Evolving Diagnostic and Treatment Strategies for Pediatric CNS Tumors: The Impact of Lipid Metabolism.

Pediatric neurological tumors are a heterogeneous group of cancers, many of which carry a poor prognosis and lack a "standard of care" therapy. While they have similar anatomic locations, pediatric neurological tumors harbor specific molecular signatures that distinguish them from adult brain and other neurological cancers. Recent advances through the application of genetics and imaging tools have reshaped the molecular classification and treatment of pediatric neurological tumors, specifically considering the molecular alterations involved. A multidisciplinary effort is ongoing to develop new therapeutic strategies for these tumors, employing innovative and established approaches. Strikingly, there is increasing evidence that lipid metabolism is altered during the development of these types of tumors. Thus, in addition to targeted therapies focusing on classical oncogenes, new treatments are being developed based on a broad spectrum of strategies, ranging from vaccines to viral vectors, and melitherapy. This work reviews the current therapeutic landscape for pediatric brain tumors, considering new emerging treatments and ongoing clinical trials. In addition, the role of lipid metabolism in these neoplasms and its relevance for the development of novel therapies are discussed.

Comparative Analysis of Primary and Monovalent Booster SARS-CoV-2 Vaccination Coverage in Adults with and without HIV in Catalonia, Spain.

People with HIV (PWH) may be more susceptible to SARS-CoV-2 infection and worse clinical outcomes. We investigated the disparity in SARS-CoV-2 vaccination coverage between PWH and those without HIV (PWoH) in Catalonia, Spain, assessing primary and monovalent booster vaccination coverage from December 2021 to July 2022. The vaccines administered were BNT162, ChAdOx1-S, mRNA-127, and Ad26.COV2.S. Using a 1:10 ratio of PWH to PWoH based on sex, age, and socioeconomic deprivation, the analysis included 201,630 individuals (183,300 PWoH and 18,330 PWH). Despite a higher prevalence of comorbidities, PWH exhibited lower rates of complete primary vaccination (78.2% vs. 81.8%, p < 0.001) but surpassed PWoH in booster coverage (68.5% vs. 63.1%, p < 0.001). Notably, complete vaccination rates were lower among PWH with CD4 <200 cells/µL, detectable HIV viremia, and migrants compared to PWoH (p < 0.001, all). However, PWH with CD4 < 200 cells/µL received more boosters (p < 0.001). In multivariable logistic regression analysis of the overall population, a prior SARS-CoV-2 diagnosis, HIV status, migrants, and mild-to-severe socioeconomic deprivation were associated with lower primary vaccination coverage, reflecting barriers to healthcare and vaccine access. However, booster vaccination was higher among PWH. Targeted interventions are needed to improve vaccine coverage and address hesitancy in vulnerable populations.

Cloning, expression, and one-step purification/immobilization of two carbohydrate-binding module-tagged alcohol dehydrogenases.

BACKGROUND: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. RESULTS: In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g- 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg- 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. CONCLUSION: CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.

Lipids in Pathophysiology and Development of the Membrane Lipid Therapy: New Bioactive Lipids.

Membranes are mainly composed of a lipid bilayer and proteins, constituting a checkpoint for the entry and passage of signals and other molecules. Their composition can be modulated by diet, pathophysiological processes, and nutritional/pharmaceutical interventions. In addition to their use as an energy source, lipids have important structural and functional roles, e.g., fatty acyl moieties in phospholipids have distinct impacts on human health depending on their saturation, carbon length, and isometry. These and other membrane lipids have quite specific effects on the lipid bilayer structure, which regulates the interaction with signaling proteins. Alterations to lipids have been associated with important diseases, and, consequently, normalization of these alterations or regulatory interventions that control membrane lipid composition have therapeutic potential. This approach, termed membrane lipid therapy or membrane lipid replacement, has emerged as a novel technology platform for nutraceutical interventions and drug discovery. Several clinical trials and therapeutic products have validated this technology based on the understanding of membrane structure and function. The present review analyzes the molecular basis of this innovative approach, describing how membrane lipid composition and structure affects protein-lipid interactions, cell signaling, disease, and therapy (e.g., fatigue and cardiovascular, neurodegenerative, tumor, infectious diseases).

