Mutating the arginine residue within the FRNK motif of telosma mosaic virus (TelMV) HC-Pro protein attenuates viral infection and confers effective protection against TelMV in passion fruit (Passiflora edulis).

BACKGROUND: Telosma mosaic virus (TelMV, Potyvirus, Potyviridae) is an emerging viral pathogen that threatens passion fruit plantations worldwide. However, an efficient strategy for controlling such a virus is not yet available. Cross protection is a phenomenon in which pre-infection of a plant with one mild strain prevents or delays subsequent infection by the same or closely related virus. HC-Pro is the potyviral encoded multifunctional protein involved in several steps of viral infection, including multiplication, movement, transmission and RNA silencing suppression. In this study, we tested whether it is possible to generate attenuated viral strains capable of conferring protection against severe TelMV infection by manipulating the HC-Pro gene. RESULTS: By introducing point mutation into the conserved motif FRNK of HC-Pro that is essential for RNA silencing suppression, we have successfully obtained three attenuated mutants of TelMV (R181K, R181D, and R181E, respectively). These attenuated TelMV mutants could systemically infect passion fruit plants without noticeable symptoms. Pre-inoculation of one of these attenuated mutants confers efficient protection against subsequent infection by severe TelMV strain. Moreover, we demonstrated that the HC-Pros harbored by the attenuated mutants exhibit reduced RNA silencing suppression activity in Nicotiana benthamiana leaves. CONCLUSION: The attenuated TelMV mutants developed in this study that are suitable for cross protection offer a practical, powerful tool to fight against TelMV for sustainable passion fruit production. © 2024 Society of Chemical Industry.

The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement.

Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

The long and short of it: a comprehensive assessment of axial length estimation in myopic eyes from ocular and demographic variables.

BACKGROUND/OBJECTIVES: Axial length, a key measurement in myopia management, is not accessible in many settings. We aimed to develop and assess machine learning models to estimate the axial length of young myopic eyes. SUBJECTS/METHODS: Linear regression, symbolic regression, gradient boosting and multilayer perceptron models were developed using age, sex, cycloplegic spherical equivalent refraction (SER) and corneal curvature. Training data were from 8135 (28% myopic) children and adolescents from Ireland, Northern Ireland and China. Model performance was tested on an additional 300 myopic individuals using traditional metrics alongside the estimated axial length vs age relationship. Linear regression and receiver operator characteristics (ROC) curves were used for statistical analysis. The contribution of the effective crystalline lens power to error in axial length estimation was calculated to define the latter's physiological limits. RESULTS: Axial length estimation models were applicable across all testing regions (p ≥ 0.96 for training by testing region interaction). The linear regression model performed best based on agreement metrics (mean absolute error [MAE] = 0.31 mm, coefficient of repeatability = 0.79 mm) and a smooth, monotonic estimated axial length vs age relationship. This model was better at identifying high-risk eyes (axial length >98th centile) than SER alone (area under the curve 0.89 vs 0.79, respectively). Without knowing lens power, the calculated limits of axial length estimation were 0.30 mm for MAE and 0.75 mm for coefficient of repeatability. CONCLUSIONS: In myopic eyes, we demonstrated superior axial length estimation with a linear regression model utilising age, sex and refractive metrics and showed its clinical utility as a risk stratification tool.

Identification of the CDPK gene family in patchouli and functional analysis in response to continuous cropping stress.

To further reveal the molecular mechanisms underlying the formation of continuous cropping disorders in patchouli, this study analyzed the function of calcium dependent protein kinase (CDPK) genes at the molecular level in patchouli continuous cropping disorders. The findings unveiled the presence of 45 PcCDPK genes within the complete patchouli genome dataset. These genes exhibited a range of molecular weights from 50.78 to 78.96 kDa and aliphatic index values spanning from 74.42 to 88.49, and are shown to be hydrophilic proteins. The evolution of 45 PcCDPK members was divided into 4 subfamilies, with a total of 65 pairs of collinear genes. Each PcCDPK contains a STKc-CAMK domain and four EF-hand structures exhibiting a certain degree of conservatism during evolution. Transcriptome data further supported the significance of PcCDPK25 and PcCDPK38 genes, showing substantial upregulation, which was corroborated by qRT-PCR results. The 1629 bp and 1716 bp CDS sequences were obtained by cloning the PcCDPK25 and PcCDPK38 genes, respectively, and subcellular localization showed that both proteins were localized on the plasma membrane. This comprehensive study provides molecular-level confirmation of the pivotal roles played by CDPK genes in the emergence of continuous cropping challenges in patchouli plants, establishing a crucial foundation for a deeper comprehension of the molecular mechanisms underpinning these obstacles.

Clinicopathological Characteristics of Three Cases with Recurrent Orbital Solitary Fibrous Tumors: A Retrospective Study and Literature Review.

Objective: Solitary fibrous tumor (SFT) is a spindle cell neoplasm that rarely occurs in orbit. This study aimed to report the clinical, imaging, and pathological features of three patients with recurrent orbital SFTs. Methods: Clinical, imaging, and pathological data of the three patients were retrospectively reviewed, and the results were compared with those of previously reported cases with recurrent orbital SFT. Results: One female and two male patients (mean age, 54 years old) were included in this study. The present cases and literature review showed that the average time to recurrence in patients who aged under 50 years old was shorter than that in those who aged over 50 years old. The most common site for recurrent orbital SFT was the retrobulbar area of the orbit (23.8%). Imaging examinations showed consistent intensity of MRI signals before and after recurrence. Immunohistochemical results of all cases revealed the expressions of CD34. The mitotic rate increased in 4/8 cases, and the percentage of Ki-67-positive cells was elevated in 5/16 cases. Conclusion: These results suggested that young patients were more likely subjected to recurrent orbital SFT. The postoperative pathological diagnosis revealed that patients with recurrent orbital SFT had more nuclear abnormalities and mitotic activity, as well as a higher percentage of Ki-67-positive cells, indicating that orbital recurrent SFT tended to be malignant according to both morphological features and immunohistochemistry results.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 µg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava.

BACKGROUND: Plant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava. RESULTS: In this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves. CONCLUSIONS: Our results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.

A Systematic Review on the Continuous Cropping Obstacles and Control Strategies in Medicinal Plants.

Continuous cropping (CC) is a common practice in agriculture, and usually causes serious economic losses due to soil degeneration, decreased crop yield and quality, and increased disease incidence, especially in medicinal plants. Continuous cropping obstacles (CCOs) are mainly due to changes in soil microbial communities, nutrient availability, and allelopathic effects. Recently, progressive studies have illustrated the molecular mechanisms of CCOs, and valid strategies to overcome them. Transcriptomic and metabolomics analyses revealed that identified DEGs (differently expressed genes) and metabolites involved in the response to CCOs are involved in various biological processes, including photosynthesis, carbon metabolism, secondary metabolite biosynthesis, and bioactive compounds. Soil improvement is an effective strategy to overcome this problem. Soil amendments can improve the microbial community by increasing the abundance of beneficial microorganisms, soil fertility, and nutrient availability. In this review, we sum up the recent status of the research on CCOs in medicinal plants, the combination of transcriptomic and metabolomics studies, and related control strategies, including uses of soil amendments, crop rotation, and intercropping. Finally, we propose future research trends for understanding CCOs, and strategies to overcome these obstacles and promote sustainable agriculture practices in medicinal plants.

The causal mutation in ARR3 gene for high myopia and progressive color vision defect.

The ARR3 gene, also known as cone arrestin, belongs to the arrestin family and is expressed in cone cells, inactivating phosphorylated-opsins and preventing cone signals. Variants of ARR3 reportedly cause X-linked dominant female-limited early-onset (age < 7 years old) high myopia (< - 6D). Here, we reveal a new mutation (c.228T>A, p.Tyr76*) in ARR3 gene that can cause early-onset high myopia (eoHM) limited to female carriers. Protan/deutan color vision defects were also found in family members, affecting both genders. Using ten years of clinical follow-up data, we identified gradually worsening cone dysfunction/color vision as a key feature among affected individuals. We present a hypothesis that higher visual contrast due to the mosaic of mutated ARR3 expression in cones contributes to the development of myopia in female carriers.

SUMOylation-modified Pelota-Hbs1 RNA surveillance complex restricts the infection of potyvirids in plants.

RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals. However, it is not known whether two other RNA quality control pathways, nonstop decay and no-go decay, are capable of restricting viruses in plants. Here, we show that the evolutionarily conserved Pelota-Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae (termed potyvirids), the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide. Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron, which is conserved in almost all potyvirids. This allows Pelota to target the virus and act as a viral restriction factor. Furthermore, Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta. Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation. These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu).

BACKGROUND: Areca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier. RESULTS: Here, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0-1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%. CONCLUSION: We have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein-protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

Clinical observations of corneal topographic and tomographic changes in congenital ptosis eyes: a study in China.

