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The Mediterranean diet is mostly based on high intakes of olive oil, vegetables, legumes, and fruits, with limited amounts of red meat and sweets, and is related to lower risk of cardiovascular diseases, mainly due to the health benefits of antioxidants of wild greens, fruits, and vegetables. Petromarula pinnata (L.) A. DC. is a unique (monotypic) and threatened local endemic wild edible green of Crete which is consumed raw in salads or cooked as an ingredient of the Mediterranean (Cretan) diet. In this study, we aimed to examine the nutritional value of P. pinnata with reference to wild-growing material; moreover, we investigated its chemical composition with 1H NMR spectra and its in vitro total phenolics and flavonoids (TPC, TF assays), and we evaluated the antioxidant (TAC, DPPH, and inhibition of lipid peroxidation), antimicrobial (MIC), and anti-inflammatory (inhibition of soybean lipoxygenase) in vitro activity during two different developmental stages (winter and summer collections), all referring to ex situ cultivated material (air-dried or frozen in liquid nitrogen). Our results showed that P. pinnata has good nutritional value, being rich in terpenoids and poor in phenolic compounds and flavonoids. Furthermore, the extracts showed high antioxidant activity for TAC and DPPH and some of the extracts had higher antioxidant activities than the standard compounds. The summer plant materials had higher antioxidant activity than the winter ones. The methanol/water extracts were the strongest inhibitors of the lipid peroxidation, and the methanol extracts were not found to be active. None of the extracts inhibited the soybean lipoxygenase, and thus they were devoid of anti-inflammatory activity. Finally, the extracts showed a moderate to strong bacterial inhibition. These findings consolidate that P. pinnata has a novel nutraceutical interest with high nutritional value and high antioxidant activity combined with bactericidal effect, thus updating the evaluation of its exploitation potential in the medicinal sector from below average (37%) to high (67%).

Heme (Fe2+-protoporphyrin IX) is a pigment of life, and as a prosthetic group in several hemoproteins, it contributes to diverse critical cellular processes. While its intracellular levels are tightly regulated by networks of heme-binding proteins (HeBPs), labile heme can be hazardous through oxidative processes. In blood plasma, heme is scavenged by hemopexin (HPX), albumin and several other proteins, while it also interacts directly with complement components C1q, C3 and factor I. These direct interactions block the classical pathway (CP) and distort the alternative pathway (AP). Errors or flaws in heme metabolism, causing uncontrolled intracellular oxidative stress, can lead to several severe hematological disorders. Direct interactions of extracellular heme with alternative pathway complement components (APCCs) may be implicated molecularly in diverse conditions at sites of abnormal cell damage and vascular injury. In such disorders, a deregulated AP could be associated with the heme-mediated disruption of the physiological heparan sulphate–CFH coat of stressed cells and the induction of local hemostatic responses. Within this conceptual frame, a computational evaluation of HBMs (heme-binding motifs) aimed to determine how heme interacts with APCCs and whether these interactions are affected by genetic variation within putative HBMs. Combined computational analysis and database mining identified putative HBMs in all of the 16 APCCs examined, with 10 exhibiting disease-associated genetic (SNPs) and/or epigenetic variation (PTMs). Overall, this article indicates that among the pleiotropic roles of heme reviewed, the interactions of heme with APCCs could induce differential AP-mediated hemostasis-driven pathologies in certain individuals.

The present work is the first report on the ingredients of the P. × commixta hybrid, a plant of the genus Phlomis. So far, thirty substances have been isolated by various chromatographic techniques and identified by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS. The compounds are classified as flavonoids: naringenin, eriodyctiol, eriodyctiol-7-O-β-D-glucoside, luteolin, luteolin-7-O-β-D-glucoside, apigenin, apigenin-7-O-β-D-glucoside, diosmetin-7-O-β-D-glucoside, quercetin, hesperetin and quercetin-3-O-β-D-glucoside; phenylpropanoids: martynoside, verbascoside, forsythoside B, echinacoside and allysonoside; chromene: 5,7-dihydroxychromone; phenolic acids: caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, chlorogenic acid methyl ester, gallic acid, p-coumaric acid and vanillic acid; aliphatic hydrocarbon: docos-1-ene; steroids: brassicasterol and stigmasterol; a glucoside of allylic alcohol, 3-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-oct-1-ene-3-ol, was fully characterized as a natural product for the first time. Two tyrosol esters were also isolated: tyrosol lignocerate and tyrosol methyl ether palmitate, the latter one being isolated as a natural product for the first time. Moreover, the biological activities of the extracts from the different polarities of the roots, leaves and flowers were estimated for their cytotoxic potency. All root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines.

