[Characterization of Microplastic Surface Bacterial Community Structure and Prediction of Ecological Risk in Poyang Lake, China].

In recent years, the environmental pollution of microplastics in Poyang Lake has received increasing attention. Baisha Lake of Poyang Lake was selected as the study area, and samples of water and sediments of Baisha Lake and the microplastics therein were collected, and the polymer types of microplastics were identified as polyethylene (PE), polyester (PET), polypropylene (PP), and polystyrene (PS) using Fourier infrared spectroscopy. We also analyzed the structural composition of bacterial communities in water, in sediments, and on microplastic surfaces using 16S high-throughput sequencing. The species richness and diversity of bacteria on the microplastic surfaces were lower than those in the surrounding water and sediments. The results of NMDS analysis showed that the bacterial community structures on the microplastic surfaces differed greatly from those in the surrounding sediments and water. The bacterial community composition in water and sediment differed from that on the microplastic surfaces, and the dominant bacterial phyla on the microplastic surfaces were Proteobacteria and Bacteroidota, and their relative abundance on the microplastic surfaces was higher than that in sediment. The relative abundance of Proteobacteria was higher than that in water. The relative abundances of Bacteroidota and Actinobacteriota were significantly lower than that of water. Massilia and Pseudomonas were the dominant genera on the microplastic surfaces, and their relative abundances were significantly higher than those in the surrounding water and sediments. BugBase phenotype prediction revealed that the relative abundance of contains mobile elements, biofilm formation, potential pathogenicity, and stress tolerance phenotypes of microplastic bacterial communities were significantly higher than those of the surrounding water and sediments. The results revealed that microplastics may have contributed to the spread of harmful bacteria, including pathogenic bacteria, and increased the potential pathogenicity of bacterial communities. Additionally, microplastic surface bacterial communities had higher phenotypes of mobile gene element content. Revealing the potential harm of microplastic pollution to wetland ecology at the micro level may provide a scientific reference for maintaining the ecological stability of wetlands.

Expression of ATP-Binding Cassette Transporter A1 (ABCA1) in Eyelid Tissues and Meibomian Gland Epithelial Cells.

Purpose: To validate the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and distribution in human eyelid tissues and meibomian gland epithelial cells. Methods: Meibomian gland tissues from human eyelids were isolated by collagenase A digestion and cultured in defined keratinocyte serum-free medium (DKSFM). Infrared imaging was used to analyze the general morphology of meibomian glands. Hematoxylin and eosin (H&E) staining and Oil Red O staining were used to observe the morphological structure and lipid secretion in the human meibomian gland tissues. Quantitative real-time polymerase chain reaction, western blotting, and immunostaining were used to detect the mRNA and protein expression and cytolocalization of ABCA1 in the meibomian gland tissues and cultured cells. Results: The degree of loss of human meibomian gland tissue was related to age. Meibomian gland lipid metabolism was also associated with age. Additionally, human meibomian gland tissues express ABCA1 mRNA and protein; glandular epithelial cells express more ABCA1 mRNA and protein than acinar cells, and their expression in acinar cells decreases with differentiation. Furthermore, the expression of ABCA1 was downregulated in abnormal meibomian gland tissues. ABCA1 was mainly localized on the cell membrane in primary human meibomian gland epithelial cells (pHMGECs), whereas it was localized in the cytoplasm of immortalized human meibomian gland epithelial cells (iHMGECs). The mRNA and protein levels of ABCA1 in pHMGECs were higher than those in iHMGECs. Conclusions: Meibomian gland tissues of the human eyelid degenerate with age. ABCA1 expression in acinar cells decreases after differentiation and plays an important role in meibomian gland metabolism.

Bacterial community structure of water, sediment and microplastics in Poyang Lake wetland.

As a new type of pollutant, microplastics accumulate continuously in the environment. The environmental problems caused by microplastics have attracted wide attention. In this study, we collected water, sediment and four types of microplastics (film, foam, fiber and fragment) from wetland in East Lake area of Poyang Lake. We used high-throughput sequencing technology to analyze the bacterial diversity and community structure of water, sediment, and microplastics surface. The results showed that the bacterial richness and diversity of water and sediment were significantly higher than that on microplastics, and the bacterial richness of foaming microplastics was significantly lower than that of the other three types of microplastics. There were significant differences of bacterial communities between water, sediment, and microplastics. There were significant differences cross different types of microplastics. Proteobacteria, Bacteroidetes, and Actinobacteria were the main bacterial communities of water, sediment, and microplastics. The relative abundance of Bacteroidetes and Actinobacteria in water was higher than that in sediments and microplastics, while the relative abundance of Bacteroidetes and Actinobacteria in foaming microplastics was higher than that in other three types. At the genus level, the dominant ones included Massilia, Flavobacteria, and Pseudomonas. The relative abundance of Massilia and Pseudomonas in water and sediments was lower than that on microplastics, and the relative abundance of Flavobacteria was not different among water, sediment and microplastics. The relative abundance of Massilia in microplastics followed an order of fragment>fiber>film>foam, and that of Pseudomonas was film>fiber>foam>fragment. The results of metabolic pathway prediction analysis showed that except for foaming microplastics, the bacterial metabolic pathways on the surface of the other three types of microplastics were significantly different from those in water and sediment. The cellular processes, organismal systems, environmental information processing, and human diseases in bacterial metabolic pathways on microplastics surface were significantly higher than those in water and sediment. Our results suggested that microbial community structure on the surface of microplastics was significantly different from that in water and sediment, and that the morphology type of microplastics affected microbial community structure on the surface.

Manufacturing of human corneal endothelial grafts.

PURPOSE: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. METHODS: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-α-tubulin, γ-tubulin, p75NTR, α-catenin, ß-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. RESULTS: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 °C or up to 1 week at 22 °C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function. CONCLUSIONS: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.

0.05% atropine on control of myopia progression in Chinese school children: a randomized 3-year clinical trial.

AIM: To evaluate the effect of 0.05% atropine on the control of myopia for 2y (phase I) and on spherical equivalent refraction (SER) progression for 1y (phase II) after its withdrawal in Chinese myopic children. METHODS: Totally 142 children with myopia were randomly assigned to the 0.05% atropine group or to the placebo group. In phase I, children received 1 treatment for each eye daily. In phase II, the patients received no treatment. Axial length (AL), SER, intraocular pressure (IOP) and atropine-related side effects were assessed at 6 months' intervals. RESULTS: During phase I, the mean change of SER was -0.46±0.30 D in the atropine group, compared to -1.72±1.12 D in the placebo group (P<0.001). The mean change of AL in the atropine group (0.26±0.30 mm) was significantly shorter than that in the placebo group (0.76±0.62 mm, P=0.002). In addition, in phase II (12mo after the withdrawal of atropine), there was no significant difference in AL change from the atropine group, when compared with that from the placebo group (0.31±0.25 mm vs 0.28±0.26 mm, P>0.05). Furthermore, the change in SER from the atropine group was 0.50±0.41 D, which was significantly lower than 0.72±0.60 D from placebo group, (P<0.05). Finally, there were no statistically significant differences in IOP between the treatment and control groups at any stages (all P>0.05). CONCLUSION: The use of 0.05% atropine for two consecutive years may effectively control elongation of AL and thus progression of myopia, without significant SER progression 1y after atropine withdrawal. Therefore, treatment with 0.05% atropine daily for 2y is effective and safe.

Deciphering Adult Neural Stem Cells with Single-Cell Sequencing.

Adult neural stem cells (NSCs) are restricted to the two neurogenic regions of the mammalian brain, where they self-renew and generate progenies of multiple lineages, including neurons, astrocytes, and oligodendrocytes. Single-cell RNA sequencing technology, which reconstructs high-resolution transcriptional landscapes, provides valuable insights into cellular heterogeneity and developmental dynamics. In this review, we overviewed recent progress in the single-cell analyses of both conventional and unconventional NSCs. We discussed the heterogeneity among the stem cell pool and characterized the transcriptional alterations in aging and brain tumors. A comprehensive understanding of NSCs in physiological and pathological settings will provide insights for the rejuvenation of the aged brain and restoration of normal brain function in multiple neurological disorders.

