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Objective: To identify and characterize concept coverage gaps of ophthalmology examination data elements within the Cerner Millennium electronic health record (EHR) implementations by the Observational Health Data Sciences and Informatics Observational Medical Outcomes Partnership (OMOP) common data model (CDM)., Design: Analysis of data elements in EHRs., Subjects: Not applicable., Methods: Source eye examination data elements from the default Cerner Model Experience EHR and a local implementation of the Cerner Millennium EHR were extracted, classified into one of 8 subject categories, and mapped to the semantically closest standard concept in the OMOP CDM. Mappings were categorized as exact, if the data element and OMOP concept represented equivalent information, wider, if the OMOP concept was missing conceptual granularity, narrower, if the OMOP concept introduced excess information, and unmatched, if no standard concept adequately represented the data element. Descriptive statistics and qualitative analysis were used to describe the concept coverage for each subject category., Main Outcome Measures: Concept coverage gaps in 8 ophthalmology subject categories of data elements by the OMOP CDM., Results: There were 409 and 947 ophthalmology data elements in the default and local Cerner modules, respectively. Of the 409 mappings in the default Cerner module, 25% (n = 102) were exact, 53% (n = 217) were wider, 3% (n = 11) were narrower, and 19% (n = 79) were unmatched. In the local Cerner module, 18% (n = 173) of mappings were exact, 54% (n = 514) were wider, 1% (n = 10) were narrower, and 26% (n = 250) were unmatched . The largest coverage gaps were seen in the local Cerner module under the visual acuity, sensorimotor testing, and refraction categories, with 95%, 95%, and 81% of data elements in each respective category having mappings that were not exact. Concept coverage gaps spanned all 8 categories in both EHR implementations., Conclusions: Considerable coverage gaps by the OMOP CDM exist in all areas of the ophthalmology examination, which should be addressed to improve the OMOP CDM's effectiveness in ophthalmic research. We identify specific subject categories that may benefit from increased granularity in the OMOP CDM and provide suggestions for facilitating consistency of standard concepts, with the goal of improving data standards in ophthalmology., Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (© 2024 by the American Academy of Ophthalmologyé.)

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., (© 2023. The Author(s).)

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., (© 2024. The Author(s).)

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.

(1) Background: mfERG testing is used to study the function of cone photoreceptors in the central retina. Various filters including "smoothing" (Smooth) and "adaptive data filtering" (Adapt) are used to simplify raw data. This study will seek to characterize the effect of data modification on raw patient data. (2) Methods: This was a retrospective study of patients with mfERG results at our institution. For each patient, raw mfERG data without filtering, with smooth level 4 modifier applied, and with adapt level 4 applied were collected and compared. (4) Conclusions: In all patients, smoothing and adaptive filter modifiers create statistically significant differences in both P1 latency and P1 amplitude values when compared to raw data. The impacts of these filters demonstrated in this study should impact physicians' decision making when interpreting mfERG results.

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., (Copyright © 2023. Published by Elsevier Inc.)

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.

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.

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., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Competing Interests: The authors have no financial or conflicts of interest to disclose.

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.

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., Competing Interests: WRF reports personal fees from Nanovision Biosciences, personal fees from Allergan, personal fees from Alcon, personal fees from Genentech, outside the submitted work. CL, AXC, CYB, CBT, SM, MHG and AND have nothing to disclose. A patent application has been filed by UC San Diego on this technology authored by MFB and WRF., (© 2021 The Author(s).)

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., Competing Interests: Disclosure: M. Cavichini, None; C. An, None; D.-U.G. Bartsch, None; M. Jhingan, None; M.J. Amador-Patarroyo, None; C.P. Long, None; J. Zhang, None; Y. Wang, None; A.X. Chan, None; S. Madala, None; T. Nguyen, None; W.R. Freeman, None, (Copyright 2020 The Authors.)

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., Competing Interests: Conflict of Interest None declared.

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.

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. 13 C metabolic flux analysis ( 13 C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for 13 C-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 13 C-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 13 C-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.