High-level production of industrially relevant oxidases by a two-stage fed-batch approach: overcoming catabolite repression in arabinose-inducible Escherichia coli systems.

With the growing interest in enzyme applications, there is an urgent demand for economic, affordable, and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose-inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a high cell density culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an eightfold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose-inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems. KEY POINTS: • Arabinose promoters are not well suited for HCDC production due to CCR effect. • This drawback has been overcome by using a two-stage Fed-batch protocol. • Protein yield has been increased by an eightfold factor, improving process economics.

Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab.

BACKGROUND: Monoclonal antibodies (mAbs) and their derivatives have become one of the most important classes of therapeutic drugs. Their multiple applications increased the interest for understanding their complex structure. In vivo, animal cells are able to fold mAbs correctly (Song et al, J Biosci Bioeng 110:135-40, 2010), whereas previous in vitro approaches were scarce and mostly unsuccessful. RESULTS: In this work, we compared in vitro assembly characteristics of trastuzumab, produced either by A) physical separation and refolding of its sub-units or B) direct joining of individually produced heavy and light chains. Native and denatured structures of trastuzumab were determined by SEC-HPLC, HIC-HPLC and SDS-PAGE. CONCLUSIONS: Our results demonstrate the requirement of correctly folded HC, forming disulfide-bonded dimers, in order to form a fully functional mAb. Otherwise, the unfolded HC tend to precipitate. We were able to assemble trastuzumab in this fashion by only mixing them to LC in pH-buffered conditions, while monomeric HC structure was too unstable to render a functional mAb. This approach has been used in the generation of homogeneous ADC, with results pending to be published.

Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly.

Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reduction - oxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG1), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays).

Hospitalizations due to exacerbations of COPD: A big data perspective.

BACKGROUND: Patients with Chronic Obstructive Pulmonary Disease (COPD) may suffer episodes of exacerbation (ECOPD) that require hospitalization and worsen their health status, and prognosis. We hypothesized that a detailed interrogation of health-care "big data" databases can provide valuable information to better understand the risk factors and outcomes of these episodes. MATERIAL AND METHODS: We interrogated four databases of the Catalan health-care system (>8,000,000 registries) to identify patients hospitalized because of ECOPD for the first time (index event) between 2010 and 2012. Analysis was carried forward since the index event until the end of 2014 or the death of the patient. The two years that preceded the index event were also investigated. RESULTS: We identified 17,555 patients, (≥50 years of age) hospitalized because of ECOPD (ICD9 v.9 codes at discharge) for the first time between 2010 and 2012. In this population we observed that: (1) 23% of patients die within a year after being discharged from their first ECOPD hospitalization; (2) in the remaining patients, all-cause mortality was related to the number of re-hospitalizations, particularly with early (<30 days) readmissions; (3) despite this being a 'respiratory' cohort, prescription and dispensation of drugs for cardiovascular diseases was higher than for obstructive airway diseases; and, finally, (4) lower winter ambient temperatures are associated with hospital admissions for ECOPD particularly in early re-admitters. CONCLUSIONS: Overall these results indicate under appreciation of the burden of COPD in patients hospitalized for the first time because ECOPD.

Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide.

Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested. Secondly, 5 different signal peptides were assessed in stable transfected cell lines. Spectrofluorometry was used to determine intra- and extracellular protein expression levels in terms of fluorescence, and the results were further confirmed by SDS-PAGE. The highest secretion efficiency was found for human IFNα2 signal peptide, achieving up to 2-fold increase in the amount of secreted protein compared to other signal peptides. The results showed that the combination of CMV promoter and IFNα2 signal peptide resulted highly efficient for recombinant protein production in HEK293 cells.