Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria.

BACKGROUND: Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2. RESULTS: In this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources. CONCLUSIONS: By the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1.

BACKGROUND: Lignocellulosic biomass as feedstock has a huge potential for biochemical production. Still, efficient utilization of hydrolysates derived from lignocellulose is challenged by their complex and heterogeneous composition and the presence of inhibitory compounds, such as furan aldehydes. Using microbial consortia where two specialized microbes complement each other could serve as a potential approach to improve the efficiency of lignocellulosic biomass upgrading. RESULTS: This study describes the simultaneous inhibitor detoxification and production of lactic acid and wax esters from a synthetic lignocellulosic hydrolysate by a defined coculture of engineered Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. A. baylyi ADP1 showed efficient bioconversion of furan aldehydes present in the hydrolysate, namely furfural and 5-hydroxymethylfurfural, and did not compete for substrates with S. cerevisiae, highlighting its potential as a coculture partner. Furthermore, the remaining carbon sources and byproducts of S. cerevisiae were directed to wax ester production by A. baylyi ADP1. The lactic acid productivity of S. cerevisiae was improved approximately 1.5-fold (to 0.41 ± 0.08 g/L/h) in the coculture with A. baylyi ADP1, compared to a monoculture of S. cerevisiae. CONCLUSION: The coculture of yeast and bacterium was shown to improve the consumption of lignocellulosic substrates and the productivity of lactic acid from a synthetic lignocellulosic hydrolysate. The high detoxification capacity and the ability to produce high-value products by A. baylyi ADP1 demonstrates the strain to be a potential candidate for coculture to increase production efficiency and economics of S. cerevisiae fermentations.

Incidence and predictors of thermal oesophageal and vagus nerve injuries in Ablation Index-guided high-power-short-duration ablation of atrial fibrillation: a prospective study.

AIMS: High-power-short-duration (HPSD) ablation is an effective treatment for atrial fibrillation but poses risks of thermal injuries to the oesophagus and vagus nerve. This study aims to investigate incidence and predictors of thermal injuries, employing machine learning. METHODS AND RESULTS: A prospective observational study was conducted at Leipzig Heart Centre, Germany, excluding patients with multiple prior ablations. All patients received Ablation Index-guided HPSD ablation and subsequent oesophagogastroduodenoscopy. A machine learning algorithm categorized ablation points by atrial location and analysed ablation data, including Ablation Index, focusing on the posterior wall. The study is registered in clinicaltrials.gov (NCT05709756). Between February 2021 and August 2023, 238 patients were enrolled, of whom 18 (7.6%; nine oesophagus, eight vagus nerve, one both) developed thermal injuries, including eight oesophageal erythemata, two ulcers, and no fistula. Higher mean force (15.8 ± 3.9 g vs. 13.6 ± 3.9 g, P = 0.022), ablation point quantity (61.50 ± 20.45 vs. 48.16 ± 19.60, P = 0.007), and total and maximum Ablation Index (24 114 ± 8765 vs. 18 894 ± 7863, P = 0.008; 499 ± 95 vs. 473 ± 44, P = 0.04, respectively) at the posterior wall, but not oesophagus location, correlated significantly with thermal injury occurrence. Patients with thermal injuries had significantly lower distances between left atrium and oesophagus (3.0 ± 1.5 mm vs. 4.4 ± 2.1 mm, P = 0.012) and smaller atrial surface areas (24.9 ± 6.5 cm2 vs. 29.5 ± 7.5 cm2, P = 0.032). CONCLUSION: The low thermal lesion's rate (7.6%) during Ablation Index-guided HPSD ablation for atrial fibrillation is noteworthy. Machine learning based ablation data analysis identified several potential predictors of thermal injuries. The correlation between machine learning output and injury development suggests the potential for a clinical tool to enhance procedural safety.

Prescribed Versus Taken Polypharmacy and Drug-Drug Interactions in Older Cardiovascular Patients during the COVID-19 Pandemic: Observational Cross-Sectional Analytical Study.

The study aimed to assess clinical pharmacology patterns of prescribed and taken medications in older cardiovascular patients using electronic health records (EHRs) (n = 704) (2019-2022). Medscape Drug Interaction Checker was used to identify pairwise drug-drug interactions (DDIs). Prevalence rates of DDIs were 73.5% and 68.5% among taken and prescribed drugs, respectively. However, the total number of DDIs was significantly higher among the prescribed medications (p < 0.05). Serious DDIs comprised 16% and 7% of all DDIs among the prescribed and taken medications, respectively (p < 0.05). Median numbers of DDIs between the prescribed vs. taken medications were Me = 2, IQR 0-7 vs. Me = 3, IQR 0-7 per record, respectively. Prevalence of polypharmacy was significantly higher among the prescribed medications compared with that among the taken drugs (p < 0.05). Women were taking significantly more drugs and had higher prevalence of polypharmacy and DDIs (p < 0.05). No sex-related differences were observed in the list of prescribed medications. ICD code U07.1 (COVID-19, virus identified) was associated with the highest median DDI number per record. Further research is warranted to improve EHR structure, implement patient engagement in reporting adverse drug reactions, and provide genetic profiling of patients to avoid potentially serious DDIs.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization.

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1.

Microbial production of intracellular compounds can be engineered by redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We first established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Second, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

Role of assist device implantation and heart transplantation in the long-term outcome of patients with structural heart disease after catheter ablation of ventricular tachycardia.

INTRODUCTION: Ablation of ventricular tachycardias (VTs) in patients with structural heart disease (SHD) has been associated with advanced heart failure and poor survival. METHODS AND RESULTS: This matched case-control study sought to assess the difference in survival after left ventricular assist device (LVAD) implantation and/or heart transplantation (HTX) in SHD patients undergoing VT ablation. From the initial cohort of 309 SHD patients undergoing VT ablation (187 ischemic cardiomyopathy, mean age 64 ± 12 years, ejection fraction of 34 ± 13%), 15 patients received an LVAD and nine patients HTX after VT ablation during a follow-up period of 44 ± 33 months. Long-term survival after LVAD did not differ from the matched control group (p = 0.761), although the cause of lethal events was different. All post-HTX patients survived during follow-up. CONCLUSION: In this matched case-control study on patients with SHD undergoing VT ablation, patients that received LVAD implantation had similar survival compared to the control group after 4­year follow-up, while the patients with HTX had a significantly better outcome.

Trends and concerns of potentially inappropriate medication use in patients with cardiovascular diseases.

Introduction: The use of potentially inappropriate medications (PIM) is an alarming social risk factor in cardiovascular patients. PIM administration may result in iatrogenic disorders and adverse consequences may be attenuated by limiting PIM intake.Areas covered: The goal of this review article is to discuss the trends, risks, and concerns regarding PIM administration with focus on cardiovascular patients. To find data, we searched literature using electronic databases (Pubmed/Medline 1966-2021 and Web of Science 1975-2021). The data search terms were cardiovascular diseases, potentially inappropriate medication, potentially harmful drug-drug combination, potentially harmful drug-disease combination, drug interaction, deprescribing, and electronic health record.Expert opinion: Drugs for heart diseases are the most commonly prescribed medications in older individuals. Despite the availability of explicit and implicit PIM criteria, the incidence of PIM use in cardiovascular patients remains high ranging from 7 to 85% in different patient categories. Physician-induced disorders often occur when PIM is administered and adverse effects may be reduced by limiting PIM intake. Main strategies promising for addressing PIM use include deprescribing, implementation of systematic electronic records, pharmacist medication review, and collaboration among cardiologists, internists, geriatricians, clinical pharmacologists, pharmacists, and other healthcare professionals as basis of multidisciplinary assessment teams.

Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1.

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 â€‹g/g glucose) and 3.15-fold improvement in titer (1.82 â€‹g/L) and productivity (0.038 â€‹g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker's yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

Co-production of 1,3 propanediol and long-chain alkyl esters from crude glycerol.

Crude glycerol is an excellent carbon source for bacterial production systems. Bacterial fermentation often generates by-products that can offer an additional carbon pool to improve the product profile for optimal valorization. In this study, the properties of two phylogenetically distinct bacteria, Acinetobacter baylyi ADP1 and Clostridium butyricum, were coupled in a one-pot batch process to co-produce 1,3 propanediol (PDO) and long-chain alkyl esters (wax esters, WEs) from crude glycerol. In the process, A. baylyi deoxidized the growth medium allowing glycerol fermentation and PDO production by C. butyricum. Reaeration of the co-cultivations enabled A. baylyi to metabolize the fermentation by-products, acetate and butyrate, and synthesize intracellular WEs. To improve PDO production and A. baylyi growth, carbon and macronutrients in the growth medium were screened and optimized using Plackett-Burman and Box-Behnken models. The validation experiment revealed a good correlation between the observed and predicted values. The salting-out method recovered 89.5% PDO from the fermentation broth and in vacuo extraction resulted in a PDO content of 5.3 g L-1. Nuclear magnetic resonance revealed a WE content and yield of 34.4 ±â€¯1.4 mg L-1 and 34.2 ±â€¯3.2 mg WE g-1 dry cell weight, respectively. A molar yield of 0.65 mol PDO mol-1 and 0.62 µmol WE mol-1 crude glycerol was achieved with the synthetic consortium. This work emphasizes the strength of response surface methodology in improving production processes from the mutualistic association of divergent bacterial species in consortium. The co-production of PDO and WEs from crude glycerol is demonstrated for the first time in this study.

Mevalonate pathway activity as a determinant of radiation sensitivity in head and neck cancer.

Radioresistance is a major hurdle in the treatment of head and neck squamous cell carcinoma (HNSCC). Here, we report that concomitant treatment of HNSCCs with radiotherapy and mevalonate pathway inhibitors (statins) may overcome resistance. Proteomic profiling and comparison of radioresistant to radiosensitive HNSCCs revealed differential regulation of the mevalonate biosynthetic pathway. Consistent with this finding, inhibition of the mevalonate pathway by pitavastatin sensitized radioresistant SQ20B cells to ionizing radiation and reduced their clonogenic potential. Overall, this study reinforces the view that the mevalonate pathway is a promising therapeutic target in radioresistant HNSCCs.

Repair-independent functions of DNA-PKcs protect irradiated cells from mitotic slippage and accelerated senescence.

The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.

Alkane and wax ester production from lignin-related aromatic compounds.

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin-related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off-line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/gcoumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /gcoumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value-added products.

Synthetic metabolic pathway for the production of 1-alkenes from lignin-derived molecules.

BACKGROUND: Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce a readily collectable product, which can reduce the production costs. Towards that end, we established a cellular platform for the production of semivolatile medium-chain α-olefins from lignin-derived molecules: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate, a lignin-derived model compound, as the sole carbon source for both cell growth and product synthesis. RESULTS: In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution to A. baylyi ADP1, resulting in a highly ferulate-tolerant strain. The adapted strain exhibited robust growth in 100 mM ferulate while the growth of the wild type strain was completely inhibited. Next, we expressed two heterologous enzymes in the wild type strain to confer 1-undecene production from glucose: a fatty acid decarboxylase UndA from Pseudomonas putida, and a thioesterase 'TesA from Escherichia coli. Finally, we constructed the 1-undecene synthesis pathway in the ferulate-tolerant strain. The engineered cells were able to produce biomass and 1-undecene solely from ferulate, and excreted the product directly to the culture headspace. CONCLUSIONS: In this study, we employed a bacterium Acinetobacter baylyi ADP1 to integrate a natural aromatics degrading pathway to a synthetic production route, allowing the upgradation of lignin derived molecules to value-added products. We developed a highly ferulate-tolerant strain and established the biosynthesis of an industrially relevant chemical, 1-undecene, solely from the lignin-derived model compound. This study reports the production of alkenes from lignin derived molecules for the first time and demonstrates the potential of lignin as a sustainable resource in the bio-based synthesis of valuable products.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Dynamic decoupling of biomass and wax ester biosynthesis in Acinetobacter baylyi by an autonomously regulated switch.

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates.

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation.

BACKGROUND: The versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape, and thus, the possibilities for more complete carbon and energy utilization. RESULTS: In this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters (wax esters) utilizing the by-products of C. butyricum, namely acetate and butyrate. The coculture produced 24.5 ± 0.8 mmol/l hydrogen (1.7 ± 0.1 mol/mol glucose) and 28 mg/l wax esters (10.8 mg/g glucose). CONCLUSIONS: The cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of 'metabolic pairing' using designed microbial consortia for more optimal electron and carbon recovery.

Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

BACKGROUND: Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. RESULTS: Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters. CONCLUSIONS: The fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.

Image-based criteria to identify the presence of epicardial arrhythmogenic substrate in patients with transmural myocardial infarction.

BACKGROUND: Patients with transmural myocardial infarction (MI) who undergo endocardial-only substrate ablation are at increased risk for ventricular tachycardia recurrence. Late gadolinium enhancement cardiac magnetic resonance (LGE-CMR) can be used to assess infarct transmurality (IT). However, the degree of IT associated with an epicardial arrhythmogenic substrate (AS) has not been determined. OBJECTIVE: The purpose of this study was to determine the degree of IT observed by LGE-CMR and multidetector computed tomography (MDCT) that predicts the presence of epicardial AS. METHODS: The study included 38 post-MI patients. Ten patients with a subendocardial infarction underwent endocardial-only mapping, and 28 with a classic transmural MI (C-TMI), defined as hyperenhancement ≥75% of myocardial wall thickness (WT), underwent endo-epicardial mapping. LGE-CMR/MDCT data were registered to high-density endocardial or epicardial maps to be analyzed for the presence of AS. RESULTS: Of the 28 post-MI patients with C-TMI, 18 had epicardial AS (64%) and 10 (36%) did not. An epicardial scar area ≥14 cm2 on LGE-CMR identified patients with epicardial AS (sensitivity 1, specificity 1). Mean WT in the epicardial scar area in these patients was lower than in patients without epicardial AS (3.14 ± 1.16 mm vs 5.54 ± 1.78 mm; P = .008). A mean WT cutoff value ≤3.59 mm identified patients with epicardial AS (sensitivity 0.91, specificity 0.93). CONCLUSION: An epicardial scar area ≥14 cm2 on LGE-CMR and mean CT-WT ≤3.59 mm predict epicardial AS in post-MI patients.