The Effect of Phenolic-Rich Extracts of Rubus fruticosus, R. ulmifolius and Morus nigra on Oxidative Stress and Caco-2 Inhibition Growth.

Currently, a clear interest has been given to berries due to their richness in active metabolites, including anthocyanins and non-coloured phenolics. Therefore, the main aim of the present work is to investigate the phenolic profile, antioxidant abilities, and antiproliferative effects on normal human dermal fibroblasts (NHDF) and human colon carcinoma cell line (Caco-2) cells of phenolic-rich extracts from three red fruits highly appreciated by consumers: two species of blackberries (Rubus fruticosus and Rubus ulmifolius) and one species of mulberry (Morus nigra). A total of 19 different phenolics were identified and quantified by HPLC-DAD-ESI/MSn and HPLC-DAD, respectively. Focusing on the biological potential of the phenolic-rich extracts, all of them revealed notable scavenging abilities. Concerning the antiproliferative properties, R. fruticosus presented a cytotoxic selectivity for Caco-2 cells compared to NHDF cells. To deeper explore the biological potential, combinations with positive controls (ascorbic acid and 5-fluorouracil) were also conducted. Finally, the obtained data are another piece of evidence that the combination of phenolic-rich extracts from natural plants with positive controls may reduce clinical therapy costs and the possible toxicity of chemical drugs.

Functional Model Beverages of Saffron Floral By-Products: Polyphenolic Composition, Inhibition of Digestive Enzymes, and Rheological Characterization.

Despite the rapid and dynamic evolution of research into dietary polyphenols, there is still a knowledge gap regarding their bioaccessibility since it could be influenced by the chemical and nutritional compositions of the food matrix. This study aimed to describe the impact of food thickeners (xanthan gum, guar gum, ß-glucan, pectin) on the bioactivity of flavonoids from saffron floral by-products in model beverages before and after thermal processing. The different beverage formulas were characterized in terms of polyphenolic composition using HPLC-DAD-ESI-MSn and rheological properties. The impact of food thickeners and thermal processing on the inhibition of digestive enzymes was also determined. The model beverages mainly presented glycosylated flavonols (of kaempferol, quercetin, and isorhamnetin), with a reduced content in some heat-treated samples. The inhibitory effect on α-amylase was only detected in heat-treated beverages, showing the formulation without any thickener to have the greatest inhibitory effect. Finally, the presence of saffron floral by-products in the beverages showed a tendency to decrease the flow consistency index (K) and an increase in the flow behavior index (n), most probably driven by the aggregation of phenolics with thickeners. Therefore, this research provides new insights into the development of flavonoid-rich beverages in order to ensure that they exert the expected beneficial effects after their ingestion.

Antinociceptive effects of Raphanus sativus sprouts involve the opioid and 5-HT1A serotonin receptors, cAMP/cGMP pathways, and the central activity of sulforaphane.

Raphanus sativus L. cv. Sango, commonly known as red radish, is widely consumed around the world as a vegetable, but its benefit in pain relief is not sufficiently investigated. This study aimed to evaluate the antinociceptive effects of R. sativus and a possible mechanism of action. An aqueous extract of R. sativus sprouts (AERSS) was investigated by parenteral (10, 30, and 100 mg kg-1, i.p.) and enteral (500 mg kg-1, p.o.) administration in the neurogenic and inflammatory phases of the formalin test, where gastric damage was also evaluated as a possible adverse effect. Ketorolac (5 mg kg-1, i.p.) was used as the reference drug. Endogenous opioid and 5-HT1A serotonin receptors, as well as the cAMP/NO-cGMP pathways, were explored in the study of a possible mechanism of action by using their corresponding antagonists: naloxone, 1 mg kg-1, i.p., WAY100635, 1 mg kg-1, i.p., and enzymatic activators or inhibitors, respectively. Sulforaphane (SFN), a known bioactive metabolite, was analyzed using electroencephalography (EEG) to evidence its central involvement. A significant and dose-dependent antinociceptive activity was observed with the AERSS resembling the antinociceptive effect of the reference drug, with an equivalent significant response with a dose of 500 mg kg-1, p.o. without causing gastric damage. The participation of the endogenous opioid and 5-HT1A serotonin receptors at central and peripheral levels was also observed, with a differential participation of cAMP/NO-cGMP. SFN as one metabolite produced significant changes in the EEG analysis, reinforcing its effects on the CNS. Our preclinical evidence supports the benefits of consuming Raphanus sativus cv. Sango sprouts for pain relief.

Endocrine Adverse Events Related To Immune Checkpoint Inhibitor Treatment: Relationship Between Antibodies and Severity of Thyroid Dysfunction.

OBJECTIVE: The purpose of this study was to identify predictive and risk factors for the development of immune-related endocrinopathies and to analyze the incidence and characteristics of immune-related endocrinopathies in our population Design: A retrospective, single-centre cohort carried out at Gregorio Marañón Hospital between January 2018 -December 2019. METHODS: A total of 163 patients were enrolled. In January 2018 and December 2019, we treated patients who underwent ICI treatment in the Medical Oncology Department of General University Hospital Gregorio Marañón, a tertiary care public hospital in Madrid, as part of an observational, retrospective, single-center cohort study. RESULTS: Endocrinopathies were diagnosed in 19.5% of the patients (n=32). The tumours with the highest incidence of endocrinopathies were non-small cell lung cancer (25,9%), kidney cell cancer (25%) and hepatocarcinoma (20%). Among the 32 patients who developed endocrinopathy, 18,8%, 19,13%, and 21,28% received anti-CTLA-4, anti-PD-1 and anti-PDL-1, respectively. Thyroid dysfunction was the most frequent endocrinopathy (12,8%). A higher percentage of patients with negative antiTPO and antiTG antibodies developed G1 hypothyroidism compared to patients with positive antibodies who developed a higher proportion of G2 hypothyroidism. The presence of an initial phase of thyrotoxicity was not related to greater severity. We observed longer progression-free survival in patients who developed thyroid dysfunction. CONCLUSION: Pre-existing antibodies were independently associated with endocrinopathies. Moreover, our study let us conclude that the presence of thyroid autoantibodies may be related to its severity. It is important to determine anti-thyroid antibodies prior to the start of immunotherapy as a risk factor for thyroid dysfunction, which in turn is a prognostic marker.

Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs.

Cell-to-cell communication strategies include extracellular vesicles (EVs) in plants and animals. The bioactive molecules in a diet rich in vegetables and fruits are associated with disease-preventive effects. Plant-derived EVs (PDEVs) are biogenetically and morphologically comparable to mammalian EVs and transport bioactive molecules, including miRNAs. However, the biological functions of PDEVs are not fully understood, and standard isolation protocols are lacking. Here, PDEVs were isolated from four foods with a combination of ultracentrifugation and size exclusion chromatography, and evaluated as vehicles for enhanced transport of synthetic miRNAs. In addition, the role of food-derived EVs as carriers of dietary (poly)phenols and other secondary metabolites was investigated. EVs from broccoli, pomegranate, apple, and orange were efficiently isolated and characterized. In all four sources, 4 miRNA families were present in tissues and EVs. miRNAs present in broccoli and fruit-derived EVs showed a reduced RNase degradation and were ferried inside exposed cells. EVs transfected with a combination of ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p showed toxic effects on human cells, as did natural broccoli EVs alone. PDEVs transport trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. Thus, PDEVs can act as nanocarriers for functional miRNAs that could be used in RNA-based therapy.

The preventive effects of broccoli bioactives against cancer: Evidence from a validated rat glioma model.

The aggressive and incurable diffuse gliomas constitute 80% of malignant brain tumors, and patients succumb to recurrent surgeries and drug resistance. Epidemiological research indicates that substantial consumption of fruits and vegetables diminishes the risk of developing this tumor type. Broccoli consumption has shown beneficial effects in both cancer and neurodegenerative diseases. These effects are partially attributed to the isothiocyanate sulforaphane (SFN), which can regulate the Keap1/Nrf2/ARE signaling pathway, stimulate detoxifying enzymes, and activate cellular antioxidant defense processes. This study employs a C6 rat glioma model to assess the chemoprotective potential of aqueous extracts from broccoli seeds, sprouts, and inflorescences, all rich in SFN, and pure SFN as positive control. The findings reveal that administering a dose of 100 mg/kg of broccoli sprout aqueous extract and 0.1 mg/kg of SFN to animals for 30 days before introducing 1 × 104 cells effectively halts tumor growth and progression. This study underscores the significance of exploring foods abundant in bioactive compounds, such as derivatives of broccoli, for potential preventive integration into daily diets. Using broccoli sprouts as a natural defense against cancer development might seem idealistic, yet this investigation establishes that administering this extract proves to be a valuable approach in designing strategies for glioma prevention. Although the findings stem from a rat glioma model, they offer promising insights for subsequent preclinical and clinical research endeavors.

CREB1 activation promotes human papillomavirus oncogene expression and cervical cancer cell transformation.

Human papillomaviruses (HPVs) infect the oral and anogenital mucosa and can cause cancer. The high-risk (HR)-HPV oncoproteins, E6 and E7, hijack cellular factors to promote cell proliferation, delay differentiation and induce genomic instability, thus predisposing infected cells to malignant transformation. cAMP response element (CRE)-binding protein 1 (CREB1) is a master transcription factor that can function as a proto-oncogene, the abnormal activity of which is associated with multiple cancers. However, little is known about the interplay between HPV and CREB1 activity in cervical cancer or the productive HPV lifecycle. We show that CREB is activated in productively infected primary keratinocytes and that CREB1 expression and phosphorylation is associated with the progression of HPV+ cervical disease. The depletion of CREB1 or inhibition of CREB1 activity results in decreased cell proliferation and reduced expression of markers of epithelial to mesenchymal transition, coupled with reduced migration in HPV+ cervical cancer cell lines. CREB1 expression is negatively regulated by the tumor suppressor microRNA, miR-203a, and CREB1 phosphorylation is controlled through the MAPK/MSK pathway. Crucially, CREB1 directly binds the viral promoter to upregulate transcription of the E6/E7 oncogenes, establishing a positive feedback loop between the HPV oncoproteins and CREB1. Our findings demonstrate the oncogenic function of CREB1 in HPV+ cervical cancer and its relationship with the HPV oncogenes.

Soft robotics-enabled large animal model of HFpEF hemodynamics for device testing.

Heart failure with preserved ejection fraction (HFpEF) is a major challenge in cardiovascular medicine, accounting for approximately 50% of all cases of heart failure. Due to the lack of effective therapies for this condition, the mortality associated with HFpEF remains higher than that of most cancers. Despite the ongoing efforts, no medical device has yet received FDA approval. This is largely due to the lack of an in vivo model of the HFpEF hemodynamics, resulting in the inability to evaluate device effectiveness in vivo prior to clinical trials. Here, we describe the development of a highly tunable porcine model of HFpEF hemodynamics using implantable soft robotic sleeves, where controlled actuation of a left ventricular and an aortic sleeve can recapitulate changes in ventricular compliance and afterload associated with a broad spectrum of HFpEF hemodynamic phenotypes. We demonstrate the feasibility of the proposed model in preclinical testing by evaluating the hemodynamic response of the model post-implantation of an interatrial shunt device, which was found to be consistent with findings from in silico studies and clinical trials. This work addresses several of the limitations associated with previous models of HFpEF, such as their limited hemodynamic fidelity, elevated costs, lengthy development time, and low throughput. By showcasing exceptional versatility and tunability, the proposed platform has the potential to revolutionize the current approach for HFpEF device development and selection, with the goal of improving the quality of life for the 32 million people affected by HFpEF worldwide.

Capnometry after an inspiratory breath hold, PLAT CO2 , as a surrogate for P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ in mild to moderate pediatric acute respiratory distress syndrome: A feasibility study.

OBJECTIVE: Accurate and reliable noninvasive methods to estimate gas exchange are necessary to guide clinical decisions to avoid frequent blood samples in children with pediatric acute respiratory distress syndrome (PARDS). We aimed to investigate the correlation and agreement between end-tidal P CO 2 ${P}_{{\mathrm{CO}}_{2}}$ measured immediately after a 3-s inspiratory-hold (PLAT CO2 ) by capnometry and P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ measured by arterial blood gases (ABG) in PARDS. DESIGN: Prospective cohort study. SETTING: Seven-bed Pediatric Intensive Care Unit, Hospital El Carmen de Maipú, Chile. PATIENTS: Thirteen mechanically ventilated patients aged ≤15 years old undergoing neuromuscular blockade as part of management for PARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: All patients were in volume-controlled ventilation mode. The regular end-tidal P CO 2 ( P ETCO 2 ) ${P}_{{\mathrm{CO}}_{2}}({P}_{{\mathrm{ETCO}}_{2}})$ (without the inspiratory hold) was registered immediately after the ABG sample. An inspiratory-hold of 3 s was performed for lung mechanics measurements, recording P ETCO 2 ${P}_{{\mathrm{ETCO}}_{2}}$ in the breath following the inspiratory-hold. (PLAT CO2 ). End-tidal alveolar dead space fraction (AVDSf) was calculated as [ ( P aCO 2 - P ETCO 2 ) / P aCO 2 ] $[({P}_{{\mathrm{aCO}}_{2}}\mbox{--}{P}_{{\mathrm{ETCO}}_{2}})/{P}_{{\mathrm{aCO}}_{2}}]$ and its surrogate (S)AVDSf as [ ( PLAT CO 2 - P ETCO 2 ) / PLAT CO 2 ] $[{(}_{\mathrm{PLAT}}{\mathrm{CO}}_{2}\mbox{--}{P}_{{\mathrm{ETCO}}_{2}}){/}_{\mathrm{PLAT}}{\mathrm{CO}}_{2}]$ . Measurements of P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ were considered the gold standard. We performed concordance correlation coefficient (ρc), Spearman's correlation (rho), and Bland-Altmann's analysis (mean difference ± SD [limits of agreement, LoA]). Eleven patients were included, with a median (interquartile range) age of 5 (2-11) months. Tidal volume was 5.8 (5.7-6.3) mL/kg, PEEP 8 (6-8), driving pressure 10 (8-11), and plateau pressure 17 (17-19) cm H2 O. Forty-one paired measurements were analyzed. P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ was higher than P ETCO 2 ${P}_{{\mathrm{ETCO}}_{2}}$ (52 mmHg [48-54] vs. 42 mmHg [38-45], p < 0.01), and there were no significant differences with PLAT CO2 (50 mmHg [46-55], p > 0.99). The concordance correlation coefficient and Spearman's correlation between P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ and PLAT CO2 were robust (ρc = 0.80 [95% confidence interval [CI]: 0.67-0.90]; and rho = 0.80, p < 0.001.), and for P ETCO 2 ${P}_{{\mathrm{ETCO}}_{2}}$ were weak and strong (ρc = 0.27 [95% CI: 0.15-0.38]; and rho = 0.63, p < 0.01). The bias between PLAT CO2 and P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ was -0.4 ± 3.5 mmHg (LoA -7.2 to 6.4), and between P ETCO 2 ${P}_{{\mathrm{ETCO}}_{2}}$ and P aCO 2 ${P}_{{\mathrm{aCO}}_{2}}$ was -8.5 ± 4.1 mmHg (LoA -16.6 to -0.5). The correlation between AVDSf and (S)AVDSf was moderate (rho = 0.55, p < 0.01), and the mean difference was -0.5 ± 5.6% (LoA -11.5 to 10.5). CONCLUSION: This pilot study showed the feasibility of measuring end-tidal CO2 after a 3-s end-inspiratory breath hole in pediatric patients undergoing controlled ventilation for ARDS. Encouraging preliminary results warrant further study of this technique.

Exploitation of ATP-sensitive potassium ion (KATP) channels by HPV promotes cervical cancer cell proliferation by contributing to MAPK/AP-1 signalling.

Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (KATP) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of KATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.

Microbiologically influenced corrosion on naval carbon steel inside the hull of tugboats: a case study of prevention and control.

Microbiologically influenced corrosion (MIC) has a significant cost to many industries, including naval engineering. In this case-of-study, three tugboats developed pitting corrosion in the carbon steel of the inner hulls. Grade A naval steel was used for the hull sheets but the inner side (corroded) showed only two protective layers of paint. The maintenance employed seawater, which ended up in the bilge and made MIC possible. Bilge's waters were submitted to physicochemical, biological and molecular tests. DNA analyses confirmed the presence of Pseudomonas spp. and Desulfovibrio spp. in water samples and, consequently, a MIC mechanism was proposed to explain the corrosion process. In addition, a biocide treatment was evaluated and a new maintenance protocol was recommended. This work highlights the importance of the engineering design to prevent MIC in marine transports and provides some guidelines to treat it.

A new surgical technique for sutureless partial nephrectomy: renal sutureless device.

BACKGROUND: Several factors impact the preservation of renal function after partial nephrectomy. Warm ischemia time is the main modifiable surgical factor. Renorrhaphy represents the key of hemostasia, but it is associated with increase of warm ischemia time and complications. The aim of this study was to describe our initial surgical experience with a new surgical technique for sutureless partial nephrectomy, based on the application of our own developed renal-sutureless-device-RSD. METHODS: Between 2020-2021, 10 patients diagnosed with renal cell carcinoma stage cT1a-b cN0M0 with an exophytic component were operated using renal-sutureless-device-RSD. Surgical technique of sutureless partial nephrectomy with renal-sutureless-device-RSD is described in a step-by-step fashion. Clinical data was collected in a dedicated database. Presurgical, intraoperative, postoperative variables, pathology and functional results were evaluated. Medians and ranges of values for selected variables were reported as descriptive statistics. RESULTS: Partial nephrectomy was carried out with the use of renal-sutureless-device-RSD without renorrhaphy in all cases (70%cT1a-30%cT1b). Median tumor size was 3.15 cm (IQR: 2.5-4.5). R.E.N.A.L Score had a range between 4a-10. Median surgical time was 97.5 minutes (IQR 75-105). Renal artery clamping was only required in 4 cases, with a median warm ischemia time of 12.5 minutes (IQR 10-15). No blood transfusion, intraoperative and postoperative complications were noted. Free-of-disease margin rate achieved was 90%. Median length of stay was 2 days (IQR 2-2). Laboratory data on hemoglobin and hematocrit levels, as well as renal function tests, remained stable after partial nephrectomy. CONCLUSIONS: Our initial experience suggests that a sutureless PN using the RSD device is feasible and safe. Further investigation is needed to determine the clinical benefit of this technique.

Anti-inflammatory potential of digested Brassica sprout extracts in human macrophage-like HL-60 cells.

Cruciferous vegetables have been reported to be a great source of anti-inflammatory compounds. Specifically, sprouts from the Brassicaceae family stand out for their high content of glucosinolates (and their bioactive derivatives, isothiocyanates), phenolic acids, and anthocyanins. Despite the evident anti-inflammatory activity of certain Brassica phytochemicals such as sulforaphane or phenolic acids, the effect of digested Brassica vegetables on inflammation remains understudied. In this work, we aimed to evaluate the anti-inflammatory potential of the bioaccessible forms of cruciferous bioactives (from red cabbage sprouts (RCS) and red radish sprouts (RRS)) obtained upon in vitro gastrointestinal digestion in the HL-60 macrophage-like differentiated human cell line. The study was performed under basal conditions or stimulated with a low dose of LPS for 24 hours as a validated in vitro model of chronic inflammation. The cell viability was determined by MTT assay. The gene expression and production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß were determined by RT-qPCR and ELISA respectively. Our results revealed no cytotoxicity with any of the treatments in LPS-stimulated macrophage-like HL60 cells. Regarding cytokine production, digestates significantly decreased the production of the three pro-inflammatory cytokines at concentrations of 50 and 100 µg mL-1 except for IL-1ß treated with RCS digestates. Furthermore, the RT-qPCR analysis showed a decrease in the relative expression of pro-inflammatory cytokines in LPS-stimulated cells treated with RRS digestates at 100 µg mL-1 but not with red cabbage digestates. In conclusion, RRS bioaccessible compounds in the extracts could be used as dietary coadjuvants given their potential anti-inflammatory effect on this in vitro model of chronic inflammation.

Bioactive, Mineral and Antioxidative Properties of Gluten-Free Chicory Supplemented Snack: Impact of Processing Conditions.

This study aimed to investigate the impact of chicory root addition (20-40%) and extrusion conditions (moisture content from 16.3 to 22.5%, and screw speed from 500 to 900 rpm) on bioactive compounds content (inulin, sesquiterpene lactones, and polyphenols) of gluten-free rice snacks. Chicory root is considered a potential carrier of food bioactives, while extrusion may produce a wide range of functional snack products. The mineral profiles were determined in all of the obtained extrudates in terms of Na, K, Ca, Mg, Fe, Mn, Zn, and Cu contents, while antioxidative activity was established through reducing capacity, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) tests. Chicory root addition contributed to the improvement of bioactive compounds and mineral contents, as well as antioxidative activities in all of the investigated extrudates in comparison to the pure-rice control sample. An increase in moisture content raised sesquiterpene lactones and minerals, while high screw speeds positively affected polyphenols content. The achieved results showed the important impact of the extrusion conditions on the investigated parameters and promoted chicory root as an attractive food ingredient in gluten-free snack products with high bioactive value.

Anti-Leukemic Activity of Brassica-Derived Bioactive Compounds in HL-60 Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells mainly treated with chemotherapy for cancer remission, but this non-selective treatment also induces numerous side effects. Investigations with bioactive compounds from plant-derived foods against cancer have increased in the last years because there is an urgent need to search for new anti-leukemic agents possessing higher efficacy and selectivity for AML cells and fewer negative side effects. In this study, we analyzed the anti-leukemic activity of several phytochemicals that are representative of the major classes of compounds present in cruciferous foods (glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols, and anthocyanins) in the human acute myeloid leukemia cell line HL-60. Our results revealed that among the different Brassica-derived compounds assayed, sulforaphane (SFN) (an aliphatic isothiocyanate) showed the most potent anti-leukemic activity with an IC50 value of 6 µM in dose-response MTT assays after 48 h of treatment. On the other hand, chlorogenic acid (a hydroxycinnamic acid) and cyanidin-3-glucoside (an anthocyanin) also displayed anti-leukemic potential, with IC50 values of 7 µM and 17 µM after 48 h of incubation, respectively. Importantly, these compounds did not show significant cell toxicity in macrophages-like differentiated cells at 10 and 25 µM, indicating that their cytotoxic effects were specific to AML cancer cells. Finally, we found that these three compounds were able to induce the NRF2/KEAP1 signaling pathway in a dose-dependent manner, highlighting SFN as the most potent NRF2 activator. Overall, the present evidence shed light on the potential for using foods and ingredients rich in anticancer bioactive phytochemicals from Brassica spp.

Potential of Sulforaphane and Broccoli Membrane Vesicles as Regulators of M1/M2 Human Macrophage Activity.

Macrophages have emerged as important therapeutic targets in many human diseases. The aim of this study was to analyze the effect of broccoli membrane vesicles and sulphoraphane (SFN), either free or encapsulated, on the activity of human monocyte-derived M1 and M2 macrophage primary culture. Our results show that exposure for 24 h to SFN 25 µM, free and encapsulated, induced a potent reduction on the activity of human M1 and M2 macrophages, downregulating proinflammatory and anti-inflammatory cytokines and phagocytic capability on C. albicans. The broccoli membrane vesicles do not represent inert nanocarriers, as they have low amounts of bioactive compounds, being able to modulate the cytokine production, depending on the inflammatory state of the cells. They could induce opposite effects to that of higher doses of SFN, reflecting its hormetic effect. These data reinforce the potential use of broccoli compounds as therapeutic agents not only for inflammatory diseases, but they also open new clinical possibilities for applications in other diseases related to immunodeficiency, autoimmunity, or in cancer therapy. Considering the variability of their biological effects in different scenarios, a proper therapeutic strategy with Brassica bioactive compounds should be designed for each pathology.

Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a Cystic Fibrosis Patient Treated with ß-Lactams.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C ß-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC ß-lactamase is the main mechanism driving resistance in this notorious pathogen to ß-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the ß-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Contribution of the diverse experimental models to unravelling the biological scope of dietary (poly)phenols.

The health benefits associated with (poly)phenols need to be supported by robust and insightful information on their biological effects. The use of in vitro, ex vivo, and in vivo models is crucial to demonstrate functionalities in specific targets. In this regard, bioaccessibility, bioavailability, and tissue/organ distribution need to be fully understood and established. In addition, the structure-function relationships, concerning both descriptive and mechanistic information, between specific compounds and therapeutic objectives, need to be supported by results obtained from in vivo studies. Nevertheless, these studies are not always possible or have some limitations, particularly concerning the mechanistic information explaining the health benefits provided that should be covered with complementary experimental models. Based on these premises, this review aims to overview the contribution of the separate experimental approaches to gain insights into the bioaccessibility, bioavailability, and bioactivity of (poly)phenols. To achieve this objective, recent evidence available on the linkage of healthy/functional foods with the incidence of non-communicable pathologies is presented. The different experimental approaches provide complementary information that allows advances to be applied to the knowledge gained on the functional properties and mechanistic facts responsible for the health attributions of polyphenols. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Nanoencapsulation of Bimi® extracts increases its bioaccessibility after in vitro digestion and evaluation of its activity in hepatocyte metabolism.

Isothiocyanates (ITCs) have low stability in aqueous conditions, reducing their bioavailability when used as food ingredients. Therefore, the aim of this work was to increase the stability of the ITCs present in extracts of Bimi® edible parts by nanoencapsulation using cauliflower-derived plasma membrane vesicles. The bioactivity of these nanoencapsulates was evaluated in a HepG2 hepatocyte cell line in a model for low-grade chronic inflammation. The vesicles showed a higher capacity of retention in the in vitro gastrointestinal digestion for 3,3-diindolylmethane (DIM), indole-3-carbinol (I3C) and sulforaphane (SFN). Furthermore, Transmission Electron Microscopy (TEM) analysis of the vesicles revealed a decreased size under acidic pH and a release of their cargo after the intestinal digestion. The HepG2 experiments revealed differences in metabolism under the condition of chronic inflammation. The cauliflower-derived plasma membrane vesicles are able to enhance the stability of ITCs through the in vitro gastrointestinal digestion, improving their bioaccesibility and potential bioavailability.

Analysis of the anti-inflammatory potential of Brassica bioactive compounds in a human macrophage-like cell model derived from HL-60 cells.

BACKGROUND: Chronic inflammatory diseases are major causes of global morbidity and mortality. Acute inflammation is meant to protect the body against foreign agents, but it also plays a major role in tissue repairment. Several mediators are involved in this process, including pro-inflammatory cytokines produced by macrophages. Occasionally, if the inflammatory response is not resolved, the acute inflammatory process can evolve into a chronic inflammation. Natural compounds from vegetables are considered as an important source of active agents with potential to treat or prevent inflammatory related pathologies and could be used as an alternative of the therapeutic agents currently in use, such as non-steroidal anti-inflammatory drugs (NSAIDs), which present several side effects. METHODS: In this research work we evaluated in vitro the anti-inflammatory activity of a series of ten phytochemicals present in Brassica, measured as the potential of those compounds to reduce the production of key pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) by a human macrophage-like cell model of HL-60 cells RESULTS: Most of the tested phytochemicals (including the most representative bioactive molecules of the major classes of compounds present in cruciferous foods such as glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols and anthocyanins) demonstrated significant anti-inflammatory activity at micromolar level in the absence of cytotoxic effects in this human macrophage-like cell model. CONCLUSION: These data confirm that phytochemicals commonly obtained from Brassica may be potential therapeutic leads to treat or prevent human chronic inflammation and related diseases.