Lens nucleus dislocation in hypermature cataract: Case report and literature review.

INTRODUCTION: Hypermature cataract is a form of late-stage cataract progression that can lead to a variety of complications. Spontaneous capsular rupture with lens nucleus displacement in hypermature cataracts has rarely been reported. We describe 2 cases of spontaneous dislocation of the lens nucleus in a hypermature cataract and perform a review of the literature on this complication. PATIENT CONCERNS: We report 2 rural men aged 50 and 76 years with deteriorating vision. DIAGNOSIS: The final diagnosis was senile hypermature cataract with dislocation of the lens nucleus in both patients and secondary glaucoma for the second patient. INTERVENTIONS AND OUTCOMES: During admission, both patients complained of deteriorating vision. Slit-lamp examination showed lens nucleus dislocation into the anterior chamber. The 50-year-old patient exhibited a residual lens capsule and a turbid cortex, with a normal anterior chamber and intraocular pressure. The 76-year-old patient presented a shrunken and ruptured capsule and no cortex in the pupillary area, mild inflammation in the anterior chamber, and high intraocular pressure. Both patients underwent intracapsular cataract extraction combined with anterior vitrectomy and achieved good postoperative recovery. CONCLUSION: Lens nucleus dislocation in hypermature cataracts can be seen in clinical practice, particularly in underdeveloped areas. Early recognition and surgery can improve vision.

Comparison of the SITA Faster-a new visual field strategy with SITA Fast strategy.

AIM: To compare visual field defects using the Swedish Interactive Thresholding Algorithm (SITA) Fast strategy with SITA Faster strategy, a newly developed time-saving threshold visual field strategy. METHODS: Ninety-three participants (60 glaucoma patients and 33 normal controls) were enrolled. One eye from each participant was selected randomly for the study. SITA Fast and SITA Faster were performed using the 24-2 default mode for each test. The differences of visual field defects between the two strategies were compared using the test duration, false-positive response errors, mean deviation (MD), visual field index (VFI) and the numbers of depressed test points at the significant levels of P<5%, <2%, <1%, and <0.5% in probability plots. The correlation between strategies was analyzed. The agreement between strategies was acquired by Bland-Altman analysis. RESULTS: Mean test durations were 246.0±60.9s for SITA Fast, and 156.3±46.3s for SITA Faster (P<0.001). The test duration of SITA Faster was 36.5% shorter than SITA Fast. The MD, VFI and numbers of depressed points at P<5%, <2%, <1%, and <0.5% in probability plots showed no statistically significant difference between two strategies (P>0.05). Correlation analysis showed a high correlation for MD (r=0.986, P<0.001) and VFI (r=0.986, P<0.001) between the two strategies. Bland-Altman analysis showed great agreement between the two strategies. CONCLUSION: SITA Faster, which saves considerable test time, has a great test quality comparing to SITA Fast, but may be not directly interchangeable.

Suppression of TGF-ß1 signaling by Matrigel via FAK signaling in cultured human trabecular meshwork cells.

The trabecular meshwork (TM) is composed of TM cells and beams of the extracellular matrix, together contributing to aqueous humor (AH) outflow resistance. Herein, we validated that our culture system on 2D Matrigel expressed putative TM markers and myocilin, of which the latter was upregulated by dexamethasone. Continuous passage of these cells on 2D Matrigel resulted in a gradual loss of expression of these markers. However, such a loss was restored by seeding cells in 3D Matrigel where expression of TM markers was further upregulated upon continuous passage. In contrast, TM cells seeded on fibronectin, collagen I/IV, or laminin lost expression of these markers and turned into myofibroblasts with expression of αSMA, which were dose-dependently upregulated by TGF-ß1/TGF-ß2. TM cells in 3D Matrigel also expressed TGF-ß1/TGF-ß3 despite challenge of TGF-ß1. The maintenance of TM phenotype by 3D Matrigel was linked to inhibition of canonical TGF-ß signaling and activation of pFAK-pSrc-pP190RhoGAP-P120RasGAP signaling. These findings indicate that basement membrane matrix with low rigidity plays an active role in maintaining TM phenotype in the presence of TGF-ß1 and shed light on its physiological role. Furthermore, abnormal matrices may perpetuate the pathological TM phenotype when the level of TGF-ß2 is elevated in glaucoma patients.

