Analyzing genetic diversity in luffa and developing a Fusarium wilt-susceptible linked SNP marker through a single plant genome-wide association (sp-GWAS) study.

BACKGROUND: Luffa (Luffa spp.) is an economically important crop of the Cucurbitaceae family, commonly known as sponge gourd or vegetable gourd. It is an annual cross-pollinated crop primarily found in the subtropical and tropical regions of Asia, Australia, Africa, and the Americas. Luffa serves not only as a vegetable but also exhibits medicinal properties, including anti-inflammatory, antidiabetic, and anticancer effects. Moreover, the fiber derived from luffa finds extensive applications in various fields such as biotechnology and construction. However, luffa Fusarium wilt poses a severe threat to its production, and existing control methods have proven ineffective in terms of cost-effectiveness and environmental considerations. Therefore, there is an urgent need to develop luffa varieties resistant to Fusarium wilt. Single-plant GWAS (sp-GWAS) has been demonstrated as a promising tool for the rapid and efficient identification of quantitative trait loci (QTLs) associated with target traits, as well as closely linked molecular markers. RESULTS: In this study, a collection of 97 individuals from 73 luffa accessions including two major luffa species underwent single-plant GWAS to investigate luffa Fusarium wilt resistance. Utilizing the double digest restriction site associated DNA (ddRAD) method, a total of 8,919 high-quality single nucleotide polymorphisms (SNPs) were identified. The analysis revealed the potential for Fusarium wilt resistance in accessions from both luffa species. There are 6 QTLs identified from 3 traits, including the area under the disease progress curve (AUDPC), a putative disease-resistant QTL, was identified on the second chromosome of luffa. Within the region of linkage disequilibrium, a candidate gene homologous to LOC111009722, which encodes peroxidase 40 and is associated with disease resistance in Cucumis melo, was identified. Furthermore, to validate the applicability of the marker associated with resistance from sp-GWAS, an additional set of 21 individual luffa plants were tested, exhibiting 93.75% accuracy in detecting susceptible of luffa species L. aegyptiaca Mill. CONCLUSION: In summary, these findings give a hint of genome position that may contribute to luffa wild resistance to Fusarium and can be utilized in the future luffa wilt resistant breeding programs aimed at developing wilt-resistant varieties by using the susceptible-linked SNP marker.

Functional divergence of a pair of Arabidopsis phospho-base methyltransferases, PMT1 and PMT3, conferred by distinct N-terminal sequences.

In seed plants, phospho-base N-methyltransferase (PMT) catalyzes a key step in the biosynthesis pathway of phosphatidylcholine (PC), the most abundant phospholipid class. Arabidopsis thaliana possesses three copies of PMT, with PMT1 and PMT3 play a primary role because the pmt1 pmt3 double mutant shows considerably reduced PC content with a pale seedling phenotype. Although the function of PMT1 and PMT3 may be redundant because neither of the parental single mutants showed a similar mutant phenotype, major developmental defects and possible functional divergence of these PMTs underlying the pale pmt1 pmt3 seedling phenotype are unknown. Here, we show the major developmental defect of the pale seedlings in xylem of the hypocotyl with partial impairments in chloroplast development and photosynthetic activity in leaves. Although PMT1 and PMT3 are localized at the endoplasmic reticulum, their tissue-specific expression pattern was distinct in hypocotyls and roots. Intriguingly, the function of PMT3 but not PMT1 requires its characteristic N-terminal sequence in addition to the promoter because truncation of the N-terminal sequence of PMT3 or substitution with PMT1 driven by the PMT3 promoter failed to rescue the pale pmt1 pmt3 seedling phenotype. Thus, PMT3 function requires the N-terminal sequence in addition to its promoter, whereas the PMT1 function is defined by the promoter.

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy.

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2ß, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.

Multiplex viral tropism assay in complex cell populations with single-cell resolution.

Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

The phospho-base N-methyltransferases PMT1 and PMT2 produce phosphocholine for leaf growth in phosphorus-starved Arabidopsis.

