Sight-Threatening Immune Retinopathy Developing Secondary to Durvalumab Treatment of Small-Cell Lung Cancer: A Case Report.

Introduction: Given the recent additions of immune checkpoint inhibitors (ICIs) to various cancer treatments, adverse effects, especially involving the eyes, have been on the rise. Here, we report an acute exacerbation of cancer-associated retinopathy (CAR) triggered by durvalumab treatment of small-cell lung cancer (SCLC). Case Presentation: An 81-year-old Asian male complained of a scotoma in the left eye after durvalumab administration, to treat SCLC. Humphrey visual field examination revealed a C-shaped temporal scotoma. Spectralis domain optical coherence tomography revealed outer retinal layer atrophy and progressive loss of the ellipsoid zone in the atrophic peripapillary area. Fundus autofluorescence (AF) images evidenced a large C-shaped hypo-AF with enhanced AF at the margin of the atrophic area, thus at the position of the scotoma. We prescribed subtenon triamcinolone injections under suspicion of CAR exacerbation, supported by positive Western blotting results for Rab6 and aldolase, and immunohistochemical staining of photoreceptor cells. The disrupted ellipsoid zone evident on OCT partially recovered, and a visual field test showed that the scotoma had improved. Conclusion: ICI-triggered exacerbation of CAR should be considered in SCLC patients before ICI treatment commences; an optimal treatment should preserve functional vision.

Development of a novel Korean reading chart.

CLINICAL RELEVANCE: Measuring reading ability is a crucial part of assessing patients who complain of reduced vision. Foreign language versions of such charts need to be developed and validated. BACKGROUND: It is difficult to measure or predict Korean reading ability due to a lack of a representative reading charts in Korean, and previous charts have limited capacity to detect deficits in reading ability among Korean patients with eye diseases. METHODS: Two printed versions of the reading chart were created. Thirty-four patients with no change in vision in the last three months and no expected change in vision in the next four weeks were included in this study. The results were validated by testing 13 normal-sighted adults (group 1), 14 patients with various macular diseases whose visual acuity was equal or better than 0.5 logarithm of the minimum angle of resolution (logMAR) (group 2), and seven patients with various macular diseases whose visual acuities were between 1.3 logMAR and 0.5 logMAR (group 3). Inter-chart and intra-subject repeatabilities were assessed for maximum reading speed (MRS) and critical print size (CPS). RESULTS: A total of 38 sentences were tested on 34 adults in three groups. Groups 1 and 2 did not differ significantly in terms of MRS and CPS. The MRS was lower in group 3, for each chart and between visits. The CPS was larger in group 3, for each chart and between visits, with the exception of chart 2 during visit one. With regard to test-retest reliability, the intraclass correlation co-efficients (ICCs) for chart 1 and chart 2 were more than 0.900. With regard to inter-chart reliability, the ICCs were more than 0.892, respectively. CONCLUSION: The reading chart developed in this study was reliable in producing consistent results among a normal Korean population and patients with various macular diseases.

Long-term safety and tolerability of subretinal transplantation of embryonic stem cell-derived retinal pigment epithelium in Asian Stargardt disease patients.

BACKGROUND: Although human embryonic stem cells (hESCs) have been considered a potential therapeutic option for regenerative medicine, there are some concerns regarding tumorigenicity, immunogenicity and ethical considerations. Stargardt macular dystrophy (SMD) is the most common form of juvenile macular degeneration that causes early onset blindness. Therapeutic options for SMD remain limited, although several treatment strategies are currently under investigation. Here, we report a 3-year assessment of a phase I clinical trial involving subretinal transplantation of hESC-retinal pigment epithelium (RPE) cells in patients with SMD. METHODS: This prospective, non-randomised clinical trial included three patients with SMD. All transplant recipients had central visual acuity no better than 20/400. Trans-pars plana vitrectomy was performed in the eye with poorer vision. RPE cells were reconstituted in balanced salt solution plus, then injected into the subretinal space using a semi-automated subretinal injection method. RESULTS: No serious adverse events occurred throughout the 3-year period following the injection of hESC-RPE cells. The functional and anatomical results were favourable, compared with the natural course of SMD reported in the ProgStar study. One patient showed best-corrected visual acuity improvement, while the other patients had stable best-corrected visual acuity during the 3-year follow-up period. CONCLUSION: These results suggest the long-term safety, tolerability, and feasibility of subretinal hESC-derived RPE cell transplantation in regenerative medicine. TRIAL REGISTRATION NUMBER: NCT01625559.