PURPOSE: To investigate the impact of drooping eyelid on corneal topographic and tomographic alterations in congenital ptosis eyes. METHODS: Seventeen Chinese patients with unilateral congenital ptosis were included in this observational study. Ptosis eyes were included in the ptosis group, while normal contralateral eyes were included in the control group. The marginal reflex distance (MRD) was used to evaluate the severity of ptosis. Topographic and tomographic parameters measured by Pentacam, including keratometric, pachymetric, volumetric parameters as well as topometric indexes and D indexes, were recorded and compared between the ptosis group and the control group. Furthermore, correlation analyses were made between MRD and all measured corneal parameters. RESULTS: The value of anterior K1, Km and posterior K2, Km was significantly decreased in the ptosis eyes (p < 0.05). Corneal thickness at the pupil center point and thinnest point was significantly thicker in ptosis group compared with the ones in control group (p < 0.05). Higher ISV, IVA, KI, IHD values were observed in ptosis eyes (p < 0.05). The intergroup difference in MRD showed significant correlation with the difference in ISV (p < 0.05). CONCLUSION: The whole corneal contour is remodeled to be "flatter" in ptosis eyes. The upper eyelid position was closely associated with the corneal irregularity in ptosis eyes. The reasons for the discrepancy in corneal topography and tomography between ptotic and normal eyes were complicated.

Ocular biometrics and uncorrected visual acuity for detecting myopia in Chinese school students.

The study is to evaluate the performance of ocular biometric measures and uncorrected visual acuity (UCVA) for detecting myopia among Chinese students. Among 5- to 18-year-old Chinese students from two cities of China, trained eye-care professionals performed assessment of ocular biometrics (axial length (AL), corneal curvature radius (CR), anterior chamber depth) under noncycloplegic conditions using NIDEK Optical Biometer AL-Scan, distance visual acuity using retro-illuminated logMAR chart with tumbling-E optotypes, and cycloplegic refractive error using NIDEK autorefractor with administration of 0.5% tropicamide. Spherical equivalent (SER) in diopters (D) was calculated as sphere plus half cylinder, and myopia was defined as SER ≤ - 0.5 D. Performances of ocular biometrics and UCVA (individually and in combination) for detecting myopia were evaluated using sensitivity and specificity, predictive values, and area under ROC curve (AUC) in both development dataset and validation dataset. Among 3436 students (mean age 9.7 years, 51% female), the mean (SD) cycloplegic SER was - 0.20 (2.18) D, and 1269 (36.9%) had myopia. Cycloplegic SER was significantly correlated with AL (Pearson Correlation coefficient r = - 0.82), AL/CR ratio (r = - 0.90), and UCVA (r = 0.79), but was not correlated with CR (r = 0.02, p = 0.15). The AL/CR ratio detected myopia with AUC 0.963 (95% CI 0.957-0.969) and combination with UCVA improved the AUC to 0.976 (95% CI 0.971-0.981). Using age-specific AL/CR cutoff (> 3.00 for age < 10 years, > 3.06 for 10-14 years, > 3.08 for ≥ 15 years) as myopia positive, the sensitivity and specificity were 87.0% (95% CI 84.4-89.6%) and 87.8% (86.0-89.6%), respectively, in the development dataset and 86.4% (95% CI 83.7-89.1%) and 89.4% (95% CI 87.3-91.4%), respectively, in the validation dataset. Combining AL/CR and UCVA (worse than 20/32 for age < 10 years, and 20/25 for ≥ 10 years) provided 91.9% (95% CI 90.4-93.4%) sensitivity and 87.0% (95% CI 85.6-88.4%) specificity, positive value of 80.6% (95% CI 78.5-82.6%) and negative value of 94.8% (95% CI 93.8-95.8%). These results suggest that AL/CR ratio is highly correlated with cycloplegic refractive error and detects myopia with high sensitivity and specificity,  AL/CR ratio alone or in combination with UCVA can be used as a tool for myopia screening or for estimating myopia prevalence in large epidemiological studies with limited resources for cycloplegic refraction.

Prediction for Cycloplegic Refractive Error in Chinese School Students: Model Development and Validation.

Purpose: To predict cycloplegic refractive error using measurements obtained under noncycloplegic conditions. Method: Refractive error was measured in 5- to 18-year-old Chinese students using a NIDEK autorefractor before and after administration of 0.5% tropicamide. Spherical equivalent (SER) in diopters (D) was calculated as sphere plus half cylinder. A multivariable prediction model for cycloplegic SER was developed using data from students in Jinyun (n = 1938) and was validated using data from students in Hangzhou (n = 1498). The performance of the prediction model was evaluated using R2, mean difference between predicted and measured cycloplegic SER, and sensitivity and specificity for predicting myopia (cycloplegic SER ≤ -0.5 D). Results: Among 3436 students (mean age, 9.7 years; 51% female), the mean (SD) noncycloplegic and cycloplegic SER values were -1.12 (1.97) D and -0.20 (2.19) D, respectively. The prediction model that included demographics, noncycloplegic SER, axial length/corneal curvature radius ratio, uncorrected visual acuity (UCVA), and intraocular pressure predicted cycloplegic SER with R2 of 0.93 in the development dataset and 0.92 in the validation dataset. The mean (SD) differences between predicted and measured cycloplegic SER were 0.0 (0.55) D in the development dataset and 0.06 (0.64) D in the validation dataset. In both the development and validation datasets, the combination of predicted SER and UCVA yielded high sensitivity (91.4% and 91.9%, respectively) and specificity (95.0% and 90.1%, respectively) for detecting myopia. Conclusions: Cycloplegic refractive error can be predicted using measurements obtained under noncycloplegic conditions. The prediction model could potentially be used to correct the myopia prevalence in epidemiological studies in which administering cycloplegic agent on all participants is not feasible. Translational Relevance: The prediction model may provide a tool for correcting the overestimation of myopia from noncycloplegic refractive error in future epidemiological studies in which administering cycloplegic agent on all participants is not feasible.

