Evidence That a Peptide-Drug/p53 Gene Complex Promotes Cognate Gene Expression and Inhibits the Viability of Glioblastoma Cells.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide-transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression-a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma.

Melon peel flour: utilization as a functional ingredient in bakery products.

Food by-products are a major concern with a direct impact on the economy, society, and environment. The valorisation of these by-products could be an advantageous approach to face the increase in food waste since it can compromise environmental health and food sustainability. On the other hand, this valorisation would allow the development of new food products with health benefits for the population. Cucumis melo L. is a highly consumed fruit all over the world since it has excellent sensory and nutritional qualities, being also a good source of bioactive compounds. However, its peel and seeds are usually discarded. The aim of this study was to evaluate the potential of melon peel flour as a functional ingredient for innovative food products. For that, two different formulations containing melon peel flour were developed (a biscuit and a muffin) by replacing a conventional flour (wheat flour) in different percentages (50% and 100%, respectively). The nutritional composition, total phenolic content, and antioxidant potential of the developed products were studied, showing a high content of fibre, high levels of phenolic compounds and good sensory acceptability. These results show that it is possible to enrich different foods with melon peel flour in order to improve their nutritional properties, contributing to improving public health, simultaneously valorising a usually rejected by-product, reducing food waste and the environmental impact.

The Health Effects of Low Glycemic Index and Low Glycemic Load Interventions on Prediabetes and Type 2 Diabetes Mellitus: A Literature Review of RCTs.

Diets with a low glycemic index (GI) and a low glycemic load (GL) can improve glycemic control, blood lipids, blood pressure and BMI in prediabetes and type 2 diabetes (T2DM), but evidence regarding other aspects of cardiometabolic health is limited. We searched the literature for RCTs published from 2013 to 2023 and reviewed the evidence on low-GI/GL diets and their effects on different aspects of health in prediabetes and T2DM, aiming to build a report on all relevant outcomes included in the studies. We included 14 RCTs with 1055 participants, who were mostly middle-aged individuals with T2DM. Interventions were mostly low GI and lasted 1-36 months. Low-GI/GL foods and diets showed benefits in terms of short-term glycemic control, weight and adiposity. Longer-term trials would be necessary to determine whether these benefits persist over time and/or lead to lower CVD risk and mortality. Effects on lipid profile were inconsistent. Some studies also reported positive effects of low-GI/GL interventions on blood pressure, inflammatory biomarkers, renal function and gut microbiota composition. Future trials should focus on some of these novel outcome measures, which may provide important insights into the metabolic effects of low-GI diets on individuals with diabetes.

Oxalate in Foods: Extraction Conditions, Analytical Methods, Occurrence, and Health Implications.

Oxalate is an antinutrient present in a wide range of foods, with plant products, especially green leafy vegetables, being the main sources of dietary oxalates. This compound has been largely associated with hyperoxaluria, kidney stone formation, and, in more severe cases, systematic oxalosis. Due to its impact on human health, it is extremely important to control the amount of oxalate present in foods, particularly for patients with kidney stone issues. In this review, a summary and discussion of the current knowledge on oxalate analysis, its extraction conditions, specific features of analytical methods, reported occurrence in foods, and its health implications are presented. In addition, a brief conclusion and further perspectives on whether high-oxalate foods are truly problematic and can be seen as health threats are shown.

The Role of Biological Rhythms in New Drug Formulations to Cross the Brain Barriers.

For brain protection, the blood-brain barrier and blood-cerebrospinal fluid barrier limit the traffic of molecules between blood and brain tissue and between blood and cerebrospinal fluid, respectively. Besides their protective function, brain barriers also limit the passage of therapeutic drugs to the brain, which constitutes a great challenge for the development of therapeutic strategies for brain disorders. This problem has led to the emergence of novel strategies to treat neurological disorders, like the development of nanoformulations to deliver therapeutic agents to the brain. Recently, functional molecular clocks have been identified in the blood-brain barrier and in the blood-cerebrospinal fluid barrier. In fact, circadian rhythms in physiological functions related to drug disposition were also described in brain barriers. This opens the possibility for chronobiological approaches that aim to use time to improve drug efficacy and safety. The conjugation of nanoformulations with chronobiology for neurological disorders is still unexplored. Facing this, here, we reviewed the circadian rhythms in brain barriers, the nanoformulations studied to deliver drugs to the brain, and the nanoformulations with the potential to be conjugated with a chronobiological approach to therapeutic strategies for the brain.