Treatment of traumatic optic neuropathy using human placenta-derived mesenchymal stem cells in Asian patients.

Aim: To assess the safety and feasibility of subtenon transplantation of human placenta-derived mesenchymal stem cells (hPMSCs) in Asian patients with traumatic optic neuropathy. Materials & methods: The survival of retinal ganglion cells in the rat retina was evaluated by monitoring the expression of Tuj1 and Gfap after optic nerve compression. Based on the preclinical data, we conducted a Phase I, open label, single center, nonrandomized clinical trial in four Asian traumatic optic neuropathy patients. The safety and ophthalmologic changes were evaluated. Results: The levels of Tuj1 and Gfap expression were significantly increased in the hPMSC treatment group compared with the sham group, suggesting a protective effect of hPMSCs on the optic nerve and retinal ganglion cells. There was no evidence of adverse proliferation, tumorigenicity, severe inflammation or other serious issues during the 12-month follow-up period. Visual acuity improved in all four patients. Conclusion: The results suggested that hPMSCs are safe and have potential utility in regenerative medicine. Clinical trial registration number: 20150196587 (Korean FDA), 2015-07-123-054 (IRB).

Endogenous Endophthalmitis Associated with Liver Abscess Successfully Treated with Vitrectomy and Intravitreal Empirical Antibiotics Injections.

PURPOSE: Klebsiella pneumoniae is the most common pathogen of endogenous endophthalmitis accompanying liver abscess in East Asia. The treatment may be different for the patients with endogenous endophthalmitis from the postoperative endophthalmitis. Prompt administration of both intraocular (vancomycin and ceftazidime) and systemic (ceftriaxone, aminoglycoside, and/or metronidazole) antibiotics have been a mainstay of treatment. However, ceftriaxone has been proven to more effectively kill K. pneumoniae than ceftazidime in in vitro studies, and the safety of intravitreal ceftriaxone has been confirmed in animal studies. METHODS: Two diabetic female patients with liver abscess presented with decreased visual acuity of the unilateral eyes. Fundus photography, ocular ultrasonography, and abdominal computed tomography were performed. RESULTS: A 50-year-old diabetic female patient with liver abscess presented decreased visual acuity of the left eye. In fundus examinations, a yellowish necrotic lesion was noted throughout the eye. The results of culture of the blood culture was positive for K. pneumoniae. She was successfully treated with intravitreal ceftazidime injections, and the remaining vitreous opacity was treated with vitrectomy. A 62-year-old female with liver abscess presented a visual symptom of floaters in the right eye. The fundus had a hazy appearance through the vitreous opacity. A yellowish-white subretinal abscess was noted at the temporal macula. Cultures of blood were negative. She underwent intravitreal injections of empirical antibiotics. However, she did not respond to intravitreal vancomycin and ceftazidime. Thus, we changed the intravitreal antibiotics from ceftazidime to ceftriaxone and performed vitrectomy. Her ocular status significantly improved after this change. CONCLUSION: Our results indicate that for cases with EE, prompt initial treatment with broad spectrum antibiotics, followed by rapid use of antibiotics selected according to culture results, and empirical use of antibiotics in cases of a negative culture may be an effective treatment. Vitrectomy also can be an effective treatment option for vitreous opacity refractory to the treatment.

Periorbital Injection of Hyaluronic Acid Gel in Patients With Deep Superior Sulcus.

