Mean first passage time analysis reveals rate-limiting steps, parallel pathways and dead ends in a simple model of protein folding

We have analyzed dynamics on the complex free energy landscape of protein folding in the FOLD-X model, by calculating for each state of the system the mean first passage time to the folded state. The resulting kinetic map of the folding process shows that it proceeds in jumps between well-defined, local free energy minima. Closer analysis of the different local minima allows us to reveal secondary, parallel pathways as well as dead ends.Comment: 7 page

Dealing with the COVID-19 pandemic: an opportunity to reflect on sustainability research

The COVID-19 pandemic has jolted societies out of normality, possibly creating new conditions for sustainability transformations. What does this mean for sustainability research? Because of the scope of the crisis, researchers have been heavily involved: not only have they had to speed up the pace of scientific production to provide urgently needed COVID-19 knowledge, but they have also been affected citizens. For sustainability science, this calls for an experience-based reflection on the positionality and orientation of research aiming to support sustainability transformations. Twenty sustainability researchers discussed their sustainability research on COVID-19 in three workshops based on the following questions: How does the pandemic—and the measures taken to deal with it—affect sustainable development? What can we learn from the pandemic from the perspective of societal transformation? The present discussion paper emerged from this multidisciplinary exchange among sustainability researchers, considering five topics: impacts of the COVID-19 crisis on sustainability transformations; learning for sustainability transformations; the role of solidarity; governance and political steering; and the role of science in society. Our discussions led to a meta-level reflection on what sustainability research can learn from research on COVID-19 regarding topics and disciplinary angles, time dimensions, the role of researchers, and how adequate preparation for both crises and long-term transformations requires interdisciplinary interaction

Model-guided metabolic engineering of Pseudomonas taiwanensis VLB120 for the production of methyl ketones

Aliphatic methyl ketones are discussed as promising novel diesel blendstocks because of their favorable cetane numbers. To achieve sustainable production of these compounds, bio-based production in engineered microbes is followed and successful synthesis in Escherichia coli1,2,3 and Pseudomonas putida4 has recently been shown. In this presentation, we report on the metabolic engineering of Pseudomonas taiwanensis VLB1205 for the production of saturated and monounsaturated medium chain methyl ketones (C11, C13, C15, C17). Major arguments for the use of this microbe are its metabolic versatility, high tolerance of organic solvents5 and ease of cultivation. P. taiwanensis VLB120 can grow on various carbon sources besides glucose such as glycerol, an important by-product of biodiesel production, as well as on major components of biomass hydrolysate such as xylose, organic acids and aromatic compounds, e.g., 4-hydroxybenzoate4. Further, its superior redox cofactor regeneration capability6 might benefit the synthesis of the reduced, aliphatic target compounds. The transformation of P. taiwanensis VLB120 into a microbial cell factory for methyl ketone production was achieved by: (i) overproduction of the fatty acyl-CoA synthetase FadB to increase acyl-CoA availability, (ii) oxidation of acyl-CoA to a trans-2-enoyl-CoA by a heterologously expressed acyl-CoA oxidase from Micrococcus luteus, (iii) conversion of this intermediate to β-hydroxyacyl-CoA and further oxidation to a β -ketoacyl-CoA by overexpression of the native fadB gene, (iv) increased thioesterase activity by overexpression of fadM to form free β -keto acids, which spontaneously decarboxylate to methyl ketones. The 1st generation production strain yielded 550 mg L-1aq methyl ketones in a batch fermentation with in situ product extraction into a second organic layer of n-decane. Further strain optimization was guided by metabolic modeling, which suggested an additional deletion of the acyl-CoA thioesterase II (tesB). TesB hydrolyzes acyl-CoA to free fatty acids, hence, reverses the desired FadD reaction. In a simple batch fermentation, the proposed gene deletion resulted in a 2.5-fold increased product titer of 1.4 g L-1aq while 9.4 g L-1aq were reached in fed-batch cultivations. Additional, successful strategies tested in parallel were the deletion of the pha operon, responsible for polyhydroxyalkanoate synthesis and deletion of a fadA homologue in the 1st generation production strain, with the later resulting in an even 4-fold improvement of the product titer. While the production of 9.4 g L-1aq is already the highest reported titer of recombinantly produced methyl ketones so far, consolidation of all successfully tested engineering strategies holds great promise to significantly boost methyl ketone production in P. taiwanensis VLB120 to even higher titers. Overall, the results of this study underline the high potential of P. taiwanensis VLB120 for the production of methyl ketones and highlight model-guided metabolic engineering as a means to rationalize and accelerate strain optimization efforts. 1Dong et al. 2018: doi:10.1101/496497 2Goh et al. 2012: doi: 10.1128/AEM.06785-11 3Goh et al. 2014: doi: 10.1016/j.ymben.2014.09.003. 4Goh et al. 2018: doi: 10.1002/bit.26558. 5Rühl et al. 2009: doi: 10.1128/AEM.00225-09 6Blank et al. 2008: doi: 10.1111/j.1742-4658.2008.06648.x