A Potential Effect of Circadian Rhythm in the Delivery/Therapeutic Performance of Paclitaxel-Dendrimer Nanosystems.

The circadian clock controls behavior and physiology. Presently, there is clear evidence of a connection between this timing system and cancer development/progression. Moreover, circadian rhythm consideration in the therapeutic action of anticancer drugs can enhance the effectiveness of cancer therapy. Nanosized drug delivery systems (DDS) have been demonstrated to be suitable engineered platforms for drug targeted/sustained release. The investigation of the chronobiology-nanotechnology relationship, i.e., timing DDS performance according to a patient's circadian rhythm, may greatly improve cancer clinical outcomes. In the present work, we synthesized nanosystems based on an octa-arginine (R8)-modified poly(amidoamine) dendrimer conjugated with the anticancer drug paclitaxel (PTX), G4-PTX-R8, and its physicochemical properties were revealed to be appropriate for in vitro delivery. The influence of the circadian rhythm on its cellular internalization efficiency and potential therapeutic effect on human cervical cancer cells (HeLa) was studied. Cell-internalized PTX and caspase activity, as a measure of induced apoptosis, were monitored for six time points. Higher levels of PTX and caspase-3/9 were detected at T8, suggesting that the internalization of G4-PTX-R8 into HeLa cells and apoptosis are time-specific/-regulated phenomena. For a deeper understanding, the clock protein Bmal1-the main regulator of rhythmic activity, was silenced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Bmal1 silencing was revealed to have an impact on both PTX release and caspase activity, evidencing a potential role for circadian rhythm on drug delivery/therapeutic effect mediated by G4-PTX-R8.

Using Machine Learning and Molecular Docking to Leverage Urease Inhibition Data for Virtual Screening.

Urease is a metalloenzyme that catalyzes the hydrolysis of urea, and its modulation has an important role in both the agricultural and medical industry. Even though numerous molecules have been tested against ureases of different species, their clinical translation has been limited due to chemical and metabolic stability as well as side effects. Therefore, screening new compounds against urease would be of interest in part due to rising concerns regarding antibiotic resistance. In this work, we collected and curated a diverse set of 2640 publicly available small-molecule inhibitors of jack bean urease and developed a classifier using a random forest machine learning method with high predictive performance. In addition, the physicochemical features of compounds were paired with molecular docking and protein-ligand fingerprint analysis to gather insight into the current activity landscape. We observed that the docking score could not differentiate active from inactive compounds within each chemical family, but scores were correlated with compound activity when all compounds were considered. Additionally, a decision tree model was built based on 2D and 3D Morgan fingerprints to mine patterns of the known active-class compounds. The final machine learning model showed good prediction performance against the test set (81% and 77% precision for active and inactive compounds, respectively). Finally, this model was employed, as a proof-of-concept, on an in-house library to predict new hits that were then tested against urease and found to be active. This is, to date, the largest, most diverse dataset of compounds used to develop predictive in silico models. Overall, the results highlight the usefulness of using machine learning classifiers and molecular docking to predict novel urease inhibitors.

Thermal Contaminants in Coffee Induced by Roasting: A Review.

Roasting is responsible for imparting the main characteristics to coffee, but the high temperatures used in the process can lead to the formation of several potentially toxic substances. Among them, polycyclic aromatic hydrocarbons, acrylamide, furan and its derivative compounds, α-dicarbonyls and advanced glycation end products, 4-methylimidazole, and chloropropanols stand out. The objective of this review is to present a current and comprehensive overview of the chemical contaminants formed during coffee roasting, including a discussion of mitigation strategies reported in the literature to decrease the concentration of these toxicants. Although the formation of the contaminants occurs during the roasting step, knowledge of the coffee production chain as a whole is important to understand the main variables that will impact their concentrations in the different coffee products. The precursors and routes of formation are generally different for each contaminant, and the formed concentrations can be quite high for some substances. In addition, the study highlights several mitigation strategies related to decreasing the concentration of precursors, modifying process conditions and eliminating/degrading the formed contaminant. Many of these strategies show promising results, but there are still challenges to be overcome, since little information is available about advantages and disadvantages in relation to aspects such as costs, potential for application on an industrial scale and impacts on sensory properties.

Processed Food: Nutrition, Safety, and Public Health.

Food processing comprises the activities involved during the transformation of raw materials from different origins (vegetable, animal) until a final product is achieved that is suitable for human consumption [...].

Development of WRAP5 Peptide Complexes for Targeted Drug/Gene Co-Delivery toward Glioblastoma Therapy.

Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood-brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma.

Circadian rhythm and disease: Relationship, new insights, and future perspectives.

The circadian system is responsible for internal functions and regulation of the organism according to environmental cues (zeitgebers). Circadian rhythm dysregulation or chronodisruption has been associated with several diseases, from mental to autoimmune diseases, and with life quality change. Following this, some therapies have been developed to correct circadian misalignments, such as light therapy and chronobiotics. In this manuscript, we describe the circadian-related diseases so far investigated, and studies reporting relevant data on this topic, evidencing this relationship, are included. Despite the actual limitations in published work, there is clear evidence of the correlation between circadian rhythm dysregulation and disease origin/development, and, in this way, clock-related therapies emerge as great progress in the clinical field. Future improvements in such interventions can lead to the development of successful chronotherapy strategies, deeply contributing to enhanced therapeutic outcomes.

The Influence of Circadian Rhythm on Cancer Cells Targeting and Transfection Efficiency of a Polycation-Drug/Gene Delivery Vector.

The conception of novel anticancer delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy. In this work, polyethylenimine (PEI) has been used to complex p53 encoded plasmid DNA (pDNA), and the anticancer drug methotrexate (MTX) has also been loaded into the vectors. To investigate the influence of circadian clock on drug/gene delivery efficiency, HeLa, C33A and fibroblast cells have been transfected with developed PEI/pDNA/MTX delivery vectors at six different time points. Phenomena as the cellular uptake/internalization, drug/gene delivery and p53 protein production have been evaluated. The cell-associated MTX fluorescence have been monitored, and p53 protein levels quantified. In HeLa and C33A cancer cells, significant levels of MTX were found for T8 and T12. For these time points, a high amount of p53 protein was quantified. Confocal microscopy images showed successful HeLa cell's uptake of PEI/pDNA/MTX particles, at T8. In comparison, poor levels of MTX and p53 protein were found in fibroblasts; nevertheless, results indicated rhythmicity. Data demonstrate the influence of circadian rhythm on both cancer-cells targeting ability and transfection performance of PEI/pDNA/MTX carriers and seemed to provide the optimum time for drug/gene delivery. This report adds a great contribution to the field of cancer chronobiology, highlighting the relationship between circadian rhythm and nanodelivery systems, and charting the path for further research on a, yet, poorly explored but promising topic.

4-hydroxy-2-alkenals in foods: a review on risk assessment, analytical methods, formation, occurrence, mitigation and future challenges.

Undoubtedly, significant advances were performed concerning 4-hydroxy-2-alkenals research on foods, and their formation by double oxidation of polyunsaturated fatty acids. But further studies are still needed, especially on their occurrence in foods enriched with n-3 and n-6 fatty acids, as well as in foods for infants and processed foods. Major factors concerning the formation of 4-hydroxy-2-alkenals were discussed, namely the influence of fatty acids composition, time/temperature, processing conditions, salt, among others. Regarding mitigation, the most effective strategies are adding phenolic extracts to foods matrices, as well as other antioxidants, such as vitamin E. Exposure assessment studies revealed 4-hydroxy-2-alkenals values that could not be considered a risk for human health. However, these toxic compounds remain unaltered after digestion and can easily reach the systemic circulation. Therefore, it is crucial to develop in vivo research, with the inclusion of the colon phase, as well as, cell membranes of the intestinal epithelium. In conclusion, according to our review it is possible to eliminate or effectively decrease 4-hydroxy-2-alkenals in foods using simple and economic practices.

Development of Tailor-Made Dendrimer Ternary Complexes for Drug/Gene Co-Delivery in Cancer.

Cancer gene therapy, mediated by non-viral systems, remains a major research focus. To contribute to this field, in this work we reported on the development of dendrimer drug/gene ternary complexes. This innovative approach explored the great capacity of both polyamidoamine (PAMAM)-paclitaxel (PTX) conjugate and polyethylenimine (PEI) polymers to complex a p53-encoding plasmid DNA (pDNA), highlighting the utility of considering two compacting agents. The pDNA complexation capacity has been investigated as function of the nitrogen to phosphate groups ratio (N/P), which revealed to be a tailoring parameter. The physicochemical properties of the conceived ternary complexes were revealed and were found to be promising for cellular transfection. Furthermore, the formulated co-delivery systems demonstrated to be biocompatible. The ternary systems were able of cellular internalization and payload intracellular release. Confocal microscopy studies showed the co-localization of stained pDNA with the nucleus of cancer cells, after transfection mediated by these carriers. From this achievement, p53 gene expression occurred with the production of protein. Moreover, the activation of caspase-3 indicated apoptosis of cancer cells. This work represents a great progress on the design of dendrimer drug/gene co-delivery systems towards a more efficient cancer therapy. In this way, it instigates further in vitro studies concerning the evaluation of their therapeutic potential, expectedly supported by the synergistic effect, in tumoral cells.

