High-efficiency production of 5-hydroxyectoine using metabolically engineered Corynebacterium glutamicum

Background: Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte fagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. Results: Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L−1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purifcation. Conclusions: C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great beneft for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains

Prospective and longitudinal evolution of postoperative periprosthetic findings on metal artifact-reduced MR imaging in asymptomatic patients after uncemented total hip arthroplasty

OBJECTIVE To prospectively assess the evolution of postoperative MRI findings in asymptomatic patients after total hip arthroplasty (THA) over 24 months (mo). METHODS This prospective cohort study included 9 asymptomatic patients (56.7 ± 15.0 years) after THA. Metal artifact-reduced 1.5-T MRI was performed at 3, 6, 12, and 24 mo after surgery. The femoral stem and acetabular cup were assessed by two readers for bone marrow edema (BME), periprosthetic bone resorption, and periosteal edema in addition to periarticular soft tissue edema and joint effusion. RESULTS BME was common around the femoral stem in all Gruen zones after 3 mo (range: 50-100%) and 6 mo (range: 33-100%) and in the acetabulum in DeLee and Charnley zone II after 3 mo (100%) and 6 mo (33%). BME decreased substantially after 12 mo (range: 0-78%) and 24 mo (range: 0-50%), may however persist in particular in Gruen zones 1 + 7. Periosteal edema along the stem was common 3 mo postoperatively (range: 63-75%) and rare after 24 mo: 13% only in Gruen zones 2 and 5. Twelve months and 24 mo postoperatively, periprosthetic bone resorption was occasionally present around the femoral stem (range: 11-33% and 13-38%, respectively). Soft tissue edema occurred exclusively along the surgical access route after 3 mo (100%) and 6 mo (89%) and never at 12 mo or 24 mo (0%). CONCLUSION Around the femoral stem, BME (33-100%) and periosteal edema (0-75%) are common until 6 mo after THA, decreasing substantially in the following period, may however persist up to 24 mo (BME: 0-50%; periosteal edema: 0-13%) in few non-adjoining Gruen zones. Soft tissue edema along the surgical access route should have disappeared 12 mo after surgery

High-efficiency production of 5-hydroxyectoine using metabolically engineered Corynebacterium glutamicum

Abstract Background Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. Results Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectD PST achieved a titre of 74 g L−1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABC opt , was successfully achieved without intermediate purification. Conclusions C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar

Lysine production from the sugar alcohol mannitol: Design of the cell factory Corynebacterium glutamicum SEA-3 through integrated analysis and engineering of metabolic pathway fluxes

The amino acid lysine is among the world’s most important biotechnological products, and enabling its manufacture from the most attractive new materials is an ever-present challenge. In this study, we describe a cell factory of Corynebacterium glutamicum, which produces lysine from mannitol. A preliminary mutant obtained by the deletion of the mannitol repressor MtlR in the glucose-based, lysine-producing C. glutamicum produced only small amounts of lysine. This limitation was due to the significant accumulation of fructose and a limited NADPH supply, which caused a low flux of only 6% into the oxidative pentose phosphate (PP) pathway. Subsequent expression of fructokinase slightly increased production but failed to substantially redirect the flux from the Emden-Meyerhof-Parnas (EMP) pathway to the PP pathway. This suggested the design C. glutamicum SEA-3, which overexpressed the NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase GapN from Streptococcus mutans and coupled the EMP pathway flux to NADPH formation. When grown on mannitol, the SEA-3 strain had a lysine yield of 0.24 mol mol-1 and a specific productivity of 1.1 mmol g-1 h-1, approximately 50% and 70% higher, respectively, than those of the basic producer. A computational pathway analysis revealed that this design would potentially enable a lysine yield of 0.9 mol mol-1, providing room for further development. Our findings open new avenues for lysine production from marine macroalgae, which is farmed globally as an attractive third-generation renewable resource. Mannitol is a major constituent of these algae (up to 30% and higher) and can be easily extracted from their biomass with hot water

MRI with state-of-the-art metal artifact reduction after total hip arthroplasty: periprosthetic findings in asymptomatic and symptomatic patients

OBJECTIVES: To assess the spectrum of periprosthetic MRI findings after primary total hip arthroplasty (THA). METHODS: This multi-center cohort study analyzed 31 asymptomatic patients (65.7 ± 12.7 years) and 27 symptomatic patients (62.3 ± 11.9 years) between 6 months and 2 years after THA. 1.5-T MRI was performed using Compressed Sensing SEMAC and high-bandwidth sequences. Femoral stem and acetabular cup were assessed for bone marrow edema, osteolysis, and periosteal reaction in Gruen zones and DeLee and Charnley zones. Student t test and Fisher's exact test were performed. RESULTS: The asymptomatic and symptomatic groups showed different patterns of imaging findings. Bone marrow edema was seen in 19/31 (61.3%) asymptomatic and 22/27 (81.5%) symptomatic patients, most commonly in Gruen zones 1, 7, and 8 (p ≥ 0.18). Osteolysis occurred in 14/31 (45.2%) asymptomatic and 14/27 (51.9%) symptomatic patients and was significantly more common in Gruen zone 7 in the symptomatic group (8/27 (29.6%)) compared to the asymptomatic group (2/31 (6.5%)) (p = 0.03). Periosteal reaction was present in 4/31 asymptomatic (12.9%) and 9/27 symptomatic patients (33.3%) and more common in Gruen zones 5 and 6 in the symptomatic group (p = 0.04 and 0.02, respectively). In the acetabulum, bone marrow edema pattern was encountered in 3/27 (11.1%) symptomatic patients but not in asymptomatic patients (p ≥ 0.21). Patient management was altered in 8/27 (29.6%) patients based on MRI findings. CONCLUSIONS: Periprosthetic bone marrow edema is common after THA both in asymptomatic and symptomatic patients. Osteolysis and periosteal reaction are more frequent in symptomatic patients. MRI findings led to altered patient management in 29.6% of patients. KEY POINTS: • Bone marrow edema pattern was frequent in both asymptomatic and symptomatic patients after THA, particularly around the proximal femoral stem in Gruen zones 1, 7, and 8. • Osteolysis was significantly more frequent in symptomatic patients in Gruen zone 7. • Periosteal reaction occurred more frequently in symptomatic patients in Gruen zones 5 and 6

Survival prediction for patients with non-resectable intrahepatic cholangiocarcinoma undergoing chemotherapy: a retrospective analysis comparing the tumor marker CA 19-9 with cross-sectional imaging

Purpose!#!Carbohydrate antigen (CA) 19-9 has been established as the main serum marker for patients with intrahepatic cholangiocarcinoma (ICC). The aim of this study was to compare the prognostic value of CA 19-9 changes versus response determined by imaging in patients with ICC undergoing chemotherapy.!##!Methods!#!Between 2003 and 2018, 151 patients with histopathologically confirmed ICC underwent chemotherapy at our tertiary care center for non-resectable or recurrent ICC, of whom 121 were included in this study. Serum CA 19-9 levels and imaging were retrospectively evaluated during chemotherapy. Log-rank testing and optimal stratification were used to classify patients into risk groups.!##!Results!#!Prior to chemotherapy, baseline serum CA 19-9 levels above the previously published cut-off of 37 U/ml were associated with poor survival (median OS 8.7 vs. 12.4 months, p = 0.003). After the beginning of chemotherapy, an increase in CA 19-9 of more than 40 U/ml resulted in impaired residual survival (median OS 5.0 vs. 12.1 months, p < 0.001). However, progressive disease at the first follow-up imaging proved the strongest predictor for poor outcome (median OS 4.6 vs. 15.5 months, p < 0.001). In contrast to prior studies, our data did not show statistically relevant differences in survival time with respect to absolute or relative decreases in serum CA 19-9 levels.!##!Conclusion!#!In our study, the disease control rate-that is, the absence of progressive disease-was the strongest predictor of prolonged residual OS. To this end, both CA 19-9 changes and progressive disease on initial follow-up showed remarkable discriminatory power, with the latter slightly outperforming the former. Therefore, imaging should remain the mainstay of patient evaluation during follow-up

Interlinkages between Climate Change Impacts, Public Attitudes, and Climate Action—Exploring Trends before and after the Paris Agreement in the EU

European member states have high emission reduction potential. They send a strong signal to the rest of the world with their action or inaction on climate change. Yet, within the EU, national-level climate policies (NLCP) lag behind the EU Commission’s overall climate goals. Transparency of and accountability for climate action requires an integrative perspective. Here, insights from diverse research disciplines address the following questions: How are climate impacts, public attitudes, and climate policies changing over time, and what are the interlinkages? Using three databases (Germanwatch (CCPI), European Extreme Events Climate Index (E3CI), and Eurobarometer Public Opinion Survey, 27 European countries were compared for NLCP (mitigation), climate change impacts (heat/drought), and public attitudes towards climate change (cognition/affect/conation). Differences among groups before and after the Paris Agreement were evaluated with ANOVA tests; trends and geographical differences were analysed with linear models. NLCP did not improve after the Paris Agreement, even displaying some deterioration in the eastern macro-region. Conversely, public awareness towards climate change is improving, with concern being higher in northern compared with southern countries. Heat stress is significantly related to NLCP, with increases in heat stress corresponding to improved climate policy in the northern region while worsening it in the southern region. Under worsening climate impacts, public attitudes increasingly favour climate action. However, overall policy responses are inadequate, although differences occur at the macro-regional level. The results provide granular insights into a persistent knowledge–action gap

Risk Stratification in Advanced Biliary Tract Cancer: Validation of the A.L.A.N. Score

Background. In addition to the clinical parameters, immune-inflammatory markers have emerged as prognostic factors in patients with advanced biliary tract cancer (ABC). The recently proposed A.L.A.N. score combines both in an easily applicable manner. The aim of this study was to perform the first external evaluation of this score. Methods. All patients from our clinical registry unit who had unresectable ABC underwent first-line chemotherapy from 2006 to 2018 and met the inclusion criteria of the original study were included (n =  74). The A.L.A.N. score comprises the following parameters: actual neutrophil count, lymphocyte-to-monocyte ratio, albumin, and neutrophil-to-lymphocyte ratio (A.L.A.N.). Univariate and multivariate hazard regression analyses were performed to evaluate the score’s parameters regarding overall survival (OS). The concordance index (C-index) and integrated Brier score (IBS) were calculated to evaluate the score’s predictive performance. Results. Low, intermediate, and high A.L.A.N. scores corresponded to median OS of 21.9, 11.4, and 4.3 months, respectively, resulting in a significant risk stratification (log-rank p=0.017). In multivariate analysis, a high-risk A.L.A.N. score remained an independent predictor of poor survival (p=0.016). Neutrophil-to-lymphocyte ratio was not a significant factor for poor OS in the analyses in the cohort. The score’s ability to predict individual patient survival was only moderate with a C-index of 0.63. Conclusions. The A.L.A.N. score can be used to identify risk groups with a poor prognosis prior to the start of chemotherapy. However, the ability of the score to predict individual patient outcome was only moderate; thus, it may only serve as a minor component in the complex interdisciplinary discussion

Quantitative washout in patients with hepatocellular carcinoma undergoing TACE: an imaging biomarker for predicting prognosis?

Background The delayed percentage attenuation ratio (DPAR) was recently identified as a novel predictor of an early complete response in patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE). In this study, we aimed to validate the role of DPAR as a predictive biomarker for short-, mid-, and long-term outcomes after TACE. Methods We retrospectively reviewed laboratory and imaging data for 103 treatment-naive patients undergoing initial TACE treatment at our tertiary care center between January 2016 and November 2020. DPAR and other washin and washout indices were quantified in the triphasic computed tomography performed before the initial TACE. The correlation of DPAR and radiologic response was investigated. Furthermore, the influence of DPAR on the 6-, 12-, 18-, and 24-month survival rates and the median overall survival (OS) was compared to other established washout indices and estimates of tumor burden and remnant liver function. Results The DPAR was significantly of the target lesions (TLs) with objective response to TACE after the initial TACE session was significantly higher compared to patients with stable disease (SD) or progressive disease (PD) (125 (IQR 118-134) vs 110 (IQR 103-116), p 120 was significantly higher in this group (n = 38 vs. n = 8; p = 0.03). However, no significant differences were observed in the 12-, 18-, and 24-month survival rates after the initial TACE. Regarding the median OS, no significant difference was observed for patients with a high DPAR compared to those with a low DPAR (18.7 months vs. 12.7 months, p = 0.260). Conclusions Our results confirm DPAR as the most relevant washout index for predicting the short-term outcome of patients with HCC undergoing TACE. However, DPAR and the other washout indices were not predictive of mid- and long-term outcomes