A citric acid cycle-deficient Escherichia coli as an efficient chassis for aerobic fermentations.

Tricarboxylic acid cycle (TCA cycle) plays an important role for aerobic growth of heterotrophic bacteria. Theoretically, eliminating TCA cycle would decrease carbon dissipation and facilitate chemicals biosynthesis. Here, we construct an E. coli strain without a functional TCA cycle that can serve as a versatile chassis for chemicals biosynthesis. We first use adaptive laboratory evolution to recover aerobic growth in minimal medium of TCA cycle-deficient E. coli. Inactivation of succinate dehydrogenase is a key event in the evolutionary trajectory. Supply of succinyl-CoA is identified as the growth limiting factor. By replacing endogenous succinyl-CoA dependent enzymes, we obtain an optimized TCA cycle-deficient E. coli strain. As a proof of concept, the strain is engineered for high-yield production of four separate products. This work enhances our understanding of the role of the TCA cycle in E. coli metabolism and demonstrates the advantages of using TCA cycle-deficient E. coli strain for biotechnological applications.

Accuracy of intraoperative aberrometry versus modern preoperative methods in post-myopic laser vision correction eyes undergoing cataract surgery with capsular tension ring placement.

PURPOSE: To assess the accuracy of intraoperative wavefront aberrometry (IWA) versus modern intraocular lens formulas in post-myopic laser vision correction (LVC) patients undergoing cataract surgery with capsular tension ring placement. METHODS: This is a retrospective chart review conducted at an academic outpatient center. All post-myopic LVC eyes undergoing cataract surgery with IWA from a single surgeon from 05/2017 to 12/2019 were included. All patients received a capsular tension ring (CTR). Mean numerical error (MNE), median numerical error (MedNE), and percentages of prediction error within 0.50D, 0.75D, and 1.00D were calculated for the above formulas. RESULTS: Twenty-seven post-myopic LVC eyes from 18 patients were included. In post-myopic LVC, MNE with Optiwave Refractive Analysis (ORA), Barrett True K (BTK), Haigis, Haigis-L, Shammas, SRK/T, Hill-RBF v3.0, and W-K AL-adjusted Holladay 1 were + 0.224, - 0.094, + 0.193, - 0.231, - 0.372, + 1.013, + 0.860, and + 0.630 (F = 8.49, p < 0.001). MedNE were + 0.125, - 0.145, + 0.175, + 0.333, + 0.333, + 1.100, + 0.880, and + 0.765 (F = 7.89, p < 0.001), respectively. BTK provided improved accuracy in both MNE (p < 0.001) and MedNE (p = .033) when compared to ORA in pairwise analysis. If the ORA vs. BTK-suggested IOL power were routinely selected, 30% and 15% of eyes would have projected hyperopic outcomes, respectively (p = 0.09). CONCLUSIONS: Our study suggests that in post-myopic LVC eyes undergoing cataract surgery with CTRs, BTK performed more accurately than ORA with regard to accuracy and yielded a lower percentage of eyes with hyperopic outcomes. Haigis, Haigis-L, and Shammas yielded similar results to ORA with regard to accuracy and percentage of eyes with hyperopic outcomes. On average, Shammas and Haigis-L suggested IOLs that would yield outcomes more myopic than expected when compared to BTK.

Retinal Ischemic Perivascular Lesions in Individuals With Atrial Fibrillation.

Background We previously demonstrated that retinal ischemic perivascular lesions (RIPLs), which are indicative of ischemia in the middle retina, may be a biomarker of ischemic cardiovascular disease. In this study, we sought to determine the relationship between RIPLs and atrial fibrillation, a common source of cardiac emboli. Methods and Results In this case-control study, we identified individuals between the ages of 50 and 90 years who had undergone macular spectral domain optical coherence tomography imaging. Individuals with atrial fibrillation were identified, and age- and sex-matched individuals from the same pool, but without a diagnosis of atrial fibrillation, were selected as controls. Spectral domain optical coherence tomography scans were reviewed by 3 independent and masked observers for presence of RIPLs. The relationship between RIPLs and atrial fibrillation was analyzed using multivariable logistic regression models. There were 106 and 91 subjects with and without atrial fibrillation, respectively. The percentage of subjects with RIPLs was higher in the atrial fibrillation group compared with the control group (57.5% versus 37.4%; P=0.005). After adjusting for age, sex, smoking history, hypertension, diabetes, coronary artery disease, carotid stenosis, stroke, and myocardial infarction, the presence of RIPLs was significantly associated with atrial fibrillation, with an odds ratio of 1.91 (95% CI, 1.01-3.59). Conclusions RIPLs are significantly associated with atrial fibrillation, independent of underlying ischemic heart disease or cardiovascular risk factors. This association may inform the diagnostic cardiovascular workup for individuals with RIPLs incidentally detected on optical coherence tomography scan of the macula.

