Simultaneous polyhydroxyalkanoates and rhamnolipids production by Thermus thermophilus HB8

The ability of Thermus thermophilus HB8 to produce simultaneously two environmentally-friendly biodegradable products, polyhydroxyalkanoates (PHAs) and rhamnolipids (RLs), using either sodium gluconate or glucose as sole carbon source, was demonstrated. The utilization of sodium gluconate resulted in higher levels of PHAs and RLs production than when glucose was used as sole carbon source. The initial phosphate concentration (as PO43-) influences both PHAs and RLs productions that were increased during cultivation time. PHAs accumulation was enhanced (> 300 mg/L) after 72 h of cultivation in an initial [PO43-] of 25 mM, while RLs production (> 200 mg/L) was started after 35 h and continued until 72 h of cultivation, in a phosphate-limited medium containing initially 5 mM of [PO43-]. In addition, the combine effect of initial [PO43-] and cultivation time on biomass, PHAs and RLs production was evaluated from 2D contour plots. The results revealed that low initial phosphate concentrations (up to 5 mM) and long incubation time (72 h) promoted RLs biosynthesis while higher initial phosphate concentrations (up to 25 mM) where favorable for biomass and PHAs production. The molecular composition of the produced bio-products was identified. The accumulated PHAs were co-polymers which mainly consisted of 3-hydroxydecanoate (3HD) as resulted by gas chromatography (GC) analysis. The secreted RLs were extracted and their total mixture contained both mono- and di- RLs identified by thin-layer chromatography (TLC). Moreover, the molecular composition of the produced RLs characterized in details by LC-MS analysis showed a plethora of diversity including mono-, and di-RLs, di-rhamno-monolipidic congeners differing in the length of the lipidic chain, which additionally were found to be saturated or unsaturated in some cases

Vaccination Coverage and Awareness of Hepatitis B Virus Among Healthcare Students at a University in Cyprus

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

Catalyzing Transcriptomics Research in Cardiovascular Disease : The CardioRNA COST Action CA17129

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu)

Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases

Micro-RNAs (miRNAs) play a pivotal role in the development and physiology of the cardiovascular system while they have been associated with multiple cardiovascular diseases (CVDs). Several cardiac miRNAs are detectable in circulation (circulating miRNAs; c-miRNAs) and are emerging as diagnostic and therapeutic biomarkers for CVDs. c-miRNAs exhibit numerous essential characteristics of biomarkers while they are extremely stable in circulation, their expression is tissue-/disease-specific, and they can be easily detected using sequence-specific amplification methods. These features of c-miRNAs are helpful in the development of non-invasive assays to monitor the progress of CVDs. Despite significant progress in the detection of c-miRNAs in serum and plasma, there are many contradictory publications on the alterations of cardiac c-miRNAs concentration in circulation. The aim of this review is to examine the pre-analytical and analytical factors affecting the quantification of c-miRNAs and provide general guidelines to increase the accuracy of the diagnostic tests in order to improve future research on cardiac c-miRNAs

The Circulating Biomarkers League: Combining miRNAs with Cell-Free DNAs and Proteins

The potential of liquid biopsy for the prognosis and diagnosis of diseases is unquestionable. Within the evolving landscape of disease diagnostics and personalized medicine, circulating microRNAs (c-miRNAs) stand out among the biomarkers found in blood circulation and other biological fluids due to their stability, specificity, and non-invasive detection in biofluids. However, the complexity of human diseases and the limitations inherent in single-marker diagnostics highlight the need for a more integrative approach. It has been recently suggested that a multi-analyte approach offers advantages over the single-analyte approach in the prognosis and diagnosis of diseases. In this review, we explore the potential of combining three well-studied classes of biomarkers found in blood circulation and other biofluids—miRNAs, DNAs, and proteins—to enhance the accuracy and efficacy of disease detection and monitoring. Initially, we provide an overview of each biomarker class and discuss their main advantages and disadvantages highlighting the superiority of c-miRNAs over the other classes of biomarkers. Additionally, we discuss the challenges and future directions in integrating these biomarkers into clinical practice, emphasizing the need for standardized protocols and further validation studies. This integrated approach has the potential to revolutionize precision medicine by offering insights into disease mechanisms, facilitating early detection, and guiding personalized therapeutic strategies. The collaborative power of c-miRNAs with other biomarkers represents a promising frontier in the comprehensive understanding and management of complex diseases. Nevertheless, several challenges must be addressed before this approach can be translated into clinical practice

Studies for the regulation of synthesis and for the structure of polymers by the bacterium thermus thermophilus: nanotechnological applications

The aim of this work was the study of the regulation of PHA biosynthesis in the thermophilic bacterium Thermus thermophilus. The influence of the initial phosphate concentration in polymer production was also investigated. In order to reduce the production cost of PHA in T.thermophilus whey, a cheap industrial by product was used as carbon source. Subsequently the extracellular PHB depolymerase of T. thermophilus was purified. The physicochemical and catalytic properties of the purified enzyme were studied and its gene was identified. Moreover, it has been shown that T. thermophilus is a flagella-motile bacterium. The biochemical properties of flagellin monomers and isolated flagella fibres were studied. In the first part of the present work the regulation of PHA production in T. thermophilus was studied. PHAs are synthesized by several bacterial species and are accumulated as energy or carbon storage materials, usually when a nutritional factor such as nitrogen or phosphorus is limited in the presence of an excess carbon source. In this work the influence of initial phosphate concentration in PHA production in T. thermophilus when it was grown in mineral salt media containing sodium gluconate as sole carbon source and excess of nitrogen was investigated. The results indicated that as the initial phosphate concentration in the culture medium was increased from 0 to 25 mM, both the PHA production as well as the enzymic activity of PHA synthase was enhanced. In contrast, when the initial phosphate concentration was increased 50 mM resulted in lower polymer concentration. The results also revealed that the time point of applying nutrient limitation had a significant effect on PHA production. The phosphate limitation in the culture medium containing initially 25 mM was achieved in the begging of stationary growth phase. Since at this time biomass was high enough, the highest PHA concentration, 392 mg/l was obtained from this culture. In contrast, lower phosphate initially concentrations resulted in poor growth and PHA production. In addition it has been shown that as the intracellular ATP levels were decreased the PHA concentration was increased. Hence, the high level of ATP in the culture containing initially 50 mM of phosphate inhibited the biosynthesis of PHA. The molecular weight of the produced polymer was approximately 280,000 and it was a heteropolymer which was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 61. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. The melting temperature and crystallization point of the produced polymer were equal to 175.6 oC and 70 oC respectively and the degree of crystallinity was approximately 40%. The ability of T. thermophilus HB8 to utilize lactose from whey based media for the biosynthesis of PHAs under nitrogen limitation was also tested. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydrohyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties. In the second part of this work, T thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. In order to improve the purification yield, a new chromatography material composed of silica matrix coated with PHB beads was used. The purified enzyme was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the TTHA0199 of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other extracellular PHB depolymerases. A catalytic triad consisting of S203, E329, and H425 and a pentapeptide sequence (GX1SX2G) characteristic for PHB depolymerases (lipase box) and for other serine hydrolases were identified. Purified extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of the purified enzyme for PHB was 53 μg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzyme acts principally as an exo-type hydrolase. In the final part of this work it has been demonstrated that T. thermophilus is a flagella-motile bacterium and it showed significant swimming motility in liquid or semisolid media. The production of flagella is enhanced under carbon and nutrient limitation. Flagellin monomer and flagella fibbers were isolated from a culture of T. thermophilus growing in mineral salt, sodium gluconate or in rich media. Western blot analysis revealed that flagellin of T. thermophilus is a 62 kDa protein, detected intacellularly and extracellularly, indicating that its formation may begin in the cytoplasm as a soluble monomer. Glycan staining of purified flagella and treatment with N-glycosidase F, suggested that flagellin of T. thermophilus is a glycosylated protein. The terminal regions of T. thermophilus flagellin were sensitive to proteolysis. Proteolytic degradation with trypsin resulted initially in a fragment of 51 kDa and subsequently in a stable fragment of 32 kDa, both detectable with flagellin antibody. N-terminal sequence analysis of T. thermophilus extracellular flagellin revealed 100% similarity with flagellins of Bacillus sp. and high similarity with flagellins of Thermotonga sp

Anti-Quorum-Sensing Potential of Ethanolic Extracts of Aromatic Plants from the Flora of Cyprus

Quorum sensing (QS) is a form of intra- and inter-species communication system employed by bacteria to regulate their collective behavior in a cell population-dependent manner. QS has been implicated in the virulence of several pathogenic bacteria. This work aimed to investigate the anti-QS potential of ethanolic extracts of eight aromatic plants of Cyprus, namely, Origanum vulgare subsp. hirtum, Rosmarinus officinalis, Salvia officinalis, Lavendula spp., Calendula officinalis, Melissa officinalis, Sideritis cypria, and Aloysia citriodora. We initially assessed the effects of the extracts on autoinducer 2 (AI-2) signaling activity, using Vibrio harveyi BB170 as a reported strain. We subsequently assessed the effect of the ethanolic extracts on QS-related processes, including biofilm formation and the swarming and swimming motilities of Escherichia coli MG1655. Of the tested ethanolic extracts, those of Origanum vulgare subsp. hirtum, Rosmarinus officinalis, and Salvia officinalis were the most potent AI-2 signaling inhibitors, while the extracts from the other plants exhibited low to moderate inhibitory activity. These three ethanolic extracts also inhibited the biofilm formation (>60%) of E. coli MG1655, as well as its swimming and swarming motilities, in a concentration-dependent manner. These extracts may be considered true anti-QS inhibitors because they disrupt QS-related activities of E. coli MG1655 without affecting bacterial growth. The results suggest that plants from the unexplored flora of Cyprus could serve as a source for identifying novel anti-QS inhibitors to treat infectious diseases caused by pathogens that are resistant to antibiotics

Alterations in Circulating miRNA Levels after Infection with SARS-CoV-2 Could Contribute to the Development of Cardiovascular Diseases: What We Know So Far

The novel coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and poses significant complications for cardiovascular disease (CVD) patients. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and influence several physiological and pathological processes, including CVD. This critical review aims to expand upon the current literature concerning miRNA deregulation during the SARS-CoV-2 infection, focusing on cardio-specific miRNAs and their association with various CVDs, including cardiac remodeling, arrhythmias, and atherosclerosis after SARS-CoV-2 infection. Despite the scarcity of research in this area, our findings suggest that changes in the expression levels of particular COVID-19-related miRNAs, including miR-146a, miR-27/miR-27a-5p, miR-451, miR-486-5p, miR-21, miR-155, and miR-133a, may be linked to CVDs. While our analysis did not conclusively determine the impact of SARS-CoV-2 infection on the profile and/or expression levels of cardiac-specific miRNAs, we proposed a potential mechanism by which the miRNAs mentioned above may contribute to the development of these two pathologies. Further research on the relationship between SARS-CoV-2, CVDs, and microRNAs will significantly enhance our understanding of this connection and may lead to the use of these miRNAs as biomarkers or therapeutic targets for both pathologies

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

Journal URL: www.elsevier.com/locate/procbioThe thermophilic bacteriumThermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3- hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties

Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-α/SPD304 Study

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a <i>K</i>_d of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins