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We analyze in detail the accessible relaxation pathways via electron emission of the Ne2+Ar states populated via the K-LL Auger decay of Ne+(1s-1)Ar. In particular, we concentrate on the “direct” interatomic Coulombic decay (ICD) of the Ne2+(2s-12p-1)Ar weakly bound doubly ionized states into the manifold of the Ne2+(2p-2)–Ar+(3p-1) repulsive triply ionized ones. To carry out the present study the potential energy curves of the NeAr ground state, the core ionized state Ne+(1s-1)Ar, the relevant dicationic and tricationic states, and the corresponding ICD transition rates have been computed using accurate ab initio methods and basis sets. The total and partial ICD electron spectra are computed within the framework of the time-dependent theory of wave packet propagation. Thereby, the impact of nuclear dynamics accompanying the electronic decay on the computed ICD-electron spectra is investigated in detail. [ABSTRACT FROM AUTHOR]

Aims To investigate the production of soluble cross-reacting material 197 ( CRM197) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. Methods and Results The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197, at a production rate ~10% of the production of insoluble CRM197, in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. Conclusions SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Significance and Impact of the Study Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197. [ABSTRACT FROM AUTHOR]

A recent DFT study of the ruthenium pincer benzoate complex [Ru(PNP)(PhCOO)2] I (PNP = 2,6-bis- (diphenylphosphanyl)lutidine) in its meridional form has revealed mer-I to be a promising catalyst lead structure for the direct insertion of CO2 into the C-H bonds of arenes, such as benzene. After the successful synthesis of I, its solid state structure interestingly and unexpectedly showed the pincer ligand to adopt the facial rather than the meridional coordination mode. Recalculation of the catalytic cycle with fac-I including all relevant local minima and transition states revealed (a) fac-I to be significantly more stable (6.1 kcal mol-1) than mer-I, (b) that the energetic span (ES; i.e. the effective activation barrier) for the cycle with fac-I amounts to 38.8 kcal mol-1, while the cycle with mer-I has an ES of 25.5 kcal mol-1 only. These results are a hint that fac-I is catalytically inactive. Experimental testing of fac-I showed indeed no product formation, which is in full accordance with the computations. To reduce the spatial flexibility of the pincer ligand, its CH2 groups were replaced by O atoms. The resulting complex [Ru(PONOP)(PhCOO)2] II (PONOP = 2,6-bis(diphenylphosphinito)pyridine) was used for the calculation of the catalytic cycle in benzene as the solvent. Gratifyingly, the starting complex mer-II is more stable than fac-II by 1.9 kcal mol-1 in benzene as the solvent. Consequently, mer-II should be available experimentally. As with fac-I, also fac-II generates a catalytic cycle with a high ES (37.1 kcal mol-1), while mer-II generates a cycle with a significantly lower ES (27.2 kcal mol-1) indicating mer-II to be a potentially active catalyst. A possible explanation of the much lower ES in the case of the meridionally coordinated species is found in the stronger interaction of the substrate with the metal center in the arene-s-bond complex. As a result the issue that is created by the mer/fac isomerism can be resolved by creating spatially less flexible structures. [ABSTRACT FROM AUTHOR]

In this paper a review of the literature on crack closure/opening load and crack tip shielding effects determination methods is presented. Commonly used ‘subjective’ (visual) and ‘non-subjective’ approaches have been included. Procedures associated with the determination of an effective crack driving force for both Elber type and that of partial (or incremental) crack closure models have been covered. Comparison among different methods of analyses based on compliance and fatigue crack growth rate measurements is discussed together with their implications and difficulties in fatigue crack growth correlations. [ABSTRACT FROM AUTHOR]

Aims To investigate the production of soluble cross-reacting material 197 ( CRM197) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. Methods and Results The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197, at a production rate ~10% of the production of insoluble CRM197, in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. Conclusions SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Significance and Impact of the Study Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197. [ABSTRACT FROM AUTHOR]

FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation., (© 2024 The Author(s). Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