Interpreting Discordant Monosomy 3 FISH and Chromosomal Microarray Analysis Results in Uveal Melanoma.

Uveal melanoma is the most common primary ocular tumor in adults and causes morbidity through lymphovascular metastasis. The presence of monosomy 3 in uveal melanomas is one of the most important prognostic indicators for metastasis. Two major molecular pathology testing modalities used to assess monosomy 3 are fluorescence in situ hybridization (FISH) and chromosomal microarray analysis (CMA). Here, we report two cases of discordant monosomy 3 test results in uveal melanoma enucleation specimens, performed using these molecular pathology tests. The first case is of uveal melanoma from a 51-year-old male that showed no evidence of monosomy 3 when assessed by CMA, but where it was subsequently detected by FISH. The second case is of uveal melanoma from a 49-year-old male that showed monosomy 3 at the limit of detection when assessed by CMA, but where it was not detected by subsequent FISH analysis. These two cases underscore the potential benefits of each testing modality for monosomy 3. Mainly, while CMA may be more sensitive to low levels of monosomy 3, FISH may be best method for small tumors with high levels of adjacent normal ocular tissue. Our cases suggest that both testing methods should be pursued for uveal melanoma, with a single positive result for either test interpreted as indicating the presence of monosomy 3.

Validation of the Prognostic Usefulness of the Gene Expression Profiling Test in Patients with Uveal Melanoma.

PURPOSE: To validate the prognostic usefulness of gene expression profile (GEP) testing in patients with uveal melanoma. To determine whether combining tumor size with the GEP classification provides additional prognostic value. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with a diagnosis of choroidal melanoma examined at Yale New Haven Hospital; University of California, San Diego; and Memorial Sloan Kettering Cancer Center. METHODS: Patients' demographic and clinical data and tumor characteristics were collected. Univariate and multivariate Cox hazard regression analysis were used to assess the association between tumor characteristics and GEP classification with metastasis as an outcome. MAIN OUTCOME MEASURES: Metastasis-free survival (MFS). RESULTS: Of the 337 individuals included in the study, 87 demonstrated metastases. The mean follow-up time was 37.2 (standard deviation [SD], 40.2) months for patients with metastases and 55.0 (SD, 49.3) months for those without metastases. Tumors of larger thickness and GEP class 2 (vs. class 1) were associated significantly with increased risk of metastasis. Tumor thickness showed better prognostic usefulness than GEP classification (Wald statistic, 40.7 and 24.2, respectively). Class 2 tumors with a thickness of 7.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 7.0 mm (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.61-6.51), whereas class 1 tumors with a thickness of 9.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 9.0 mm (HR, 2.07; 95% CI, 0.86-4.99). No difference in MFS was found between patients with class 1A tumors compared with those with class 1B tumors (P = 0.8). Patients with class 2 tumors showed an observed 5-year MFS of 47.5% (95% CI, 36.0%-62.8%). CONCLUSIONS: Tumor size was the most significant predictor of metastasis and provided additional prognostic value independent of GEP classification. In addition, rates of metastasis for class 2 tumors were lower than estimates reported by Castle Bioscience, and no difference in rates of metastasis were found between class 1A and 1B tumors. This indicates that tumor size should be accounted for when relying on GEP for prognostication and that patients with GEP class 1A or 1B tumors may benefit from the same metastatic surveillance protocols. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Experimental Evolution Reveals Unifying Systems-Level Adaptations but Diversity in Driving Genotypes.

Genotype-fitness maps of evolution have been well characterized for biological components, such as RNA and proteins, but remain less clear for systems-level properties, such as those of metabolic and transcriptional regulatory networks. Here, we take multi-omics measurements of 6 different E. coli strains throughout adaptive laboratory evolution (ALE) to maximal growth fitness. The results show the following: (i) convergence in most overall phenotypic measures across all strains, with the notable exception of divergence in NADPH production mechanisms; (ii) conserved transcriptomic adaptations, describing increased expression of growth promoting genes but decreased expression of stress response and structural components; (iii) four groups of regulatory trade-offs underlying the adjustment of transcriptome composition; and (iv) correlates that link causal mutations to systems-level adaptations, including mutation-pathway flux correlates and mutation-transcriptome composition correlates. We thus show that fitness landscapes for ALE can be described with two layers of causation: one based on system-level properties (continuous variables) and the other based on mutations (discrete variables). IMPORTANCE Understanding the mechanisms of microbial adaptation will help combat the evolution of drug-resistant microbes and enable predictive genome design. Although experimental evolution allows us to identify the causal mutations underlying microbial adaptation, it remains unclear how causal mutations enable increased fitness and is often explained in terms of individual components (i.e., enzyme rate) as opposed to biological systems (i.e., pathways). Here, we find that causal mutations in E. coli are linked to systems-level changes in NADPH balance and expression of stress response genes. These systems-level adaptation patterns are conserved across diverse E. coli strains and thus identify cofactor balance and proteome reallocation as dominant constraints governing microbial adaptation.

Dry Eye Symptom Severity and Visual Field Reliability Metrics.

PRCIS: Tracking failure frequency (TFF) increases with dry eye symptom severity and in the left eye. PURPOSE: Symptoms of dry eye disease are commonly encountered in glaucoma patients and can be exacerbated by topical glaucoma medications. Dry eye disease may influence the reliability of visual field (VF) tests and impact the accurate interpretation of the results. PATIENTS AND METHODS: Patients at the Veterans Administration Medical Center San Diego completed the 5-item Dry Eye Questionnaire before VF testing between December 2018 and February 2019. VF reliability metrics were recorded for each patient. Standard reliability metrics included fixation losses, false positive, and false negative rates. Gaze tracking (GT) metrics included percent of stimuli with gaze deviations between 1 and 2 degrees, 3 and 5 degrees, 6 degrees or greater, and percent of stimuli with tracking failure (TFF). The use of glaucoma medications and artificial tears was also recorded. RESULTS: A total of 494 patients completed the 5-item Dry Eye Questionnaire and VF testing. There was no association between dry eye symptom severity and standard reliability metrics or most GT metrics. However, TFF increased as dry eye symptom severity increased (P=0.015). TFF was also greater in the left eye, which was tested second (P=0.012); no other reliability metrics were related to laterality. Patients were more likely to use artificial tears with increased dry eye symptom severity (P<0.001), but there was no relationship between symptom severity and glaucoma medication use. DISCUSSION: Dry eye symptom severity may influence the acceptable range or threshold of TFF when using GT metrics to determine VF reliability. Likewise, the acceptable range or threshold for TFF may be different between eyes.

Standard Reliability and Gaze Tracking Metrics in Glaucoma and Glaucoma Suspects.

PURPOSE: To compare standard reliability metrics and gaze tracking (GT) metrics on the Humphrey field analyzer (HFA). DESIGN: Retrospective cross-sectional study. METHODS: The study was performed at the VA Medical Center, San Diego, and included 494 glaucoma and glaucoma suspect patients who had an HFA 24-2 SITA Fast visual field (VF) performed in both eyes. Standard reliability metrics (fixation loss [FL], false-positive [FP], and false-negative [FN]) were compared to GT metrics (deviations of 1°-2° [M1], deviations of 3°-5° [M3], deviations >6° [M6], and tracking failure frequency [TFF]). The main outcome measures were Spearman rank-based correlation coefficient and area under the receiver operating characteristic (AUROC) curves between standard and GT reliability metrics. RESULTS: The 95th percentile limits for GT metrics were 66.7% for M1, 67.5% for M3, 49.5% for M6, and 79.8% for TFF. There were statistically significant correlations between standard and GT reliability metrics using the 95th percentile as a binary cutoff for GT metrics. However, low Spearman correlation values and AUROC calculations suggest little clinical significance of the associations. FN increased as VF severity worsened (P < .001). M6 was lower in eyes with mild compared to moderate and advanced VF loss (P = .012). CONCLUSIONS: GT metrics do not have a clinically significant association with standard reliability metrics. Both FN and M6 are influenced by VF severity. Aggregate GT metrics do not aid in reliability assessment. These findings suggest that GT metrics may provide an alternative or complementary measure of VF reliability.

Prevalence of subclinical retinal ischemia in patients with cardiovascular disease - a hypothesis driven study.

BACKGROUND: Cardiovascular disease is the leading cause of mortality and disability worldwide. A noninvasive test that can detect underlying cardiovascular disease has the potential to identify patients at risk prior to the occurrence of adverse cardiovascular events. We sought to determine whether an easily observed imaging finding indicative of retinal ischemia, which we term 'retinal ischemic perivascular lesions' (RIPLs), could serve as a biomarker for cardiovascular disease. METHODS: We reviewed optical coherence tomography (OCT) scans of individuals, with no underlying retinal pathology, obtained at UC San Diego Health from July 2014 to July 2019. We identified 84 patients with documented cardiovascular disease and 76 healthy controls. OCT scans were assessed for evidence of RIPLs. In addition, the 10-year atherosclerotic cardiovascular disease (ASCVD) risk calculator was used to risk-stratify the subjects into four different categories. FINDINGS: Patients with documented cardiovascular disease had higher number of RIPLs compared to healthy controls (2.8 vs 0.8, p < 0.001). After adjusting for age, sex, smoking history, systolic blood pressure and triglycerides, cholesterol and hemoglobin A1C levels, each RIPL was associated with an odds ratio of having cardiovascular disease of 1·60 (1.09-2>37). The number of RIPLs in individuals with intermediate and high 10-year ASCVD risk scores was higher than in those with low ASCVD risk scores (1.7 vs 0.64, p = 0.02 and 2.9 vs 0.64, p 0.002, respectively). INTERPRETATION: The presence of RIPLs, which are anatomical markers of prior retinal ischemic infarcts, is suggestive of coexisting cardiovascular disease. RIPLs detection, obtained from routine retinal scans, may thus provide an additional biomarker to identify patients at risk of developing adverse cardiovascular events. FUNDING: None.

Colorectal carcinoma presenting in the orbit: mass effect from an uncommon cause.

An 84-year-old male with previously documented poor medical follow-up presented with progressive painless proptosis of the right eye. Right upper eyelid ptosis, limited motility, proptosis, and inferomedial displacement of the right globe were noted on the exam. Computed tomography (CT) imaging revealed a right retrobulbar extraconal heterogenous mass with ill-defined borders. Biopsy revealed a malignant adenocarcinoma with tumor markers suggestive of a colorectal primary. A rectal mass was identified during a systemic workup. After biopsy, the patient was diagnosed with stage IV metastatic rectal adenocarcinoma. He began palliative radiation therapy shortly following diagnosis.

Electronic Health Record Use among Ophthalmology Residents while on Call.

BACKGROUND: As electronic health record (EHR) use becomes more widespread, detailed records of how users interact with the EHR, known as EHR audit logs, are being used to characterize the clinical workflows of physicians including residents. After-hours EHR use is of particular interest given its known association with physician burnout. Several studies have analyzed EHR audit logs for residents in other fields, such as internal medicine, but none thus far in ophthalmology. Here, we focused specifically on EHR use during on-call shifts outside of normal clinic hours. METHODS: In this retrospective study, we analyzed raw EHR audit log data from on-call shifts for 12 ophthalmology residents at a single institution over the course of a calendar year. Data were analyzed to characterize total time spent using the EHR, clinical volume, diagnoses of patients seen on call, and EHR tasks. RESULTS: Across all call shifts, the median and interquartile range (IQR) of the time spent logged into the EHR per shift were 88 and 131 minutes, respectively. The median (IQR) unique patient charts accessed per shift was 7 (9) patients. When standardized to per-hour measures, weekday evening shifts were the busiest call shifts with regard to both EHR use time and clinical volume. Total EHR use time and clinical volume were greatest in the summer months (July to September). Chart review comprised a majority (63.4%) of ophthalmology residents' on-call EHR activities. CONCLUSION: In summary, EHR audit logs demonstrate substantial call burden for ophthalmology residents outside of regular clinic hours. These data and future studies can be used to further characterize the clinical exposure and call burden of ophthalmology residents and could potentially have broader implications in the fields of physician burnout and education policy.

Artificial Intelligence for Automated Overlay of Fundus Camera and Scanning Laser Ophthalmoscope Images.

