries combined with other injuries. The incidence of open ocular injuries (4585 eyes, 76.9%) was higher than closed ocular injuries (939 eyes, 15.7%). The incidences of chemical and thermal ocular injuries were 1.2% and 0.6%. Ocular trauma score (OTS) predicted final visual acuity at non light perception (NLP), 20/200-20/50 and 20/40 with a sensitivity of 100%, and light perception (LP)/hand motion (HM) and 1/200-19/200 with a specificity of 100%., Conclusions: This study provides recent epidemiological data of patients hospitalized for ocular trauma in central China. Some factors influencing the visual outcome include time interval between injury and visit to the clinic, wound location, open or closed globe injury, initial visual acuity, and OTS.

Objective: To study the morphological change of Schistosoma japonicum eggs processed by calcium cyanamide synthetic drug, so as to provide the basis for further study of the mechanism that calcium cyanamide synthetic drug to schistosome eggs., Methods: The calcium cyanamide synthetic drug was added to the cattle feces containing schistosome eggs and mixed up, and then the cattle feces was stacked as original shape on the marshland. Blank controls were set at the same time. The cattle feces samples were collected and.the schistosome eggs were observed under a microscope on the 1st, 2nd, 3rd, 7th day after the experiment., Results: By the effect of calcium cyanamide synthetic drug, the color of eggs was deepening gradually, the miracidia were atrophied, and the shells of eggs were thickened. The embryonic membrane of miracidia was no longer completed 3 days later, and the miracidia were deformed severely 7 days later. The atrophy of miracidia was not obvious in the blank controls., Conclusion: The schistosome miracidia and embryonic membrane can be damaged by the calcium cyanamide synthetic drug, and worse damaged with time extending.

Aim: To evaluate the accuracy of 20 MHz immersion B-scan ultrasonography in observing lens and to investigate the value of this noninvasive preoperative diagnosis method in alkali burn eyes., Methods: It was a comparative study. Fifty-six cases (56 eyes) of alkali burn eyes were examined by ultrasound biomicroscopy (UBM) and immersion 20 MHz B-scan ultrasonography from June 2011 to April 2013, the images were analyzed, and the ultrasonographic diagnosis compared with the operation results., Results: In 56 alkali burn eyes examined by UBM, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; the anterior lens capsule surface or/and the front lens could be detected in 18 eyes, and lens opacification in 3 eyes of them; suspected abnormal lens were detected in the other 20 eyes. In all the same eyes examined by immersion 20 MHz B-scan ultrasonography, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; 24 abnormal lens (opacity, lens expansion, shrinkage) and 14 normal lens were found. Compared with the intraoperative findings, the diagnostic accordance rate of the immersion 20 MHz B-scan appearance of lens was 100% (56/56), which was significantly higher than examined by UBM 57.14% (32/56) (χ(2)=30.55, P=0.0000)., Conclusion: Immersion 20 MHz B-scan ultrasonography can observe the lens accurately in alkali burn eyes. It has important clinical value to combine with UBM in eyes of alkali burn.

Aim: To evaluate the accuracy of axial length (AL) measurements obtained from immersion B-scan ultrasonography (immersion B-scan) for intraocular lens (IOL) power calculation in patients with high myopia and cataracts., Methods: Immersion B-scan, contact A-scan ultrasonography (contact A-scan), and the IOLMaster were used to preoperatively measure the AL in 102 eyes from 102 patients who underwent phacoemulsification and IOL implantation. Patients were divided into two groups according to the AL: one containing patients with 22 mm≤AL<26 mm(group A) and the other containing patients with AL≥26 mm (group B). The mean error (ME) was calculated from the difference between the AL measurement methods predicted refractive error and the actual postoperative refractive error., Results: In group A, ALs measured by immersion B-scan (23.48±1.15) didn't differ significantly from those measured by the IOLMaster (23.52±1.17) or from those by contact A-scan (23.38±1.20). In the same group, the standard deviation (SD) of the mean error (ME) of immersion B-scan (-0.090±0.397 D) didn't differ significantly from those of IOLMaster (-0.095±0.411 D) and contact A-scan (-0.099±0.425 D). In group B, ALs measured by immersion B-scan (27.97±2.21 mm) didn't differ significantly from those of the IOLMaster (27.86±2.18 mm), but longer than those measured by Contact A-scan (27.75±2.23 mm, P=0.009). In the same group, the standard deviation (SD) of the mean error (ME) of immersion B-scan (-0.635±0.157 D) didn't differ significantly from those of the IOLMaster (-0.679±0.359 D), but differed significantly from those of contact A-scan (-0.953±1.713 D, P=0.028)., Conclusion: Immersion B-scan exhibits measurement accuracy comparable to that of the IOLMaster, and is thus a good alternative in measuring AL in eyes with high myopia when the IOLMaster can't be used, and it is more accurate than the contact A-scan.

Objective: To understand the endemic regulation of acute schistosomiasis in Nanchang City of Jiangxi Province from 2005 to 2012 so as to provide the reference for policy-making of schistosomiasis control., Methods: The data of acute schistosomiasis were collected and analyzed with the descriptive epidemiology method in Nanchang City from 2005 to 2012., Results: A total of 52 acute schistosomiasis cases were reported in Nanchang City from 2005 to 2012. The morbidity of acute schistosomiasis was 0.003 46% in 2005, and it was 0 in 2012. The morbidity of acute schistosomiasis in different years was significant different (chi2 = 61.077, P < 0.01). The duration of incidence was from May to November, but the peak of the incidence was July. The sex ratio between males and females was 4.78:1 (43:9, chi2 = 22.231, P < 0.01). The ages of cases showed double humps, namely the 6-15 years' group and 30-40 years' group. Most of the acute cases were farmers (65.38%), students (25.00%), and fishermen (3.85%). Fishing and swimming were the main infection ways, with the infection rates of 63.46% and 26.92%, respectively., Conclusion: The susceptible populations are peasants and young students; therefore, we should strengthen health education for these high-risk groups.

Rod and cone photoreceptor neurons in the mammalian retina possess specialized cellular architecture and functional features for converting light to a neuronal signal. Establishing and maintaining these characteristics requires appropriate expression of a specific set of genes, which is tightly regulated by a network of photoreceptor transcription factors centered on the cone-rod homeobox protein CRX. CRX recruits transcription coactivators p300 and CBP to acetylate promoter-bound histones and activate transcription of target genes. To further elucidate the role of these two coactivators, we conditionally knocked out Ep300 and/or CrebBP in differentiating rods or cones, using opsin-driven Cre recombinase. Knockout of either factor alone exerted minimal effects, but loss of both factors severely disrupted target cell morphology and function: the unique nuclear chromatin organization seen in mouse rods was reversed, accompanied by redistribution of nuclear territories associated with repressive and active histone marks. Transcription of many genes including CRX targets was severely impaired, correlating with reduced histone H3/H4 acetylation (the products of p300/CBP) on target gene promoters. Interestingly, the presence of a single wild-type allele of either coactivator prevented many of these defects, with Ep300 more effective than Cbp. These results suggest that p300 and CBP play essential roles in maintaining photoreceptor-specific structure, function and gene expression.

Objective: To understand the dynamics of human infection and reinfection of Schistosoma japonicum after chemotherapy in marshland and lake endemic regions so as to provide the evidence for developing a cost-effective control strategy., Methods: From 2007 to 2011, the schistosome infection, intensities and reinfection of population were investigated and the results were analyzed dynamically in Caohui Village, a national schistosomiasis surveillance site in Changyi Township, Xinjian County, Jiangxi Province, China., Results: The schistosome infection rate was 3.55% and the mean intensity was 1.56 in 2007, and the schistosome infection rate was 1.52% and the mean intensity was 1.60 in 2011. The reinfection rates were 2.70%, 9.09% and 2.47% in 2009, 2010, and 2011, respectively., Conclusion: The schistosomiasis endemic situation shows a downtrend in the village. However, the reinfection is still serious; therefore, we should strengthen the research of risk factors of reinfection and control them.

Mitochondrial 12S rRNA A1555AG mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. We report here the clinical, genetic and molecular characterization of 25 Chinese families carrying the A1555G mutation.Clinical and genetic characterizations of these Chinese families exhibited a wide range of penetrance, severity and age-at-onset of hearing impairment. The average penetrances of deafness were 28.1% and 21.5%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 1 and 15 years old. Their mitochondrial genomes exhibited distinct sets of polymorphisms including 16 novel variants, belonging to ten Eastern Asian haplogroups A, B, D, F, G, M, N and R, respectively. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, 7 known secondary mutations and 21 variants resided at the highly conservative residues may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in GJB2 gene suggested that GJB2 may not be a modifier for the phenotypic expression of the A1555G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes and other modifiers may modulate the variable penetrance and expressivity of deafness among these Chinese families.

Combinatorial binding of transcription factors (TFs) and cofactors to specific regulatory regions of target genes in vivo is an important mechanism of transcriptional regulation. Chromatin immunoprecipitation (ChIP) is a powerful technique to detect protein binding to specific regions of target genes in vivo. However, conventional ChIP analysis for individual factors (single ChIP) does not provide information on co-occupancy of two interacting TFs on target genes, even if both bind to the same chromatin regions. Double ChIP analysis involves sequential (double) immunoprecipitation of two chromatin-binding proteins and can be used to study co-occupancy of two or more factors on specific regions of the same DNA allele. Furthermore, by including a cell type-specific protein in double-ChIP, target co-occupancy in a specific cell type can be studied even if the other partner is more widely expressed. In this chapter, we describe a detailed protocol for double ChIP analysis in mouse retinas. Using the rod-specific transcription factor NR2E3 and the cone/rod homeobox protein CRX as examples, we show that NR2E3 and CRX are co-enriched on the promoter of active Rho and Rbp3 genes in rods, but are present to a much lesser degree on the promoters of silent cone opsin genes. These results suggest a new mechanism by which rod and cone genes are differentially regulated by these transcription factors in rod photoreceptors.

Objective: To investigate mutational spectrum and frequency of the mitochondrial 12S rRNA gene in Chinese subjects with aminoglycoside-induced and non-syndromic hearing loss., Methods: Total of 456 subjects with non-syndromic hearing loss were recruited from seven schools for deaf-mutes in Zhejiang province. Genomic DNA was extracted from the whole blood, and then the DNA fragment was amplified spanning the 12S rRNA gene, followed by sequencing and analyzed., Results: Thirty-one variants were identified by mutation analysis of 12S rRNA gene in these subjects. The frequency of the known 1555A > G mutation was 4.4% (20/456). Prevalence of other putative deafness-associated mutation at positions 961 and 1095 were 2.0% (9/456) and 0.7% (3/456) respectively. Furthermore, the 1027A > G, 1109T > C and 1431G > A variants conferred increased sensitivity to ototoxic drugs or non-syndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this 12S rRNA gene. Moreover, clinical data showed a wide range of age-of-onset, variety of severity and various audiometric configurations in subjects carrying the 1555A > G mutation., Conclusions: Our data demonstrated that the mitochondrial 12S rRNA gene is the hot spot for mutations associated with aminoglycoside ototoxicity and non-syndromic hearing loss. Nuclear modifier genes, mitochondrial haplotypes and environmental factors might play a role in the phenotypic manifestation of these mutations.

Objective: To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations, haplotypes, GJB2 gene mutations on phenotype of 1494C>T mutation, and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss., Methods: Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected. The two probands and their family members underwent clinical, genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene., Results: Clinical evaluation revealed wide range of severity, age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families, for which the penetrance of hearing loss was respectively 42.9% and 28.6% when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss were 14.3% and 14.3%. Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation, in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c, respectively., Conclusion: Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families. Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression. However, aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families. The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation. The 1494C>T mutation seems to have occurred sporadically through evolution.

Mitochondrial DNA (mtDNA) mutations are one of the important causes of deafness. In particular, the 12S rRNA gene is the hot spots for mutations associated with both aminoglycoside ototoxicity and nonsyndromic deafness. In this report, a total of 318 Chinese pediatric hearing-impaired subjects were recruited from otology clinics in the Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of 12S rRNA gene. Mutational analysis identified 34 variants in the 12S rRNA gene in this cohort. The incidences of the known deafness-associated 1555A>G, 1494C>T and 1095T>C mutations were 9.1%, 0.6% and 1.25% in this cohort, respectively. Other mtDNA variants were evaluated by structural and phylogenetic analysis. Of these, the 839A>G and 1452T>C variants could confer increased sensitivity to aminoglycosides or nonsyndromic deafness as they were not present in 449 Chinese controls and localized at highly conserved nucleotides of the 12S rRNA. However, other variants appeared to be polymorphisms. These data further support the idea that mitochondrial 12S rRNA is one of major targets for aminoglycoside ototoxicity. These data have been providing valuable information to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

