Recent advances on cell culture platforms for In vitro drug screening and cell therapies: from conventional to microfluidic strategies

The clinical translations of drugs and nanomedicines depend on coherent pharmaceutical research based on biologically accurate screening approaches. Since establishing the 2D in vitro cell culture method, the scientific community has improved cell-based drug screening assays and models. Those advances result in more informative biochemical assays and the development of 3D multicellular models to describe the biological complexity better and enhance the simulation of the in vivo microenvironment. Despite the overall dominance of conventional 2D and 3D cell macroscopic culture methods, they present physicochemical and operational challenges that impair the scale-up of drug screening by not allowing a high parallelization, multidrug combination, and high-throughput screening. Their combination and complementarity with microfluidic platforms enable the development of microfluidics-based cell culture platforms with unequivocal advantages in drug screening and cell therapies. Thus, this review presents an updated and consolidated view of cell culture miniaturization's physical, chemical, and operational considerations in the pharmaceutical research scenario. It clarifies advances in the field using gradient-based microfluidics, droplet-based microfluidics, printed-based microfluidics, digital-based microfluidics, SlipChip, and paper-based microfluidics. Finally, it presents a comparative analysis of the performance of cell-based methods in life research and development to achieve increased precision in the drug screening process.This work was supported by FCT - Fundação para a Ciência e Tecnolo gia (FCT) under the scope of the strategic funding UIDB/04650/2020, UIDP/04650/2020, UIDB/04436/2020, UIDP/04436/2020. The authors also thank FCT for financial support under grant SFRH/BD/141936/2018 (B.D.C.) and the contract under the Stimulus of Scientific Employment 2020.02304.CEECIND (V.C.). The authors also acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019-106099RBC43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program

Solid magnetoliposomes as multi-stimuli-responsive systems for controlled release of doxorubicin: assessment of lipid formulations

Stimuli-responsive liposomes are a class of nanocarriers whose drug release occurs, preferentially, when exposed to a specific biological environment, to an external stimulus, or both. This work is focused on the design of solid magnetoliposomes (SMLs) as lipid-based nanosystems aiming to obtain multi-stimuli-responsive vesicles for doxorubicin (DOX) controlled release in pathological areas under the action of thermal, magnetic, and pH stimuli. The effect of lipid combinations on structural, colloidal stability, and thermodynamic parameters were evaluated. The results confirmed the reproducibility for SMLs synthesis based on nine lipid formulations (combining DPPC, DSPC, CHEMS, DOPE and/or DSPE-PEG), with structural and colloidal properties suitable for biological applications. A loss of stability and thermosensitivity was observed for formulations containing dioleoylphosphatidylethanolamine (DOPE) lipid. SMLs PEGylation is an essential step to enhance both their long-term storage stability and stealth properties. DOX encapsulation (encapsulation efficiency ranging between 87% and 96%) in the bilayers lowered its pK(a), which favors the displacement of DOX from the acyl chains to the surface when changing from alkaline to acidic pH. The release profiles demonstrated a preferential release at acidic pH, more pronounced under mimetic mild-hyperthermia conditions (42 degrees C). Release kinetics varied with the lipid formulation, generally demonstrating hyperthermia temperatures and acidic pH as determining factors in DOX release; PEGylation was shown to act as a diffusion barrier on the SMLs surface. The integrated assessment and characterization of SMLs allows tuning lipid formulations that best respond to the needs for specific controlled release profiles of stimuli-responsive nanosystems as a multi-functional approach to cancer targeting and therapy.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UIDB/04650/2020, UIDB/04436/2020, UIDP/04436/2020 and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), cofinanced by European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The authors also thank FCT for financial support under grants SFRH/BD/141936/2018 (B.D.C.) and 2020.02304.CEECIND (V.F.C.) Finally, the authors acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program

β2-adrenergic agonists do not improve physical performance in healthy individuals

All beta-2 agonists are prohibited by the World Anti-Doping Agency (WADA, www.wada-ama.org). However, its use may be permitted in athletes who are granted a therapeutic use exemption (TUE). [...]info:eu-repo/semantics/publishedVersio

β2-adrenergic agonists and doping: Where do we stand?