MiR-15a Participated in the Pathogenesis of Pterygium via Targeting BCL-2: An Experimental Research.

PURPOSE: To compare the expression levels of miR-15a between pterygium and normal conjunctiva, and further investigate the potential role of miR-15a in the progression of pterygium. METHODS: 21 cases of primary pterygium were enrolled in our study. The length of the pterygium invaded into the cornea and the total thickness of the pterygium were measured with anterior segment optical coherence tomography (AS-OCT). The pterygial and adjacent normal conjunctival samples of the 21 patients were collected. Expressions of miR-15a, BCL-2, Bax in both pterygium and normal conjunctiva were measured, and correlations between miR-15a and BCL-2, miR-15a and Bax, miR-15a and clinical parameters were made. Pterygium epithelial cells (PECs) were isolated, cultured and transfected with miR-15a mimic or miR-15a inhibitor to interfere the miR-15a expression levels. The regulation of BCL-2 expression by miR-15a was examined with Real-Time PCR (RT-PCR), Western blot and immunofluorescence. The regulation of Bax expression by miR-15a was also examined with Real-Time PCR (RT-PCR) and Western blot. The cell viability of the transfected PECs was measured with the CCK-8 assay and the apoptosis in these cells was detected using the TUNEL assay. RESULTS: The expression of miR-15a, Bax were significantly decreased while the BCL-2 was significantly increased in pterygium (p < .05). There was a negative correlation in expression between miR-15a and BCL-2 in pterygium tissues (r = -0.516, p < .05). We also found that relative miR-15a level was positively correlated with the length of pterygium invaded into the cornea (r = -0.570, p < .05). In cultured PECs, miR-15a could downregulate the expression of BCL-2 and upregulate the expression of Bax. Promotion of miR-15a could suppress cell proliferation and promote cell apoptosis in cultured PECs. CONCLUSIONS: Our study demonstrated that decreased expression of miR-15a in pterygium might be associated with the apoptosis and proliferation of abnormal cell via regulating BCL-2, which could subsequently contribute to the development of pterygium. Downregulation of miR-15a might also contribute to the pathogenesis of pterygium by other mechanisms including abnormal proliferation and neovascularization, which remain to be investigated.

Effect of Cycloplegia on Refractive Error Measure in Chinese School Students.

PURPOSE: To determine differences in cycloplegic vs. non-cycloplegic refractive error and factors associated with these differences in Chinese school students. METHOD: In this cross-sectional school-based study, refractive error was measured in school students using a NIDEK autorefractor before and after administration of 0.5% tropicamide. Spherical equivalent (SER) in diopters (D) was calculated as sphere plus half cylinder. SER differences before vs. after cycloplegia were evaluated using mean, standard deviation (SD), 95% limits of agreement. Univariable and multivariable regression models were used to determine factors associated with SER differences. RESULTS: Among 3604 students, 3450 (95.7%) provided data for analysis. Mean age (SD) was 9.7 (3.6) years. The mean SER (SD) was -1.12 (1.97) D before cycloplegia, and -0.20 (2.19) D after cycloplegia, with a mean difference of 0.92 D (95% limits of agreement: -0.93 to 2.78 D). Among 196 eyes with non-cycloplegic SER -6.0 D or worse (e.g., met high myopia definition), 71.4% had cycloplegic SER -6.0 D or worse, and among 3607 eyes with non-cycloplegic SER -0.5 D or worse (e.g., met myopia definition), 62.1% eyes had cycloplegic SER -0.5 D or worse. Cycloplegic SER was more correlated with axial length than non-cycloplegic SER (Pearson r = 0.82 vs. 0.72, p < .0001). In multivariable analysis, larger SER differences were associated with more hyperopic refractive error and smaller axial length (all p < .0001). CONCLUSION: Non-cycloplegic refractive error overestimates myopia by approximately one diopter. This overestimation increases with more hyperopic refractive error and smaller axial length. Non-cycloplegic refractive error should not be used for evaluating pediatric myopia. ABBREVIATIONS: BCVA = best corrected visual acuity; D = diopter; SD = standard deviation; SE = standard error; SER = spherical equivalent; CI = confidence interval.