Dechlorinated water (100 ml, 30 degrees C) was put into a plate (diameter 15 cm), and 1% niclosamide spreading oil 5 microl was added, then a ring of Schistosoma japonicum cercariae were picked up to the plate. The time of killing all the cercariae was observed at three time points (immediately, 24, 48 h), and the dechlorinated water was used as control. The results showed that schistosome cercariae were all killed in three minutes by 1% niclosamide spreading oil at the three time points. The cercaria-killing effects of each time point were not significantly different (F = 0.062, P > 0.05). The cercariae were alive in the control in 48 h.

Objective: To explore the influencing factors of the significant decrease of snail densities in Poyang Lake region, Nanchang City, 2011., Methods: According to the elevation of the environment, 5 grasslands where there were high snail densities and their elevations were from 14 to 17 m were selected as study areas. The data of snails in the past three years and the hydrological information in the past five years were collected and analyzed. The current snail status was surveyed and the fishermen were interviewed., Results: The snail density of Poyang Lake region in Jinxian County decreased by 91.2% in 2011 compared with that in 2009 (P < 0.01). In 2010, the water levels were 13.90 m in April and 16.07 m in May respectively in Xingzi Station, and the rainfalls were 312 mm in April and 356 mm in May in Poyang Lake region; in 2011, the water levels were 8.73 m in April and 10.31 m in May respectively in Xingzi Station, and the rainfalls were 90 mm in April and 145 mm in May respectively in Poyang Lake region, which were lower than those during the past five years. There was no any other apparently unusual phenomenon., Conclusion: The significant decrease of snail densities in Poyang Lake region in 2011 may be related to the long soaking time of marshland in 2010 and low rainfall in the first half of 2011.

Chromosome conformation capture (3C) is a biochemical assay to reveal higher order chromosomal organizations mediated by physical contact between discrete DNA segments in vivo. Chromosomal organizations are involved in transcriptional regulation of a number of genes in various cell types. We have adapted 3C for analyzing the intrachromosomal looping organization of rod and cone photoreceptor genes in the mammalian retina. Here, we describe a detailed protocol for 3C assays on whole mouse retinas. Using the M-cone opsin gene as an example, we demonstrate how to genetically distinguish 3C signals from cones versus rods in retinal 3C assays. We also describe the challenges and key points of 3C design and performance as well as appropriate controls and result interpretations.

Rod and cone opsin genes are expressed in a mutually exclusive manner in their respective photoreceptor subtypes in the mammalian retina. Previous transgenic mouse studies showed that functional interactions between the distal enhancer and proximal promoter of rhodopsin and long/medium-wavelength (L/M) opsin genes are essential for regulating their cell-type-specific transcription. We have used chromosomal conformation capture assays in mouse retinas to investigate the molecular mechanism responsible for this interaction. Here we show that each opsin gene forms intrachromosomal loops in the appropriate photoreceptor subtype, while maintaining a linear configuration in other cell types where it is silent. The enhancer forms physical contacts not only with the promoter but also with the coding regions of each opsin locus. ChIP assays showed that cell-type-specific target binding by three key photoreceptor transcription factors-cone--rod homeobox (CRX), neural retina leucine zipper (NRL), and nuclear receptor subfamily 2, group E, member 3 (NR2E3)--is required for the appropriate local chromosomal organization and transcription of rod and cone opsins. Similar correlations between chromosomal loops and active transcription of opsin genes were also observed in human photoreceptors. Furthermore, quantitative chromosomal conformation capture on human retinas from two male donors showed that the L/M enhancer locus control region (LCR) loops with either the L or M promoter in a near 3:1 ratio, supporting distance-dependent competition between L and M for LCR. Altogether, our results suggest that the photoreceptor transcription factor network cooperatively regulates the chromosomal organization of target genes to precisely control photoreceptor subtype-specific gene expression.

The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl(-/-) rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORβ can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORβ directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORβ and later maintained at high levels by CRX and RORβ.