PD-L1 and HIF-2α Upregulation in Head and Neck Paragangliomas after Embolization

Hypoxia activates pathways associated with tumor progression, metastatic spread, and alterations in the immune microenvironment leading to an immunosuppressive phenotype. In particular, the upregulation of PD-L1, a target for therapy with checkpoint inhibitors, is well-studied in several tumors. However, the relationship between hypoxia and PD-L1 regulation in pheochromocytomas and paragangliomas (PPGL), and especially in paragangliomas treated with embolization, is still largely unexplored. We investigated the expression of the hypoxia-marker HIF-2α and of PD-L1 in a PPGL-cohort with and without embolization as potential biomarkers that may predict the response to treatment with HIF-2α and checkpoint inhibitors. A total of 29 tumor samples from 25 patients who were operated at a single center were included and analyzed utilizing immunohistochemistry (IHC) for PD-L1 and HIF-2α. Embolization prior to surgery was performed in seven (24%) tumors. PD-L1 expression in tumor cells of head and neck paragangliomas (HNPGLs) receiving prior embolization (median PD-L1 positivity: 15%) was significantly higher as compared to PD-L1 expression in HNPGLs without prior embolization (median PD-L1 positivity: 0%) (p = 0.008). Consistently, significantly more HNPGLs with prior embolization were positive for HIF-2α (median nuclear HIF-2α positivity: 40%) as compared to HNPGLs without prior embolization (median nuclear HIF-2α positivity: 0%) (p = 0.016). Our results support the hypothesis that embolization with subsequent hypoxia leads to the upregulation of both PD-L1 and HIF-2α in HNPGLs, and could thus facilitate targeted treatment with HIF-2α and checkpoint inhibitors in the case of inoperable, locally advanced, or metastatic disease

Adjuvant mitotane versus surveillance in low-grade, localised adrenocortical carcinoma (ADIUVO): an international, multicentre, open-label, randomised, phase 3 trial and observational study

