Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough.

Stop codon readthrough events give rise to longer proteins, which may alter the protein's function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells.

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments.

Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation.

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

Different Material States of Pub1 Condensates Define Distinct Modes of Stress Adaptation and Recovery.

How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery.

RNA buffers the phase separation behavior of prion-like RNA binding proteins.

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.

Phase separation of a yeast prion protein promotes cellular fitness.

Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.

A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy.

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Quality of life, weight loss and improvement of co-morbidities after primary and revisional laparoscopic roux Y gastric bypass procedure-comparative match pair study.

BACKGROUND: The prevalence of morbid obesity and its co-morbidities is dramatically increasing, as is the extent of weight loss surgery. A large number of patients after various bariatric procedures need revisional intervention for various reasons. We investigated the efficacy and the safety of revisional laparoscopic Roux Y gastric bypass (LRYGB) among our patients, who were revised as a consequence of inadequate weight loss or weight regain after previous bariatric interventions. METHODS: A comparative, double-centre, match pair study was performed comparing the data of 44 patients after revisional surgery with 44 patients after primary gastric bypasses, focusing on weight loss, life quality and improvement of co-morbidities. Matching criteria were age, gender, preoperative BMI and follow-up period. Previous procedures consisted of 23 gastric bandings, 13 sleeve resections, 4 LRYGB and 4 vertical banded gastroplasties. RESULTS: Extra weight loss (EWL) was significantly reduced after revisional gastric bypasses compared to primary intervention (EWL 66 vs. 91 %, p<0.05). Life quality scores were also decreased in the revisional group compared to the control group without statistical significance (SF 36 score 635 vs. 698.5, p=0.22; Moorehead-Aldert II score 1.4 vs. 2.0, p=0.10). The resolution rate of co-morbidities (T2DM, hypertension, gastro-oesophageal reflux (GER), osteoarthrosis, sleep apnoea) was also higher after primary gastric bypasses. CONCLUSIONS: Revisional LRYGB is an effective and safe method for patients with inadequate weight loss after previous bariatric surgery concerning weight reduction, life quality and improvement of co-morbidities. Our results indicate lower efficacy of revisional compared to primary LRYGB reaching statistical significance in regard to weight loss.

Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation.

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

Comfort response of three silicone hydrogel daily disposable contact lenses.

PURPOSE: To evaluate subjective ocular comfort across the day with three silicone hydrogel daily disposables (SHDDs) in a group of adapted lens wearers. METHODS: Masked subjects (asymptomatic or symptomatic of end-of-day (EOD) dryness with habitual lenses) wore three SHDDs: DAILIES TOTAL1 (DT1), Clariti 1day (C1D), or 1-DAY ACUVUE TRUEYE (AVTE), each for 3 days. On day 2, wearing time (WT) and comfort ratings after insertion, at 4, 8, and 12 hours, and at EOD were recorded. Because not all subjects wore lenses for 12 hours, comfort was analyzed across the day (up to 8 hours, 8 to 12 hours), and a new variable ("cumulative comfort" [CC]) was calculated for EOD. RESULTS: One hundred four subjects completed the study (51 asymptomatic, 53 symptomatic). The two groups had different WTs (mean WT, 14.0 and 12.7 hours, respectively; p < 0.001). Ocular comfort was rated higher in the asymptomatic group throughout the day (p < 0.001). One hundred four subjects wore all three SHDDs for at least 8 hours, whereas 74 (45 asymptomatic, 29 symptomatic) subjects wore them for 12 hours or longer. Comfort ratings were higher with DT1 (least square means [LSM] = 91.0) than with C1D (LSM = 86.5; p < 0.001) and AVTE (LSM = 87.7; p = 0.011) for the first 8 hours and lower with C1D compared with DT1 (p = 0.012) from 8 to 12 hours. Mean EOD (± SD) comfort with the C1D lens was 72 ± 21, lower than both DT1 (mean, 79 ± 17; p = 0.001) and AVTE (mean, 78 ± 21; p = 0.010). Mean CC was higher in the asymptomatic group (mean, 1261 ± 59) compared with that in the symptomatic group (mean, 1009 ± 58; p < 0.001) and higher for DT1 (mean, 1184 ± 258) than C1D (mean, 1094 ± 318; p = 0.002) and AVTE (mean, 1122 ± 297; p = 0.046). CONCLUSIONS: All three SHDDs had average WTs of 12 hours or longer for 1 day. Comfort during the first 12 hours was highest with DT1 (similar to AVTE between 8 and 12 hours) and lowest with C1D. End-of-day comfort was lowest with C1D, and CC was highest for DT1. Cumulative comfort may be a valuable new metric to assess ocular comfort during the day.

A multi-country assessment of compliance with daily disposable contact lens wear.