Ganoderma is a genus of wood-degrading mushrooms with medicinal importance. Most Ganoderma species have been studied extensively for their secondary metabolites, biological activities, and ecological value. In this study, the biological activities of the extracts of G. adspersum growing wild on Morus alba trees in the region of Western Thrace (Greece) were evaluated, and the petroleum ether, dichloromethanolic, and methanolic extracts were studied further for their secondary metabolites. Six substances were isolated by chromatographic (Clumn Chromatography (C.C.), High Performance Liquid Chromatography (HPLC)) and spectroscopic methods (Nuclear Magnetic Resonance (NMR)), which were classified in the following categories: (a) unsaturated fatty acids: cis-oleic acid (1); (b) sterols: ergosta-7,22-dien-3-one (2), ergosta-7,22-dien-3-ol (3), and ergosta-5,7,22-trien-3-ol (4); and (c) lanostane-type triterpenoids: applanoxidic acid G (5) and applanoxidic acid A (6). Finally, the biological activities of the extracts were estimated for their antioxidant, antimicrobial, and cytotoxic potential. The methanolic extract of G. adspersum showed the highest total antioxidant activity. The results of the antimicrobial activities indicated that all of the extracts had a minimum inhibitory concentration (MIC) ranging between 39.1 and 312.5 μg/mL. The evaluation of the cytotoxic activity of the samples showed once again that the methanolic extract was the most potent among the examined extracts, with half-maximal inhibitory concentration (IC50) values of 19.22 μg/mL (Hep2c cells), 32.9 μg/mL (RD cells), and 8.94 μg/mL (L2OB cells). Moreover, the bioactivity scores of the isolated secondary metabolites were calculated using the online computer software program Molinspiration. The compounds showed promising bioactivity scores for drug targets.

Glutathione (GSH) is a ubiquitous tripeptide that is biosynthesized in situ at high concentrations (1–5 mM) and involved in the regulation of cellular homeostasis via multiple mechanisms. The main known action of GSH is its antioxidant capacity, which aids in maintaining the redox cycle of cells. To this end, GSH peroxidases contribute to the scavenging of various forms of ROS and RNS. A generally underestimated mechanism of action of GSH is its direct nucleophilic interaction with electrophilic compounds yielding thioether GSH S-conjugates. Many compounds, including xenobiotics (such as NAPQI, simvastatin, cisplatin, and barbital) and intrinsic compounds (such as menadione, leukotrienes, prostaglandins, and dopamine), form covalent adducts with GSH leading mainly to their detoxification. In the present article, we wish to present the key role and significance of GSH in cellular redox biology. This includes an update on the formation of GSH-S conjugates or GSH adducts with emphasis given to the mechanism of reaction, the dependence on GST (GSH S-transferase), where this conjugation occurs in tissues, and its significance. The uncovering of the GSH adducts’ formation enhances our knowledge of the human metabolome. GSH–hematin adducts were recently shown to have been formed spontaneously in multiples isomers at hemolysates, leading to structural destabilization of the endogenous toxin, hematin (free heme), which is derived from the released hemoglobin. Moreover, hemin (the form of oxidized heme) has been found to act through the Kelch-like ECH associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor-2 (Nrf2) signaling pathway as an epigenetic modulator of GSH metabolism. Last but not least, the implications of the genetic defects in GSH metabolism, recorded in hemolytic syndromes, cancer and other pathologies, are presented and discussed under the framework of conceptualizing that GSH S-conjugates could be regarded as signatures of the cellular metabolism in the diseased state.

Complement dysregulation has been documented in adults with COVID-19 and implicated in relevant pediatric inflammatory responses against SARS-CoV-2. We propose that signatures of complement missense coding SNPs associated with dysregulation could also be identified in children with multisystem inflammatory syndrome (MIS-C). We investigated 71 pediatric patients with RT-PCR validated SARS-CoV-2 hospitalized in pediatric COVID-19 care units (November 2020–March 2021) in three major groups. Seven (7) patients suffered from MIS-C (MIS-C group), 32 suffered from COVID-19 and were hospitalized (admitted group), whereas 32 suffered from COVID-19, but were sent home. All patients survived and were genotyped for variations in the C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MASP1, MASP2, MBL2, COLEC11, FCN1, and FCN3 genes. Upon evaluation of the missense coding SNP distribution patterns along the three study groups, we noticed similarities, but also considerably increased frequencies of the alternative pathway (AP) associated with SNPs rs12614 CFB, rs1061170, and rs1065489 CFH in the MIS-C patients. Our analysis suggests that the corresponding substitutions potentially reduce the C3b-inactivation efficiency and promote slower and weaker AP C3bBb pre-convertase assembly on virions. Under these circumstances, the complement AP opsonization capacity may be impaired, leading to compromised immune clearance and systemic inflammation in the MIS-C syndrome.

The Mediterranean diet is mostly based on high intakes of olive oil, vegetables, legumes, and fruits, with limited amounts of red meat and sweets, and is related to lower risk of cardiovascular diseases, mainly due to the health benefits of antioxidants of wild greens, fruits, and vegetables. Petromarula pinnata (L.) A. DC. is a unique (monotypic) and threatened local endemic wild edible green of Crete which is consumed raw in salads or cooked as an ingredient of the Mediterranean (Cretan) diet. In this study, we aimed to examine the nutritional value of P. pinnata with reference to wild-growing material; moreover, we investigated its chemical composition with 1H NMR spectra and its in vitro total phenolics and flavonoids (TPC, TF assays), and we evaluated the antioxidant (TAC, DPPH, and inhibition of lipid peroxidation), antimicrobial (MIC), and anti-inflammatory (inhibition of soybean lipoxygenase) in vitro activity during two different developmental stages (winter and summer collections), all referring to ex situ cultivated material (air-dried or frozen in liquid nitrogen). Our results showed that P. pinnata has good nutritional value, being rich in terpenoids and poor in phenolic compounds and flavonoids. Furthermore, the extracts showed high antioxidant activity for TAC and DPPH and some of the extracts had higher antioxidant activities than the standard compounds. The summer plant materials had higher antioxidant activity than the winter ones. The methanol/water extracts were the strongest inhibitors of the lipid peroxidation, and the methanol extracts were not found to be active. None of the extracts inhibited the soybean lipoxygenase, and thus they were devoid of anti-inflammatory activity. Finally, the extracts showed a moderate to strong bacterial inhibition. These findings consolidate that P. pinnata has a novel nutraceutical interest with high nutritional value and high antioxidant activity combined with bactericidal effect, thus updating the evaluation of its exploitation potential in the medicinal sector from below average (37%) to high (67%). [ABSTRACT FROM AUTHOR]

The potential clinical applications of the powerful in vitro-transcribed (IVT)-mRNAs, to restore defective protein functions, strongly depend on their successful intracellular delivery and transient translation through the development of safe and efficient delivery platforms. In this study, an innovative (international patent-pending) methodology was developed, combining the IVT-mRNAs with the protein transduction domain (PTD) technology, as an efficient delivery platform. Based on the PTD technology, which enables the intracellular delivery of various cargoes intracellularly, successful conjugation of a PTD to the IVT-mRNAs was achieved and evaluated by band-shift assay and NMR spectroscopy. In addition, the PTD-IVT-mRNAs were applied and evaluated in two protein-disease models, including the mitochondrial disorder fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency (attributed to SCO2 gene mutations) and β-thalassemia. The PTD-IVT-mRNA of SCO2 was successfully transduced and translated to the corresponding Sco2 protein inside the primary fibroblasts of a SCO2/COX-deficient patient, whereas the PTD-IVT-mRNA of β-globin was transduced and translated in bone marrow cells, derived from three β-thalassemic patients. The transducibility and the structural stability of the PDT-IVT-mRNAs, in both cases, were confirmed at the RNA and protein levels. We propose that our novel delivery platform could be clinically applicable as a protein therapy for metabolic/genetic disorders.

This study aims to approximate the optimum sulfate content of cement, applying maximization of compressive strength as a criterion for cement produced in industrial mills. The design includes tests on four types of cement containing up to three main components and belonging to three strength classes. We developed relationships correlating to 7- and 28-day strength with the sulfate and clinker content of the cement (CL), as well as the clinker mineral composition (tricalcium silicate, C3S, tricalcium aluminate, C3A). We correlated strength with the ratio %SO3/CL and the molecular ratios MSO3/C3S and MSO3/C3A. The data processing stage proved that artificial neural networks (ANNs) fit the results’ distribution better than a parabolic function, providing reliable models. The optimal %SO3/CL value for 7- and 28-day strength was 2.85 and 3.00, respectively. Concerning the ratios of SO3 at the mineral phases for 28-day strength, the best values were MSO3/C3S = 0.132–0.135 and MSO3/C3A = 1.55. We implemented some of the ANNs to gain a wide interval of input variables’ values. Thus, the approximations of SO3 optimum using ANNs had a relatively broad application in daily plant quality control, at least as a guide for experimental design. Finally, we investigated the impact of SO3 uncertainty on the 28-day strength variance using the error propagation method.

This study aims at the isolation and structural determination of the secondary metabolites of the herbaceous perennial plant Achillea grandifolia Friv. (Asteraceae). The examination of the non-volatile content of the leaves and flowers of A. grandifolia afforded the isolation of sixteen secondary metabolites. On the basis of NMR spectra, the identified compounds included ten sesquiterpene lactones; three guaianolides—rupicolin A (1), rupicolin B (2), and (4S,6aS,9R,9aS,9bS)-4,6a,9-trihydroxy-9-methyl-3,6-dimethylene-3a,4,5,6,6a,9,9a,9b-octahydro-3H-azuleno [4,5-b]furan-2-one (3); two eudesmanolides—artecalin (4) and ridentin B (5); two sesquiterpene methyl esters—(1S,2S,4αR,5R,8R,8αS)-decahydro-1,5,8-trihydroxy-4α,8-dimethyl–methylene-2-naphthaleneacetic acid methylester (6) and 1β, 3β, 6α-trihydroxycostic acid methyl ester (7); three secoguaianolides—acrifolide (8), arteludovicinolide A (9), and lingustolide A (10); and an iridoid—loliolide (11). Moreover, five known flavonoids, i.e., apigenin, luteolin, eupatolitin, apigenin 7-O-glucoside, and luteolin 7-O-glucoside (12-16) were also purified from the aerial parts of the plant material. We also investigated the effect of rupicolin A (1) and B (2) (main compounds) on U87MG and T98G glioblastoma cell lines. An MTT assay was performed to define cytotoxic effects and to calculate the IC50, while flow cytometry was employed to analyze the cell cycle. The IC50 values of reduced viability during the 48 h treatment for compound (1) and (2) were 38 μM and 64 μM for the U87MG cells and 15 μM and 26 μM for the T98G cells, respectively. Both rupicolin A and B induced a G2/M cell cycle arrest.

Glioblastoma is one of the most malignant and lethal forms of primary brain tumors in adults. Linearol, a kaurane diterpene isolated from different medicinal plants, including those of the genus Sideritis, has been found to possess significant anti-oxidant, anti-inflammatory and anti-microbial properties. In this study, we aimed to determine whether linearol could exhibit anti-glioma effects when given alone or in combination with radiotherapy in two human glioma cell lines, U87 and T98. Cell viability was examined with the Trypan Blue Exclusion assay, cell cycle distribution was tested with flow cytometry, and the synergistic effects of the combination treatment were analyzed with CompuSyn software. Linearol significantly suppressed cell proliferation and blocked cell cycle at the S phase. Furthermore, pretreatment of T98 cells with increasing linearol concentrations before exposure to 2 Gy irradiation decreased cell viability to a higher extent than linearol or radiation treatment alone, whereas in the U87 cells, an antagonistic relationship was observed between radiation and linearol. Moreover, linearol inhibited cell migration in both tested cell lines. Our results demonstrate for the first time that linearol is a promising anti-glioma agent and further studies are needed to fully understand the underlying mechanism of this effect.

