Proteomic analysis of aqueous humor reveals novel regulators of diabetic macular edema.

Diabetic macular edema (DME) is the most common cause of blindness in patients with diabetic retinopathy. To investigate the proteomic profiles of the aqueous humor (AH) of individuals with diabetic macular edema (DME), AH samples were collected from patients with non-diabetes mellitus (NDM), DM, nonproliferative diabetic retinopathy (NPDR), and DME. We performed comparative proteomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analyses. We identified 425 proteins in these AH samples, of which 113 showed changes in expression in DME compared with NDM, 95 showed changes in expression in DME vs. DM, and 84 showed changes in expression in DME compared with NPDR. The bioinformatics analysis suggested that DME is closely associated with platelet degranulation, oxidative stress-related pathway, and vascular-related pathways. Upregulation of haptoglobin (HP) and downregulation of fibrillin 1 (FBN1) were validated by ELISA. Receiver operating characteristic (ROC) analysis showed that HP and FBN1 could distinguish DME from NPDR with areas under the curve of 0.987 (p = 0.00608) and 0.791 (p = 0.00629), respectively. The findings provide potential clues for further analysis of the molecular mechanisms and the development of new treatments for DME. HP and FBN1 may be potential key proteins and therapeutic targets in human DME. The proteomics dataset generated has been deposited to the ProteomeXchange/iProX Consortium with Identifier: PXD033404/IPX0004353001.

Mechanistic insights into mitochondrial tRNAAla 3'-end metabolism deficiency.

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.

Complex I mutations synergize to worsen the phenotypic expression of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.

Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber's hereditary optic neuropathy.

Mitochondrial DNA (mtDNA) mutations have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we investigated the pathophysiology of a LHON susceptibility allele (m.3394T>C, p.30Y>H) in the Mitochondrial (MT)-ND1 gene. The incidence of m.3394T>C mutation was 2.7% in the cohort of 1741 probands with LHON. Extremely low penetrances of LHON were observed in 26 pedigrees carrying only m.3394T>C mutation, while 21 families bearing m.3394T>C, together with m.11778G>A or m.14484T>C mutation, exhibited higher penetrance of LHON than those in families carrying single mtDNA mutation(s). The m.3394T>C mutation disrupted the specific electrostatic interactions between Y30 of p.MT-ND1 with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1) of complex I, thereby altering the structure and function of complex I. We demonstrated that these cybrids bearing only m.3394T>C mutation caused mild mitochondrial dysfunctions and those harboring both m.3394T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions than cybrids carrying only m.11778G>A mutation. In particular, the m.3394T>C mutation altered the stability of p.MT-ND1 and complex I assembly. Furthermore, the m.3394T>C mutation decreased the activities of mitochondrial complexes I, diminished mitochondrial ATP levels and membrane potential and increased the production of reactive oxygen species in the cybrids. These m.3394T>C mutation-induced alterations aggravated mitochondrial dysfunctions associated with the m.11778G>A mutation. These resultant biochemical defects contributed to higher penetrance of LHON in these families carrying both mtDNA mutations. Our findings provide new insights into the pathophysiology of LHON arising from the synergy between mitochondrial ND1 and ND4 mutations.

The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families.

To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON), a cohort of 1164 Han Chinese subjects with LHON were screened for ND1 G3460A mutation. A total of 295 subjects from 16 Han Chinese families carrying the G3460A mutation underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. The incidence of G3460A mutation was 1.4% in this cohort of Chinese subjects with LHON. Twenty-seven (20 males/7 females) of 109 matrilineal relatives among 10 Chinese pedigrees carrying this mutation exhibited a wide range of severity and age-at-onset in visual impairment. Penetrances of optic neuropathy ranged from 7.1% to 50%, with the average of 24.5%. The age-at-onset of 27 affected matrilineal relatives varied from 10 to 40 years, with the average of 22 years. Molecular analysis identified the homoplasmic G3460A mutation and distinct sets of variants belonging to eight haplogroups. Haplogroup M with G3460A mutation was of higher frequency than those in controls. The penetrances of visual loss in families carrying mitochondrial DNA haplogroups A, B and M were higher than those in other families. Furthermore, haplogroup-specific variants tRNA(Ser(AGY)) A12223G, tRNA(Thr) G15927A and tRNA(Glu) A14693G may enhance the penetrance of visual loss in these families. The G3460A mutation occurred through recurrent origins and founder events in Chinese population. Mitochondrial modifiers may modulate the penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the G3460A mutation. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

Spectrum of rhodopsin gene mutations in Chinese patients with retinitis pigmentosa.

PURPOSE: This study was to analyze the spectrum and frequency of rhodopsin gene (RHO) mutations in Chinese patients with retinitis pigmentosa (RP). METHODS: Patients were given physical examinations, and blood samples were collected for DNA extraction. The RHO mutations were screened with direct sequencing. RESULTS: Eight heterozygous nucleotide changes were detected in eight of 300 probands with RP, including six novel mutations and two known mutations. p.R21C, p.C110S, p.G182V, p.C187G, c.409-426delGTGGTGGTGTGTAAGCCC, and p.P347L were found in six autosomal dominant families. p.T92I and p.Y178C were found in two isolated cases. CONCLUSIONS: The results reveal the spectrum and frequency of RHO mutations in Chinese patients with different forms of RP and demonstrate that RHO mutations account for a high proportion of autosomal dominant RP (adRP) cases.

[The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss.

Bilateral endogenous fungal endophthalmitis: A case report.

RATIONALE: Endogenous fungal endophthalmitis is a challenging condition. There are no universally accepted diagnostic or management protocols. We share a case of endogenous fungal endophthalmitis who was successfully treated, focusing on the diagnostic and treatment procedures. PATIENT CONCERNS: A 31-year-old female with a history of fungal vaginitis and tinea corporis presented with progressive visual decrease in both eyes after having an induced abortion. Her best corrected visual acuity at presentation was 20/1000 in her right eye and 20/250 in her left eye. Upon slit lamp examination, mild inflammatory reaction in the anterior chamber was found. Dilated fundus examination revealed a hazy view of the optic disc and posterior retina, and there was a whitish mass with "string and pearls" just in front of the macular region in each eye. DIAGNOSES: Bilateral fungal endogenous endophthalmitis was diagnosed empirically, which was confirmed later by deoxyribonucleic acid sequencing and culture of intraocular fluid. INTERVENTIONS: Oral itraconazole and intravitreal voriconazole were administered to the patient at first. The intraocular inflammation was partially responsive to the medication, yet the visual acuity persisted to deteriorate and the vitreous whitish masses became more prominent. Then vitrectomy procedures were carried out and oral itraconazle was switched to intravenous fluconazole. The antifungal treatment lasted for 8 weeks. OUTCOMES: The intraocular inflammation alleviated and visual acuity improved after vitrectomy. At the 9-month follow-up visit, the patient's best corrected visual acuity was 20/40 in the right eye and 20/30 in the left eye. There was no intraocular inflammatary reaction, and retinal scar was noticed in each eye. LESSON: Early and correct diagnosis, coupled with prompt and aggressive treatment, is crucial for cases of fungal endogenous endophthalmitis. Deoxyribonucleic acid sequencing techniques can contribute to early diagnosis, while vitrectomy may be necessary when antifungal medication is insufficient in controlling the condition.

Deferoxamine attenuates visual impairment in retinal ischemia‒reperfusion via inhibiting ferroptosis.

Retinal ischemia‒reperfusion (I/R) injury can cause significant damage to human retinal neurons, greatly compromising their functions. Existing interventions have been proven to have little effect. Ferroptosis is a newly discovered type of programmed cell death that has been found to be involved in the process of ischemia‒reperfusion in multiple organs throughout the body. Studies have shown that it is also present in retinal ischemia‒reperfusion injury. A rat model of retinal ischemia‒reperfusion injury was constructed and treated with deferoxamine. In this study, we found the accumulation of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), and the consumption of glutathione (GSH) via ELISA testing; increased expression of transferrin; and decreased expression of ferritin, SLC7A11, and GPX4 via Western blotting (WB) and real-time PCR testing. Structural signs of ferroptosis (mitochondrial shrinkage) were observed across multiple cell types, including retinal ganglion cells (RGCs), photoreceptor cells, and pigment epithelial cells. Changes in visual function were detected by F-VEP and ERG. The results showed that iron and oxidative stress were increased in the retinal ischemia‒reperfusion injury model, resulting in ferroptosis and tissue damage. Deferoxamine protects the structural and functional soundness of the retina by inhibiting ferroptosis through the simultaneous inhibition of hemochromatosis, the initiation of transferrin, and the degradation of ferritin and activating the antioxidant capacity of the System Xc-GSH-GPX4 pathway.

Mitochondrial tRNA variants in 811 Chinese probands with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 71 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in < 1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G > A, ND4 11778G > A or ND6 14484 T > C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families. METHODS: One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA). RESULTS: Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families. CONCLUSION: The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families.

[The mitochondrial tRNA(Thr) A15951G mutation may be associated with Leber's hereditary optic neuropathy in two Chinese families].

OBJECTIVE: To explore clinical, genetic and molecular features of two Chinese Han families with Leber's hereditary optic neuropathy (LHON). METHODS: Ophthalmologic examinations revealed variable severity and age-at-onset of visual loss among probands and other matrilineal relatives of both families. The families exhibited extremely low penetrance of visual impairment. The entire mitochondrial genome of two probands was amplified by PCR in 24 overlapping fragments using sets of oligonucleotide primers. RESULTS: Sequence analysis of complete mitochondrial genome in the pedigrees excluded three common LHON associated mutations G11778A, G3460A and T14484C, but revealed the presence of a known homoplasmic tRNA(Thr) A15951G mutation. It also showed distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroup D4b1. The A15951G mutation is located at the extremely conserved nucleotide (conventional position 71) of tRNA(Thr). Thus, this mutation may alter the structure and stability of mitochondrial tRNA(Thr), thereby leading to a failure in the tRNA metabolism and mitochondrial dysfunction, causing visual impairment. CONCLUSION: The results suggested that the A15951G mutation might be involved in the pathogenesis of Leber's hereditary optic neuropathy in the two families.

Clinical assessment and FGFR2 mutation analysis in a Chinese family with Crouzon syndrome: A case report.

RATIONALE: Crouzon syndrome is an autosomal dominant genetic disorder caused by mutations in fibroblast growth factor receptor 2 (FGFR2) and one of the most common types of craniosynostosis. Here we report the detection of FGFR2 mutation and its related clinical findings in 2 patients with Crouzon syndrome from a Chinese family. PATIENT CONCERNS: We report a case of a 28-year-old male patient presented with the chief complaint of gradually blurring of his eyes over the last 6 months before visiting our clinics. History revealed low visual acuity in his right eye since childhood. Physical examination showed that both the patient and his mother have the appearance of craniofacial dysostosis, mandibular prognathism, ocular proptosis, short superior lip, scoliosis, and thoracic deformity. DIAGNOSIS: Auxiliary examinations lead to the diagnosis of Crouzon syndrome with binocular optic atrophy, myelinated retina nerve fibers, and ametropia in both eyes, and amblyopia in the right eye of the male patient. The molecular genetic analysis confirmed the diagnosis by detecting a heterozygous pathogenic mutation c.1026C > G (C342W) in exon 10 of FGFR2 in both the patient and his mother, but not in any of the unaffected family members. INTERVENTIONS AND OUTCOMES: None. LESSONS: Our study confirms the presence of optic nerve atrophy in patients with Crouzon syndrome carrying FGFR2 C342W mutations and indicates that MRI and funduscopy should be performed to examine the optic nerve changes for patients with Crouzon syndrome.

A Novel BBS9 Mutation Identified via Whole-Exome Sequencing in a Chinese Family with Bardet-Biedl Syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by polydactyly, obesity, rod-cone dystrophy, and mental retardation. Twenty-one genes have been identified as causing BBS. This study collected a BBS pedigree from two patients and performed whole-exome sequencing on one patient. We identified a novel homozygous variant c.1114C>T (p.Q372X) in the BBS9 of the two siblings. This variant was confirmed and completely cosegregated with the disease of this family by Sanger sequencing. We report a novel homozygous variant c.1114C>T in the BBS9 gene in a Chinese family.

Mitochondrial haplogroup M9a specific variant ND1 T3394C may have a modifying role in the phenotypic expression of the LHON-associated ND4 G11778A mutation.

We report here the clinical, genetic and molecular characterization of four Han Chinese families with Leber's hereditary optic neuropathy (LHON). The penetrances of optic neuropathy in these Chinese pedigrees were 38%, 38%, 44% and 56%. This observation is in contrast with the previously identified 14 Chinese families with very low penetrance of LHON. The age-at-onset for visual impairment in matrilineal relatives in these Chinese families varied from 18 to 30years. Furthermore, the ratios between affected male and female matrilineal relatives in these families were 3:0, 3:0, 3:1 and 2:3, respectively. Molecular analysis of mitochondrial genomes identified the known ND4 G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M9a. Of these, the ND1 T3394C mutation caused the substitution of a highly conserved histidine for tyrosine (Y30H) at amino acid position 30. This mutation was associated with LHON in other families with low penetrance of optic neuropathy and other clinical abnormalities. The presence of both G11778A and T3394C mutations appears to contribute to higher penetrance of optic neuropathy in these four Chinese families than other Chinese families carrying only the G11778A mutation. Therefore, the mitochondrial haplogroup M9a specific variant T3394C may modulate the phenotypic manifestation of LHON-associated G11778A mutation in these Chinese pedigrees.

Huoxue-Tongluo-Lishui-Decoction is visual-protective against retinal ischemia-reperfusion injury.

Retinal ischemia reperfusion injury (IRI) is a leading cause of visual impairment or blindness, and an effective way to prevent the visual loss needs to be developed. Although decades of clinical application of Huoxue-Tongluo-Lishui-Decoction (HTLD) has demonstrated its reliable clinical efficacy against retinal IRI, no convincing randomized controlled trials were conducted in humans or animals, and the associated mechanism still needs to be explored. To confirm the protective effect of HTLD against retinal IRI and to explore its underlying mechanisms, a standard retinal IRI animal model, randomized controlled trials, objective evaluation and examination methods were adopted in this study. Flash visual evoked potentials (F-VEP) was performed 8 weeks post-reperfusion. The results showed that the medium dose of HTLD had better treatment effects than low dose of HTLD. High dose of HTLD did not further improve visual function relative to medium dose of HTLD, but had poor performance in the latency of P2 wave. The angio-optical coherence tomography (angio-OCT) examination showed that retinal nerve fiber layer (RNFL) became edematous in the early stage, then the edema subsided, and RNFL became thinning in the late stage. HTLD reduced the swelling of RNFL in the early stage and prevented the thinning of RNFL in the late stage. Similar to F-VEP, medium dose of HTLD has the best neural-protective effects against retinal IRI. In mechanisms, HTLD treatment not only enhanced autophagy at 6 h after reperfusion, but extended the enhancing effect until at least 24 h. HTLD treatment significantly reduced the cleaved Caspase-3, cleaved PARP and Caspase-3 activity at 48 h after reperfusion. HTLD inhibited neuro-toxic cytokines expression in retinal IRI by modulating Akt/NF-kB signaling. HTLD treatment enhanced the expressions of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS), and lower the concentration of free glutamate in retina after reperfusion. The phosphorylation of iNOS increased significantly in retinal IRI at 6 h, and HTLD treatment suppressed the phosphorylation of Inducible nitric oxide synthetase (iNOS). In conclusion, HTLD is visual-protective against retinal IRI, and the regulation of autophagy, apoptosis and neuro-toxic mediators may be the underlying mechanisms. These findings may provide new ideas for the clinical treatment of retinal IRI related diseases.

Efficacy and Safety of Houttuynia Eye Drops Atomization Treatment for Meibomian Gland Dysfunction-Related Dry Eye Disease: A Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

PURPOSE: To evaluate the efficacy and safety of Houttuynia eye drops (a Chinese traditional medicine) atomization treatment in meibomian gland dysfunction (MGD)-related dry eye disease (DED) patients. METHODS: A total of 240 eligible patients diagnosed with MGD-related DED were assigned either Houttuynia eye drops or placebo for atomization once daily for four weeks in a multi-center, randomized, double-blind, placebo-controlled clinical study. Primary outcome evaluations used included eye symptom score (using the Chinese Dry Eye Questionnaire), meibum quality, and tear break-up time (TBUT), while safety evaluations included adverse events (AEs), visual acuity, and intraocular pressure monitoring. Indicators were measured at baseline as well as one week, two weeks, and four weeks after treatment. RESULTS: Primary outcome measures of the Houttuynia group were improved compared with their placebo counterparts following four-week treatment. Eye symptom scores were significantly reduced relative to the baseline in the Houttuynia group (mean ± standard error of the mean, 9.00 ± 0.61) compared with the placebo group (6.29 ± 0.55; p = 0.0018). Reduction in meibum quality score in the Houttuynia group (0.91 ± 0.10) was also significantly higher compared with the placebo group (0.57 ± 0.10; p = 0.0091), while TBUT in the treatment group (6.30 ± 0.22) was also longer than in the latter (5.60 ± 0.24; p = 0.0192). No medication-related adverse events were observed. CONCLUSIONS: Atomization treatment with Houttuynia eye drops is both clinically and statistically effective for the treatment of mild to moderate MGD-related DED patients. This approach is generally safe and was tolerated well by patients.

PRICKLE3 linked to ATPase biogenesis manifested Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease. X-linked nuclear modifiers were proposed to modify the phenotypic manifestation of LHON-associated mitochondrial DNA (mtDNA) mutations. By whole-exome sequencing, we identified the X-linked LHON modifier (c.157C>T, p.Arg53Trp) in PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase in 3 Chinese families. All affected individuals carried both ND4 11778G>A and p.Arg53Trp mutations, while subjects bearing only a single mutation exhibited normal vision. The cells carrying the p.Arg53Trp mutation exhibited defective assembly, stability, and function of ATP synthase, verified by PRICKLE3-knockdown cells. Coimmunoprecipitation indicated the direct interaction of PRICKLE3 with ATP synthase via ATP8. Strikingly, cells bearing both p.Arg53Trp and m.11778G>A mutations displayed greater mitochondrial dysfunction than those carrying only a single mutation. This finding indicated that the p.Arg53Trp mutation acted in synergy with the m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON. Furthermore, we demonstrated that Prickle3-deficient mice exhibited pronounced ATPase deficiencies. Prickle3-knockout mice recapitulated LHON phenotypes with retinal deficiencies, including degeneration of retinal ganglion cells and abnormal vasculature. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutations and X-linked nuclear modifiers.

Clinical Features of Combined Central Retinal Artery and Vein Occlusion.

PURPOSE: To describe the clinical features of combined central retinal artery and vein occlusion (CCRAVO). METHODS: This retrospective study included 33 admitted patients (33 eyes) who had CCRAVO. Clinical data, such as age, gender, best-corrected visual acuity (BCVA), intraocular pressure (IOP), findings on fundus color photography and fundus fluorescein angiography (FFA), and information about follow-up, were collected and analyzed. RESULTS: The age of the patients with CCRAVO ranged from 22 to 78 years, with a mean of 48.8 ± 14.1 years. At presentation, BCVA of the involved eyes ranged from no light perception (NLP) to 20/20. In addition, 45.5% (15/33) of the eyes had BCVA of finger counting (FC) or below, whereas 12.1% (4/33) had BCVA of 20/60 or above. The IOP was lower in the involved eyes than in the fellow eyes (15.0 ± 3.0 mmHg vs. 16.4 ± 2.3 mmHg, p=0.03). Ophthalmoscopic examination showed changes in both central retinal artery occlusion (CRAO) and central retinal vein occlusion (CRVO), including retinal hemorrhage, retinal ischemic whitening, optic disc hyperemia and/or edema, venous dilation and tortuosity, cotton wool spot (CWS), and Roth's spot. FFA showed prolonged arm-to-retina time (ART) and retinal arteriovenous passage time (RAP) (17.1 ± 4.9 s and 12.1 ± 8.8 s, respectively). Capillary nonperfusion (CNP) was seen in 21 eyes (63.6%), and in 14 (42.2%) of these, CNP was larger than 10 disc areas. At 2 to 3 weeks after presentation, BCVA improved in 23 eyes (71.9%) and further deteriorated in 5 eyes (15.6%). Retinal ischemic whitening improved in more than half of the eyes, whereas retinal hemorrhage increased in nearly half of the eyes. Follow-up ranged from 6 to 56 months. Seven patients were lost to follow-up. At final follow-up, six eyes had a visual acuity of 20/60 or greater, but 6 eyes had FC or worse. Four eyes developed neovascularization on follow-up. CONCLUSION: CCRAVO is a sight-threatening entity. Manifestations of CRAO and CRVO can be seen simultaneously in the early stage of disease, and CRVO may play a more important role in the development of CCRAVO.