Near Infrared (NIR) Light Therapy of Eye Diseases: A Review.

Near infrared (NIR) light therapy, or photobiomodulation therapy (PBMT), has gained persistent worldwide attention in recent years as a new novel scientific approach for therapeutic applications in ophthalmology. This ongoing therapeutic adoption of NIR therapy is largely propelled by significant advances in the fields of photobiology and bioenergetics, such as the discovery of photoneuromodulation by cytochrome c oxidase and the elucidation of therapeutic biochemical processes. Upon transcranial delivery, NIR light has been shown to significantly increase cytochrome oxidase and superoxide dismutase activities which suggests its role in inducing metabolic and antioxidant beneficial effects. Furthermore, NIR light may also boost cerebral blood flow and cognitive functions in humans without adverse effects. In this review, we highlight the value of NIR therapy as a novel paradigm for treatment of visual and neurological conditions, and provide scientific evidence to support the use of NIR therapy with emphasis on molecular and cellular mechanisms in eye diseases.

COX-2 Signaling in the Tumor Microenvironment.

Tumorigenesis is a multistep, complicated process, and many studies have been completed over the last few decades to elucidate this process. Increasingly, many studies have shifted focus toward the critical role of the tumor microenvironment (TME), which consists of cellular players, cell-cell communications, and extracellular matrix (ECM). In the TME, cyclooxygenase-2 (COX-2) has been found to be a key molecule mediating the microenvironment changes. COX-2 is an inducible form of the enzyme that converts arachidonic acid into the signal transduction molecules (thromboxanes and prostaglandins). COX-2 is frequently expressed in many types of cancers and has been closely linked to its occurrence, progression, and prognosis. For example, COX-2 has been shown to (1) regulate tumor cell growth, (2) promote tissue invasion and metastasis, (3) inhibit apoptosis, (4) suppress antitumor immunity, and (5) promote sustainable angiogenesis. In this chapter, we summarize recent advances of studies that have evaluated COX-2 signaling in TME.

Two-year outcome of Trabeculo-Canalectomy for Chinese Glaucoma Patients.

To evaluate the efficacy of trabeculo-canalectomy in treating glaucoma patients, a retrospective investigation of 53 glaucoma patients (53 eyes) who underwent trabeculo-canalectomy was conducted at the First Affiliated Hospital of Nanjing Medical University, China, from April 2017 to January 2019. Intraocular pressure (IOP), visual acuity, surgical success rates, medications, and complications were monitored at post-operative 1 day, 1 week, 1, 3, 6, 12 and 24 months. Surgical success criteria were defined as 6 mm Hg≤IOP≤21 mmHg with or without additional medications. Our results showed that average IOP was statistically significant between pre-operative visit and each follow-up visit (all P <0.05). The total success rate of trabeculo-canalectomy at 1, 3, 6, 12 and 24 months was 92.5%, 86.8%, 94.3%, 92.5% and 90.6% respectively. After 3 months post-operatively, all patients had no obvious filtering blebs. The main early complications included postoperative hyphema (7.5%), elevated IOP (5.7%) and anterior chamber exudation (3.8%), which were all cured after conservative treatment. No blebitis, shallow anterior chamber, choroidal detachment and endophthalmitis were observed. Logistic regression analysis showed that patients with secondary glaucoma were more likely to undergo surgical failure 24 months post-operatively (P= 0.008). Thus, we conclude that trabeculo-canalectomy is effective and safe for the treatment of glaucoma.

Effect of intraocular lens implantation on visual field in glaucoma and comorbid cataracts.