PURPOSE: Hyaluronic acid gel (HAG) fillers provide a versatile and safe correction method for a sunken superior sulcus (SS) resulting from soft tissue volume loss occurring with aging, previous surgery, or enophthalmos related to trauma or phthisis bulbi. The purpose of this study was to report the long-term clinical outcomes of filler injection for soft-tissue augmentation in patients with a deep SS. DESIGN: Retrospective, interventional case series METHODS:: The medical records of 27 patients (32 eyes) with deep superior sulci were reviewed. Twenty injections of hyaluronic acid fillers (Restylane; Q-med AB, Uppsala, Sweden) were performed in the retrobulbar (RB) area, and 15 injections were performed in the SS. Pre- and post-treatment photographs were taken. MAIN OUTCOMES MEASURES: Visual acuity for seeing eyes, intraocular pressure, exophthalmos measurement, marginal reflex distance 1, interpalpebral fissure, tarsal platform show, and SS hollowing depth grade RESULTS:: Enophthalmos corrections were 1.3 ±â€Š0.8 mm (P < 0.001) and the SS hollowing grade decrease was 1.2 ±â€Š0.9 (P < 0.01) after 1 month of RB injection. Enophthalmos corrections were 0.1 ±â€Š0.4 mm (P = 0.317) and the SS sunkeness grade decrease was 1.7 ±â€Š0.8 (P < 0.001) after 1 month of SS injection. The amount of filler to correct enophthalmos of 1 mm was 0.83 ±â€Š0.53 mL using the RB injection. Using Kaplan-Meier survival analysis, the mean period of maintenance for deep SS correction was 9.1 months for RB injections and 8.5 months for SS injections. CONCLUSION: Retrobulbar (RB) hyaluronic acid gel (HAG) injections corrected enophthalmos and a deep SS, while SS injections corrected only a deep SS. These 2 techniques of RB and direct sulcus injection are safe and effective methods for the correction of a deep SS.

Epiblepharon correction in Korean children based on the epicanthal pathology.

PURPOSE: This study aims to investigate the surgical outcomes of a novel epiblepharon correction method based on the pathologic features of epicanthal tissue. The correlation between the pathology and clinical presentations was also studied. METHODS: This retrospective study included 72 eyelids of 36 children with epiblepharon and epicanthal folds. From July 2013 to March 2016, epicanthal tension-releasing procedure of subdermal fibrinolysis with orbicularis oculi ring myotomy were performed in 36 children. Photographs before and after operation were analyzed with Image J program. A clinical severity of the epiblepharon was scored based on the lower eyelid skin fold height (mild to severe). Subdermal fibrosis index was defined as the portion of fibrosis in low power field (× 40) and eyelid contour was assessed with custom software (MATLAB). Eighty eyelids without eyelid pathology were analyzed to establish the normal eyelid contour. The epicanthal tissues were classified into three categories: mild, moderate, and severe fibrosis. RESULTS: The mean age of the patients was 5.5 years. Intercanthal distance (ICD) ratio was defined as Inner ICD (IICD)/outer ICD (OICD). The IICD/OICD was 0.41 in the severe epiblepharon group that is significantly longer compared to that of the mild epiblepharon group (0.38). Subdermal fibrosis index was higher in severe epiblepharon group. Eyelid contour was normalized and the cilia touch was resolved after the epicanthal tension-releasing procedure. CONCLUSION: Epicanthal tension-releasing procedure with orbicularis oculi ring myotomy achieved favorable surgical outcomes. Clinical correlations with pathologic findings and satisfactory results of the procedure suggest that medial epicanthal fibrosis is the main pathologic feature causing epiblepharon and epicanthal folds.

Clinical and Genetic Characteristics Analysis of Korean Patients with Stargardt Disease Using Targeted Exome Sequencing.