PURPOSE: To investigate compliance with daily disposable contact lens (DDCL) wear and investigate re-use of lenses according to country and DDCL material worn. METHODS: Optometrists invited eligible DDCL patients from their practices to participate in a survey on DDCL wear in Australia, Norway, the United Kingdom (UK) and the United States (US). Eligible participants completed an online or paper version of the survey. RESULTS: 805 participants completed the survey (96% online): Australia 13%, Norway 32%, UK 17%, US 38%. The median age was 38 years; 66% were female. Silicone hydrogel (SiHy) DDCLs were worn by 14%. Overall, 9% were non-compliant with DDCL replacement; Australia 18%, US 12%, UK 7% and Norway 4%. There were no differences with respect to sex, years of contact lens wear experience or DDCL material (SiHy versus hydrogels). The primary reason for re-use was "to save money" (60%). Re-use of DDCLs resulted in inferior comfort at insertion and prior to lens removal (p=0.001). 75% reported occasional napping and 28% reported sleeping overnight for at least one night in the preceding month, while wearing their DDCLs. CONCLUSION: Non-compliance with replacement of DDCLs occurred in all countries investigated; the rate was highest in Australia and lowest in Norway. Re-use of DDCLs was associated with reduced comfort. DDCL wearers often reported wearing lenses overnight. It is important for optometrists to counsel their patients on the importance of appropriate lens wear and replacement for DDCLs.

Compliance with lens replacement and the interval between eye examinations.

PURPOSE: Eye care practitioners (ECPs) acknowledge that their patients do not always follow recommendations for lens replacement, but many may not realize the possible implications for their offices. The study was conducted to investigate whether there is a relationship between contact lens compliance and the interval between full eye examinations (IEE). METHODS: The study was conducted in ECP offices in the United States. Eye care practitioners and patients independently completed linked questionnaires, evaluating their contact lens wear and care. Patients were required to be current wearers of daily disposable (DD) lenses or reusable silicone hydrogel lenses with a manufacturer-recommended replacement frequency (MRRF) of 2 weeks (2WR) or 1 month (1MR). RESULTS: A total of 2147 questionnaires from 141 offices were eligible. Fifty-four percent of patients were wearing 2WR, 37% 1MR, and 9% DD lenses. Wearers of 2WR lenses were significantly less compliant with replacement than wearers of both DD and 1MR lenses (34% vs. 74% and 67%, both p < 0.001); patients purchasing an annual supply were more compliant (55% vs. 45%, p < 0.001). The mean IEE was 16 months and was longer for wearers who were noncompliant with the MRRF (17.4 months vs. 14.5 months, p < 0.001). Other factors affecting IEE were household income (p = 0.030), insurance (p < 0.001), purchase source (p < 0.001), and sex (p = 0.007). CONCLUSIONS: Patients who were not compliant with the MRRF had longer IEEs and were less likely to purchase an annual supply of lenses. Patients who purchased lenses from their ECP, had a higher household income, had eye examination insurance, and were female had shorter IEEs. Patients failing to replace their lenses when scheduled were also found to be less compliant with lens care procedures. Eye care practitioners should reinforce the importance of all aspects of lens wear and care with their patients, with the overall aim of reducing possible complications and retaining successful contact lens wearers in their offices.

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

Exogenous ether lipids predominantly target mitochondria.

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.

Examination of the efficacy and safety of intraoperative gastroscopic testing of the gastrojejunal anastomosis in laparoscopic Roux Y gastric bypass surgery.

The laparoscopic Roux Y gastric bypass (LRYGB) is one of the most often performed bariatric surgical intervention. Intraoperative gastroscopy (IOG) seems to be reliable to decrease the leakage rate of gastrojejunal anastomosis (GJA) and of gastric pouch (GP). Our aim was to test the efficacy and the safety of this method. Two hundred fifty-two LRYGB operations were performed in our institution between 1 January 2008 and 1 January 2010. IOG is routinely made to test the integrity of GJA and of GP. Patients' dates were retrospectively analysed. The intragastric pressure developed during gastroscopy in humans was measured and compared with pressure values led to destruction (positive air test) of the GJA and/or GP in animal models (hybrid pigs). Stomach and bowel wall samples from the test animals without pressure strain, with pressure strain developed at gastroscopy in humans and with pressure strains led to destruction of GJA and/or GP were histologically examined. IOG resulted in six of our cases (2.3%) positive air test. There was no anastomosis insufficiency in postoperative period. Mean pressure during IOG was 32 mmHg, mean time of examination was 3.8 min and mean maximal pressure was 43 mmHg in humans. The mean pressure leading to positive air test in pigs was 150 mmHg. We could not detect any microscopical difference between stomach and jejunum samples without pressure strain and after pressure strain developed in humans during the gastroscopy. We conclude that intraoperative gastroscopy is an effective and safe method to test the integrity of GJA and GP in LRYGB surgery.

Distribution of overnight corneal swelling across subjects with 4 different silicone hydrogel lenses.