Despite remarkable progress in mapping biochemistry and gene-protein-reaction relationships, quantitative systems-level understanding of microbial metabolism remains a persistent challenge. Here, 13 C-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., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

A 63-year-old woman with common variable immunodeficiency (CVID) presented with 1 year of insidious onset lower extremity pain and weakness. She underwent a circuitous workup, failed to improve despite treatment for various presumed diagnoses. She presented to a University physical medicine and rehabilitation clinic with continued symptoms. Electrophysiologic testing was recommended revealing a lower extremity motor greater than sensory axonal neuropathy. While CVID has known central nervous system complications, to our knowledge, this represents the second known reported case of peripheral neuropathy. We review the literature on CVID and summarize neurological disease mechanisms and manifestations. Although peripheral neuropathy is a rarely documented complication of CVID, providers need to be aware of potential peripheral nervous system complications of primary immune deficiencies such as CVID due to its significant impact on physical performance, balance, and fall risks., Competing Interests: Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Adaptive laboratory evolution (ALE) has emerged as a powerful tool in basic microbial research and strain development. In the context of metabolic science and engineering, it has been applied to study gene knockout responses, expand substrate ranges, improve tolerance to process conditions, and to improve productivity via designed growth coupling. In recent years, advancements in ALE methods and systems biology measurement technologies, particularly genome sequencing and 13 C metabolic flux analysis ( 13 C-MFA), have enabled detailed study of the mechanisms and dynamics of evolving metabolism. In this review, we discuss a range of studies that have applied flux analysis to adaptively evolved strains, as well as modeling frameworks developed to predict and interpret evolved fluxes. These efforts link mutations to fitness-enhanced phenotypes, identify bottlenecks and approaches to resolve them, and address systems concepts such as optimality., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest.

In this study, we have investigated for the first time the metabolism of E. coli grown on agar using 13 C metabolic flux analysis ( 13 C-MFA). To date, all 13 C-MFA studies on microbes have been performed with cells grown in liquid culture. Here, we extend the scope of 13 C-MFA to biological systems where cells are grown in dense microbial colonies. First, we identified new optimal 13 C tracers to quantify fluxes in systems where the acetate yield cannot be easily measured. We determined that three parallel labeling experiments with the tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, and [4,5,6- 13 C]glucose permit precise estimation of not only intracellular fluxes, but also of the amount of acetate produced from glucose. Parallel labeling experiments were then performed with wild-type E. coli and E. coli ΔackA grown in liquid culture and on agar plates. Initial attempts to fit the labeling data from wild-type E. coli grown on agar did not produce a statistically acceptable fit. To resolve this issue, we employed the recently developed co-culture 13 C-MFA approach, where two E. coli subpopulations were defined in the model that engaged in metabolite cross-feeding. The flux results identified two distinct E. coli cell populations, a dominant cell population (92% of cells) that metabolized glucose via conventional metabolic pathways and secreted a large amount of acetate (~40% of maximum theoretical yield), and a second smaller cell population (8% of cells) that consumed the secreted acetate without any glucose influx. These experimental results are in good agreement with recent theoretical simulations. Importantly, this study provides a solid foundation for future investigations of a wide range of problems involving microbial biofilms that are of great interest in biotechnology, ecology and medicine, where metabolite cross-feeding between cell populations is a core feature of the communities., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

In the originally published version of this Article, the colours of the bars in Fig. 4b were inadvertently switched during the production process, such that 'HK2-Dox' and 'HK2+Dox' were depicted in red and 'Nt-Dox' and 'Nt+Dox' were depicted in blue. These errors have now been corrected in both the PDF and HTML versions of the Article.

Hepatocellular carcinoma (HCC) cells are metabolically distinct from normal hepatocytes by expressing the high-affinity hexokinase (HK2) and suppressing glucokinase (GCK). This is exploited to selectively target HCC. Hepatic HK2 deletion inhibits tumor incidence in a mouse model of hepatocarcinogenesis. Silencing HK2 in human HCC cells inhibits tumorigenesis and increases cell death, which cannot be restored by GCK or mitochondrial binding deficient HK2. Upon HK2 silencing, glucose flux to pyruvate and lactate is inhibited, but TCA fluxes are maintained. Serine uptake and glycine secretion are elevated suggesting increased requirement for one-carbon contribution. Consistently, vulnerability to serine depletion increases. The decrease in glycolysis is coupled to elevated oxidative phosphorylation, which is diminished by metformin, further increasing cell death and inhibiting tumor growth. Neither HK2 silencing nor metformin alone inhibits mTORC1, but their combination inhibits mTORC1 in an AMPK-independent and REDD1-dependent mechanism. Finally, HK2 silencing synergizes with sorafenib to inhibit tumor growth.

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and high-resolution 13 C-metabolic flux analysis were performed on 10 adaptively evolved pgi knockouts of Escherichia coli Pgi catalyzes the first reaction in glycolysis, and its loss results in major physiological and carbon catabolism pathway changes, including an 80% reduction in growth rate. Following adaptive laboratory evolution (ALE), the knockouts increase their growth rate by up to 3.6-fold. Through combined genomic-fluxomic analysis, we characterized the mutations and resulting metabolic fluxes that enabled this fitness recovery. Large increases in pyridine cofactor transhydrogenase flux, correcting imbalanced production of NADPH and NADH, were enabled by direct mutations to the transhydrogenase genes sthA and pntAB The phosphotransferase system component crr was also found to be frequently mutated, which corresponded to elevated flux from pyruvate to phosphoenolpyruvate. The overall energy metabolism was found to be strikingly robust, and what have been previously described as latently activated Entner-Doudoroff and glyoxylate shunt pathways are shown here to represent no real increases in absolute flux relative to the wild type. These results indicate that the dominant mechanism of adaptation was to relieve the rate-limiting steps in cofactor metabolism and substrate uptake and to modulate global transcriptional regulation from stress response to catabolism., Competing Interests: The authors declare no conflict of interest.

Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metabolic engineering tools to these organisms. To address this concern, we applied high resolution 13 C metabolic flux analysis to quantify fluxes for three divergent extremely thermophilic bacteria from separate phyla: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252. We performed 18 parallel labeling experiments, using all singly labeled glucose tracers for each strain, reconstructed and validated metabolic network models, measured biomass composition, and quantified precise metabolic fluxes for each organism. In the process, we resolved many uncertainties regarding gaps in pathway reconstructions and elucidated how these organisms maintain redox balance and generate energy. Overall, we found that the metabolisms of the three thermophiles were highly distinct, suggesting that adaptation to growth at high temperatures did not favor any particular set of metabolic pathways. All three strains relied heavily on glycolysis and TCA cycle to generate key cellular precursors and cofactors. None of the investigated organisms utilized the Entner-Doudoroff pathway and only one strain had an active oxidative pentose phosphate pathway. Taken together, the results from this study provide a solid foundation for future model building and engineering efforts with these and related thermophiles., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V. natriegens metabolism. We constructed a detailed model of V. natriegens core metabolism, measured the biomass composition, and performed high-resolution 13 C metabolic flux analysis ( 13 C-MFA) to estimate intracellular fluxes using parallel labeling experiments with the optimal tracers [1,2- 13 C]glucose and [1,6- 13 C]glucose. During exponential growth in glucose minimal medium, V. natriegens had a growth rate of 1.70 1/h (doubling time of 24min) and a glucose uptake rate of 3.90g/g/h, which is more than two 2-fold faster than E. coli, although slower than the fast-growing thermophile Geobacillus LC300. 13 C-MFA revealed that the core metabolism of V. natriegens is similar to that of E. coli, with the main difference being a 33% lower normalized flux through the oxidative pentose phosphate pathway. Quantitative analysis of co-factor balances provided additional insights into the energy and redox metabolism of V. natriegens. Taken together, the results presented in this study provide valuable new information about the physiology of V. natriegens and establish a solid foundation for future metabolic engineering efforts with this promising microorganism., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Adaptive laboratory evolution (ALE) is a widely-used method for improving the fitness of microorganisms in selected environmental conditions. It has been applied previously to Escherichia coli K-12 MG1655 during aerobic exponential growth on glucose minimal media, a frequently used model organism and growth condition, to probe the limits of E. coli growth rate and gain insights into fast growth phenotypes. Previous studies have described up to 1.6-fold increases in growth rate following ALE, and have identified key causal genetic mutations and changes in transcriptional patterns. Here, we report for the first time intracellular metabolic fluxes for six such adaptively evolved strains, as determined by high-resolution 13 C-metabolic flux analysis. Interestingly, we found that intracellular metabolic pathway usage changed very little following adaptive evolution. Instead, at the level of central carbon metabolism the faster growth was facilitated by proportional increases in glucose uptake and all intracellular rates. Of the six evolved strains studied here, only one strain showed a small degree of flux rewiring, and this was also the strain with unique genetic mutations. A comparison of fluxes with two other wild-type (unevolved) E. coli strains, BW25113 and BL21, showed that inter-strain differences are greater than differences between the parental and evolved strains. Principal component analysis highlighted that nearly all flux differences (95%) between the nine strains were captured by only two principal components. The distance between measured and flux balance analysis predicted fluxes was also investigated. It suggested a relatively wide range of similar stoichiometric optima, which opens new questions about the path-dependency of adaptive evolution., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.

Introduction: In contrast to previous research that has primarily examined how psychological disorders (e.g., post-traumatic stress disorder [PTSD], anxiety) are affected by and affect individuals' threat perceptions, this study examines the relationship between combat experience and threat-monitoring in psychologically healthy Soldiers. Existing research has established how prolonged or intense experiences with war-related stressors can lead individuals to undergo an unconscious fear-conditioning process that affects the circuitry of the prefrontal cortex, hippocampus, amygdala, and anterior cingulate cortex regions of the brain. We predict that the intensity of one's combat experience positively influences Soldiers' attention to environmental threats., Materials and Methods: Participants included U. S. Army Soldiers with a score of 50 or below on the PTSD Checklist-Military Version. Participants completed the Combat Exposure Scale and the State-Trait Anxiety Inventory. The experimental prediction task we employed assesses the expectation of an intrusively loud white noise sound that occurred on three variable patterns in a pseudorandomized order. Each tone pattern was used 20 times over a total of 60 trials. The experimental prediction task included two neutral tones (700 and 1,300 Hz) that were presented in a repeated pattern along with a 100-dB burst of white noise (0.5-second duration). In each trial, one of three possible tone combinations was presented. To assess their attentiveness to threats, participants were asked to continuously rate their expectancy of the burst of white noise using a visual analogue scale (VAS) ranging from 0 to 100. VAS ratings were collected at controlled points throughout the task., Results: None of the participants reported scores on any of the diagnostic surveys that met standards for clinical significance. A repeated-measures analysis of variance was conducted to assess the overall effect of the three prediction conditions on participants' VAS ratings. There was a significant main effect for Combat Exposure Scale scores on VAS ratings [F (1, 27) = 5.19, p = 0.031], with high scorers demonstrating a generally higher expectancy of the white noise burst throughout the entire experimental sequence. Results suggest that within subclinical populations of Soldiers, the intensity of one's combat experience is positively associated with their attention to threats., Conclusion: These findings suggest that Soldiers who experience combat should be observed for signs of increased threat-attention bias, as this may indicate that their capacities for information processing, decision-making, and emotion regulation could be compromised. The positive relationship we observe between a level of combat experience and attentional biases toward threatening stimuli may also help to explain why these veterans engage in "externalizing" behaviors that are risky, aggressive, or violent as well as relational problems and antisocial behaviors that are reported in higher-than-average rates among these populations of Soldiers. Acknowledging that increased threat attention may be a preclinical indication of developing PTSD or other related psychological conditions (e.g., depression, anxiety) should motivate clinicians to more actively diagnose and treat this condition., (Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.)