PURPOSE: To investigate genetic mutations in Korean patients with Stargardt disease (STGD) using exome sequencing, and to analyze the correlations between genetic mutations and clinical phenotypes. METHODS: Peripheral venous blood was obtained from 24 clinically diagnosed Korean STGD patients, followed by extraction of genomic DNAs. Using exome sequencing we investigated gene mutations for the adenosine triphosphate-binding cassette, subfamily A, member 4 (ABCA4) elongation of very-long-chain fatty acids 4 (ELOVL4), and prominin 1 (PROM1), and confirmed gene mutations by the direct sequencing of polymerase chain reaction products. RESULTS: ABCA4 mutations were confirmed in 17 of 24 patients, and 12 novel mutations were identified. ELOVL4 and PROM1 gene mutations were not identified in this study. We also identified 16 previously reported mutations related to STGD1. In patients whose disease symptoms occurred before 20 years of age, visual acuity was poorer and atrophic flecks were more frequently found. In addition, more ABCA4 mutations were found in patients who had choroidal silence or atrophic flecks. CONCLUSIONS: Novel ABCA4 gene mutations were found in Korean patients with STGD1. This study will facilitate better understanding of the relationships between ABCA4 gene mutations and clinical symptoms in Korean patients.

Measurement of lacrimal punctum using spectralis domain anterior optical coherence tomography.

PURPOSE: Anterior segment optical coherence tomography (AS-OCT) was used to image the lacrimal punctum and establish normal punctal parameters including the tear volume inside the punctum. METHODS: This study included totally 67 eyes from 38 normal subjects. Punctal parameters and tear meniscus height (TMH) were measured using spectralis OCT anterior-segment scans. Outer punctal diameter (OPD), punctal depth, tear well diameter (TWD), tear well depth, punctal reserve (PR) and lower eyelid TMH were assessed. Authors defined a 'tear well' as a pool of standing tears in the punctum. Punctal reserve was defined as the difference between punctal depth and tear well depth. RESULTS: Outer punctal diameter was 614.6 ± 195.6 µm, the punctal depth was 545.8 ± 270.1 µm, TWD was 229.9 ± 133.8 µm, the tear well depth was 434.9 ± 247.8 µm, the PR was 110.9 ± 72.9 µm, the PR ratio was 0.20 ± 0.17, and the TMH was 222.9 ± 88.8 µm. The punctal depth decreased significantly according to the patient's age (correlation coefficient = -0.352, respectively). CONCLUSION: AS-OCT is useful for improving anatomical understanding of the lacrimal punctal structure. Quantitative punctum and tear well measurements were correlated with TMH.

Clinical Outcomes of Frontalis Sling Using Silicone Rod With Two-point Brow Incisions in Blepharoptosis.

PURPOSE: To investigate the functional and cosmetic outcomes of the modified frontalis sling operation using silicone rod with two-point brow incisions. METHODS: A retrospective medical record review of 17 patients who underwent frontalis suspension surgery was performed. Functional outcomes were measured using habitual margin reflex distance. Cosmetic outcomes were investigated with contour analysis and a Patient Scar Assessment Questionnaire. RESULTS: At the final visit, good and fair eyelid heights were achieved in 75% of patients in the unilateral group and 61.1% in the bilateral group. The average increase in margin reflex distance 1 was 1.9 mm in the unilateral group and 1.8 mm in the bilateral group. Satisfaction scores were 14.6, 8.2, and 11.8 for appearance, symptoms, and consciousness, respectively. In contour analysis, the normal arch generally increased. CONCLUSIONS: Frontalis suspension with two-point brow incisions resulted in similar functional and cosmetic outcomes as the three-point brow incision technique. This study showed patient satisfaction with the aesthetic result after the frontalis sling procedure. [J Pediatr Ophthalmol Strabismus. 2016;53(4):224-232.].

Novel technique to measure horizontal corneal diameter using a Scheimpflug image on the Pentacam.

PURPOSE: A new method to measure the horizontal corneal diameter using a Scheimpflug image (SFI) was introduced and compared with the automated iris camera photo (ICP) method on the Pentacam. METHODS: Each one eye of 88 subjects without significant abnormality in the cornea was included. In SFI, diameters were measured manually using the built-in software tool with the edge-filter option. ICP value read from the overview display. Repeatability was determined from the first two measurements of each eye. The accuracy was assessed using Bland-Altman plot in comparison with caliper measurements. RESULTS: The measurement failure rate of ICP was aggravated by pupil dilation, while that of SFI was unrelated to pupil dilation. Repeatability coefficients of ICP and SFI were 0.1mm and 0.12mm, respectively. The mean difference between ICP values and caliper values was not significant. SFI values were greater than caliper measurements by a mean of 0.13mm. CONCLUSIONS: New SFI was reliable and could complement ICP in dilated pupil. The measurement failure rates of Pentacam need to be improved.

Semi-automated counting method of axons in transmission electron microscopic images.

OBJECTIVE: To evaluate the accuracy of a new semi-automated method for counting axons in transmission electron microscopic (TEM) images. PROCEDURES: Optic nerve cross sections were obtained from both eyes of Sprague Dawley rats after unilateral induction of chronic ocular hypertension. TEM images (3000× magnification) of cross sections were evaluated by both semi-automated and manual counting methods. The semi-automated counting method was performed using ImageJ software after simple image optimization, and the resulting estimate of axon damage was compared with semiquantitative damage grading scale from light microscopic (LM) images. RESULTS: Axon counts obtained from the semi-automated method were strongly correlated with those obtained from the manual counting method (Pearson's correlation coefficient r = 0.996, P < 0.001) and from the full manual count from LM images (Spearman's ρ = 0.973, P < 0.001). The semi-automated method measured axonal damage with an error of 0.94 ± 3.16% (mean ± standard deviation), with worse axonal damage associated with greater error. Interobserver and intra-observer variability in axons counts were low (Spearman's ρ = 0.999, P < 0.005). The results of the semi-automated counting method were highly correlated with semiquantitative damage grading scale (Spearman's ρ = 0.965, P < 0.001). CONCLUSION: Results of our semi-automated method for counting axons in TEM images were strongly correlated with those of conventional counting methods and showed excellent reproducibility.

Bleb Analysis and Short-Term Results of Biodegradable Collagen Matrix-Augmented Ahmed Glaucoma Valve Implantation: 6-Month Follow-up.

PURPOSE: To evaluate the short-term efficacy of a biodegradable collagen matrix (BCM) as an adjuvant for Ahmed valve implantation surgery to prevent the hypertensive phase. METHODS: This prospective study included 43 refractory glaucoma eyes, all followed for 6 months. Refractory glaucoma was defined as an IOP higher than 20 mm Hg with antiglaucoma eye drops without previous glaucoma surgery. Conventional method was performed in 21 eyes and BCM-augmented Ahmed valve implantation (BAAVI) in 22 eyes. In the BAAVI group, a 10 × 10 × 2-mm BCM was sutured on an Ahmed glaucoma valve FP7 model. Complete success was defined as an IOP of 21 mm Hg or lower (target IOP 1) or 17 mm Hg or lower (target IOP 2) without antiglaucoma medications and qualified success as an IOP of 21 mm Hg or lower with or without medications. Maximal bleb thickness was measured using anterior segment optical coherence tomography images. RESULTS: The preoperative IOPs and numbers of preoperative antiglaucoma medications were similar for both groups. Complete target IOP 1 success rates were 38.1% and 86.4%, complete target IOP 2 success rates were 19.0% and 59.1%, and qualified success rates were 52.4% and 90.9% in the conventional and BAAVI groups, respectively (P < 0.05). The hypertensive phase rate was lower in the BAAVI group (4.5% vs. 47.6%, P = 0.002). Maximal bleb thickness was increased in the BAAVI group on postoperative days 30 and 180 (P < 0.05). CONCLUSIONS: Success rates were higher in the BAAVI group than in the conventional group with the change of bleb morphology. Furthermore, use of BCM significantly decreased the need for antiglaucoma medications for at least 6 months postoperatively.

Clinical outcomes of individualized botulinum neurotoxin type A injection techniques in patients with essential blepharospasm.

PURPOSE: To assess the clinical outcomes following botulinum neurotoxin type A (BoNT-A) treatment with an individualized injection technique based on the types of spasms and to compare the results of the individualized injection technique with those of the conventional injection technique in the same patients. METHODS: From November 2011 to July 2013, 77 BoNT-A injections were performed in 38 patients. Eighteen patients were treated with conventional BoNT-A injections before 2011, and 20 patients were referred to our hospital for unsatisfactory results after a conventional injection technique. We classified the patients by spasm-dominant sites: the lateral orbital area, representing the orbital orbicularis-dominant group (ODG); the glabella, representing the corrugator-dominant group (CDG); and the ptosis, representing the palpebral part of the orbicularis-dominant group (PDG). We increased the injection dose into the spasm-dominant sites of the blepharospasm groups. We assessed subjective symptom scores (functional disability score, FDS) after treatment. RESULTS: This study included 38 patients (26 women, 12 men; mean age, 60.6 ± 10.9 years). There were 21 patients in the ODG, 10 patients in the CDG, and 7 patients in the PDG. Mean ages were 59.7 ± 12.6, 59.8 ± 8.5, and 66.8 ± 9.0 years, and mean BoNT-A injection dose was 38.8 ± 11.2, 38.8 ± 11.2, and 38.8 ± 10.8 U in each group, respectively (p = 0.44, 0.82 Kruskal-Wallis test). Mean FDS after injection was 1.7 ± 0.7 in the ODG, 1.4 ± 0.8 in the CDG, and 1.2 ± 0.3 in the PDG. There were significant differences in reading and job scale among the three groups. In a comparison between the conventional and individualized injection techniques, there was a significant improvement in mean FDS and in the reading scale in the PDG with the individualized injection technique. The success rate was 92.1% in the conventional injection group and 94.1% in the individualized injection group. CONCLUSIONS: The individualized injection technique of BoNT-A according to the spasm-dominant site is an effective and safe treatment method for essential blepharospasm patients.

Scleral fixation technique using a hydrophobic foldable intraocular lens with ring-shaped connecting bridges.

UNLABELLED: We introduce a new method of scleral fixation using a hydrophobic foldable intraocular lens (IOL) with ring-shaped connecting bridges. A double-armed 10-0 polypropylene suturing needle is inserted using the routine ab externo technique. The midportion of the thread is pulled out through a main wound and cut. Using another 10-0 polypropylene needle, the IOL is sutured at both inner ring-shaped connecting bridges (inner shorter rim). The ring-shaped connecting-bridges IOL was carefully injected using the IOL cartridge with gentle pulling out of the thread. The externalized sutures were tied at the corresponding sites. All ties between 2 cut threads were performed using a European death knot. A scleral fixation outcome with the ring-shaped connecting-bridges IOL was similar to a poly(methyl methacrylate) IOL outcome; however, the surgeon should consider the possibility of a postoperative myopic shift. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Improvement of diagnostic performance regarding retinal nerve fiber layer defect using shifting of the normative database according to vessel position.

PURPOSE: To evaluate the clinical efficacy of early detection of retinal nerve fiber layer (RNFL) defect in a Korean population using shifting of the normative RNFL thickness database according to vessel position. METHODS: Retinal nerve fiber layer thickness data of 151 healthy eyes (normative group) and 120 validation subjects (validation group; additional healthy controls plus early glaucoma) were prospectively obtained using spectral-domain optical coherence tomography (SD-OCT) measurement. Clinical profiles and position of major retinal artery peaks (superotemporal, STa; inferotemporal, ITa) were investigated with position of RNFL peaks. Three different criteria for the lower 1% limit were adopted for the validation. Criterion 1 used normative data from the manufacturer. Criterion 2 used data from healthy volunteers. Criterion 3 used four combinations of two subgroups from data for volunteers, which were divided by the median value of STa in the superior region and that of ITa in the inferior region. The κ value was used to determine the diagnostic performance of each criterion (agreement with standard answer). RESULTS: Assessment of the validation group using criterion 3 showed greater accuracy than with criterion 1 or criterion 2 (κ = 0.571, 0.774, and 0.979). Although SD-OCT specificity for RNFL defect detection was similar among the criteria (100%, 98.8%, and 98.9%), sensitivity was highest with criterion 3 (42.4%, 72.7%, and 100%) (all values; criteria 1, 2, and 3, respectively). CONCLUSIONS: Shifting of the lower 1% reference line, according to vessel position, could remarkably improve the diagnostic performance regarding RNFL defect detection with SD-OCT.

Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering.

The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes.