Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude

Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to (R)-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme\u27s activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid

Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae

The use of lignocellulosic-based fermentation media will be a necessary part of the transition to a circular bio-economy. These media contain many inhibitors to microbial growth, including acetic acid. Under industrially relevant conditions, acetic acid enters the cell predominantly through passive diffusion across the plasma membrane. The lipid composition of the membrane determines the rate of uptake of acetic acid, and thicker, more rigid membranes impede passive diffusion. We hypothesized that the elongation of glycerophospholipid fatty acids would lead to thicker and more rigid membranes, reducing the influx of acetic acid. Molecular dynamics simulations were used to predict the changes in membrane properties. Heterologous expression of Arabidopsis thaliana genes fatty acid elongase 1 (FAE1) and glycerol-3-phosphate acyltransferase 5 (GPAT5) increased the average fatty acid chain length. However, this did not lead to a reduction in the net uptake rate of acetic acid. Despite successful strain engineering, the net uptake rate of acetic acid did not decrease. We suggest that changes in the relative abundance of certain membrane lipid headgroups could mitigate the effect of longer fatty acid chains, resulting in a higher net uptake rate of acetic acid

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity

The small G protein MglA and its cognate GAP MglB exemplify a new type of GTPase activation mechanism. In contrast to other Ras-like proteins, the key 'arginine finger' is provided not by the GAP, but by MglA itself

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Computational approaches for drug repurposing against cancer

Cancer remains a leading cause of death worldwide, particularly in its advanced and metastatic stages, where resistance to current treatments is common. In response to this challenge, drug repurposing offers a viable approach for cancer therapy, leveraging existing drugs approved for treating other conditions to reduce development risks, costs, and time. Notably, cationic amphiphilic drugs (CADs), commonly used for allergies and psychiatric disorders, have shown potential as anti-cancer agents. CADs target and destabilize lysosomal membranes in cancer cells, inhibiting enzymes such as acid sphingomyelinases (ASM) and other lysosomal lipases. This vulnerability leads to cell death, making them promising candidates for cancer treatment. In the context of lysosomal function, the role of ASM is particularly significant in Niemann-Pick disease, a genetic disorder characterized by sphingomyelin accumulation in the lysosomes of several tissues due to deficient ASM activity. Indeed, as for cancer, the growing number of variants of uncertain significance (VUS) identified in patient data highlights the need for an in-depth understanding of these variants and their roles in this disease. Another example of successful repurposing is disulfiram (DSF), traditionally used for treating alcoholism. One of the metabolites of DSF, the diethyldithiocarbamate-copper complex (CuET), specifically accumulates in cancer cells and targets the NPL4 protein, a crucial component of the p97 segregase complex involved in protein degradation within the proteasome. The interaction between CuET and NPL4 disrupts the function of the protein function, leading to cancer cell death.These cases emphasize the potential of repurposed drugs in developing new cancer treatments, providing a novel approach to combating this global health challenge.This thesis represents the first step in investigating the molecular mechanisms underlying the tumor-suppressing action of CADs and DSF that our collaborators in the Unit of Cell Death and Metabolism (CDM) and in the Unit of Genome Integrity (GI) at the Danish Cancer Institute (DCI) have identified, using computational methods from the field of molecular modeling and simulations.Five manuscripts are included. Manuscript I laid the groundwork for the future investigation of CADs and lysosomal membranes. We developed a Python-based pipeline to streamline the analysis of molecular dynamics (MD) simulations, which can handle biological membranes with diverse compositions and account for the presence of proteins. The analyses include different biophysical membrane properties that can be better rationalized using supporting plotting tools. Manuscript II presents the MAVISp framework, specifically designed for interpreting VUS, which holds particular relevance in cancer research. This extensive framework includes several modules, each capable of analyzing individual protein structures, their complexes, and ensembles of structures.These modules are uniquely configured to predict the impact of specific mutations on protein function or structural stability. My contribution to this work included curating and validating the MAVISp database, focusing particularly on a case study on the NPL4 protein, and developing two Python-based pipelines for protein alignments and computation of EVE pathogenicity scores, used for assessing the potential disease-causing impact of human variants. In Manuscript III, we applied the MAVISp framework focusing on the ASM protein, one of the targets of the thesis. Over 400 ASM variants, identified from ClinVar, literature curation, or cancer samples, were studied, creating a comprehensive atlas of their structural effects on ASM. Additionally, the study offers a reassessment of several previously known variants. Manuscript IV focuses on MD simulations to characterize the interaction between the ASM protein and a lysosomal-like membrane. This study aimed to understand the impact of ebastine, chosen as representative of CADs, on both the lysosomal membrane and the ASM protein. Ultimately, Manuscript V presents a thorough comparison of various force-field parameters, including CHARMM36m, ff99SB-ILDN, and f99SB*-ILDN, evaluating their efficacy in MD simulations for depicting the structure and dynamics of yeast NPL4. The study encompasses the modeling of zinc ions, a cofactor of NPL4, and cupric ions, which are hypothesized to play a role in the mechanism where copper might replace zinc in the Zinc Finger (ZF) domains of NPL4.Overall, these manuscripts collectively contribute to the field of drug repurposing by providing innovative computational tools to evaluate MD simulations of biological membrane and protein, and in-depth analyses of relevant protein structures, protein-drug interactions, and the structural effect of specific variants

Simulations of yeast membranes

Collections of simulations for the publication Maertens JM, Scrima S, Lambrughi M, Genheden S, Trivellin C, Eriksson LA, Papaleo E, Olsson L, Bettiga M. Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae. Sci Rep. 2021 Aug 30;11(1):17333. doi: 10.1038/s41598-021-96757-y. The script and input files can be found in the associated Github repository of our group: https://github.com/ELELAB/YEAST_MEMBRANE_M

Simulations

Collections of simulations for the publication Maertens JM, Scrima S, Lambrughi M, Genheden S, Trivellin C, Eriksson LA, Papaleo E, Olsson L, Bettiga M. Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae. Sci Rep. 2021 Aug 30;11(1):17333. doi: 10.1038/s41598-021-96757-y. The script and input files can be found in the associated Github repository of our group: https://github.com/ELELAB/YEAST_MEMBRANE_M