Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A.

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

Trust in and Acceptance of Artificial Intelligence Applications in Medicine: Mixed Methods Study.

BACKGROUND: Artificial intelligence (AI)-powered technologies are being increasingly used in almost all fields, including medicine. However, to successfully implement medical AI applications, ensuring trust and acceptance toward such technologies is crucial for their successful spread and timely adoption worldwide. Although AI applications in medicine provide advantages to the current health care system, there are also various associated challenges regarding, for instance, data privacy, accountability, and equity and fairness, which could hinder medical AI application implementation. OBJECTIVE: The aim of this study was to identify factors related to trust in and acceptance of novel AI-powered medical technologies and to assess the relevance of those factors among relevant stakeholders. METHODS: This study used a mixed methods design. First, a rapid review of the existing literature was conducted, aiming to identify various factors related to trust in and acceptance of novel AI applications in medicine. Next, an electronic survey including the rapid review-derived factors was disseminated among key stakeholder groups. Participants (N=22) were asked to assess on a 5-point Likert scale (1=irrelevant to 5=relevant) to what extent they thought the various factors (N=19) were relevant to trust in and acceptance of novel AI applications in medicine. RESULTS: The rapid review (N=32 papers) yielded 110 factors related to trust and 77 factors related to acceptance toward AI technology in medicine. Closely related factors were assigned to 1 of the 19 overarching umbrella factors, which were further grouped into 4 categories: human-related (ie, the type of institution AI professionals originate from), technology-related (ie, the explainability and transparency of AI application processes and outcomes), ethical and legal (ie, data use transparency), and additional factors (ie, AI applications being environment friendly). The categorized 19 umbrella factors were presented as survey statements, which were evaluated by relevant stakeholders. Survey participants (N=22) represented researchers (n=18, 82%), technology providers (n=5, 23%), hospital staff (n=3, 14%), and policy makers (n=3, 14%). Of the 19 factors, 16 (84%) human-related, technology-related, ethical and legal, and additional factors were considered to be of high relevance to trust in and acceptance of novel AI applications in medicine. The patient's gender, age, and education level were found to be of low relevance (3/19, 16%). CONCLUSIONS: The results of this study could help the implementers of medical AI applications to understand what drives trust and acceptance toward AI-powered technologies among key stakeholders in medicine. Consequently, this would allow the implementers to identify strategies that facilitate trust in and acceptance of medical AI applications among key stakeholders and potential users.

Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations.

To accelerate the development of Advanced Therapy Medicinal Products (ATMPs) for patients suffering from life-threatening cancer with limited therapeutic options, regulatory approaches need to be constantly reviewed, evaluated and adjusted, as necessary. This includes utilizing science and risk-based approaches to mitigate and balance potential risks associated with early clinical research and a more flexible manufacturing paradigm. In this paper, T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores opportunities to expedite the development of CAR and TCR engineered T cell therapies in the EU by leveraging tools within the existing EU regulatory framework to facilitate an iterative and adaptive learning approach across different product versions with similar design elements or based on the same platform technology. As understanding of the linkage between product quality attributes, manufacturing processes, clinical efficacy and safety evolves through development and post licensure, opportunities are emerging to streamline regulatory submissions, optimize clinical studies and extrapolate data across product versions reducing the need to perform duplicative studies. It is worth noting that this paper is focusing on CAR- and TCR-engineered T cell therapies but the concepts may be applied more broadly to engineered cell therapy products (e.g., CAR NK cell therapy products).

Lymphodepletion - an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an "a secco" technique.

The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii's archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient "a secco" technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides.

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder's protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.

Analysis of nickel-binding peptides in a human hepidermoid cancer cell line by Ni-NTA affinity chromatography and mass spectrometry.

To elucidate whether eukaryotic elongation factor 1A (eEF-1A) in a human hepidermoid cancer cell line (H1355) belonged to the family of the Ni-interacting protein, we analyzed the sequence of peptides obtained by on-Ni-NTA-agarose tryptic digestion of proteins from H1355 cell extract. LC/MS analysis showed the presence of several peptides mainly from abundant cellular proteins corresponding to eEF-1A, tubulin and actin. The results indicated that F-actin strongly binds to Ni-NTA-agarose whereas the other proteins are indirectly bound to the resin because of the formation of a protein-protein complex with actin.

Porous Silicon Based Resonant Mirrors for Biochemical Sensing.

We report on our preliminary results in the realization and characterization of a porous silicon (PSi) resonant mirror (RM) for optical biosensing. We have numerically and experimentally studied the coupling between the electromagnetic field, totally reflected at the base of a high refractive index prism, and the optical modes of a PSi waveguide. This configuration is very sensitive to changes in the refractive index and/or in thickness of the sensor surface. Due to the high specific area of the PSi waveguide, very low DNA concentrations can be detected confirming that the RM could be a very sensitive and labelfree optical biosensor.

New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A.

The eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms.

Raf kinases in signal transduction and interaction with translation machinery.

In recent years, a large amount of evidence has given a central role to translational control in diseases such as cancer, tissue hypertrophy and neurodegeneration. Its deregulation can directly modulate cell cycling, transformation and survival response. The aim of this review is to describe the interaction between Raf activation and the main characters of the translational machinery, such as the elongation factor 1A (eEF1A), which has been recognized in recent years as one of the most interesting putative oncogenes. A particular emphasis is given to an intriguing non-canonical role that eEF1A can play in the relationship between the Ras→Raf-1→MEK1→ERK-1/2 and PI3K→Akt signaling pathways. Recently, our group has described a C-Raf kinase-mediated phosphorylation of eEF1A triggered by a survival pathway induced upon interferon alpha (IFNα) treatment in the human epidermoid cancer cell line (H1355). This phosphorylation seems to be the center of the survival pathway that counteracts the well-known pro-apoptotic function of IFNα. Furthermore, we have identified two new phosphorylation sites on eEF1A (Ser21 and Thr88) that are substrates for Raf kinases in vitro and, likely, in vivo as well. These residues seem to have a significant functional role in the control of cellular processes, such as cell proliferation and survival. In fact, overexpression of eEF1A2 in gemcitabine-treated cancer cells caused the upregulation of phosphoAkt and an increase in cell viability, thereby suggesting that eEF1A2 could exert its oncogenic behavior by participating in the regulation of PI3K pathway.

Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics.

The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein-protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it's N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.