AIM: To investigate the effects of intraocular lens (IOL) implantation on visual field (VF) in patients with glaucoma and comorbid cataracts (G&C) with different disease severities. METHODS: Totally 56 eyes of 50 patients with primary G&C were included. All patients were divided into three groups based on the severity of the VF defect: the mild, moderate, and severe stage. Phacoemulsification was performed for cataract removal combined with IOL implantation. Visual acuity (VA) and VF tests were performed for all enrolled patients, up to 3mo after surgery. Changes in VF threshold and global VF index in various groups were also recorded before and after surgery. The mean light sensitivity (MS) values and the changes following surgery (DMS) were compared between the three groups. Advanced Glaucoma Intervention Study (AGIS) scoring was analyzed on all VF results for analysis of changes in VF before and after surgery. RESULTS: Following surgery, the MS values of the three groups of G&C increased significantly, while the AGIS scores decreased statistically in all groups. The DMS values for the three zones in moderate and severe stage but not mild stage were statistically different between zones. The DMS value was significantly higher in zone I than those in zone II and III (zone I>zone II>zone III; P<0.05). The DMS was significantly higher in zone I than that in zone III in moderate stage patients (zone I>zone II>zone III; P<0.01), while the DMS values in the severe stage patients was significantly higher in zone I than those in zone II and III (zone I>zone II>zone III; P<0.01). CONCLUSION: The mean VF sensitivity of glaucoma patients increased significantly after cataract removal and IOL implantation. Variations in the severity and distribution of characteristics of VF defects result in differences in postoperative VF improvements after cataract surgery. The magnitude of increase in VF sensitivity is associated with VF defect characteristic in glaucoma.

HC-HA/PTX3 Purified From Human Amniotic Membrane Reverts Human Corneal Fibroblasts and Myofibroblasts to Keratocytes by Activating BMP Signaling.

Purpose: Fibrosis or scarring is a pathological outcome of wound healing and is characterized by terminally differentiated myofibroblasts. Heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) is a unique matrix component purified from amniotic membrane that exerts an anti-inflammatory effect. Herein, we investigate whether HC-HA/PTX3 can also exert an antiscarring effect. Methods: Human corneal fibroblasts and myofibroblasts were seeded on plastic, immobilized HA or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM+10% FBS, with or without AMD3100 or SB431542 in DMEM+ITS with or without transforming growth factor-ß1 (TGF-ß1). Transcript expression of keratocyte and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-SMA. Western blotting was used to measure relative protein level. Results: Human corneal fibroblasts and myofibroblasts cultured in or on HC-HA/PTX3, but not HA, were refrained from cytoplasmic expression of αSMA and nuclear translocation of pSMAD2/3 when challenged with exogenous TGF-ß1. Such an antiscarring action by suppressing canonical TGF-ß1 signaling was surprisingly accompanied by phenotypic reversal to keratocan-expressing keratocytes through activation of BMP signaling. Further investigation disclosed that such phenotypic reversal was initiated by cell aggregation mediated by SDF1-CXCR4 signaling highlighted by nuclear translocation of CXCR4 and upregulation of CXCR4 transcript and protein followed by activation of canonical BMP signaling. Conclusions: These findings collectively provide mechanistic understanding explaining how amniotic membrane transplantation exerts an antiscarring action. In addition, HC-HA/PTX3 and derivatives may be developed into a new biologic to treat corneal blindness caused by stromal scar or opacity in the future.

The Role of Cyclooxygenase-2 in Colorectal Cancer.

Colorectal cancer is the third common cancer in this world, accounting for more than 1 million cases each year. However, detailed etiology and mechanism of colorectal cancer have not been fully understood. For example, cyclooxygenase-2 (COX-2) and its product prostaglandin E2 (PGE2) have been closely linked to its occurrence, progression and prognosis. However, the mechanisms on how COX-2 and PGE2-mediate the pathogenesis of colorectal cancer are obscure. In this review, we have summarized recent advances in studies of pathogenesis and control in colorectal cancer to assist further advances in the research for the cure of the cancer. In addition, the knowledge gained may also guide the audiences for reduction of the risk and control of this deadly disease.

Pax 6 Controls Neural Crest Potential of Limbal Niche Cells to Support Self-Renewal of Limbal Epithelial Stem Cells.

On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.

Effects of chronic elevated intraocular pressure on parameters of optical coherence tomography in rhesus monkeys.