The bacterial phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) consists of cascading phosphotransferases that couple the simultaneous import and phosphorylation of a variety of sugars to the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. As the primary route of glucose uptake in E. coli, the PTS plays a key role in regulating central carbon metabolism and carbon catabolite repression, and is a frequent target of metabolic engineering interventions. Here we show that Enzyme I, the terminal phosphotransferase responsible for the conversion of PEP to pyruvate, is responsible for a significant in vivo flux in the reverse direction (pyruvate to PEP) during both gluconeogenic and glycolytic growth. We use 13 C alanine tracers to quantify this back-flux in single and double knockouts of genes relating to PEP synthetase and PTS components. Our findings are relevant to metabolic engineering design and add to our understanding of gene-reaction connectivity in E. coli., Competing Interests: The authors declare no competing financial interests.

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13 C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U- 13 C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH 2 , NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

13 C metabolic flux analysis ( 13 C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now, 13 C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for 13 C-MFA. Specifically, we demonstrate that isotopic labeling of glucose moiety of glycogen and ribose moiety of RNA greatly enhances resolution of metabolic fluxes in the upper part of metabolism; importantly, these measurements allow precise quantification of net and exchange fluxes in the pentose phosphate pathway. To demonstrate the practical importance of these measurements for 13 C-MFA, we have used Escherichia coli as a model microbial system and CHO cells as a model mammalian system. Additionally, we have applied this approach to determine metabolic fluxes of glucose and xylose co-utilization in the E. coli ΔptsG mutant. The convenience of measuring glycogen and RNA, which are stable and abundant in microbial and mammalian cells, offers the following key advantages: reduced sample size, no quenching required, no extractions required, and GC-MS can be used instead of more costly LC-MS/MS techniques. Overall, the presented approach for 13 C-MFA will have widespread applicability in metabolic engineering and biomedical research., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

13 C-Metabolic flux analysis ( 13 C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13 C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13 C-MFA. The best single tracers were doubly 13 C-labeled glucose tracers, including [1,6- 13 C]glucose, [5,6- 13 C]glucose and [1,2- 13 C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6- 13 C]glucose and [1,2- 13 C]glucose. Combined analysis of [1,6- 13 C]glucose and [1,2- 13 C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1- 13 C]glucose +20% [U- 13 C]glucose., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The green microalga Chlorella vulgaris has been widely recognized as a promising candidate for biofuel production due to its ability to store high lipid content and its natural metabolic versatility. Compartmentalized genome-scale metabolic models constructed from genome sequences enable quantitative insight into the transport and metabolism of compounds within a target organism. These metabolic models have long been utilized to generate optimized design strategies for an improved production process. Here, we describe the reconstruction, validation, and application of a genome-scale metabolic model for C. vulgaris UTEX 395, iCZ843. The reconstruction represents the most comprehensive model for any eukaryotic photosynthetic organism to date, based on the genome size and number of genes in the reconstruction. The highly curated model accurately predicts phenotypes under photoautotrophic, heterotrophic, and mixotrophic conditions. The model was validated against experimental data and lays the foundation for model-driven strain design and medium alteration to improve yield. Calculated flux distributions under different trophic conditions show that a number of key pathways are affected by nitrogen starvation conditions, including central carbon metabolism and amino acid, nucleotide, and pigment biosynthetic pathways. Furthermore, model prediction of growth rates under various medium compositions and subsequent experimental validation showed an increased growth rate with the addition of tryptophan and methionine., (© 2016 American Society of Plant Biologists. All rights reserved.)

13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth.