Phosphorus (P) is an essential nutrient for plants. Membrane lipid remodeling is an adaptive mechanism for P-starved plants that replaces membrane phospholipids with non-P galactolipids, presumably to retrieve scarce P sources and maintain membrane integrity. Whereas metabolic pathways to convert phospholipids to galactolipids are well-established, the mechanism by which phospholipid biosynthesis is involved in this process remains elusive. Here, we report that phospho-base N-methyltransferases 1 and 2 (PMT1 and PMT2), which convert phosphoethanolamine to phosphocholine (PCho), are transcriptionally induced by P starvation. Shoots of seedlings of pmt1 pmt2 double mutant showed defective growth upon P starvation; however, membrane lipid profiles were unaffected. We found that P-starved pmt1 pmt2 with defective leaf growth had reduced PCho content, and the growth defect was rescued by exogenous supplementation of PCho. We propose that PMT1 and PMT2 are induced by P starvation to produce PCho mainly for leaf growth maintenance, rather than for phosphatidylcholine biosynthesis, in membrane lipid remodeling.

Three-month Chan-Chuang qigong program improves physical performance and quality of life of patients with cognitive impairment: A randomized controlled trial.

This randomized controlled trial was conducted to evaluate the effects of a 3-month-long Chan-Chuang qigong program on patients' physical performance and quality of life while excluding the influence caused by the progression of their cognitive impairment. Patients with mild to moderate cognitive impairment were recruited from two dementia daycare centers in Taiwan. The control group (n = 41) received the standardized plan of treatment, and the qigong group (n = 39) received the standardized plan of treatment plus the Chan-Chuang qigong program. The outcomes were muscle strength, muscle endurance, exercise capacity, and quality of life. After controlling for the progression of cognitive impairment, the qigong group showed significant improvements over the control group and baseline in muscle strength and exercise capacity at Months 2 and 3 (p < 0.05) and in muscle endurance at Months 1, 2, and 3 (p < 0.05). The Cognitron test scores were significantly associated with muscle strength (p = 0.03), whereas the Corsi block-tapping test scores were significantly associated with exercise capacity (p = 0.001). Furthermore, a significant between-group difference was detected in the physical (p = 0.01), not mental (p = 0.83), component of quality of life. The 3-month Chan-Chuang qigong program can be applied for patients with mild to moderate cognitive impairment as complementary therapy to improve their muscle strength, muscle endurance, exercise capacity, and physical quality of life. This program should be practiced for at least 2 months to achieve satisfactory results.

Human SMILE-Derived Stromal Lenticule Scaffold for Regenerative Therapy: Review and Perspectives.

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients' vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.

Efficacy of Modified Amnion-Assisted Conjunctival Epithelial Redirection (ACER) for Partial Limbal Stem Cell Deficiency.

Background and objectives: the aim of this study was to analyze the efficacy of a modified "amnion-assisted conjunctival epithelial redirection (ACER)" technique for the treatment of partial limbal stem cell deficiency (LSCD). Materials and methods: the medical records of three patients with partial LSCD who underwent corneal surface reconstruction with modified ACER following superficial keratectomy were retrospectively studied. Briefly, in this technique, an inner amniotic membrane (AM) layer was applied on the corneal surface to promote corneal re-epithelialization. The outer AM layer was applied as a barrier to prevent the invasion of conjunctival epithelial cells into the cornea before the corneal surface was completely covered by corneal epithelial cells derived from the remaining intact limbal stem cells. Results: in all three cases, the outer AM layer successfully kept the conjunctival epithelium away from the corneal surface and prevented an admixture of conjunctival epithelial cells with corneal epithelial cells. In all three patients, the cornea was completely re-epithelized with epithelial cells derived from the remaining healthy limbal stem cells, and a clear visual axis was maintained without recurrence for a mean follow-up period of 37.3 ± 8.6 months. Conclusions: the preliminary results suggest that modified ACER appears to be a viable option for patients with partial LSCD.

A Methyltransferase Trio Essential for Phosphatidylcholine Biosynthesis and Growth.

Phosphatidylcholine (PC) is a primary class of membrane lipids in most eukaryotes. In plants, the primary PC biosynthetic pathway and its role in plant growth and development remain elusive due to lack of a mutant model with substantially decreased PC content. Recently, a double mutant of Arabidopsis (Arabidopsis thaliana) PHOSPHO-BASE N-METHYLTRANSFERASE 1 (PMT1) and PMT3 was reported with reduced PC content and defective plant growth. However, residual PC content as well as the nonlethal phenotype of the mutant suggests an additional enzyme contributes to PC biosynthesis. In this article, we report on the role of three PMTs in PC biosynthesis and plant development, with a focus on PMT2. PMT2 had the highest expression level among the three PMTs, and it was highly expressed in roots. The pmt1 pmt2 double mutant enhanced the defects in root growth, cell viability, and PC content of pmt1, suggesting that PMT2 functions together with PMT1 in roots. Chemical inhibition of PMT activity in wild-type roots reproduced the short root phenotype observed in pmt1 pmt2, suggesting that PMT1 and PMT2 are the major PMT isoforms in roots. In shoots, pmt1 pmt2 pmt3 enhanced the phenotype of pmt1 pmt3, showing seedling lethality and further reduced PC content without detectable de novo PC biosynthesis. These results suggest that PMTs catalyze an essential reaction step in PC biosynthesis and that the three PMTs have differential tissue-specific functions in PC biosynthesis and plant growth.

Reanalysis and optimisation of bioinformatic pipelines is critical for mutation detection.

Rapid advances in genomic technologies have facilitated the identification pathogenic variants causing human disease. We report siblings with developmental and epileptic encephalopathy due to a novel, shared heterozygous pathogenic 13 bp duplication in SYNGAP1 (c.435_447dup, p.(L150Vfs*6)) that was identified by whole genome sequencing (WGS). The pathogenic variant had escaped earlier detection via two methodologies: whole exome sequencing and high-depth targeted sequencing. Both technologies had produced reads carrying the variant, however, they were either not aligned due to the size of the insertion or aligned to multiple major histocompatibility complex (MHC) regions in the hg19 reference genome, making the critical reads unavailable for variant calling. The WGS pipeline followed different protocols, including alignment of reads to the GRCh37 reference genome, which lacks the additional MHC contigs. Our findings highlight the benefit of using orthogonal clinical bioinformatic pipelines and all relevant inheritance patterns to re-analyze genomic data in undiagnosed patients.

A pair of phospho-base methyltransferases important for phosphatidylcholine biosynthesis in Arabidopsis.

Phosphatidylcholine (PtdCho) is a predominant membrane lipid class in eukaryotes. Phospho-base N-methyltransferase (PMT) catalyzes a critical step in PtdCho biosynthesis. However, in Arabidopsis thaliana, the discovery of involvement of the specific PMT isoform in PtdCho biosynthesis remains elusive. Here, we show that PMT1 and PMT3 redundantly play an essential role in phosphocholine (PCho) biosynthesis, a prerequisite for PtdCho production. A pmt1 pmt3 double mutant was devoid of PCho, which affected PtdCho biosynthesis in vivo, showing severe growth defects in post-embryonic development. PMT1 and PMT3 were both highly expressed in the vasculature. The pmt1 pmt3 mutants had specifically affected leaf vein development and showed pale-green seedlings that were rescued by exogenous supplementation of PCho. We suggest that PMT1 and PMT3 are the primary enzymes for PCho biosynthesis and are involved in PtdCho biosynthesis and vascular development in Arabidopsis seedlings.

Cataracts.

An estimated 95 million people worldwide are affected by cataract. Cataract still remains the leading cause of blindness in middle-income and low-income countries. With the advancement of surgical technology and techniques, cataract surgery has evolved to small-incisional surgery with rapid visual recovery, good visual outcomes, and minimal complications in most patients. With the development of advanced technology in intraocular lenses, the combined treatment of cataract and astigmatism or presbyopia, or both, is possible. Paediatric cataracts have a different pathogenesis, surgical concerns, and postoperative clinical course from those of age-related cataracts, and the visual outcome is multifactorial and dependent on postoperative visual rehabilitation. New developments in cataract surgery will continue to improve the visual, anatomical, and patient-reported outcomes. Future work should focus on promoting the accessibility and quality of cataract surgery in developing countries.

A pair of nonspecific phospholipases C, NPC2 and NPC6, are involved in gametophyte development and glycerolipid metabolism in Arabidopsis.

Phospholipases play crucial roles in plant membrane lipid homeostasis. Nonspecific phospholipase C (NPCs) establish a unique class of phospholipases found only in plants and certain bacteria. Here, we show that two previously uncharacterized NPC isoforms, NPC2 and NPC6, are required for male and female gametophyte development in Arabidopsis. Double mutant plants of npc2-1 npc6-2 could not be retrieved because npc2-1 npc6-2 ovule and pollen development is affected. Genetic complementation, reciprocal crossing and microscope observation of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants suggest that NPC2 and NPC6 are redundant and are required for normal gametophyte development. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate, being consistent with the reported substrate selectivity of NPCs. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol were increased in buds but not in flowers of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants, presumably due to reduced phospholipid hydrolysis activity in developing flowers. Our results demonstrate that NPC2 and NPC6 play crucial roles in gametogenesis during flower development.

The Choline/Ethanolamine Kinase Family in Arabidopsis: Essential Role of CEK4 in Phospholipid Biosynthesis and Embryo Development.

Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylethanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoethanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/ethanolamine kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis.

Long-term outcomes of hemi-automated lamellar keratoplasty.

IMPORTANCE: To describe long-term outcomes of hemi-automated lamellar keratoplasty (HALK). BACKGROUND: HALK is a hybrid anterior lamellar keratoplasty technique for corneas with anterior to mid-stromal scars and topographical irregularities. DESIGN: Prospective interventional case series. PARTICIPANTS: Thirty-five eyes of 35 consecutive patients undergoing HALK at a single tertiary referral centre from 2007 to 2016. METHODS: Patients were followed up for a mean period of 61.4 ± 29.2 months. MAIN OUTCOME MEASURE: Uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), spherical equivalent (SE) and cylinder, endothelial cell density (ECD), central corneal thickness (CCT), graft survival and complications were analysed. RESULTS: The most common indications for HALK were scars because of contact lens associated infectious keratitis (29%), unknown origin (26%) or corneal dystrophies (14%). Five patients had a previous keratoplasty (n = 4; deep anterior lamellar keratoplasty, n = 1). Two HALKs failed at 22 and 32 months follow-up. No graft rejections occurred. UCVA improved from 0.91 ± 0.31 to 0.58 ± 0.35 and BSCVA from 0.66 ± 0.30 to 0.21 ± 0.20 logMAR (P < 0.001) at the last follow-up. Astigmatism (P = 0.2), SE (P = 0.8) and ECD (P = 0.4) did not change significantly during follow-up. CCT increased from 490 to 560 µm (P = 0.004). Kaplan-Meier estimated survival for all HALK cases was 90.6 (95% confidence interval 82.6-98.5) months with a survival probability of 96% at 12 months and 92% at 3, 5 and 7 years of follow-up. CONCLUSIONS AND RELEVANCE: HALK provides excellent graft survival in primary cases and in patients with prior keratoplasty as well as significant improvement in visual acuity with low complication rates.

The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana.

In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

Corneal lenticule storage before reimplantation.

Purpose: To explore the optimal lenticule storage conditions that maintain lenticule integrity and clarity. Methods: A total of 99 lenticules obtained from myopic patients undergoing small incision lenticule extraction (SMILE) were divided into four combinations for short-term storage conditions: PBS, Dulbecco's Modified Eagle's Medium (DMEM), Optisol GS, or anhydrous glycerol. Two thirds of the lenticules were further stored for 4 weeks under eight different conditions. Clarity evaluation with transmittance measurements, cell-death assays with terminal deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL), collagen fibril spacing and necrotic response assessed with transmission electron microscopy (TEM), and immunohistochemistry analysis for human leukocyte antigens (HLAs) and CD45 for immunogenicity, and matrix metalloproteinase (MMP)-2 for keratocyte response, were undertaken at baseline, 48 h (short term), and 4 weeks (long term). Results: The TUNEL and immunogenicity results were comparable among the groups. The mean percentage of TUNEL-positive cells across all groups was 24.3% ± 11.8% and 62.9% ± 20.7% at the 48 h and 4 week time points, respectively. HLA-ABC+, HLA-DR+, and CD45+ cells were extremely rare, and MMP-2 expression ranged from non-detectable to minimal, under all conditions at all time points. Transmittance at 4 weeks was significantly different among groups with the greatest maintenance of clarity seen in the lenticules stored initially in DMEM at 4 °C for 48 h followed by cryopreservation in serum-free medium or glycerol at 4 °C followed by storage at room temperature. At TEM analysis at 4 weeks, the lenticules cryopreserved in liquid nitrogen, regardless of storage solutions, had significantly narrower inter-fibrillar distance than controls, while glycerol-preserved lenticules, at either room temperature or -80 °C, maintained the inter-fibrillar distance. Conclusions: Clarity, structural integrity, and low immunogenicity under various conditions, at 4 °C or room temperature for short-term storage, offer encouragement for lenticule storage. It can be undertaken without access to s specialized and potentially expensive laboratory setup at least within the first 48 h before transportation to larger facilities for long-term storage.

Enhancement after Small-Incision Lenticule Extraction: Incidence, Risk Factors, and Outcomes.

PURPOSE: To report the incidence, risk factors, and outcomes of enhancement after small-incision lenticule extraction (SMILE). DESIGN: Retrospective cohort study. PARTICIPANTS: Five hundred twenty-four eyes of 307 patients who underwent SMILE at Singapore National Eye Center between February 2012 and March 2016. METHODS: The data collected included patient age at primary SMILE, gender, race, preoperative and postoperative manifest refraction spherical equivalent (MRSE), preoperative and postoperative uncorrected distance visual acuity and corrected distance visual acuity, the occurrence of suction loss during the procedure, and the need for enhancement. All enhancements were carried out by performing an alcohol-assisted photorefractive keratectomy (PRK) procedure with application of mitomycin C (MMC). MAIN OUTCOME MEASURES: Incidence, prevalence, preoperative and intraoperative risk factors for enhancement, and outcomes after enhancement. RESULTS: The prevalence of enhancement was 2.7%, and 71.4% eyes had enhancement within 1 year of primary SMILE. The incidence of enhancement was 2.1% and 2.9% at 1 and 2 years, respectively. Age older than 35 years, preoperative MRSE more than -6.00 diopters (D), preoperative myopia more than 6.00 D, preoperative astigmatism more than 3.00 D, and intraoperative suction loss were significant risk factors for enhancement after SMILE after adjusting for all other covariates (odds ratios, 5.58, 4.80, 1.41, 3.06, and 2.14, respectively; P = 0.004, 0.021, 0.022, 0.002, and 0.020, respectively). In the patients who underwent bilateral SMILE, the first-operated eye had a marginal trend toward significance for enhancement (P = 0.054). There was no gender or racial difference. In the 14 eyes requiring enhancement, the uncorrected distance visual acuity before enhancement ranged from 20/80 to 20/25, and the mean attempted enhancement spherical equivalent was -0.50±0.86 D. The uncorrected distance visual acuity improved in most patients (92.9%) after enhancement. CONCLUSIONS: The 2-year incidence of enhancement after SMILE was 2.9%. Risk factors associated with enhancement included older age at SMILE procedure, greater preoperative MRSE, greater preoperative myopia, greater preoperative astigmatism, and the occurrence of intraoperative suction loss. Clinical outcomes of using PRK with application of MMC for enhancement were good.