Hostrup et al1 kindly commented on our previous paper related to the enhancement of physical performance by β2-agonists.2 First, we would like to thank the authors for their review and acknowledge that our title and conclusions may have been misleading because, in fact, they only refer to endurance/aerobic-dominated performances [...]info:eu-repo/semantics/publishedVersio

Keeping the eIF2 alpha kinase Gcn2 in check

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. in mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2 alpha, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity. (C) 2014 Published by Elsevier B.V.Massey University Research FundAuckland Medical Research FoundationMaurice & Phyllis Paykel TrustNutricia Research FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Massey UniversityInstitute of Natural and Mathematical SciencesUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilMassey Univ, Inst Nat & Math Sci, Auckland 0745, Albany, New ZealandUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilAuckland Medical Research Foundation: 4109024Auckland Medical Research Foundation: 4113010Nutricia Research Foundation: 2010-35FAPESP: 2009/52047-5CNPq: 478903/2012-0CNPq: 309860/2011-3CNPq: 153660/2010-4CAPES: 1915-13-4Web of Scienc

Apple pomace aqueous extract as a food ingredient

The present work aimed to assess the potential of apple pomace (AP) as a low priced source of food ingredients rich in bioactive compounds. In order to determine its safety, AP was evaluated regarding Total Aerobic Mesophiles (TAM), Yeasts and Moulds (YM) and Enterobacteriaceae (ENT). A hot water extract of AP was investigated concerning total phenolic content (TPC) by the Folin-Ciocalteu method and its antioxidant capacity by the hydroxyl scavenging (OH˙) and ABTS˙+ methods. The extract was incorporated into a yoghurt and its microbiological parameters, antioxidant activity and sensorial acceptability were evaluated. AP had very acceptable microbial levels respecting the TAM, YM and ENT, potentiating its valuation as a source of food ingredients. The AP extract presented a TPC of ≈11 µg of GAE/g, which resulted in the capability to inhibit OH˙ radical and the ABTS˙+ radicals. These properties were reflected by an increased antioxidant activity of the yoghurts fortified with the extract, reaching more than twice of the controls. This was achieved without affecting the native yoghurt lactic acid bacteria and sensorial acceptance. Although the present strategy proves to be very promising for the valuation of AP, further studies are required in order to better elucidate the extracts’ bioactivity.info:eu-repo/semantics/publishedVersio

Two-step purification of epilactose produced by cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus

Epilactose is a functional sugar that can be produced from lactose using cellobiose 2-epimerase and it is considered a developing prebiotic. In that sense, the development of strategies to produce and purify epilactose is key for its wider use in the food industry. The aim of this work was to establish a food-grade purification strategy suitable to be scaled-up to an industrial level. Firstly, the epilactose was produced by enzymatic epimerization of lactose in a reaction catalyzed by the recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus produced by Saccharomyces cerevisiae. Then, to remove the unreacted lactose, a screening study was performed to find a suitable -galactosidase enzyme with high lactose hydrolysis capacity but low ability to convert the epilactose. The elimination of the generated monosaccharides was then attempted by microbial treatment using different microorganisms and using activated charcoal. The bakers yeast S. cerevisiae was proven to be the most suitable microorganism for glucose and galactose removal from the reaction mixture. Overall, an attractive and food-grade two-step process for epilactose recovery was established, resulting in a purity and yield of 87% and 76.4%, respectively. Additionally, the INFOGEST 2.0 static in vitro simulation of gastrointestinal food digestion was used, for the first time, to assess the resistance of epilactose (77% resistance) to the upper gastrointestinal tract conditions, reinforcing its potential to be used as prebiotic.BBC and JMF acknowledge their doctoral grants (SFRH/BD/ 132324/2017 and SFRH/BD/147286/2019, respectively) from the Portuguese Foundation of Science and Technology (FCT). This study was supported by FCT under the scope of the strategic funding of UIDB/ 04469/2020 unit.info:eu-repo/semantics/publishedVersio