Exploring the link between chronobiology and drug delivery: effects on cancer therapy.

Circadian clock is an impressive timing system responsible for the control of several metabolic, physiological and behavioural processes. Nowadays, the connection between the circadian clock and cancer occurrence and development is consensual. Therefore, the inclusion of circadian timing into cancer therapy may potentially offer a more effective and less toxic approach. This way, chronotherapy has been shown to improve cancer treatment efficacy. Despite this relevant finding, its clinical application is poorly exploited. The conception of novel anticancer drug delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy, instigating the incorporation of the circadian timing into clinical practice towards a more personalized drug delivery. This review focuses on the recent advances in the field of cancer chronobiology, on the link between cancer and the disruption of circadian rhythms and on the promising targeted drug nanodelivery approaches aiming the clinical application of cancer chronotherapy.

Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine.

DNA vaccines still represent an emergent area of research, giving rise to continuous progress towards several biomedicine demands. The formulation of delivery systems to specifically target mannose receptors, which are overexpressed on antigen presenting cells (APCs), is considered a suitable strategy to improve the DNA vaccine immunogenicity. The present study developed binary and ternary carriers, based on polyethylenimine (PEI), octa-arginine peptide (R8), and mannose ligands, to specifically deliver a minicircle DNA (mcDNA) vaccine to APCs. Systems were prepared at various nitrogen to phosphate group (N/P) ratios and characterized in terms of their morphology, size, surface charge, and complexation capacity. In vitro studies were conducted to assess the biocompatibility, cell internalization ability, and gene expression of formulated carriers. The high charge density and condensing capacity of both PEI and R8 enhance the interaction with the mcDNA, leading to the formation of smaller particles. The addition of PEI polymer to the R8-mannose/mcDNA binary system reduces the size and increases the zeta potential and system stability. Confocal microscopy studies confirmed intracellular localization of targeting systems, resulting in sustained mcDNA uptake. Furthermore, the efficiency of in vitro transfection can be influenced by the presence of R8-mannose, with great implications for gene expression. R8-mannose/PEI/mcDNA ternary systems can be considered valuable tools to instigate further research, aiming for advances in the DNA vaccine field.

Opuntia ficus-indica (L.) Mill.: A Multi-Benefit Potential to Be Exploited.

Consumer interest in foods with enhanced nutritional quality has increased in recent years. The nutritional and bioactive characterization of fruits and their byproducts, as well as their use in the formulation of new food products, is advisable, contributing to decrease the global concerns related to food waste and food security. Moreover, the compounds present in these raw materials and the study of their biological properties can promote health and help to prevent some chronic diseases. Opuntia ficus-indica (L.) Mill. (prickly pear) is a plant that grows wild in the arid and semi-arid regions of the world, being a food source for ones and a potential for others, but not properly valued. This paper carries out an exhaustive review of the scientific literature on the nutritional composition and bioactive compounds of prickly pear and its constituents, as well as its main biological activities and applications. It is a good source of dietary fiber, vitamins and bioactive compounds. Many of its natural compounds have interesting biological activities such as anti-inflammatory, hypoglycemic and antimicrobial. The antioxidant power of prickly pear makes it a good candidate as an ingredient of new food products with fascinating properties for health promotion and/or to be used as natural extracts for food, pharmaceutic or cosmetic applications. In addition, it could be a key player in food security in many arid and semi-arid regions of the world, where there are often no more plants.

Compliance of declared vs. analysed values with EU tolerance limits for mandatory nutrients in prepacked foods.

This study assessed compliance between declared and analysed values on prepacked foods, considering the tolerance limits for salt, fat and saturated fatty acids. Foods were distributed by food categories (e.g. snacks, ready-to-eat meals, potato and potato-products, bakery and pastry products) and a total of 209 products were analysed. Only half of the samples with a declared value ≥1.25 g/100 g were within tolerance limits for salt content. The lowest number of samples outside tolerance limits was observed for fat content; for saturated fatty acids, 27% of the samples were outside of tolerance limits. Only amongst "cereal products" were 100% of products compliant for fat and salt declarations. It is of utmost importance that manufacturers update continuously values declared for prepacked foods, because this information is crucial for consumers and food policy-makers as well as being a legal requirement.

Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network.

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.