Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain

We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3\u2019-5\u2019 exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring \u394G and C M equal to (15.7 \ub1 1.9) kJ/mol and (3.5 \ub1 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs

Preliminary engineering assessment of alternative magnetic divertor configurations for EU-DEMO

One of the main challenges in the roadmap to the realization of fusion energy is to develop a heat and power exhaust system able to withstand the large loads expected in the divertor of a fusion power plant. The challenge of reducing the heat load on the divertor targets is addressed, within the mission 2 \u2018Heat-exhaust systems\u2019, through the investigation of divertor configurations alternative to the standard Single Null (SN), such as the Snowflake (SF), Double Null (DN), X and Super-X (SX) divertors. This paper focuses on a preliminary engineering assessment of the alternative configurations proposed for the EU DEMO reactor. Starting from the description of the optimized plasma shape developed for each configuration, the 3D geometrical description of the Magnet System and of the main Mechanical Structures (Vacuum Vessel and in-vessel components) is presented. Based on the 3D geometry, the compatibility of the location and dimensions of ports with Remote Maintenance needs is discussed and possible design optimizations are proposed both for the Magnets system and the mechanical structures design. Finally, the various configurations are compared with regard to the engineering and feasibility aspects

Chemical composition, in vitro bioaccessibility and antioxidant activity of polyphenolic compounds from nutraceutical fennel waste extract

Fennel (Foeniculum vulgare Mill.) waste contains a broad range of bioactive molecules, including polyphenols, which have poor bioaccessibility during gastrointestinal digestion. This work aimed to investigate the bioaccessibility of total phenolic compounds and the antioxidant capacity during simulated gastrointestinal digestion using two nutraceutical formulations based on non-acid-resistant (NAR) and acid-resistant (AR) capsules containing aqueous-based extracts from fennel waste. Moreover, to obtain a comprehensive investigation of the polyphenolic constituents of the fennel waste extract, a high-resolution mass spectrometry (Q-Orbitrap) analysis was performed. Notably, chlorogenic acids, such as 4-caffeoylquinic acid and 3,4-dicaffeoylquinic acid, were the most detected compounds found in assayed samples (1.949 and 0.490 mg/g, respectively). After in vitro gastrointestinal digestion, the extract contained in AR capsules displayed higher bioaccessibility in both the duodenal and colonic stages (1.96 and 5.19 mg GAE/g, respectively) than NAR capsules (1.72 and 3.50 mg GAE/g, respectively), suggesting that the acidic gastric conditions negatively affected the polyphenol compounds released from the NAR capsules. Therefore, the aqueous extract of fennel waste could be proposed as an innovative and easily available source of dietary polyphenols. Furthermore, the use of an AR capsule could improve the polyphenol bioaccessibility and can be proposed as a nutraceutical formulation

Ageritin—The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine

In vitro bioaccessibility and antioxidant activity of polyphenolic compounds from spent coffee grounds-enriched cookies

Spent coffee ground (SCG) is a significant by-product generated by the coffee industry. It is considered a great source of bioactive molecules well-recognized for exerting biological properties. This study aimed to implement SCG in a baked foods, such as cookies (SCGc), to increase their bioactive potential. A comprehensive study of the polyphenolic fraction of the SCG and SCGc using a high-resolution mass spectrometry analysis was performed. Moreover, the polyphenol bioaccessibility and change in antioxidant activity during simulated gastrointestinal digestion (GiD) were assessed. Data showed that SCGc provided 780 mg of melanoidins, 16.2 mg of chlorogenic acid (CGA), 6.5 mg of caffeine, and 0.08 mg of phenolic acids per 100 g of sample. Moreover, the 5-caffeoylquinic acid was the most relevant CGA found in SCG (116.4 mg/100 g) and SCGc (8.2 mg/100 g) samples. The antioxidant activity evaluated through three spectrophotometric tests, and the total phenolic compounds of SCGc samples exhibited significantly higher values than the control samples. Furthermore, during simulated GiD, the highest bioaccessibility of SCGc polyphenols was observed after the colonic stage, suggesting their potential advantages for human health. Therefore, SCG with high content in bioactive molecules could represent an innovative ingredient intended to fortify baked food formulations

Antioxidant and anti-inflammatory activity of coffee brew evaluated after simulated gastrointestinal digestion

Coffee contains human health-related molecules, namely polyphenols that possess a wide range of pharmacological functions, and their intake is associated with reduced colon cancer risk. This study aimed to assess the changes in the anti-inflammatory and antioxidant activity of coffee after simulated gastrointestinal digestion. The evaluation of intracellular reactive oxygen species (ROS) levels in the HT-29 human colon cancer cell line and three in vitro spectrophotometric assays were performed to determine the antioxidant activity of the samples. Characterization of coffee composition was also assessed through a Q-Orbitrap high-resolution mass spectrometry analysis. The results highlighted that the levels of polyphenols in the digested coffee brews were higher than those of the non-digested ones. All assayed samples decreased the levels of intracellular ROS when compared to untreated cells, while digested coffee samples exhibited higher antioxidant capacity and total phenolic content than not-digested coffee samples. Digested coffee samples showed a higher reduction in interleukin-6 levels than the not-digested samples in lipopolysaccharide-stimulated HT-29 cells treated for 48 h and fewer cytotoxic effects in the MTT assay. Overall, our findings suggest that coffee may exert antioxidant and anti-inflammatory properties, and the digestion process may be able to release compounds with higher bioactivity

Cytotoxicity effect of quinoin, type 1 ribosome-inactivating protein from quinoa seeds, on glioblastoma cells

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblas-toma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients’ gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), cur-rently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells’ growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs

Tenascin Expression in Human Placentas during FGR Affected Pregnancies and Umbilical Doppler Velocimetry Correlation

Objective: The aim of this study was to evaluate the expression of some non collagenous extracellular matrix proteins, in particular tenascin, in human placentas of intrauterine growth restricted fetuses with abnormal umbilical Doppler velocimetry. Study Design: Study group (group A) consisted of 23 pregnant women with intrauterine growth restricted fetuses, with or without preeclampsia. Control group (group B) consisted of 10 pregnant women with appropriate fetal weight for gestational age. Placental specimens were collected from biopsies obtained after cesarean delivery. Umbilical artery Doppler velocimetry was performed within four hours from delivery in all patients. Tenascin expression was studied by immunohistochemistry and western blot techniques. Results: A difference in birth weight and placental weight was found in the two groups, being lower in the study group. Umbilical artery Doppler velocimetry showed abnormal patterns in the study group and normal findings in the control one. Tenascin was strongly expressed in placentas from growth restricted fetuses, as shown by immunohistochemistry and by RT-PCR, while it was almost absent in placentas from group B. Conclusion: A relationship between abnormal Doppler patterns and tenascin distribution in growth restricted fetuses has been observed. The presence of tenascin might be considered as a placental compensatory mechanism in FGR fetuses with abnormal umbilical artery Doppler velocimetry

An Italian survey on dietary habits and changes during the COVID-19 lockdown

The World Health Organization has declared the coronavirus outbreak a Public Health Emergency of International Concern; the outbreak has led to lockdowns in several parts of the world, and sudden changes in people’s lifestyles. This study explores the impact of the first coronavirus disease 2019 (COVID-19) pandemic period on dietary habits, lifestyle changes, and adherence to the Mediterranean diet among the Italian population, through an online questionnaire, conducted from April to May 2020, involving 1519 participants. The 14-point Mediterranean Diet Adherence Screener (MEDAS) highlighted a medium Mediterranean diet adherence in 73.5% of responders, which principally included the younger population, aged 18–30 years (p < 0.05). In regards to changes in eating habits, 33.5% of responders declared an influence of the pandemic period on nutritional practice. A decrease in alcohol consumption was reported by 81% of responders, while an increase in frozen food consumption was reported by 81.3% of responders. In addition, 58.8% reported positive weight modification (40.8%, +1–3 kg); physical activity reduction was reported for 70.5% of responders. Our study contributes toward amplifying the investigation on the dietary habits and changes of the Italian population during the COVID-19 lockdown, although the pandemic is ongoing. Similar studies should be performed around the world to understand how the emergency has impacted people’s habits