Heterotrophic Proteobacteria and Actinobacteria were isolated from Lake Matano, Indonesia, a stratified, ferruginous (iron-rich), ultra-oligotrophic lake with phosphate concentrations below 50 nM. Here, we describe the growth of eight strains of heterotrophic bacteria on a variety of soluble and insoluble sources of phosphorus. When transferred to medium without added phosphorus (P), the isolates grow slowly, their RNA content falls to as low as 1% of cellular dry weight, and 86-100% of the membrane lipids are replaced with amino- or glycolipids. Similar changes in lipid composition have been observed in marine photoautotrophs and soil heterotrophs, and similar flexibility in phosphorus sources has been demonstrated in marine and soil-dwelling heterotrophs. Our results demonstrate that heterotrophs isolated from this unusual environment alter their macromolecular composition, which allows the organisms to grow efficiently even in their extremely phosphorus-limited environment., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h(-1) on glucose and 1.52 h(-1) on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio's RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The use of parallel labeling experiments for (13)C metabolic flux analysis ((13)C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-(13)C]glucose and mixtures of [1-(13)C]glucose and [U-(13)C]glucose, four novel tracers were applied in this study: [2,3-(13)C]glucose, [4,5,6-(13)C]glucose, [2,3,4,5,6-(13)C]glucose and a mixture of [1-(13)C]glucose and [4,5,6-(13)C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-(13)C]glucose+20% [U-(13)C]glucose, while [4,5,6-(13)C]glucose and [5-(13)C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

We developed a set of methods for the quantification of four major components of microbial biomass using gas chromatography/mass spectrometry (GC/MS). Specifically, methods are described to quantify amino acids, RNA, fatty acids, and glycogen, which comprise an estimated 88% of the dry weight of Escherichia coli. Quantification is performed by isotope ratio analysis with fully (13)C-labeled biomass as internal standard, which is generated by growing E. coli on [U-(13)C]glucose. This convenient, reliable, and accurate single-platform (GC/MS) workflow for measuring biomass composition offers significant advantages over existing methods. We demonstrate the consistency, accuracy, precision, and utility of this procedure by applying it to three metabolically unique E. coli strains. The presented methods will have widespread applicability in systems microbiology and bioengineering.

Cellular metabolic and regulatory systems are of fundamental interest to biologists and engineers. Incomplete understanding of these complex systems remains an obstacle to progress in biotechnology and metabolic engineering. An established method for obtaining new information on network structure, regulation and dynamics is to study the cellular system following a perturbation such as a genetic knockout. The Keio collection of all viable Escherichia coli single-gene knockouts is facilitating a systematic investigation of the regulation and metabolism of E. coli. Of all omics measurements available, the metabolic flux profile (the fluxome) provides the most direct and relevant representation of the cellular phenotype. Recent advances in (13)C-metabolic flux analysis are now permitting highly precise and accurate flux measurements for investigating cellular systems and guiding metabolic engineering efforts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Taeniasis, caused by Taenia species, is one of the common zoonoses in China, particularly in the western region of China. Up to now, not enough attention has been given in the high prevalence and high burden of the diseases. In order to study the endemic patterns and control strategies of taeniasis, a series of epidemiological investigations, molecular researches and pilot control activities have been conducted in recent years. This paper reviews the relevant publications in taeniasis research over the last 10 years.

Background: Procedures such as venepuncture or heel prick are painful and may cause considerable stress to newborn infants. Topical local anaesthetics are effective for venepuncture but need to be applied for at least 60 min and the delivered dose will vary. We assessed a novel tetracaine-based self-adhesive patch in providing controlled local anaesthesia before venepuncture., Methods: A placebo-controlled, double-blind trial was conducted using a tetracaine patch formulated from hydroxypropylcellulose discs (0.283 cm(2)) containing tetracaine (1 mg x cm(-2)) surrounded by a low tack pressure-sensitive adhesive backing layer. Thirty-two newborn infants of gestation 32-42 weeks (median 36 weeks), aged 3-18 days (median 6 days) were randomized to receive a tetracaine-containing patch or a placebo device applied to the dorsum of the hand 30 min before venepuncture to obtain blood samples. Pain was assessed in response to needle insertion using a validated adaptation of the neonatal facial coding score (NFCS) and the presence of crying., Results: Of 15 tetracaine-treated neonates, 14 (93%) presented little or no pain in response to the procedure compared with six of 17 (35%) who had the placebo patch applied (P=0.01)., Conclusions: The tetracaine patch produced effective pain relief during the venepuncture procedure in both term and pre-term infants. There were no adverse effects, either local or systemic.