Neurovascular dysfunction is a central process in the pathogenesis of the stroke and most neurode... more Neurovascular dysfunction is a central process in the pathogenesis of the stroke and most neurodegenerative diseases, including Alzheimer's disease. The multi-cell neurovascular unit (NVU) combines the components of the neural, vascular and extracellular matrix (ECM) into an important interface whose proper function is critical to maintaining brain health. Tissue engineering now offers new tools and information to promote understanding of NVU's operation. A promising area for the development of NVU models is their bio-production through 3D bio-printing to produce a multi-layered NVU in which the contribution of the different cell types to neurovascular function and dysfunction can be studied at molecular and cellular levels. Nerve and vascular cells are encapsulated in a construct suitable for their viability and growth. This construct, called "bioink", is a pre-gelled biomaterial, usually with encapsulated cells, which can be bio-printed and gelled to successfully form a solid construct. Bio-printing allows accurate placement of the neural and vascular cells to form appropriate interactions mimicking the in vivo state. Individual NVU cell types interact with the other cellular components of NVU through biochemical and physical markers, with direct and indirect interactions between neural and vascular components. The cell line sources, either derived from AD patients or healthy individuals, can be developed with the IPSCs technology. IPSCs can be obtained by different somatic cells via reprogramming strategies and further on differentiated into various cell lines that can be used to model disease, to discover new drugs and to treat cell replacement. Last but not least, the availability of 3D NVU models can also facilitate screening of drugs to correct neural dysfunction due to stroke, Alzheimer's disease and other dementia.
During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate T... more During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl – PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) – PHAs. The optimum pH range was 7.5–9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.
Abstract The constantly changing bacterial resistance to antibiotics is a well-known problem and ... more Abstract The constantly changing bacterial resistance to antibiotics is a well-known problem and the scientific community is seeking to exploit new approaches through nanotechnology. The potential of copper as an antimicrobial agent has long been recognized and thus metallic copper as well as cupric oxide (CuO) or cuprous oxide (Cu2O) nanoparticles, called copper-based nanoparticles (Cu-based NPs), have attracted the attention of biomedical applications. Indeed, from a number of studies it is illustrated that Cu-based NPs display particular characteristics that can be properly handled to offer an attractive alternative, since they have higher retention times and can penetrate the microbial cells. In this chapter we focus on the antibacterial activity of CuO, Cu2O NPs, and copper composites Cu/Cu2O covered by polymers or embedded into matrices, since the composition of the NPs results in different mechanisms of action. The size- and shape-dependent effects are highlighted as well as the synthetic conditions that have been applied for the preparation of the NPs. Special attention is given to the antifungal activity of Cu-based NPs, since fungal cells are among the eukaryotic cells that are most similar to animal cells.
Objective: Although Mediterranean diet is connected with longevity and lower rate of many disorde... more Objective: Although Mediterranean diet is connected with longevity and lower rate of many disorders including Alzheimer's disease (AD), the effect of olive oil, which is the principal component of the Mediterranean diet, on fibrinolytic system related to AD and especially on plasminogen activator inhibitor-1 (PAI-1) and a2-antiplasmin in aged participants are not yet examined. This study was performed on 108 aged participants allocated into 5 groups: Mild Cognitive Impairment (MCI) (36) patients subjected to 1-year therapy with extra virgin olive oil (EVOO), MCI without therapy patients (26), MCI without therapy 1-year later patients (11), AD patients (30) and healthy individuals (16). Hypothesis/Purpose: To examine the effect of EVOO therapy on the fibrinolytic factors PAI-1 and a2-antiplasmin, on hallmarks of AD, tau and Aβ amyloid fragments and on an oxidative stress biomarker, MDA in the serum of MCI patients aiming to be exploited as a future preventive therapy. Results: Using ELISA method, the levels of both fibrinolytic factors PAI-1 and a2- antiplasmin in the serum of MCI patients were reduced notably in the EVOO treated patients versus the control group and were lower than those of all other groups. For better determination of AD from other pathological conditions the ratio Aβ1-42/Aβ1-40 was measured in serum of all participants. The more lessened the ratio is, the more cognitive impairment is observed in patients. The MCI group with one-year EVOO therapy displayed a ratio similar to this of healthy individuals. Moreover, patients with EVOO therapy showed decreased tau protein levels in comparison with all the other groups. The levels of the oxidative stress's biomarker, malondialdehyde (MDA) showed a significant decrease in MCI patients subjected to EVOO therapy revealing the involvement of the beneficial antioxidative properties of EVOO in the progression of AD. Conclusion: We demonstrated that EVOO therapy may prevent the risk of patients with MCI to progress to AD via decreasing fibrinolytic factors PAI-1 and a2 antiplasmin that reflecting in the diminution of the hallmarks proteins of AD, tau and Aβ amyloid as well and in a biomarker of oxidative stress, MDA.
As an antioxidant, Vit C is involved in a number of biological processes such as cancer resistanc... more As an antioxidant, Vit C is involved in a number of biological processes such as cancer resistance, reduction of DNA damage and chromosomal breakage caused by several carcinogens and other agents (Odin, 1997; Konopacka et al., 2002; Dusinska et al., 2003). However, in experimental studies (Odin, 1997), administration of large amounts of Vit C had a genotoxic effect. More specifically, Vit C has been reported to induce sister chromatid exchanges (SCEs) in CHO cells and human lymphocytes (Weitberg & Weitzman, 1985; Lialiaris et al., 1987), chromosomal aberrations, unscheduled DNA synthesis, DNA fragmentation (Rosin et al., 1980) and site-specific DNA cleavage (Kazakov et al., 1988; Wang & Van Ness, 1989). Other studies have also reported that mitomycin C, doxorubicin, cisplatin and hyperoxia, induced chromosomal aberrations and furthermore micronuclei pretreated with certain Vit C concentrations exhibited either protection or induction of chromosomal damage (Gille et al., 1991; Greggi...
Copper based nanoparticles (Cu-based NPs) of different compositions and sizes have been hydrother... more Copper based nanoparticles (Cu-based NPs) of different compositions and sizes have been hydrothermally synthesized by varying the reaction time in the presence of the biocompatible surfactants polyoxyethylene (20) sorbitan laurate (Tween 20) and polyethylene glycol 8000 (PEG 8000). Effective control of the above synthetic parameters gave rise to Cu, Cu2O and Cu/Cu2O NPs of 10-44 nm. The antibacterial activity of the NPs was screened against Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) and Gram-negative (Xanthomonas campestris, Escherichia coli) bacteria. The Cu-based NPs induce pDNA degradation in a dose-dependent manner as well as extensive ds CT-DNA degradation. Cu2O NPs of 16 nm and 12 nm exhibit the lowest IC50 values (2.13 μg/mL and 3.7 μg/mL) against B. cereus and B. subtilis, respectively. The agarose gel electrophoresis of ds CT-DNA treated with Cu2O NPs demonstrated degradation at high concentration. In lower concentrations, viscosity measurements indicated groove binding. In regard to the enhanced antibacterial effect and specificity of Cu2O NPs against the Gram-positive strains, the activity pathway was further explored and ROS production and lipid peroxidation verified. The released copper ions 5.15 mg/L in distilled water and 16.32 mg/L in nutrient medium, found below the critical value to inhibit bacterial growth and thus nanosized composition effect is predominant.
Oxidative/nitrative stress that results from the unbalance of the overproduction/clearance of rea... more Oxidative/nitrative stress that results from the unbalance of the overproduction/clearance of reactive oxygen/nitrogen species (ROS/NOS), originated from a variety of endo- and/or exo-genous sources, can have detrimental effects on DNA and is involved in Alzheimer's disease (AD) pathology. An excellent marker of oxidative DNA lesions is 8-hydroxy-2'-deoxyguanosine (8-OHdG) while of nitrative stress the enzyme NOS2 (Nitric oxide synthase 2). Under massive oxidative stress, poly(ADP-ribose)polymerase 1 (PARP-1) enzyme activity, responsible for restoration of DNA damage, is augmented, DNA repair enzymes are recruited, and cell survival/or death is ensued through PARP-1 activation, which is correlated positively with neurodegenerative diseases. In this biochemical study the levels of PARP-1, 8-oxo-dG, and NOS2, Aβ1-42, and p-tau in their sera determined using Enzyme-Linked Immunosorbent Assay (ELISA). Patients diagnosed with Mild Cognitive Impairment participated in MICOIL clinical trial, were daily administered with 50 ml Extra Virgin Olive Oil (EVOO) for one year. All MCI patients' biomarkers that had consumed EVOO were tantamount to those of healthy participants, contrary to MCI patients who were not administered. EVOO administration in MCI patients resulted in the restoration of DNA damage and of the well-established "hallmarks" AD biomarkers, thanks probably to its antioxidant properties exhibiting a therapeutic potentiality against AD. Molecular docking simulations of the EVOO constituents on the crystal structure of PARP-1 and NOS-2 target enzymes were also employed, to study in silico the ability of the compounds to bind to these enzymes and explain the observed in vitro activity. In silico analysis has proved the binding of EVOO constituents on PARP-1and NOS-2 enzymes and their interaction with crucial amino acids of the active sites. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT03362996. MICOIL GOV IDENTIFIER: NCT03362996.
Neurovascular dysfunction is a central process in the pathogenesis of the stroke and most neurode... more Neurovascular dysfunction is a central process in the pathogenesis of the stroke and most neurodegenerative diseases, including Alzheimer's disease. The multi-cell neurovascular unit (NVU) combines the components of the neural, vascular and extracellular matrix (ECM) into an important interface whose proper function is critical to maintaining brain health. Tissue engineering now offers new tools and information to promote understanding of NVU's operation. A promising area for the development of NVU models is their bio-production through 3D bio-printing to produce a multi-layered NVU in which the contribution of the different cell types to neurovascular function and dysfunction can be studied at molecular and cellular levels. Nerve and vascular cells are encapsulated in a construct suitable for their viability and growth. This construct, called "bioink", is a pre-gelled biomaterial, usually with encapsulated cells, which can be bio-printed and gelled to successfully form a solid construct. Bio-printing allows accurate placement of the neural and vascular cells to form appropriate interactions mimicking the in vivo state. Individual NVU cell types interact with the other cellular components of NVU through biochemical and physical markers, with direct and indirect interactions between neural and vascular components. The cell line sources, either derived from AD patients or healthy individuals, can be developed with the IPSCs technology. IPSCs can be obtained by different somatic cells via reprogramming strategies and further on differentiated into various cell lines that can be used to model disease, to discover new drugs and to treat cell replacement. Last but not least, the availability of 3D NVU models can also facilitate screening of drugs to correct neural dysfunction due to stroke, Alzheimer's disease and other dementia.
During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate T... more During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl – PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) – PHAs. The optimum pH range was 7.5–9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.
Abstract The constantly changing bacterial resistance to antibiotics is a well-known problem and ... more Abstract The constantly changing bacterial resistance to antibiotics is a well-known problem and the scientific community is seeking to exploit new approaches through nanotechnology. The potential of copper as an antimicrobial agent has long been recognized and thus metallic copper as well as cupric oxide (CuO) or cuprous oxide (Cu2O) nanoparticles, called copper-based nanoparticles (Cu-based NPs), have attracted the attention of biomedical applications. Indeed, from a number of studies it is illustrated that Cu-based NPs display particular characteristics that can be properly handled to offer an attractive alternative, since they have higher retention times and can penetrate the microbial cells. In this chapter we focus on the antibacterial activity of CuO, Cu2O NPs, and copper composites Cu/Cu2O covered by polymers or embedded into matrices, since the composition of the NPs results in different mechanisms of action. The size- and shape-dependent effects are highlighted as well as the synthetic conditions that have been applied for the preparation of the NPs. Special attention is given to the antifungal activity of Cu-based NPs, since fungal cells are among the eukaryotic cells that are most similar to animal cells.
Objective: Although Mediterranean diet is connected with longevity and lower rate of many disorde... more Objective: Although Mediterranean diet is connected with longevity and lower rate of many disorders including Alzheimer's disease (AD), the effect of olive oil, which is the principal component of the Mediterranean diet, on fibrinolytic system related to AD and especially on plasminogen activator inhibitor-1 (PAI-1) and a2-antiplasmin in aged participants are not yet examined. This study was performed on 108 aged participants allocated into 5 groups: Mild Cognitive Impairment (MCI) (36) patients subjected to 1-year therapy with extra virgin olive oil (EVOO), MCI without therapy patients (26), MCI without therapy 1-year later patients (11), AD patients (30) and healthy individuals (16). Hypothesis/Purpose: To examine the effect of EVOO therapy on the fibrinolytic factors PAI-1 and a2-antiplasmin, on hallmarks of AD, tau and Aβ amyloid fragments and on an oxidative stress biomarker, MDA in the serum of MCI patients aiming to be exploited as a future preventive therapy. Results: Using ELISA method, the levels of both fibrinolytic factors PAI-1 and a2- antiplasmin in the serum of MCI patients were reduced notably in the EVOO treated patients versus the control group and were lower than those of all other groups. For better determination of AD from other pathological conditions the ratio Aβ1-42/Aβ1-40 was measured in serum of all participants. The more lessened the ratio is, the more cognitive impairment is observed in patients. The MCI group with one-year EVOO therapy displayed a ratio similar to this of healthy individuals. Moreover, patients with EVOO therapy showed decreased tau protein levels in comparison with all the other groups. The levels of the oxidative stress's biomarker, malondialdehyde (MDA) showed a significant decrease in MCI patients subjected to EVOO therapy revealing the involvement of the beneficial antioxidative properties of EVOO in the progression of AD. Conclusion: We demonstrated that EVOO therapy may prevent the risk of patients with MCI to progress to AD via decreasing fibrinolytic factors PAI-1 and a2 antiplasmin that reflecting in the diminution of the hallmarks proteins of AD, tau and Aβ amyloid as well and in a biomarker of oxidative stress, MDA.
As an antioxidant, Vit C is involved in a number of biological processes such as cancer resistanc... more As an antioxidant, Vit C is involved in a number of biological processes such as cancer resistance, reduction of DNA damage and chromosomal breakage caused by several carcinogens and other agents (Odin, 1997; Konopacka et al., 2002; Dusinska et al., 2003). However, in experimental studies (Odin, 1997), administration of large amounts of Vit C had a genotoxic effect. More specifically, Vit C has been reported to induce sister chromatid exchanges (SCEs) in CHO cells and human lymphocytes (Weitberg & Weitzman, 1985; Lialiaris et al., 1987), chromosomal aberrations, unscheduled DNA synthesis, DNA fragmentation (Rosin et al., 1980) and site-specific DNA cleavage (Kazakov et al., 1988; Wang & Van Ness, 1989). Other studies have also reported that mitomycin C, doxorubicin, cisplatin and hyperoxia, induced chromosomal aberrations and furthermore micronuclei pretreated with certain Vit C concentrations exhibited either protection or induction of chromosomal damage (Gille et al., 1991; Greggi...
Copper based nanoparticles (Cu-based NPs) of different compositions and sizes have been hydrother... more Copper based nanoparticles (Cu-based NPs) of different compositions and sizes have been hydrothermally synthesized by varying the reaction time in the presence of the biocompatible surfactants polyoxyethylene (20) sorbitan laurate (Tween 20) and polyethylene glycol 8000 (PEG 8000). Effective control of the above synthetic parameters gave rise to Cu, Cu2O and Cu/Cu2O NPs of 10-44 nm. The antibacterial activity of the NPs was screened against Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) and Gram-negative (Xanthomonas campestris, Escherichia coli) bacteria. The Cu-based NPs induce pDNA degradation in a dose-dependent manner as well as extensive ds CT-DNA degradation. Cu2O NPs of 16 nm and 12 nm exhibit the lowest IC50 values (2.13 μg/mL and 3.7 μg/mL) against B. cereus and B. subtilis, respectively. The agarose gel electrophoresis of ds CT-DNA treated with Cu2O NPs demonstrated degradation at high concentration. In lower concentrations, viscosity measurements indicated groove binding. In regard to the enhanced antibacterial effect and specificity of Cu2O NPs against the Gram-positive strains, the activity pathway was further explored and ROS production and lipid peroxidation verified. The released copper ions 5.15 mg/L in distilled water and 16.32 mg/L in nutrient medium, found below the critical value to inhibit bacterial growth and thus nanosized composition effect is predominant.
Oxidative/nitrative stress that results from the unbalance of the overproduction/clearance of rea... more Oxidative/nitrative stress that results from the unbalance of the overproduction/clearance of reactive oxygen/nitrogen species (ROS/NOS), originated from a variety of endo- and/or exo-genous sources, can have detrimental effects on DNA and is involved in Alzheimer's disease (AD) pathology. An excellent marker of oxidative DNA lesions is 8-hydroxy-2'-deoxyguanosine (8-OHdG) while of nitrative stress the enzyme NOS2 (Nitric oxide synthase 2). Under massive oxidative stress, poly(ADP-ribose)polymerase 1 (PARP-1) enzyme activity, responsible for restoration of DNA damage, is augmented, DNA repair enzymes are recruited, and cell survival/or death is ensued through PARP-1 activation, which is correlated positively with neurodegenerative diseases. In this biochemical study the levels of PARP-1, 8-oxo-dG, and NOS2, Aβ1-42, and p-tau in their sera determined using Enzyme-Linked Immunosorbent Assay (ELISA). Patients diagnosed with Mild Cognitive Impairment participated in MICOIL clinical trial, were daily administered with 50 ml Extra Virgin Olive Oil (EVOO) for one year. All MCI patients' biomarkers that had consumed EVOO were tantamount to those of healthy participants, contrary to MCI patients who were not administered. EVOO administration in MCI patients resulted in the restoration of DNA damage and of the well-established "hallmarks" AD biomarkers, thanks probably to its antioxidant properties exhibiting a therapeutic potentiality against AD. Molecular docking simulations of the EVOO constituents on the crystal structure of PARP-1 and NOS-2 target enzymes were also employed, to study in silico the ability of the compounds to bind to these enzymes and explain the observed in vitro activity. In silico analysis has proved the binding of EVOO constituents on PARP-1and NOS-2 enzymes and their interaction with crucial amino acids of the active sites. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT03362996. MICOIL GOV IDENTIFIER: NCT03362996.
Uploads
Papers by Anastasia A Pantazaki