PURPOSE: To determine distribution of central corneal swelling (CCS) across subjects after 8 hr of sleep in eyes wearing silicone hydrogel lenses with various oxygen transmissibility (Dk/t) values and in eyes without lenses. METHODS: Twenty-nine neophytes wore lotrafilcon A (Dk, 140), balafilcon A (Dk, 91), galyfilcon A (Dk, 60), and senofilcon A (Dk, 103) lenses in powers -3.00, -10.00, and +6.00 diopters on separate nights, in random order, and on 1 eye only. The contralateral eye (no lens) served as the control. Central corneal thickness was measured using a digital optical pachometer before lens insertion and immediately after lens removal on waking. RESULTS: The average difference between the mean (7%) and the median (6.8%) CCS of all lenses was only 0.2%, suggesting a normal distribution. There was no correlation between the mean and the range of the CCS (r=0.058, P=0.766). Normal CCS distributions were also found with each lens-wearing eye and the control eye (P>0.20 for all). There was a significant correlation between lens-wearing eye and control eye (r=0.895, P<0.001) and between lotrafilcon A and each of the other 3 lenses for mean CCS across the study participants (P<0.001 for all). CONCLUSIONS: Distribution of corneal swelling in both lens-wearing eye and control eye followed a normal curve. An individual's corneal swelling response seems to be independent of lens type.

Comfort and vision with silicone hydrogel lenses: effect of compliance.

PURPOSE: Silicone hydrogel (SH) lenses are usually replaced after 2 weeks (2W) or 1 month (1M); however, many patients do not comply with the manufacturers' recommended replacement frequency (MRRF). The purpose of this analysis was to investigate the effect of compliance with MRRF on comfort and vision in SH wearers. METHODS: As a part of a larger study investigating compliance with MRRF, patients were asked to rate their subjective comfort and vision from 0 (very poor) to 10 (excellent) in the morning, at the end of the day (EVE), when lenses were new, and needed replacing (NR). RESULTS: One thousand three hundred forty-four patients wore 2W replacement modality (2WR) (n = 717) or 1M replacement modality (1MR) (n = 617) SH lenses. Comfort and vision in the morning and when lenses are new were significantly higher than for EVE and NR (p < 0.001). Twenty-nine percent (95% confidence interval 25.3-32.4) of 1MR and 59% (95% confidence interval 55.5-62.7) of 2WR wearers were non-compliant with the MRRF. Compliance had a significant effect on EVE (p = 0.002, p = 0.008) and NR (p < 0.001, p < 0.001) comfort and vision. After accounting for compliance, EVE and NR comfort and EVE vision were higher for 1MR than 2WR (p = 0.015, p = 0.044, p = 0.019). CONCLUSIONS: Compliant patients had better EVE and NR comfort and vision than non-compliant patients, regardless of replacement modality. Optimal subjective performance with SH lenses seems to be facilitated by replacing lenses as recommended.

Compliance with contact lens replacement in Canada and the United States.

PURPOSE: To assess eye care practitioners (ECPs) recommendations for replacement frequency (RF) of silicone hydrogel (SH) and daily disposable (DD) lenses in Canada and the U.S. and to compare noncompliance (NC) with manufacturer recommended RF by the ECP and patient, and the reasons given for NC. METHODS: Invitations to participate were sent by e-mail to ECPs in Canada and the U.S. Twenty patient surveys were sent to 420 ECPs, and 2232 eligible surveys were received from 216 ECPs (26% Canada, 74% U.S.). Questions related to patient demographics, lens type, wearing patterns, ECP instructions for RF, and actual patient RF. ECPs provided lens information and their recommendation for RF after the surveys were completed and sealed in envelopes. Responses were anonymous. RESULTS: DD accounted for 18% (Canada) vs. 16% (U.S.) of wearers (p > 0.05); 35% (Canada) vs. 45% (U.S.) wore 2-week replacement SH (2WR; p = 0.011); and 47% (Canada) vs. 39% (U.S.) wore 1-month replacement SH (1MR) lenses (p = 0.025). Thirty-four percent (Canada) vs. 18% (U.S.) of ECPs recommended longer RFs than the manufacturer recommended RF for 2WR lens wearers (p < 0.001); 6% (Canada) vs. 4% (U.S.) for DD wearers; and 2% (Canada) vs. 1% (U.S.) for 1MR lens wearers. NC rates for actual RFs reported by patients were not different between countries (p > 0.05) and were lowest for DD (13% Canada, 12% U.S.), followed by 1MR (33% Canada, 28% U.S.). The highest NC rates were with 2WR (50% Canada, 52% U.S.). The most frequent reason for NC with 2WR and 1MR was "forgetting which day to replace lenses" (54% Canada, 53% U.S.) and in DD wearers "to save money" (56% Canada, 29% U.S., p < 0.001). CONCLUSIONS: 1MR lenses are more frequently prescribed in Canada. ECPs in Canada were NC with 2WR lenses more frequently than U.S. ECPs, but patient NC rates were the same in both countries for all lens types. ECP and patient NC rates were highest for 2WR lens wearers.