Papers by Christos Papaneophytou
Nutrients, Feb 26, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Modern Applications in the Determination of Food and Feed Additives
Many factors must be considered during the optimization of an enzyme assay. These include the cho... more Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.
Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been consi... more Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.
Biochemistry and Molecular Biology Education, Jun 30, 2020
This article describes a straightforward approach to deliver an enzyme assay and kinetics laborat... more This article describes a straightforward approach to deliver an enzyme assay and kinetics laboratory via online delivery methods in the time of COVID-19.
Synthesis and biological evaluation of potential small moleculeinhibitors of tumor necrosis factor
MedChemComm, 2015
Resistance of bacteria, fungi, and parasites to antibiotics or natural substances of botanical origin
Elsevier eBooks, 2020
Abstract The discovery of antibiotics led to a reduction in mortality and morbidity due to infect... more Abstract The discovery of antibiotics led to a reduction in mortality and morbidity due to infectious diseases. However, their overuse has been contributed to rapidly increasing bacterial resistance. Antibiotic resistance is the ability of microorganisms, including bacteria, to resist the effects of an antibiotic to which they were once sensitive. It is well known that bacteria, parasites, protozoa, and fungi develop resistance when exposed to antibiotics. The microorganisms that survive and multiply cause more harm, even when treated with certain antimicrobials. Moreover, the emergence of resistance has been rapid and limited the useful life of many antibiotics or other antiparasitic drugs. Concerns about the increased antibiotic resistance of microorganisms commonly found in human patients, due to excessive use of antibiotics in humans and/or animals for the treatment of diseases or in animals as dietary growth promoters have been raised worldwide. The aim of this chapter is to further elucidate the matter of antibiotic or natural substances resistance and explore possible mechanisms of action of herbal compounds.
Materia socio-medica, 2019
Introduction: The risk for healthcare students to get infected by transmitting infectious viruses... more Introduction: The risk for healthcare students to get infected by transmitting infectious viruses, including hepatitis B virus (HBV), in a hospital setting is extremely high through exposure to blood and/or body secretions. Aim: The aim of this work was to evaluate both the vaccination history of healthcare students at a University in Cyprus and their serologic immunity against HBV. In addition, we assessed their knowledge and behaviors towards the transmission and prevention of hepatitis B (HB). Results: Total amount of 168 students participated in this study and more than 50% of them provided complete documentation of vaccination history against HBV. Antibodies levels 10 mIU/mL to HB surface antigen (HBsAg) were detected for the 98.8% of healthcare students while 1.2% of the participants tested positive for HBsAg and antibodies to HB core antigen indicating chronic infection. Our study also revealed significant gaps in the knowledge of healthcare students on the efficiency of the vaccine against HBV and in terms of the HBV transmission. Conclusions: More information needs to be provided to healthcare students in Cyprus regarding HBV transmission and vaccination. In addition, there is a need for intervention to provide a safer workplace environment.
Molecular Biotechnology, Oct 29, 2019
In this review, the basic concepts and applications of design of experiments (DoE) in recombinant... more In this review, the basic concepts and applications of design of experiments (DoE) in recombinant protein biotechnology will be discussed. The production of recombinant proteins usually begins with the construction of an expression vector that is then introduced into a microbial host. The target protein is overexpressed in the host's cells and subsequently, it is isolated using a suitable purification method, its activity is assessed using a biological assay, while its crystallization is often required. Because each protein is unique and due to the complex interactions among the reagents in experiments, it is impossible that one set of reaction conditions would be optimal for all cases. Optimization of experimental conditions is usually carried out by the inefficient one-factor-at-a-time approach that does not take into account the combined effects of factors on a process. On the other hand, DoE approaches with a carefully selected small set of experiments, and therefore with a reduced cost and in a limited amount of time predict the effect of each factor and the effects of their interactions on a process. Importantly, several software packages are available that facilitate the choice of the DoE approach, design of the experiments, and analysis of the results.
Μελέτη ρύθμισης της παραγωγής και της δομής πολυμερών στο βακτήριο thermus thermophilus
Molecular Biotechnology, Aug 11, 2017
Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various a... more Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25°C in a variety of buffers.
International Journal of Molecular Sciences
Receptor activator of nuclear factor-κB ligand (RANKL) has been actively pursued as a therapeutic... more Receptor activator of nuclear factor-κB ligand (RANKL) has been actively pursued as a therapeutic target for osteoporosis, given that RANKL is the master mediator of bone resorption as it promotes osteoclast differentiation, activity and survival. We employed a structure-based virtual screening approach comprising two stages of experimental evaluation and identified 11 commercially available compounds that displayed dose-dependent inhibition of osteoclastogenesis. Their inhibitory effects were quantified through TRAP activity at the low micromolar range (IC50 < 5 μΜ), but more importantly, 3 compounds displayed very low toxicity (LC50 > 100 μΜ). We also assessed the potential of an N-(1-aryl-1H-indol-5-yl)aryl-sulfonamide scaffold that was based on the structure of a hit compound, through synthesis of 30 derivatives. Their evaluation revealed 4 additional hits that inhibited osteoclastogenesis at low micromolar concentrations; however, cellular toxicity concerns preclude their...
International journal of molecular sciences, Mar 17, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Good laboratory and experimental practices for microRNA analysis in cardiovascular research
Elsevier eBooks, 2021
Abstract Cardiovascular diseases (CVDs) are a major cause of death worldwide, especially in devel... more Abstract Cardiovascular diseases (CVDs) are a major cause of death worldwide, especially in developed countries. Noncoding RNA molecules and particularly circulating microRNAs (miRNAs) have become potential diagnostic, prognostic, and therapeutic biomarkers for CVDs. In the cardiovascular system, miRNAs regulate numerous biological functions while they have been implicated in the pathogenesis of several CVDs. miRNAs exhibit several advantages over other biological molecules (proteins, cytokines, and peptides), especially due to their high stability in circulation and under extreme pH and temperature conditions. However, it is still challenging to accurately determine miRNA levels and several preanalytical and analytical issues need to be addressed. Methodological heterogeneity in sample storage and handling, extraction and quantification of miRNAs, and data normalization resulted in inconsistency among different studies. In this chapter, we highlight the factors that must be taken into account when studying circulating miRNAs and we propose laboratory and experimental practices to ensure valid scientific inference.
International Journal of Molecular Sciences, Jan 25, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Dairy Research, Aug 1, 2022
In this research communication we describe a straightforward triplex-PCR protocol able to differe... more In this research communication we describe a straightforward triplex-PCR protocol able to differentiate the origin of milk from three closely related species (goat, sheep and cow) in Halloumi, a cheese with Protected Designation of Origin (PDO), and yogurts. Halloumi must contain at least 51% sheep or goat milk, therefore, the fraudulent adulteration of this cheese with excess of cow milk must be routinely tested. The assay employs one universal forward primer and three species-specific reverse primers giving rise to 287 bp (cow), 313 bp (goat), and 336 bp (sheep) amplicons, under the same amplification conditions. This protocol, when used to test a small number of Cyprus commercial products, correctly detected mislabeling in Halloumi (2 out of 6 samples were adulterated) and yogurt brands (1 out of 4 was adulterated). The suggested protocol is a reliable tool for identifying the origin of milk in Halloumi cheeses and yogurts and can be used in any laboratory equipped with a thermocycler and an agarose gel electrophoresis apparatus.
Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8
Polymer Degradation and Stability, Apr 1, 2011
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
Nutrients, Mar 21, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Research Square (Research Square), Feb 10, 2022
Background: Four vaccines that have been authorized in the European Union offer different levels ... more Background: Four vaccines that have been authorized in the European Union offer different levels of protection against SARS-CoV-2 by generating immune responses against the spike receptor-binding domain (RBD) of the virus. Monitoring the levels of IgG antibodies against the SARS-CoV-2 is important during the coronavirus disease 2019 (COVID-19) pandemic to plan an adequate and evidence-based public health response. Methods: We compared the levels of serum IgG antibodies against SARS-CoV-2 spike protein in three groups: i) individuals without evidence of prior infection with SARS-CoV-2 who received one or two doses of either an mRNA-based (Comirnaty BNT162b2/P zer-BioNTech or Spikevax mRNA-1273/Moderna) or an adenoviral-based vaccine (Vaxzervia ChAdOx1 nCoV-19 /Oxford-Astra Zeneca) (n=227), ii) unvaccinated individuals with evidence of prior infection with SARS-CoV-2 (n=109), and iii) individuals with evidence of prior infection with SARS-CoV-2 who received at least one dose of a vaccine (n=30). Unvaccinated individuals without evidence of prior infection with SARS-CoV-2 were used as a control group (n=211). Results: The levels of speci c SARS-CoV-2 IgG antibodies in all three groups were signi cantly higher (p<0.001) compared to the control group. The highest levels of virus-speci c IgG antibodies were observed in individuals who were infected with SARS-CoV-2 and received at least one dose of a vaccine. Our analyses also revealed that the levels of speci c anti-SARS-CoV-2 IgG levels were higher in individuals who received two doses of a licensed vaccine, regardless of the vaccine technology (mRNAbased and adenoviral vector-based). Furthermore, anti-SARS-CoV-2 IgG levels were higher in previously uninfected participants who received at least one dose of a vaccine compared to the unvaccinated individuals with evidence of prior infection. Conclusions: Our results indicate that vaccine-induced responses lead to higher levels of IgG antibodies compared to those produced following infection with the virus. In agreement with previous studies, our results suggest that among individuals previously infected with SARS-CoV-2, even a single dose of a vaccine is adequate to elicit high levels of humoral immunity.
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Papers by Christos Papaneophytou