Effects of fluorescent protein tdTomato on mouse retina.

Fluorescent proteins (FPs) have been widely used to investigate cellular and molecular interactions and trace biological events in many applications. Some of the FPs have been demonstrated to cause undesirable cellular damage by light-induced ROS production in vivo or in vitro. However, it remains unknown if one of the most popular FPs, tdTomato, has similar effects in neuronal cells. In this study, we discovered that tdTomato expression led to unexpected retinal dysfunction and ultrastructural defects in the transgenic mouse retina. The retinal dysfunction mainly manifested in the reduced photopic electroretinogram (ERG) responses and decreased contrast sensitivity in visual acuity, caused by mitochondrial damages characterized with cellular redistribution, morphological modifications and molecular profiling alterations. Taken together, our findings for the first time demonstrated the retinal dysfunction and ultrastructural defects in the retinas of tdTomato-transgenic mice, calling for a more careful design and interpretation of experiments involved in FPs.

Chromone-deferiprone hybrids as novel MAO-B inhibitors and iron chelators for the treatment of Alzheimer's disease.

A series of chromone-deferiprone hybrids were designed, synthesized, and evaluated as inhibitors of human monoamine oxidase B (hMAO-B) with iron-chelating activity for the treatment of Alzheimer's disease (AD). The majority exhibited moderate inhibitory activity towards hMAO-B and potent iron-chelating properties. Particularly, compound 25c demonstrated remarkable selectivity against hMAO-B with an IC50 value of 1.58 µM and potent iron-chelating ability (pFe3+ = 18.79) comparable to that of deferiprone (pFe3+ = 17.90). Molecular modeling and kinetic studies showed that 25c functions as a non-competitive hMAO-B inhibitor. According to the predicted results, compound 25c can penetrate the blood-brain barrier (BBB). Additionally, it has been proved to display significant antioxidant activity and the ability to inhibit neuronal ferroptosis. More importantly, compound 25c reduced the cognitive impairment induced by scopolamine and showed significant non-toxicity in short-term toxicity assays. In summary, compound 25c was identified as a potential anti-AD agent with hMAO-B inhibitory, iron-chelating and anti-ferroptosis activities.

Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin.

Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate <30%. We have developed a cancerous model of Rb in retinal organoids derived from genetically engineered human embryonic stem cells (hESCs) with a biallelic mutagenesis of the RB1 gene. These organoid Rbs exhibit properties highly consistent with Rb tumorigenesis, transcriptome, and genome-wide methylation. Single-cell sequencing analysis suggests that Rb originated from ARR3-positive maturing cone precursors during development, which was further validated by immunostaining. Notably, we found that the PI3K-Akt pathway was aberrantly deregulated and its activator spleen tyrosine kinase (SYK) was significantly up-regulated. In addition, SYK inhibitors led to remarkable cell apoptosis in cancerous organoids. In conclusion, we have established an organoid Rb model derived from genetically engineered hESCs in a dish that has enabled us to trace the cell of origin and to test novel candidate therapeutic agents for human Rb, shedding light on the development and therapeutics of other malignancies.

Antioxidant and anti-tyrosinase activity of a novel stilbene analogue as an anti-browning agent.

BACKGROUND: Tyrosinase inhibitors find potential application in food, cosmetic and medicinal products, but most of the identified tyrosinase inhibitors are not suitable for practical use because of safety regulations or other problems. For the purpose of development of novel tyrosinase inhibitors that meet the requirement for practical application, a novel stilbene analogue (SA) was designed. RESULTS: SA was found to possess a potent inhibitory effect against both mono- and diphenolase activities of mushroom tyrosinase, with IC50 values of 1.56 and 7.15 µmol L-1 , respectively. Compared with a natural tyrosinase inhibitor - kojic acid - the anti-tyrosinase effect of SA was significantly improved. Analysis of inhibition kinetics indicated that SA was a reversible and competitive-noncompetitive mixed-type inhibitor. SA was also found to possess more potent antioxidant activities (DPPH, superoxide anion radical and hydroxyl radical scavenging ability) than those of kojic acid. Cell viability studies revealed that SA was non-toxic to two cell lines. Furthermore, an anti-browning test demonstrated that SA effectively delayed the blackening of shrimp. CONCLUSION: SA has potential as an anti-browning agent in foods. © 2021 Society of Chemical Industry.

The road to restore vision with photoreceptor regeneration.