BACKGROUND: Adjuvant treatment with mitotane is commonly used after resection of adrenocortical carcinoma; however, treatment remains controversial, particularly if risk of recurrence is not high. We aimed to assess the efficacy and safety of adjuvant mitotane compared with surveillance alone following complete tumour resection in patients with adrenocortical carcinoma considered to be at low to intermediate risk of recurrence. METHODS: ADIUVO was a multicentre, open-label, parallel, randomised, phase 3 trial done in 23 centres across seven countries. Patients aged 18 years or older with adrenocortical carcinoma and low to intermediate risk of recurrence (R0, stage I-III, and Ki67 ≤10%) were randomly assigned to adjuvant oral mitotane two or three times daily (the dose was adjusted by the local investigator with the target of reaching and maintaining plasma mitotane concentrations of 14-20 mg/L) for 2 years or surveillance alone. All consecutive patients at 14 study centres fulfilling the eligibility criteria of the ADIUVO trial who refused randomisation and agreed on data collection via the European Network for the Study of Adrenal Tumors adrenocortical carcinoma registry were included prospectively in the ADIUVO Observational study. The primary endpoint was recurrence-free survival, defined as the time from randomisation to the first radiological evidence of recurrence or death from any cause (whichever occurred first), assessed in all randomly assigned patients by intention to treat. Overall survival, defined as time from the date of randomisation to the date of death from any cause, was a secondary endpoint analysed by intention to treat in all randomly assigned patients. Safety was assessed in all patients who adhered to the assigned regimen, which was defined by taking at least one tablet of mitotane in the mitotane group and no mitotane at all in the surveillance group. The ADIUVO trial is registered with ClinicalTrials.gov, NCT00777244, and is now complete. FINDINGS: Between Oct 23, 2008, and Dec 27, 2018, 45 patients were randomly assigned to mitotane and 46 to surveillance alone. Because the study was discontinued prematurely, 5-year recurrence-free and overall survival are reported instead of recurrence-free and overall survival as defined in the protocol. 5-year recurrence-free survival was 79% (95% CI 67-94) in the mitotane group and 75% (63-90) in the surveillance group (hazard ratio 0·74 [95% CI 0·30-1·85]). Two people in the mitotane group and five people in the surveillance group died, and 5-year overall survival was not significantly different (95% [95% CI 89-100] in the mitotane group and 86% [74-100] in the surveillance group). All 42 patients who received mitotane had adverse events, and eight (19%) discontinued treatment. There were no grade 4 adverse events or treatment-related deaths. INTERPRETATION: Adjuvant mitotane might not be indicated in patients with low-grade, localised adrenocortical carcinoma considering the relatively good prognosis of these patients, and no significant improvement in recurrence-free survival and treatment-associated toxicity in the mitotane group. However, the study was discontinued prematurely due to slow recruitment and cannot rule out an efficacy of treatment. FUNDING: AIFA, ENSAT Cancer Health F2-2010-259735 programme, Deutsche Forschungsgemeinschaft, Cancer Research UK, and the French Ministry of Health

Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation

Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity

Additive Anti-Tumor Effects of Lovastatin and Everolimus In Vitro through Simultaneous Inhibition of Signaling Pathways

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis work was supported by a research grant from the Ludwig-Maximilians University of Munich (Förderprogramm für Forschung und Lehre [FöFoLe], grant number 865/829)

Validation of a Novel Immunoline Assay for Patient Stratification according to Virulence of the Infecting Helicobacter pylori Strain and Eradication Status

Helicobacter pylori infection shows a worldwide prevalence of around 50%. However, only a minority of infected individuals develop clinical symptoms or diseases. The presence of H. pylori virulence factors, such as CagA and VacA, has been associated with disease development, but assessment of virulence factor presence requires gastric biopsies. Here, we evaluate the H. pylori recomLine test for risk stratification of infected patients by comparing the test score and immune recognition of type I or type II strains defined by the virulence factors CagA, VacA, GroEL, UreA, HcpC, and gGT with patient's disease status according to histology. Moreover, the immune responses of eradicated individuals from two different populations were analysed. Their immune response frequencies and intensities against all antigens except CagA declined below the detection limit. CagA was particularly long lasting in both independent populations. An isolated CagA band often represents past eradication with a likelihood of 88.7%. In addition, a high recomLine score was significantly associated with high-grade gastritis, atrophy, intestinal metaplasia, and gastric cancer. Thus, the recomLine is a sensitive and specific noninvasive test for detecting serum responses against H. pylori in actively infected and eradicated individuals. Moreover, it allows stratifying patients according to their disease state

Method for Determining the Electron Number in Charge-coupled Measurement Devices

The conversion factor in a CCD device is determined by the photon transfer technique in a region where pure shot noise is not dominating, i. e. where noise of the readout electronics must be taken into account