Various species of the genus Achillea L. (Asteraceae) are traditionally used worldwide for wound healing against diarrhea, flatulence, and abdominal pains, as diuretic and emmenagogue agents. In the present study, the essential oils (EOs) obtained separately from the leaves and inflorescences of wild-growing Achillea grandifolia Friv. from Mt. Menoikio and Mt. Pelion (Greece) were analyzed by Gas Chromatography–Mass Spectrometry. The major compounds found in EOs of A. grandifolia inflorescences from Mt. Menoikio were as follows: cis-thujone (36.9%), 1,8-cineole (11.9%), camphor (10.0%), ascaridole (7.3%), α-terpinene (6.4%), sabinene (4.1%), trans-thujone (3.6%), and cis-jasmone (3.4%). In leaves from Mt. Menoikio, they were as follows: cis-thujone (50.8%), 1,8-cineole (20.0%), trans-thujone (5.5%), camphor (5.5%), borneol (3.6%), and α-terpineol (3.1%). In inflorescences from Mt. Pelion, they were as follows: camphor (70.5%), camphene (5.9%), cis-jasmone (3.2%), bornyl acetate (3.2%). In leaves from Mt. Pelion, they were as follows: camphor (83.2%), camphene (3.9%), and borneol (3.7%). Subsequently, the samples were first time tested for their antioxidant activities with the interaction of EOs with DPPH (2,2-diphenyl-1-picrylhydrazyl) and their inhibition of lipid peroxidation, as well as for their anti-inflammatory activity through the soybean LOX (lipoxygenase) inhibition. All of the examined samples were found effective. A. grandifolia leaves presented the highest antioxidant potential according to the DPPH method, and the highest percentage of LOX inhibition. The study herein investigated for the first time the leaves and the inflorescences of A. grandifolia separately, and the results generally align with similar studies from neighboring countries (Turkey and Serbia) in terms of the yields and categorization of main EO compounds (oxygenated monoterpenes). However, the findings were not in agreement with previously studied Greek material, as a higher amount of cis-thujone and lower antioxidant activity are reported herein. Both the EOs of inflorescences and the leaves of the wild-growing population collected from Mt. Menoikio were characterized by a high quantity of cis-thujone (36.9% and 50.8%, respectively).

Thymus holosericeus Čelak. (Lamiaceae) is a taxonomically isolated and endangered local endemic thyme species which is geographically isolated in four Ionian Islands (West Greece). The present study investigated the essential oil (EO) composition, the ecological preferences, and their correlations in three T. holosericeus wild-growing populations from Zakynthos (ΤH-Z), Cephalonia (ΤH-C) and Lefkada (ΤH-L). The variations in essential oil yield and the composition of T. holosericeus populations were evaluated using hydrodistillation, GC/MS, TLC and NMR analysis. The climatic conditions of each sample were organized and analyzed in RStudio with the raster package and in SPSS with Pearson’s Canonical Correlation Analysis (CCA), respectively. The aerial parts of the plants varied in EO yields from 1.92 to 2.28% w/v. The analysis of EO constituents revealed noteworthy qualitative and quantitative inter-population variation. The composition of EOs revealed the presence of linalool (82.77%) and borneol (5.95%) as major compounds in ΤH-Z, while carvacrol (35.34%), geraniol (23.98%), linalool (14.37%), borneol (5.66%), thymol (4.27%) and p-cymene (4.08%) were the main compounds in ΤH-C and linalool (40.37%), geraniol (39.42%) and borneol (5.20%) were dominant components in ΤH-L. The results of the gas chromatography procedure have also been confirmed by 1H and 13C-NMR and TLC analysis. The ecological profile showed an average annual precipitation of 942 ± 18.33 mm and the temperature limits in which T. holosericeus seems to adapt to are 6.80± 1.08 °C 27.70 ± 0.70 °C. Regarding the examined samples, TH-C was adapted to the driest summer and coldest winter conditions, TH-Z was adapted to the lowest annual precipitation with the most complex climatic conditions, and TH-L was adapted to the highest summer temperatures with the lowest precipitation in the wettest period of the year. For each sampled population, the CCA identified the association of the samples’ EOs composition with the prevailing local environmental conditions.

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.

Hemin, an oxidized form of heme, acts as potent oxidant to regulate glutathione (GSH) content in pro-erythroid K562 nucleated cells, via activation of the KEAP1/NRF2 defensive signaling pathway. Moreover, GSH, as an essential metabolite, is involved in the regulation of cell-redox homeostasis and proposed to scavenge cytotoxic free heme, which is released from hemoglobin of damaged red blood cells (RBCs) during different hemolytic disorders. In the present study, we aimed to uncover the molecular mechanism by which GSH inhibits hemin-induced cytotoxicity (HIC) by affecting hemin’s structural integrity in K562 cells and in RBC hemolysates. GSH, along with other thiols (cysteine, thioglycolic acid, and mercaptoethanol) altered the spectrum of hemin, while each of them co-added with hemin in cultures of K562 cells prevented HIC and growth arrest and markedly reduced the intracellular level of hemin. In addition, GSH endogenous levels served as a barrier to HIC in K562 cells, as shown by the depletion in GSH. LC-MS/MS analysis of the in vitro reaction between hemin and GSH revealed at least five different isomers of GSH–hemin adducts, as well as hydroxy derivatives as reaction products, which are characterized by unique mass spectra (MS). The latter allowed the detection of adducts in human RBC hemolysates. Based on these findings, we proposed a molecular mechanism via which GSH prevents HIC and structurally disintegrates heme. An analogous reaction was observed in RBC hemolysates via direct inter-reaction between hematin (ferric and hydroxide heme) released from hemoglobin and GSH. Overall, GSH–hematin adducts could be considered as novel entities of the human metabolome of RBCs in hemolytic disorders.

Ganoderma is a genus of wood-degrading mushrooms with medicinal importance. Most Ganoderma species have been studied extensively for their secondary metabolites, biological activities, and ecological value. In this study, the biological activities of the extracts of G. adspersum growing wild on Morus alba trees in the region of Western Thrace (Greece) were evaluated, and the petroleum ether, dichloromethanolic, and methanolic extracts were studied further for their secondary metabolites. Six substances were isolated by chromatographic (Clumn Chromatography (C.C.), High Performance Liquid Chromatography (HPLC)) and spectroscopic methods (Nuclear Magnetic Resonance (NMR)), which were classified in the following categories: (a) unsaturated fatty acids: cis-oleic acid (1); (b) sterols: ergosta-7,22-dien-3-one (2), ergosta-7,22-dien-3-ol (3), and ergosta-5,7,22-trien-3-ol (4); and (c) lanostane-type triterpenoids: applanoxidic acid G (5) and applanoxidic acid A (6). Finally, the biological activities of the extracts were estimated for their antioxidant, antimicrobial, and cytotoxic potential. The methanolic extract of G. adspersum showed the highest total antioxidant activity. The results of the antimicrobial activities indicated that all of the extracts had a minimum inhibitory concentration (MIC) ranging between 39.1 and 312.5 μg/mL. The evaluation of the cytotoxic activity of the samples showed once again that the methanolic extract was the most potent among the examined extracts, with half-maximal inhibitory concentration (IC50) values of 19.22 μg/mL (Hep2c cells), 32.9 μg/mL (RD cells), and 8.94 μg/mL (L2OB cells). Moreover, the bioactivity scores of the isolated secondary metabolites were calculated using the online computer software program Molinspiration. The compounds showed promising bioactivity scores for drug targets. [ABSTRACT FROM AUTHOR]

The rapid progress achieved in the development of many biopharmaceuticals had a tremendous impact on the therapy of many metabolic/genetic disorders. This type of fruitful approach, called protein replacement therapy (PRT), aimed to either replace the deficient or malfunctional protein in human tissues that act either in plasma membrane or via a specific cell surface receptor. However, there are also many metabolic/genetic disorders attributed to either deficient or malfunctional proteins acting intracellularly. The recent developments of Protein Transduction Domain (PTD) technology offer new opportunities by allowing the intracellular delivery of recombinant proteins of a given therapeutic interest into different subcellular sites and organelles, such as mitochondria and other entities. Towards this pathway, we applied successfully PTD Technology as a protein therapeutic approach, in vitro, in SCO2 deficient primary fibroblasts, derived from patient with mutations in human SCO2 gene, responsible for fatal, infantile cardioencephalomyopathy and cytochrome c oxidase deficiency.In this work, we radiolabeled the recombinant TAT-L-Sco2 fusion protein with technetium-99 m to assess its in vivo biodistribution and fate, by increasing the sensitivity of detection of even low levels of the transduced recombinant protein. The biodistribution pattern of [99mTc]Tc-TAT-L-Sco2 in mice demonstrated fast blood clearance, significant hepatobiliary and renal clearance. In addition, western blot analysis detected the recombinant TAT-L-Sco2 protein in the isolated mitochondria of several mouse tissues, including heart, muscle and brain. These results pave the way to further consider this PTD-mediated Protein Therapy Approach as a potentially alternative treatment of genetic/metabolic disorders.

Human mesenchymal stem cells (MSC) are multipotent stem cells, which are isolated from various sources. Currently, there is a worldwide interest for dental MSC to be used against neurodegenerative diseases, since they derive from the neural crest and express embryonic stem cell markers. This fact prompted us to explore their potential for neural trans-differentiation in culture. We employed all-trans-retinoic acid (ATRA) and 2-(3-ethylureido)-6-methylpyridine (UDP-4) to induce neural differentiation of human MSC from the dental apical papilla (SCAP). The SCAP were exposed to either agent separately and assessed for proliferation, viability, morphology, and gene expression of the following neural-specific markers: neuron-specific enolase (ENO2), neurofibromin 1 (NF1), choline acetyltransferase (CHAT), tyrosine hydroxylase (TH), and the vesicular GABA transporter (SLC32A1). They were also assessed for the expression of glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) by immunofluorescence. ATRA or UDP-4 treatment inhibited the cell growth and promoted limited cell death, but to a different extent. The addition of the neuroprotective agent recombinant human erythropoietin-alpha (rhEPO-α) enhanced the UDP-4-inducing capacity for more than three weeks. ATRA or UDP-4 treatment significantly upregulated ENO2 and NF1 expression, indicating neuronal differentiation. Moreover, the ATRA treatment significantly induced the upregulation of the GABAergic-specific SLC32A1, while the UDP-4 treatment led to the significant upregulation of the adrenergic-specific TH. The UDP-4 treatment induced the expression of NeuN and GFAP after four and three weeks, respectively, while the ATRA-treatment did not. Our findings indicate that SCAP can be differentiated into neural-like cells after treatment with ATRA or UDP-4 by exhibiting a disparate pattern of differentiation. Therefore, UDP-4 is suggested here as a new potent neural-differentiation-inducing compound, which, when combined with rhEPO-α, could lay the foundation for robust stem-cell-based therapies of neurodegeneration.

The inflammatory cytokine Stem Cell Factor (SCF, ligand of c-kit receptor) has been implicated as a pro-oncogenic driver and an adverse prognosticator in several human cancers. Increased SCF levels have recently been reported in a small series of patients with chronic lymphocytic leukemia (CLL), however its precise role in CLL pathophysiology remains elusive. In this study, CLL cells were found to predominantly express the membrane isoform of SCF that is known to elicit a more robust activation of the c-kit receptor. SCF was significantly overexpressed in CLL cells compared to healthy tonsillar B cells whilst it correlated with adverse-prognostic biomarkers, shorter time-to-first treatment and shorter overall survival. Activation of immune receptors and long-term cell-cell interactions with the mesenchymal stroma led to an elevation of SCF primarily in adverse-prognostic CLL cases. On the contrary, suppression of oxidative stress and the BTK inhibitor Ibrutinib negated SCF levels. Interestingly, SCF significantly correlated with mitochondrial dynamics and HIF-1α which have previously been linked with clinical aggressiveness in CLL. SCF was able to elicit direct biological effects in CLL cells affecting redox homeostasis and cell proliferation. Overall, the aberrantly expressed SCF in CLL cells emerges as a key response regulator to microenvironmental stimuli whilst correlating with poor prognosis. On these grounds, specific targeting of this inflammatory molecule could serve as a novel therapeutic approach in CLL.

Human erythropoietin (EPO) is an N-linked glycoprotein consisting of 166 aa that is produced in the kidney during the adult life and acts both as a peptide hormone and hematopoietic growth factor (HGF), stimulating bone marrow erythropoiesis. EPO production is activated by hypoxia and is regulated via an oxygen-sensitive feedback loop. EPO acts via its homodimeric erythropoietin receptor (EPO-R) that increases cell survival and drives the terminal erythroid maturation of progenitors BFU-Es and CFU-Es to billions of mature RBCs. This pathway involves the activation of multiple erythroid transcription factors, such as GATA1, FOG1, TAL-1, EKLF and BCL11A, and leads to the overexpression of genes encoding enzymes involved in heme biosynthesis and the production of hemoglobin. The detection of a heterodimeric complex of EPO-R (consisting of one EPO-R chain and the CSF2RB β-chain, CD131) in several tissues (brain, heart, skeletal muscle) explains the EPO pleotropic action as a protection factor for several cells, including the multipotent MSCs as well as cells modulating the innate and adaptive immunity arms. EPO induces the osteogenic and endothelial transdifferentiation of the multipotent MSCs via the activation of EPO-R signaling pathways, leading to bone remodeling, induction of angiogenesis and secretion of a large number of trophic factors (secretome). These diversely unique properties of EPO, taken together with its clinical use to treat anemias associated with chronic renal failure and other blood disorders, make it a valuable biologic agent in regenerative medicine for the treatment/cure of tissue de-regeneration disorders.

The effect of a diet supplemented with a novel cornus extract, enriched with essential oils of oregano and thyme, on the performance of Chios cross-bred dairy sheep was investigated during the summer period. The plant extracts were prepared using a “green” method based on aqueous extraction. A total of 45 lactating ewes were allocated into three equal groups in a randomized block design. The three groups were fed the same feed allowance, roughage based on Lucerne hay and wheat straw and a concentrate based on cereals and oil cakes (the control diet). The diet of two groups was fortified with cornus extract, with or without oregano and thyme essential oils, at a level 0.515 g of plant extract/essential oils per kg of concentrate. Individual milk yield was recorded weekly and feed refusals were recorded on a pen basis daily, during a six-week period of lactation. Milk samples were analyzed for the chemical composition of protein, fat, lactose and solids-not-fat constituents, somatic cell counts and total viable bacteria counts. Moreover, the milk of each group was used for yoghurt and Feta cheese production. The lipid oxidative stability, protein carbonyl content and fatty acid composition of milk, yoghurt and cheese samples were also evaluated. The results showed that the incorporation of novel plant extracts and essential oils increased the milk production per ewe. Dietary supplementation with cornus extracts and essential oils lowered lipid and protein oxidation in milk, yoghurt and cheese samples, compared to the control. However, diet supplementation with herbal extracts did not affect the fatty acid profile in milk, cheese and yoghurt or the serum biochemical parameters. In conclusion, dietary supplementation with cornus in combination with oregano and thyme has the potential to improve feed utilization and the performance of high-yield dairy Chios cross-bred ewes reared under heat stress.

BACKGROUND: Prevention of bone metastases is a major issue for breast cancer patients, as it would improve quality of life in a population where long survival is anticipated. PATIENTS AND METHODS: Early breast cancer patients, who had been treated with anthracycline-based chemotherapy within two randomized trials, were included in the study. We evaluated, by quantitative reverse transcription–polymerase chain reaction, 819 formalin-fixed paraffin-embedded tumor tissue samples for mRNA expression of RANK, OPG, and RANKL, as well as their ratios, for potential prognostic significance for the development of bone metastases and also for disease-free survival (DFS) and overall survival. RESULTS: Median age was 52.7 years, whereas 54.2% of the patients were postmenopausal and 78.3% estrogen receptor/progesterone receptor positive. After a median follow-up of 119.9 months, 226 patients (27.6%) had died and 291 patients (35.5%) had disease progression. Low mRNA expression of RANKL was associated with postmenopausal status and greater number of positive lymph nodes (P = .002 and P

This study aims to approximate the optimum sulfate content of cement, applying maximization of compressive strength as a criterion for cement produced in industrial mills. The design includes tests on four types of cement containing up to three main components and belonging to three strength classes. We developed relationships correlating to 7- and 28-day strength with the sulfate and clinker content of the cement (CL), as well as the clinker mineral composition (tricalcium silicate, C3S, tricalcium aluminate, C3A). We correlated strength with the ratio %SO3/CL and the molecular ratios MSO3/C3S and MSO3/C3A. The data processing stage proved that artificial neural networks (ANNs) fit the results' distribution better than a parabolic function, providing reliable models. The optimal %SO3/CL value for 7- and 28-day strength was 2.85 and 3.00, respectively. Concerning the ratios of SO3 at the mineral phases for 28-day strength, the best values were MSO3/C3S = 0.132–0.135 and MSO3/C3A = 1.55. We implemented some of the ANNs to gain a wide interval of input variables' values. Thus, the approximations of SO3 optimum using ANNs had a relatively broad application in daily plant quality control, at least as a guide for experimental design. Finally, we investigated the impact of SO3 uncertainty on the 28-day strength variance using the error propagation method. [ABSTRACT FROM AUTHOR]