Purpose: The purpose of this study was to evaluate the ability to align two types of retinal images taken on different platforms; color fundus (CF) photographs and infrared scanning laser ophthalmoscope (IR SLO) images using mathematical warping and artificial intelligence (AI). Methods: We collected 109 matched pairs of CF and IR SLO images. An AI algorithm utilizing two separate networks was developed. A style transfer network (STN) was used to segment vessel structures. A registration network was used to align the segmented images to each. Neither network used a ground truth dataset. A conventional image warping algorithm was used as a control. Software displayed image pairs as a 5 × 5 checkerboard grid composed of alternating subimages. This technique permitted vessel alignment determination by human observers and 5 masked graders evaluated alignment by the AI and conventional warping in 25 fields for each image. Results: Our new AI method was superior to conventional warping at generating vessel alignment as judged by masked human graders (P < 0.0001). The average number of good/excellent matches increased from 90.5% to 94.4% with AI method. Conclusions: AI permitted a more accurate overlay of CF and IR SLO images than conventional mathematical warping. This is a first step toward developing an AI that could allow overlay of all types of fundus images by utilizing vascular landmarks. Translational Relevance: The ability to align and overlay imaging data from multiple instruments and manufacturers will permit better analysis of this complex data helping understand disease and predict treatment.

Evaluation of Electronic Health Record Implementation in an Academic Oculoplastics Practice.

PURPOSE: Despite increasing electronic health record (EHR) adoption, perceptions of EHRs are negative among ophthalmologists due to concerns about productivity, costs, and documentation. The authors evaluated the effects of EHR adoption in an oculoplastics practice, which had not been previously studied. METHODS: Clinical volume, documentation time, time spent with patients, reimbursement, relative value units, and patient satisfaction were examined for 2 academic oculoplastics attendings between April 2018 and April 2019, with EHR implementation in September 2018. RESULTS: The mean number of patients seen in a half-day clinic was 31.8 versus 27.7 (p = 0.018) pre- and post-EHR implementation, respectively. EHR implementation had no effect on total monthly reimbursement (p = 0.88) or total monthly relative value units (p = 0.54). Average reimbursement (p = 0.004) and relative value units (p = 0.001) per patient encounter were significantly greater with EHR use. Patient satisfaction scores improved (p = 0.018). Mean physician time per patient increased from 6.4 to 9.0 minutes (p < 0.001). Mean documentation time per patient increased from 1.7 to 3.6 minutes (p < 0.001). Average patient wait times decreased by 9 minutes (p = 0.03) with EHR use. No scribes were used. CONCLUSIONS: EHR implementation was associated with decreased patient volume without significant differences in total reimbursement. Although EHR adoption was associated with increased physician time devoted to patients and greater time expenditure on documentation, patients experienced decreased wait times. This suggests that EHR use streamlined the overall clinic flow without sacrificing physicians' time with the patient. The author's findings suggest that EHR implementation can be accomplished in an academic oculoplastics setting without negative impact on patient experience or reimbursement considerations.

High-resolution 13C metabolic flux analysis.

Precise quantification of metabolic pathway fluxes in biological systems is of major importance in guiding efforts in metabolic engineering, biotechnology, microbiology, human health, and cell culture. 13C metabolic flux analysis (13C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for 13C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with 13C-labeled glucose tracers, followed by gas chromatography-mass spectrometry (GC-MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for 13C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of fluxes. The presented protocol can be completed in 4 d and quantifies metabolic fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms.

Metabolic flux responses to deletion of 20 core enzymes reveal flexibility and limits of E. coli metabolism.

Despite remarkable progress in mapping biochemistry and gene-protein-reaction relationships, quantitative systems-level understanding of microbial metabolism remains a persistent challenge. Here, 13C-metabolic flux analysis was applied to interrogate metabolic responses to 20 genetic perturbations in all viable Escherichia coli single gene knockouts in upper central metabolic pathways. Strains with severe growth defects displayed highly altered intracellular flux patterns and were the most difficult to predict using current constraint-based modeling approaches. In the ΔpfkA strain, an unexpected glucose-secretion phenotype was identified. The broad range of flux rewiring responses that were quantified suggest that some compensating pathways are more flexible than others, resulting in a more robust physiology. The fact that only 2 out of 20 strains displayed an increased net pathway-flux capacity points to a fundamental rate limitation of E. coli core metabolism. In cataloguing the various cellular responses, our results provide a critical resource for kinetic model development and efforts focused on genotype-to-phenotype predictions.