In sub-Saharan Africa (SSA), the (sub)genotypes A1, D3, and E of the hepatitis B virus (HBV) prevail. Individuals infected with subgenotype A1 have a 4.5-fold increased risk of HCC compared to those infected with other (sub)genotypes. The effect of (sub)genotypes on protein expression and host signalling has not been studied. Mass spectrometry was used to analyse the proteome of Huh7 cells transfected with replication-competent clones. Proteomic analysis revealed significantly differentially expressed proteins between SSA (sub)genotypes. Different (sub)genotypes have the propensity to dysregulate specific host signalling pathways. Subgenotype A1 resulted in dysregulation within the Ras pathway. Ras-associated protein, RhoC, was significantly upregulated in cells transfected with subgenotype A1 compared to those transfected with other (sub)genotypes, on both a proteomic (>1.5-fold) and mRNA level ( p < 0.05). Two of the main cellular signalling pathways involving RHOC, MAPK and PI3K/Akt/mTOR, regulate cell growth, motility, and survival. Downstream signalling products of these pathways have been shown to increase MMP2 and MMP9 expression. An extracellular MMP2 and MMP9 ELISA revealed a non-significant increase in MMP2 and MMP9 in the cells transfected with A1 compared to the other (sub)genotypes ( p < 0.05). The upregulated Ras-associated proteins have been implicated as oncoproteins in various cancers and could contribute to the increased hepatocarcinogenic potential of A1.

Objectives: It has been estimated that vaccines can accrue a relatively large part of their value from patient and carer productivity. Yet, productivity value is not commonly or consistently considered in health economic evaluations of vaccines in several high-income countries. To contribute to a better understanding of the potential impact of including productivity value on the expected cost-effectiveness of vaccination, we illustrate the extent to which the incremental costs would change with and without productivity value incorporated., Methods: For two vaccines currently under development, one against Cloistridioides difficile (C. difficile) infection and one against respiratory syncytial disease (RSV), we estimated their incremental costs with and without productivity value included and compared the results., Results: In this analysis, reflecting a UK context, a C. difficile vaccination programme would prevent £12.3 in productivity costs for every person vaccinated. An RSV vaccination programme would prevent £49 in productivity costs for every vaccinated person., Conclusions: Considering productivity costs in future cost-effectiveness analyses of vaccines for C. difficile and RSV will contribute to better-informed reimbursement decisions from a societal perspective., (© 2024. The Author(s).)

Tumour cell haematogenous dissemination is predicated on molecular changes that enhance their capacity for invasion and preparation of the pre-metastatic niche. It is increasingly evident that platelets play an essential role in this transformation. The systemic nature of signalling molecules and extravascular factors that participate in mediating platelet-tumour cell interactions led to the development of an in vitro co-culture using whole blood and breast tumour cells, allowing us to decipher the impact of hormone-therapy on tumour cells and associated changes in the plasma proteome. Using mass spectrometry, we determined dysregulation of proteins associated with maintaining an invasive tumour phenotype. Tumour changes in genes associated with EMT and survival were documented. This is postulated to be induced via tumour cell interactions with the coagulatory and immune systems. Results highlight tumour cell adaptability to both treatment and blood resulting in a pro-tumorigenic response and a hypercoagulatory state. We illustrate that the breast cancer cell secretome can be altered by hormone-therapy, subject to the tumour subphenotype and linked to platelet activation. More sophisticated co-culture systems are required to recapitulate these interactions to better understand tumorigenesis. Moreover, deeper plasma profiling, using abundant protein depleted and/or vesicle enriched strategies, will likely reveal additional secretory proteins related to tumour cell-platelet interactions., (© 2024. The Author(s).)

Genomic profiling has identified therapeutic targets for precision treatment of certain cancers, but many patients lack actionable mutations. Additional omics approaches, like proteomics and phosphoproteomics, are essential for comprehensive mapping of cancer-associated molecular phenotypes. In vivo models, such as cell line and patient-derived xenografts (PDX), offer valuable insights into cancer biology and treatment strategies.This chapter presents a semiautomated high-throughput workflow for integrated proteomics and phosphoproteomics analysis on the Kingfish platform coupled with MagReSyn ® Zr-IMAC HP. It enhances protein extraction from in vivo xenograft samples and provides better insights into cancers with poor prognosis. The approach successfully identified over 11,000 unique phosphosites and ~6000 proteins in SJSA-1 pediatric osteosarcoma xenografts, demonstrating its efficacy. This workflow is a valuable tool for studying tumor biology and developing precision oncology strategies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)