Residual metabolic burden in young psoriasis patients successfully treated with biologics.

Metabolic disorders are common in patients with psoriasis and contribute significantly to an increased cardiovascular risk. While biologic therapy is very successful in clearing skin lesions, its impact on metabolic parameters is uncertain. Our aim was to investigate the residual metabolic burden in psoriasis patients successfully treated with biologic therapy. We conducted a cross-sectional study of 80 young patients (54 men, 26 women, aged 30-45 years) successfully treated with either adalimumab, secukinumab or guselkumab and topical therapy or methotrexate, and 20 healthy controls. Anthropometric parameters, lipid levels and metabolic indices (HOMA-IR, TyG index and FIB-4 index) were measured. Patients did not receive any other treatments to exclude confounding effects. After analysis, we found that patients treated with three different biologics had similar metabolic status, only the FIB-4 index was higher in the adalimumab group than in the secukinumab and guselkumab treatment groups. There were no significant differences between the patients treated with biologics and the control group. The comparison with patients treated topically or with methotrexate showed that only triglyceride levels, HOMA-IR, TyG index, and FIB-4 index were elevated in patients treated with adalimumab compared to patients treated with topical therapy. Finally, metabolic status was also similar in patients treated with methotrexate or topical therapy. In conclusion, this study suggests that psoriasis patients successfully treated with biologics have similar metabolic parameters to the control group and patients treated with topical therapy or methotrexate. This indicates that there is no significant residual metabolic burden in young patients successfully treated with biologics. These results are clinically relevant and should be considered in the treatment of psoriasis patients.The study is registered at http://clinicaltrials.gov (identifier: NCT05957120). Date of registration: 24th of July 2023.

Contextualized Metabolic Modelling Revealed Factors Affecting Isoflavone Accumulation in Soybean Seeds.

Isoflavones, secondary metabolites with numerous health benefits, are predominantly found in legume seeds, especially soybean; however, their contents in domesticated soybean seeds are highly variable. Wild soybeans are known for higher seed isoflavone contents than cultivars. Here we used experimental and modelling approaches on wild soybean (W05) and cultivated soybean (C08) to delineate factors influencing isoflavone accumulation. We found imported nutrients were converted into storage compounds, with isoflavone accumulation in W05 seeds being faster than in C08 ones. The isoflavone accumulation during seed development was simulated using context-specific cotyledon metabolic models of four developmental stages on cultivar C08, and the metabolic burden imposed by increasing biomass was evaluated. Trade-off analyses between biomass and isoflavone suggest that high biomass requirement in cultivars could limit the reallocation of resources for secondary metabolite production. Isoflavone production in mature seeds was also influenced by biomass compositions. Seeds with higher carbohydrate contents favour isoflavone production, while those with highest protein and oil contents had lowest isoflavone contents. Although seeds could synthesize isoflavones on their own, the predicted fluxes from biosynthesis alone were lower than the empirical levels. Shadow price analyses indicated that isoflavone accumulation depended on both intrinsic biosynthesis and direct contribution from the plant.

The Predictive Role of Metabolic Volume Segmentation Compared to Semiquantitative PET Parameters in Diagnosis of LVAD Infection using [18F]FDG Imaging.

PURPOSE: Left ventricular assisting device (LVAD) is a vital mechanical circulatory assist device for patients with end-stage heart disease, serving as either a bridge to transplantation or palliative destination therapy. Yet device infection represents a major lethal complication, warranting a multi-step, complex therapy approach including an urgent device exchange or heart transplantation. Still, timely diagnosis of site and extent of VAD-specific infection for a proper therapy planning poses challenges in regular clinical care. This single-center, retrospective study aimed to evaluate the impact of volumetric PET parameters with different thresholding compared to semiquantitative PET parameters for accurate diagnosis of VAD-specific infection. PROCEDURES: Seventeen patients (1 female, 16 males; mean age 57 ± 11 years) underwent [18F]FDG imaging for suspected VAD-specific infection between April 2013 and October 2023. Various metabolic and volumetric PET parameters with different thresholding were collected for specific LVAD components including driveline entry point, subcutaneous driveline, pump pocket, inner cannula and outflow tract. Microbiology and clinical follow-up were used as the final diagnosis standard. RESULTS: Nine of eleven patients with VAD-specific infection underwent urgent heart transplantation, and one had a surgical revision of LVAD. Two patients had non-VAD specific infections, and two had non-VAD related infections. Metabolic burden determination using a fixed absolute threshold provided the best outcome compared to relative thresholding or other metabolic SUV parameters. The total metabolic tumor volume (MTV) cutoff value was 9.3 cm3, and the corresponding sensitivity, specificity, accuracy, and AUC were 90.0%, 71.43%, 82.5%, and 0.814 (95% CI 0.555-0.958), respectively. The total lesion glycolysis (TLG) was 30.6, and the corresponding sensitivity, specificity, accuracy, and AUC were 90.0%, 71.4%, 82.5%, and 0.829 (95% CI 0.571-0.964), respectively. CONCLUSIONS: Volumetric PET parameters with fixed absolute thresholding appear to be a valuable auxiliary tool in the evaluation of [18F]FDG imaging to enhance the diagnostic accuracy of VAD-specific infection.

Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli.

The impact of recombinant protein production (RPP) on host cells and the metabolic burden associated with it undermine the efficiency of the production system. This study utilized proteomics to investigate the dynamics of parent and recombinant cells induced at different time points for RPP. The results revealed significant changes in both transcriptional and translational machinery that may have impacted the metabolic burden, growth rate of the culture and the RPP. The timing of protein synthesis induction also played a critical role in the fate of the recombinant protein within the host cell, affecting protein and product yield. The study identified significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways between two E. coli host strains (M15 and DH5⍺), with the E. coli M15 strain demonstrating superior expression characteristics for the recombinant protein. Overall, these findings contribute to the knowledge base for rational strain engineering for optimized recombinant protein production.

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

Context-dependent redesign of robust synthetic gene circuits.

Cells provide dynamic platforms for executing exogenous genetic programs in synthetic biology, resulting in highly context-dependent circuit performance. Recent years have seen an increasing interest in understanding the intricacies of circuit-host relationships, their influence on the synthetic bioengineering workflow, and in devising strategies to alleviate undesired effects. We provide an overview of how emerging circuit-host interactions, such as growth feedback and resource competition, impact both deterministic and stochastic circuit behaviors. We also emphasize control strategies for mitigating these unwanted effects. This review summarizes the latest advances and the current state of host-aware and resource-aware design of synthetic gene circuits.

Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress.

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner-Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer's productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

Synergistic PAH biodegradation by a mixed bacterial consortium: based on a multi-substrate enrichment approach.

Polycyclic aromatic hydrocarbon (PAH) contamination in the environment involves multiple PAHs and various intermediates produced during the microbial metabolic process. A multi-substrate enrichment approach was proposed to develop a mixed bacterial community (MBC) from the activated sludge of a coking wastewater plant. The degradation performance of MBC was evaluated under different initial concentrations of PAHs (25-200 mg/L), temperature (20-35 °C), pH (5.0-9.0), salinity (0-10 g/L NaCl), and coexisting substrates (catechol, salicylic acid, and phthalic acid). The results showed that the degradation rates of phenanthrene and pyrene in all treatments were up to (99 ± 0.71)% and (99 ± 0.90)% after incubation of 5 days, respectively, indicating excellent biodegradation ability of PAHs by MBC. Furthermore, 16S rRNA gene amplicon sequencing analysis revealed that Pseudomonas was dominant, while Burkholderia had the largest proportion in acidic (pH = 5.0) and saline (10 g/L NaCl) environments. However, the proportion of dominant bacteria in MBC was markedly affected by intermediate metabolites. It was shown that MBC had a higher degradation rate of PAHs in the coexisting matrix due to the timely clearance of intermediates reducing the metabolic burden. Overall, our study provided valuable information to help design an effective strategy for the bioremediation of PAHs in complex environments.