Neuroretinal diseases are the predominant cause of irreversible blindness worldwide, mainly due to photoreceptor loss. Currently, there are no radical treatments to fully reverse the degeneration or even stop the disease progression. Thus, it is urgent to develop new biological therapeutics for these diseases on the clinical side. Stem cell-based treatments have become a promising therapeutic for neuroretinal diseases through the replacement of damaged cells with photoreceptors and some allied cells. To date, considerable efforts have been made to regenerate the diseased retina based on stem cell technology. In this review, we overview the current status of stem cell-based treatments for photoreceptor regeneration, including the major cell sources derived from different stem cells in pre-clinical or clinical trial stages. Additionally, we discuss herein the major challenges ahead for and potential new strategy toward photoreceptor regeneration.

miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance.

MicroRNAs (miRNAs) are known to be essential for retinal maturation and functionality; however, the role of the most abundant miRNAs, the miR-183/96/182 cluster (miR-183 cluster), in photoreceptor cells remains unclear. Here we demonstrate that ablation of two components of the miR-183 cluster, miR-183 and miR-96, significantly affects photoreceptor maturation and maintenance in mice. Morphologically, early-onset dislocated cone nuclei, shortened outer segments and thinned outer nuclear layers are observed in the miR-183/96 double-knockout (DKO) mice. Abnormal photoreceptor responses, including abolished photopic electroretinography (ERG) responses and compromised scotopic ERG responses, reflect the functional changes in the degenerated retina. We further identify Slc6a6 as the cotarget of miR-183 and miR-96. The expression level of Slc6a6 is significantly higher in the DKO mice than in the wild-type mice. In contrast, Slc6a6 is down-regulated by adeno-associated virus-mediated overexpression of either miR-183 or miR-96 in wild-type mice. Remarkably, both silencing and overexpression of Slc6a6 in the retina are detrimental to the electrophysiological activity of the photoreceptors in response to dim light stimuli. We demonstrate that miR-183/96-mediated fine-tuning of Slc6a6 expression is indispensable for photoreceptor maturation and maintenance, thereby providing insight into the epigenetic regulation of photoreceptors in mice.

Twin delivery after IVF-ET with variable dose letrozole-FSH protocol of lower estradiol in a patient previously treated for breast cancer: a case report.

OBJECTIVE: To present a case of twin pregnancy obtained by in vitro fertilization and embryo transfer (IVF-ET) with variable dose letrozole-FSH protocol of lower peak estradiol level, after treatment of carcinoma of the breast. MATERIALS AND METHODS: A 34-year-old patient diagnosed with mucinous breast carcinoma undergoing assisted fertilization treatment after breast cancer operation and treatment including controlled ovarian stimulation (COS), oocyte retrieval, IVF, and embryo culture and transfer. RESULTS: Four oocytes were obtained in three COS procedures in the three IVF cycle. All oocytes were fertilized. In the third cycle, two fresh embryos were transferred, and two healthy girls were born at 37 gestational weeks. CONCLUSION: Variable dose letrozole-FSH protocol can maintain lower peak estradiol levels and reduce estrogen exposure after breast cancer operation and chemotherapy.

[Association study of telomere length with idiopathic male infertility].

Telomeres are evolutionary conserved, multifunctional DNA-protein complexes located at the ends of eukaryotic chromosomes. Telomeres maintain chromosome stability and genome integrity and also play an important role in meiosis which aid in synapsis, homologous recombination, and segregation. Sperm telomere has been reported to play an important role in fertilization and embryo development. Nowadays, the association between telomere and reproduction is one of the major areas of interest, however whether sperm telomere associated with male infertility is not clear. In this study, in order to find out the association between Chinese idiopathic infertility and sperm telomere length, we analyzed the difference of sperm telomere length between idiopathic infertile men and normal fertile men, as well as the correlations between sperm telomere length and human semen characteristics. We analyzed 126 Chinese idiopathic infertile men and 138 normal fertile men for sperm telomere length by using quantitative PCR. We found that the relative sperm mean telomere length of infertile men was significantly shorter than that of fertile men (2.894 ± 0.115 vs. 4.016 ± 0.603, P=5.097 x 10⁻5). Both sperm count and semen progressive motility are related with telomere length. Our results suggest that sperm telomere length is associated with idiopathic male infertility of China and we proposed the possibility that shorter telomeres in sperm chromosome will reduce spermatogenesis and sperm functions, which finally affected the fertility of male.

[Strategies of sperm collection from men with temporary penile erectile dysfunction on the day of oocyte retrieval and the outcomes of IVF-ET].

OBJECTIVE: To search for the optimal strategies for sperm collection from the patient with temporary penile erectile dysfunction (ED) on the day of oocyte pick-up ( OPU) in in vitro fertilization embryo transfer (IVF-ET). METHODS: We retrospectively analyzed 93 cases of temporary ED on the OPU day of IVF-ET from January 2011 to May 2014, with fresh semen for 45 cases (group A), cryopreserved sperm before oocyte retrieval for 30 cases (group B), and frozen oocytes for 18 cases (group C). Group A was again subdivided into A1 (n = 18) and A2 n = 27) , the former intervened with oral sildenafil while the latter left untreated. We compared the rates of fertilization, high-quality embryo, and pregnancy among different groups. RESULTS: No statistically significant differences were found among groups A, B and C in the age of the males and females, duration of infertility, numbers of obtained and mature oocytes, and rates of cleavage, or in the percentages of normal fertilization (80.78% vs 80.43% vs 84.77%), high-quality embryo (53.27% vs 52.97% vs 47.69%) and pregnancy (60.00% vs 56.77% vs 44.44%) (all P > 0.05). The rate of 3PN was markedly lower in group C (0.63%) than in A (9. 61%) and B (4.34%) (P < 0.05). There were no significant differences between groups A1 and A2 in the age of the males and females, duration of infertility, numbers of obtained and mature oocytes, and the rates of fertilization, cleavage, high-quality embryo, and pregnancy (all P > 0.05). CONCLUSION: On the OPU day of IVF-ET, oral sildenafil can help temporary ED men to achieve penile erection and ejaculation without affecting the outcomes of assisted reproduction. Cryopreserved sperm can be used in case of predicted temporary ED and frozen oocytes can also be employed if sperm retrieval fails. However, to avoid puncture injury to the epididymis or testis, fresh semen should be the first choice.

REG1A protects retinal photoreceptors from blue light damage.

With the increased use of artificial light and the prolonged use of optoelectronic products, light damage (LD) to the human retina has been identified as a global vision-threatening problem. While there is evidence of a significant correlation between light-induced retinal damage and age-related vision impairment in age-related macular degeneration, it is unclear how light-induced retinal degeneration manifests itself and whether there are agents capable of preventing the development of LD in the retina. This study investigated a mechanism by which blue light leads to photoreceptor death. By observing blue light exposure in retinal organoids and photoreceptor cells, we concluded that there could be significant apoptosis of the photoreceptors. We demonstrate that regenerating islet-derived 1 alpha (REG1A) prevents photoreceptors from undergoing this LD-induced apoptosis by increasing expression of the anti-apoptotic gene Bcl2 and downregulating expression of the pro-apoptotic gene Bax, resulting in reduced mitochondrial damage and improved aerobic capacity in photoreceptor cells. For the first time, REG1A has been shown to restore mitochondrial function and cell apoptosis after LD-induced damage, suggesting its potential application in the prevention and treatment of retinal vision loss.

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

[Association between single nucleotide polymorphisms of 5'-untranslated region of GPx4 gene and male infertility].

OBJECTIVE: To study the association between the single nucleotide polymorphisms (SNPs) of the 5'-untranslated region (5'-UTR) of phospholipid hydroperoxide glutathione peroxidase (GPx4 or PHGPx) gene and oligo- or asthenozoospermic male infertility. METHODS: The 5'-UTR region of the GPx4 gene was amplified from infertile men and controls using the polymerase chain reaction and was analyzed for polymorphisms by direct sequencing. RESULTS: A total of 9 SNPs were present in the cohort, however there were no significant differences in these 9 SNPs between the case and control groups. According to the results of linkage disequilibrium analysis and haplotype construction, one haplotype (rs757229-rs757230-rs4588110-rs3746165-rs3746166: C-G-G-T-A) was present only in the control men, and significant difference was detected(P< 0.01). CONCLUSION: The SNPs of 5'-UTR region of the GPx4 gene might not be associated with oligo- or asthenozoospermic male infertility. However, the haplotype (rs757229-rs757230-rs4588110- rs3746165-rs3746166: C-G-G-T-A) might be a protective haplotype.

[(E)-N'-(5-Chloro-2-oxidobenzyl-idene-κO)-3,4,5-trimeth-oxy-benzohydrazidato-κN',O](pyridine-κN)copper(II).

In the title compound, [Cu(C(17)H(15)ClN(2)O(5))(C(5)H(5)N)], the Cu(II) atom is coordinated by one N atom and two O atoms from an anionic salicyl-aldehyde benzoyl-hydrazone ligand and one pyridine N atom in a distorted square-planar geometry. The bonds displays the usual elongation with mean Cu-O and Cu-N bond lengths of 1.926 and 1.976 Å, respectively. The pyridine ring makes dihedral angles of 26.12 (13) and 11.08 (12)°, respectively, with the trimeth-oxy-phenyl and phenolate rings, which make a dihedral angle of 16.05 (12)° with one another.

Retinal Degeneration Caused by Ago2 Disruption.

Purpose: Argonaute proteins are key players in small RNA-guided gene silencing processes. Ago2 is the member of the Argonaute subfamily with slicer endonuclease activity and is critical for microRNA homeostasis and indispensable for biological development. However, the impact of Ago2 dysregulation in the retina remains to be fully explored. In this study, we studied the role of Ago2 in mouse retina. Methods: We explored the function of Ago2 in the mouse retina through an adeno-associated virus-mediated Ago2 disruption mouse model. An ERG was carried out to determine the retinal function. Spectral domain optical coherence tomography, fundus photographs, and immunostaining were performed to investigate the retinal structure. A quantitative RT-PCR assay was used to determine the expression of noncoding RNAs. Results: Both silencing and overexpression of Ago2 in mouse retina resulted in significant retinal morphological alterations and severe impairment of retinal function, mainly with a thinned outer nuclear layer, shortened inner segment/outer segment, and diminished ERG responses. Furthermore, Ago2 disruption resulted in alterations of noncoding RNAs in retina. Conclusions: Our finding demonstrated that Ago2 interruption led to severe retinal degeneration, suggested that Ago2 homeostasis contributed to retinal structural and functional maintenance.

Transplantation of GMP-grade human iPSC-derived retinal pigment epithelial cells in rodent model: the first pre-clinical study for safety and efficacy in China.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly due in large part to age-dependent atrophy of retinal pigment epithelium (RPE) cells. RPE cells form a monolayer located between the choroid and the outer segments of photoreceptors, playing multifarious roles in maintenance of visual function. Allogeneically induced pluripotent stem cell-derived RPE (iPSC-RPE or iRPE) has become a potential approach for providing an abundant source of donors for clinical cell products. Transplantation of iRPE has been proven effective in rescuing impaired retinas in Royal College of Surgeons (RCS) rats after approximately 5 to 6 weeks. Here, we explore the long-term (19 weeks) safety and efficacy of human iRPE cell transplantation in pre-clinical animal models. METHODS: The expression of human RPE-specific markers in iRPE cells was determined using immunofluorescence staining. For the proliferative test, Ki-67 expression was also verified by immunofluorescence and flow cytometric analysis. Then, iRPE cells were transplanted into the subretinal space of immune-deficient NOD/SCID/IL-2Rgcnull (NSG) mice to assess their safety. To evaluate whether the transplanted cells could survive and rescue visual function, we performed color fundus photography, focal electroretinogram and immunostaining after delivering iRPE cells into the subretinal space of RCS rats. RESULTS: Human iRPE cells expressed native RPE-specific markers, such as microphthalmia-associated transcription factor (MiTF), retinal pigment epithelium-specific 65-kDa protein (RPE65) and tight-junction associated structural protein (ZO-1), and their proliferative capacity (Ki-67 expression) was poor after 25 days of induction. A tumorigenicity test revealed no tumor formation or abnormal proliferation in the immunodeficient mice after subretinal injection of 5×105 iRPE cells. The transplanted iRPE cells survived for at least 19 weeks and maintained visual function for 15 weeks. CONCLUSIONS: In the present study, we provided further evidence for the use of human iRPE transplantation to treat retinal degenerative disease in pre-clinical animal models. Therefore, we consider human iRPE cells a promising source of cell replacement therapy for AMD.

Generation of Nonhuman Primate Model of Cone Dysfunction through In Situ AAV-Mediated CNGB3 Ablation.

A major challenge to the development of therapies for human retinal degenerative diseases is the lack of an ideal preclinical model because of the physiological differences between humans and most model animals. Despite the successful generation of a primate model through germline knockout of a disease-causing gene, the major issues restricting modeling in nonhuman primates (NHPs) are their relatively long lifespan, lengthy gestation, and dominant pattern of singleton births. Herein, we generated three cynomolgus macaques with macular in situ knockout by subretinal delivery of an adeno-associated virus (AAV)-mediated CRISPR-Cas9 system targeting CNGB3, the gene responsible for achromatopsia. The in vivo targeting efficiency of CRISPR-Cas9 was 12%-14%, as shown by both immunohistochemistry and single-cell transcriptomic analysis. Through clinical ophthalmic examinations, we observed a reduced response of electroretinogram in the central retina, which corresponds to a somatic disruption of CNGB3. In addition, we did not detect CRISPR-Cas9 residue in the heart, liver, spleen, kidney, brain, testis, or blood a year after administration. In conclusion, we successfully generated a NHP model of cone photoreceptor dysfunction in the central retina using an in situ CNGB3-knockout strategy.

