High-Yield Biosynthesis of trans-Nerolidol from Sugar and Glycerol.

Isoprenoids, or terpenoids, have wide applications in food, feed, pharmaceutical, and cosmetic industries. Nerolidol, an acyclic C15 isoprenoid, is widely used in cosmetics, food, and personal care products. Current supply of nerolidol is mainly from plant extraction that is inefficient, costly, and of inconsistent quality. Here, we screened various nerolidol synthases from bacteria, fungi, and plants and found that the strawberry nerolidol synthase was most active in Escherichia coli. Through systematic optimization of the biosynthetic pathways, carbon sources, inducer, and genome editing, we constructed a series of deletion strains (single mutants ΔldhA, ΔpoxB, ΔpflB, and ΔtnaA; double mutants ΔadhE-ΔldhA; and triple mutants and beyond ΔadhE-ΔldhA-ΔpflB and ΔadhE-ΔldhA-ΔackA-pta) that produced high yields of 100% trans-nerolidol. In flasks, the highest nerolidol titers were 1.8 and 3.3 g/L in glucose-only and glucose-lactose-glycerol media, respectively. The highest yield reached 26.2% (g/g), >90% of the theoretic yield. In two-phase extractive fed-batch fermentation, our strain produced ∼16 g/L nerolidol within 4 days with about 9% carbon yield (g/g). In a single-phase fed-batch fermentation, the strain produced >6.8 g/L nerolidol in 3 days. To the best of our knowledge, our titers and productivity are the highest in the literature, paving the way for future commercialization and inspiring biosynthesis of other isoprenoids.

Total enzymatic synthesis of cis-α-irone from a simple carbon source.

Metabolic engineering has become an attractive method for the efficient production of natural products. However, one important pre-requisite is to establish the biosynthetic pathways. Many commercially interesting molecules cannot be biosynthesized as their native biochemical pathways are not fully elucidated. Cis-α-irone, a top-end perfumery molecule, is an example. Retrobiosynthetic pathway design by employing promiscuous enzymes provides an alternative solution to this challenge. In this work, we design a synthetic pathway to produce cis-α-irone with a promiscuous methyltransferase (pMT). Using structure-guided enzyme engineering strategies, we improve pMT activity and specificity towards cis-α-irone by >10,000-fold and >1000-fold, respectively. By incorporating the optimized methyltransferase into our engineered microbial cells, ~86 mg l-1 cis-α-irone is produced from glucose in a 5 l bioreactor. Our work illustrates that integrated retrobiosynthetic pathway design and enzyme engineering can offer opportunities to expand the scope of natural molecules that can be biosynthesized.

Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up.

BACKGROUND: α-Ionone is highly valued in cosmetics and perfumery with a global usage of 100-1000 tons per year. Metabolic engineering by microbial fermentation offers a promising way to produce natural (R)-α-ionone in a cost-effective manner. Apart from optimizing the metabolic pathways, the approach is also highly dependent on generating a robust strain which retains productivity during the scale-up process. To our knowledge, no study has investigated strain robustness while increasing α-ionone yield. RESULTS: Built on our previous work, here, we further increased α-ionone yield to 11.4 mg/L/OD in 1 mL tubes by overexpressing the bottleneck dioxygenase CCD1 and re-engineering the pathway, which is > 65% enhancement as compared to our previously best strain. However, the yield decreased greatly to 2.4 mg/L/OD when tested in 10 mL flasks. Further investigation uncovered an unexpected inhibition that excessive overexpression of CCD1 was accompanied with increased hydrogen peroxide (H2O2) production. Excessive H2O2 broke down lycopene, the precursor to α-ionone, leading to the decrease in α-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of α-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved α-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H2O2. The strain exhibited improved robustness and produced ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature. CONCLUSION: Our study provides an insight on the importance of mediating the oxidative stress to improve strain robustness and microbial production of α-ionone during scaling up. This new strategy may be inspiring to the biosynthesis of other high-value apocarotenoids such as retinol and crocin, in which oxygenases are also involved.

Patterns of failure in limited-stage small cell lung cancer: Implications of TNM stage for prophylactic cranial irradiation.

BACKGROUND AND PURPOSE: The relationship between tumor-node-metastasis (TNM) stage and patterns of failure in limited-stage small cell lung cancer (LS-SCLC) remains unclear. We hypothesized that TNM stage predicts brain metastasis risk, and could inform the use of prophylactic cranial irradiation. MATERIAL AND METHODS: We reviewed 283 patients with stage I-IIIB SCLC. Competing-risks regression was used to analyze local, distant, and brain failure. Multivariate analysis was used to evaluate the effect of treatment and clinical factors on failure and OS. RESULTS: Patients with stage I or II SCLC (35% of cohort) had significantly better survival and lower risk of distant and brain metastasis, compared with stage III patients. The 5-year cumulative incidence of brain metastasis for stage I/II and III were 12% and 26%, respectively. Stage had no correlation with local failure. On multivariate analysis, stage was independently prognostic for survival, distant metastasis risk, and brain metastasis risk. CONCLUSIONS: TNM staging predicts likelihood of distant metastasis, brain metastasis, and survival in LS-SCLC. This supports the routine use of TNM staging in clinical practice. The lower risk of brain metastasis in stage I and II SCLC suggests that prophylactic cranial irradiation could play a more limited role in treatment of early-stage disease.

Comparison of FDG-PET/CT and contrast-enhanced CT for monitoring therapy response in patients with metastatic breast cancer.

PURPOSE: The aim of this study was to compare fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) and contrast-enhanced computed tomography (CE-CT) for the prediction of progression-free survival (PFS) and disease-specific survival (DSS) in patients with stage IV breast cancer undergoing systemic therapy. METHODS: Sixty-five patients with metastatic breast cancer treated with first- or second-line systemic therapy in prospective clinical trials were included. Response to treatment was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 for CE-CT and by PET Response Criteria in Solid Tumors (PERCIST), respectively. RESULTS: All responders by RECIST (n = 22) were also responders by PERCIST, but 40% (17/43) of non-responders by RECIST were responders by PERCIST. Responses according to RECIST and PERCIST both correlated with PFS, but PERCIST showed a significantly higher predictive accuracy (concordance index for PFS: 0.70 vs. 0.60). One-year PFS for responders vs. non-responders by RECIST was 59% vs. 27%, compared to 63% vs. 0% by PERCIST. Four-year DSS of responders and non-responders by RECIST was 50% and 38%, respectively (p = 0.2, concordance index: 0.55) as compared to 58% vs. 18% for PERCIST (p < 0.001, concordance index: 0.65). Response on PET/CT was also a significantly better predictor for DSS than disease control on CE-CT. CONCLUSIONS: In patients with metastatic breast cancer, tumor response on PET/CT appears to be a superior predictor of PFS and DSS than response on CE-CT. Monitoring tumor response by PET/CT may increase the power of clinical trials using tumor response as an endpoint, and may improve patient management in clinical routine.

Implementation of a Point-of-Care Radiologist-Technologist Communication Tool in a Quality Assurance Program.

OBJECTIVE: We implemented an Image Quality Reporting and Tracking Solution (IQuaRTS), directly linked from the PACS, to improve communication between radiologists and technologists. MATERIALS AND METHODS: IQuaRTS launched in May 2015. We compared MRI issues filed in the period before IQuaRTS implementation (May-September 2014) using a manual system with MRI issues filed in the IQuaRTS period (May-September 2015). The unpaired t test was used for analysis. For assessment of overall results in the IQuaRTS period alone, all issues filed across all modalities were included. Summary statistics and charts were generated using Excel and Tableau. RESULTS: For MRI issues, the number of issues filed during the IQuaRTS period was 498 (2.5% of overall MRI examination volume) compared with 78 issues filed during the period before IQuaRTS implementation (0.4% of total examination volume) (p = 0.0001), representing a 625% relative increase. Tickets that documented excellent work were 8%. Other issues included images not pushed to PACS (20%), film library issues (19%), and documentation or labeling (8%). Of the issues filed, 55% were MRI-related and 25% were CT-related. The issues were stratified across six sites within our institution. Staff requiring additional training could be readily identified, and 80% of the issues were resolved within 72 hours. CONCLUSION: IQuaRTS is a cost-effective online issue reporting tool that enables robust data collection and analytics to be incorporated into quality improvement programs. One limitation of the system is that it must be implemented in an environment where staff are receptive to quality improvement.

The Impact That Number of Analyzed Metastatic Breast Cancer Lesions Has on Response Assessment by 18F-FDG PET/CT Using PERCIST.

UNLABELLED: The PET Response Criteria in Solid Tumors (PERCIST) are not specific regarding the number of lesions that should be analyzed per patient. This study evaluated how the number of analyzed lesions affects response assessment in metastatic breast cancer. METHODS: In 60 patients, response was assessed by the change in SUVpeak, normalized to lean body mass, of the most (18)F-FDG-avid lesion (PERCIST 1) and by the change in the sum of normalized SUVpeak for up to 5 lesions (PERCIST 5). The correlation between response by PERCIST and progression-free and disease-specific survival was evaluated. RESULTS: In responders and nonresponders, the respective progression-free survival at 2 y was 37.26% and 6.43% for PERCIST 1 (P < 0.0001) and 33.65% and 7.14% for PERCIST 5 (P < 0.0001) and the respective disease-specific survival at 4 y was 58.96% and 25.44% for PERCIST 1 (P < 0.012) and 59.12% vs 20.01% for PERCIST 5 (P < 0.002). CONCLUSION: The number of analyzed lesions does not appear to have a major impact on the prognostic value of response assessment with (18)F-FDG PET/CT in metastatic breast cancer.

A phase 2 trial of everolimus and pasireotide long-acting release in patients with metastatic uveal melanoma.

The aim of this study was to test the hypothesis that inhibiting mammalian target of rapamycin and insulin-like growth factor-1 receptor would be efficacious in metastatic uveal melanoma. This was a phase 2 trial of everolimus 10 mg daily plus pasireotide long-acting release 60 mg every 28 days enrolling patients with progressive, metastatic uveal melanoma to treatment until progression by Response Evaluation Criteria In Solid Tumors 1.1 (RECIST 1.1) or unacceptable toxicity. The primary endpoint was clinical benefit rate, defined as any objective response or RECIST 1.1 stable disease at 16 weeks. A subset of patients underwent baseline indium-111-octreotide scans. A total of 14 patients were enrolled, of which 13 were evaluable for the primary endpoint, before the study was terminated due to poor accrual. Three of 13 (26%) patients obtained clinical benefit. Seven of 13 (54%) had stable disease lasting for a median of 8 weeks (range: 8-16 weeks). Grade 3 adverse events deemed at least possibly related to study drugs were hyperglycemia (n=7), oral mucositis (n=2), diarrhea (n=1), hypophosphatemia (n=1), and anemia (n=1). Seven of 14 (50%) patients required at least one dose reduction due to toxicity. Seven of eight (88%) patients with baseline indium-111-octreotide scans had at least one avid lesion, with significant intrapatient heterogeneity. There was a trend toward an association between octreotide avidity and cytostatic response to therapy (P=0.078). The combination of everolimus and pasireotide has limited clinical benefit in this small metastatic uveal melanoma cohort. Dose reductions for side effects were common. Further investigation into the relationship between somatostatin receptor expression and cytostatic activity of somatostatin analogues is warranted.

Prognostic Value of Preradiotherapy (18)F-FDG PET/CT Volumetrics in Limited-Stage Small-Cell Lung Cancer.

BACKGROUND: Whether (18)F-fludeoxyglucose positron emission tomography (PET)/computed tomography (CT) metrics predict outcome in limited-stage (LS) small-cell lung cancer (SCLC) has not been well established; most previous reports have only analyzed maximal standardized uptake values (SUVmax). We investigated multiple pretreatment PET metrics, including SUVmax, mean SUV (SUVmean), total lesion glycolysis, and metabolic tumor volume (MTV) in LS-SCLC patients undergoing chemoradiotherapy (CRT) and correlated them with survival and disease control outcomes. PATIENTS AND METHODS: All patients received platinum-based chemotherapy and a median radiation dose of 45 Gy. Kaplan-Meier and competing-risks analyses were performed to assess the prognostic value of PET metrics with respect to overall survival (OS), distant failure (DF), disease-free survival (DFS), and locoregional failure (LRF). Univariate and multivariate analyses were performed to account for the effect of other clinical factors on outcomes. RESULTS: A total of 120 patients with LS-SCLC had analyzable pre-CRT PET/CTs. The median follow up was 34 months. Median OS was 26.9 months. OS was 53.2% at 2 years and 33.1% at 5 years. SUVmax, SUVmean, MTV, and total lesion glycolysis of the primary tumor were not significantly associated with OS, LRF, and DFS on univariate analysis. MTV was significantly associated with DF (P = .024) on univariate but not multivariate analysis. CONCLUSION: This is the largest reported series to date evaluating the prognostic value of baseline PET metrics in LS-SCLC. Neither SUVmax nor other analyzed PET metrics demonstrated significant correlation with OS or LRF. MTV was correlated with DF and DFS, but this association was no longer significant after adjustment for other clinical factors. This analysis suggests that pretreatment PET scans, even with the use of advanced metrics, do not have independent prognostic value for outcomes in LS-SCLC patients after CRT.

Prognostic significance of PET assessment of metabolic response to therapy in oesophageal squamous cell carcinoma.

OBJECTIVES: The role of maximum standard uptake value (SUVmax) at baseline and after induction chemotherapy (CT) on positron emission tomography (PET) as an imaging biomarker has not been well established in oesophageal squamous cell carcinoma (SCC). In this retrospective analysis, we investigated the prognostic significance of various PET metrics in oesophageal SCC patients treated with induction chemotherapy followed by concurrent chemoradiotherapy (CRT). METHODS: A total of 57 patients were treated with CRT; 52 patients received induction chemotherapy and 10 patients underwent surgery following CRT. Scans were independently analysed by a nuclear medicine physician blinded to patient outcome. Using region of interest analysis, SUVmax and metabolic tumour volume (MTV) were calculated for the index lesion and lymph node metastases in each patient. Kaplan-Meier analysis was used to evaluate overall survival (OS), disease-free survival (DFS), local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS). Cox proportional hazards regression was used to assess correlation between outcomes and PET metrics. RESULTS: Median follow-up for those who are alive was 4.4 years, with a median survival for all patients of 2.9 years. The 3-year OS, DFS, DMFS and LRFS rates were 47, 40, 44 and 36%, respectively. Using a pre-established cutoff of a 35% decrease in SUVmax from baseline to post-induction PET, 3-year OS for responders (⩾35% decrease from baseline) was 64%, whereas non-responders (<35% decrease from baseline) had a 3-year OS of 15% (P=0.004). CONCLUSIONS: The pre-specified 35% decrease in SUVmax after induction chemotherapy was prognostic for OS. Baseline and post-induction PET metrics provide prognostic information for oesophageal SCC.

Corneal endothelial abnormalities after selective laser trabeculoplasty (SLT).

PURPOSE: Whitish spots are sometimes observed in the corneal endothelium after selective laser trabeculoplasty (SLT). To evaluate the corneal endothelium after SLT, corneal specular microscopy was performed. METHODS: A total of 142 eyes with open angle glaucoma that underwent SLT during the period of 9 months from February 10 to November 10, 2012, had their corneal endothelium examined with specular microscopy before and after SLT. RESULTS: Dark spots were observed in the corneal endothelium on corneal specular microscopy immediately after SLT. Seventy-one of the 142 eyes had no significant dark spots or increase in dark spots after SLT. Thirty-seven of the 142 eyes showed few dark spots after SLT. Thirty-four of the 142 eyes showed numerous dark spots after SLT on specular microscopy imaging, which resolved by 1 month. The last group had the greatest decrease in the corneal endothelial cell count at 1 month after SLT, but this was not statistically significant in this study (P=0.1). CONCLUSIONS: The corneal endothelial abnormalities after SLT shown in this study may be transient, and long-term effects are probably negligible in normal corneas or single treatments. However, in corneas with reduced transparency of the endothelium, such as compromised corneas and corneas with pigment deposits on the endothelium, there may be a risk of further corneal endothelial compromise, especially after repeated SLT. Larger and longer term studies with histopathologic evaluation would be useful to evaluate the effect of SLT on normal and subnormal corneal endothelium. Until further studies are done, it would be wise to minimize the number and energy of SLT laser shots.

Dual-isotope SPECT/CT impact on hospitalized patients with suspected diabetic foot infection: saving limbs, lives, and resources.

BACKGROUND: Foot ulcer with suspected infection is one of the most common reasons for hospitalization and a major factor contributing to morbidity and high healthcare-related expenses among diabetic patients. Many patients will require amputation; however, major amputation is associated with an alarmingly high 5-year mortality rate. In this study, we assess the diagnosis and management of suspected foot infection in diabetic patients using dual-isotope (DI) single-photon emission computed tomography/computed tomography (SPECT/CT) compared with conventional imaging. METHODS: The diagnostic accuracy in and management of 227 patients who had undergone DI SPECT/CT was compared with that of 232 similar patients who had undergone conventional imaging including plain radiography, CT, planar bone scanning, planar indium-111 white blood cell scanning, and MRI. The duration of hospitalization was additionally compared between these two groups of patients after excluding patients with other active comorbidities. RESULTS: Soft-tissue infection, osteomyelitis with or without soft-tissue infection, and other bony pathologies were more accurately and confidently identified with DI SPECT/CT than with conventional imaging. DI SPECT/CT use was associated with significantly fewer major amputations and more selective bony resection as well as with shorter duration of hospitalization when compared with conventional imaging. CONCLUSION: In this large population of diabetic patients with suspected foot infection DI SPECT/CT was more accurate in diagnosing and localizing infection compared with conventional imaging. In addition, DI SPECT/CT provided clear guidance and promoted many limb salvage procedures. Of equal importance to health economics, DI SPECT/CT use was associated with considerably reduced length of hospitalization compared with conventional imaging.

Corneal endothelial changes after selective laser trabeculoplasty.

BACKGROUND: Whitish spots are sometimes noted in the corneal endothelium after selective laser trabeculoplasty. To evaluate the corneal endothelium after selective laser trabeculoplasty, corneal specular microscopy was performed. DESIGN: Prospective observational study in a private practice. PARTICIPANTS: Fifteen consecutive patients with open-angle glaucoma undergoing selective laser trabeculoplasty in February 2012 had their corneal endothelium examined with specular microscopy before and after treatment. METHODS: Selective laser trabeculoplasty was done with 50 ± 5 shots to 180 degrees of meshwork using 0.3-0.9 mJ of power to achieve the reaction of a hint of microbubbles in 80% of laser shots. MAIN OUTCOME MEASURES: Corneal specular microscopy photographs (0.25 × 0.54 mm) were taken prior to, immediately after and 1 month after selective laser trabeculoplasty. These photographs were assessed and graded. RESULTS: Of the 15 patients, three showed numerous, four showed few and eight showed no increase of dark spots after selective laser trabeculoplasty. All the dark spots resolved by 1 month. CONCLUSION: The effect of selective laser trabeculoplasty on the corneal endothelium may be transient, and long-term effects are probably negligible in normal corneas. However, in compromised corneas and corneas with pigment deposits on endothelium, there may be a risk of corneal endothelial compromise, especially after repeated selective laser trabeculoplasty. Larger and longer term studies with histopathological evaluation would be useful to evaluate the effect of selective laser trabeculoplasty on normal and subnormal corneal endothelium. Until further studies are done, it would be wise to minimize the number and energy of laser shots.