AIM: To determine the progression of parameters from optical coherence tomography (OCT) in chronic elevated intraocular pressure (IOP) monkeys. METHODS: A chronic elevated IOP model of rhesus monkeys was induced by laser photocoagulation. Representative OCT parameters, including the average and four-quadrant retinal nerve fiber layer (RNFL) thickness, and parameters from optic nerve head (ONH) analysis were collected before and after laser treatments biweekly for up to 28wk. The performance of each parameter for early progression detection was analyzed. The progressive trends toward elevated IOP were analyzed using a linear mixed-effects model. RESULTS: There were 10 successfully maintained high IOP eyes in 7 monkeys. The follow-up time was 24±5.37wk. With cumulative IOP elevation, the cup area, rim area and C/D area ratio were statistically significantly changed as early as 2wk after elevated IOP induction (P<0.05). The quadrant RNFL thickness changed at 6wk after high IOP induction, and the superior and inferior RNFL thicknesses exhibited more obvious reductions than other quadrants. The average RNFL thickness was the last one to show a significant decrease at 8wk. CONCLUSION: The parameters of ONH are most sensitive to elevated IOP in a primate glaucomatous model. These findings suggest that we should focus on those parameters instead of RNFL thickness in patients with elevated IOP, as they might present with earlier glaucomatous changes.

Phenotypic variability of SLC7A14 mutations in patients with inherited retinal dystrophy.

BACKGROUND: Inherited retinal dystrophy (IRD) is a group of retinal disorders that are both clinically and genetically diverse, typically with loss of photoreceptor function. Herein, we aimed to identify the underlying genetic defect in IRD patients with mutations in the SLC7A14 gene. METHODS: A targeted exome capture panel was applied for mutational screening of SLC7A14. Targeted exome sequencing (TES) was performed on 200 non-syndromic and unrelated autosomal recessive or sporadic IRD families. Candidate variants were validated by direct sequencing and further examined using bioinformatics analyses for determination of their potential effect. RESULTS: We identified compound heterozygous missense mutations (c.988G>A, p.G330R; c.1970G>A, p.R657Q) in an autosomal recessive retinitis pigmentosa (RP) case and a homozygous mutation (c.988G>A, p.G330R) in a simplex case with Leber congenital amaurosis (LCA) in the SLC7A14 gene. Both G330R and R657Q were deleterious based on in silico predictive tools. Our proposed topological model of the SLC7A14 polypeptide suggested that both G330R and R657Q affected evolutionarily highly conserved amino acid residues in SLC7A14 that occurred in transmembrane helixes. Structural modeling revealed a broken arginine and aspartic acid connection between residues 657 and 406. CONCLUSIONS: We applied TES to the molecular diagnosis of patients with IRD and for the first time identified SLC7A14 mutations in two unrelated families with RP and LCA separately. Our findings uniquely add the knowledge of the phenotypic variability of SLC7A14 mutations.

Isolation and Expansion of Multipotent Progenitors from Human Trabecular Meshwork.

To expand multi-potent progenitors from human trabecular meshwork (TM), we have created a new optimized method on two-dimensional (2D) followed by three-dimensional (3D) Matrigel in modified embryonic stem cell medium supplemented with 5% fetal bovine serum (MESCM + 5% FBS). The expanded TM cells were small cuboidal cells expressing TM markers such as AQP1, MGP, CHI3L1, and AnkG, embryonic stem cell (ESC) markers such as Oct4, Sox2, Nanog, and ABCG2, and neural crest (NC) markers such as p75NTR, FOXD3, Sox9, Sox10, and MSX1. Although expanded cells lost expression of these markers after passage, the cells regained the markers when Passage 2 cells were seeded on 3D Matrigel through activation of canonical BMP signaling. Such restored progenitors could differentiate into corneal endothelial cells, adipocytes, and chondrocytes but not keratocytes or osteocytes. Therefore, we have concluded that human TM harbors multipotent progenitors that can be effectively isolated and expanded using 2D Matrigel in MESCM + 5% FBS. This unique in vitro model system can be used to understand how TM is altered in glaucoma and whether such TM progenitor cells might one day be used for treating glaucoma or corneal endothelial dysfunction.

Descemet's Membrane Supports Corneal Endothelial Cell Regeneration in Rabbits.

Descemet's membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet's membrane. Descemet's membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.