Abundant Neural circRNA Cdr1as Is Not Indispensable for Retina Maintenance.

Cdr1as is the abundant circular RNA (circRNA) in human and vertebrate retinas. However, the role of Cdr1as in the retina remains unknown. In this study, we aimed to generate a Cdr1as knockout (KO) mouse model and investigate the retinal consequences of Cdr1as loss of function. Through in situ hybridization (ISH), we demonstrated that Cdr1as is mainly expressed in the inner retina. Using CRISPR/Cas9 targeting Cdr1as, we successfully generated KO mice. We carried out ocular examinations in the KO mice until postnatal day 500. Compared with the age-matched wild-type (WT) siblings, the KO mice displayed increased b-wave amplitude of photopic electrophysiological response and reduced vision contrast sensitivity. Through small RNA profiling of the retinas, we determined that miR-7 was downregulated, while its target genes were upregulated. Taken together, our results demonstrated for the first time that Cdr1as ablation led to a mild retinal consequence in mice, indicating that Cdr1as abundance is not indispensable for retinal development and maintenance.

Ablation of Mature miR-183 Leads to Retinal Dysfunction in Mice.

Purpose: The microRNA cluster miR-183C, which includes miR-183 and two other genes, is critical for multiple sensory systems. In mouse retina, removal of this cluster results in photoreceptor defects in polarization, phototransduction, and outer segment elongation. However, the individual roles of the three components of this cluster are not clearly known. We studied the separate role of mouse miR-183 in in vivo. Methods: miR-183 knockout mice were generated using the CRISPR/Cas9 genome-editing system. Electroretinography were carried out to investigate the changes of retinal structures and function. miR-183 was overexpressed by subretinal adeno-associated virus (AAV) injection in vivo. Rnf217, a target of miR-183 was overexpressed by cell transfection of the photoreceptor-derived cell line 661W in vitro. RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to compare the gene expression changes in AAV-injected mice and transfected cells. Results: The miR-183 knockout mice showed progressively attenuated electroretinogram responses. Over- or under-expression of Rnf217, a direct target of miR-183, misregulated expression of cilia-related BBSome genes. Rnf217 overexpression also led to compromised electroretinography responses in WT mice, indicating that it may contribute to functional abnormalities in miR-183 knockout mice. Conclusions: miR-183 is essential for mouse retinal function mediated directly and indirectly through Rnf217 and cilia-related genes. Our findings provide valuable insights into the explanation and analysis of the regulatory role of the individual miR-183 in miR-183C.

The Circular RNome of Developmental Retina in Mice.

Circular RNAs (circRNAs) represent a class of noncoding RNAs with a wide expression pattern, and they constitute an important layer of the genome regulatory network. To date, the expression pattern and regulatory potency of circRNAs in the retina, a key part of the central nervous system, are not yet well understood. In this study, RNAs from five stages (E18.5, P1, P7, P14, and P30) of mouse retinal development were sequenced. A total of 9,029 circRNAs were identified. Most circRNAs were expressed in different stages with a specific signature, and their expression patterns were different from those of their host linear transcripts. Some circRNAs could act as sponges for several retinal microRNAs (miRNAs). Furthermore, circTulp4 could function as a competitive endogenous RNA (ceRNA) to regulate target genes. Remarkably, silencing circTulp4 in vivo led to mice having a thin outer nuclear layer (ONL) and defective retinal function. In addition, we found that circRNAs were dysregulated at a much earlier time point than that of disease onset in a retinal degeneration model (rd8 mice). In summary, we provide the first circRNA expression atlas during retinal development and highlight a key biological role for circRNAs in retinal development and degeneration.

[Establishment of immortalized cell lines and genetic stability evaluation for a family with spinocerebellar ataxia type 2].

OBJECTIVE: Immortalized cell lines of spinocerebellar ataxia type 2 (SCA2) with Parkinson disease symptoms were established in order to provide experimental material for future study. METHODS: The immortalized cell lines were constructed by using Epstein Barr virus and cyclosporine A. Microsatellite markers were detected to see whether there is any change between the cell lines and the original blood samples, and the genetic stability of the cell lines were evaluated. RESULTS: Twenty-five immortalized cell lines were established successfully from the family and the microsatellite markers were unchanged. CONCLUSION: The karyotypes of the immortal cell lines were normal and the cell lines were genetically stable.