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

A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli

During a discovery project of potential inhibitors for three proteins, TNF-α, RANKL and HO-1, implicated in the pathogenesis of rheumatoid arthritis, significant amounts of purified proteins were required. The application of statistically designed experiments for screening and optimization of induction conditions allows rapid identification of the important factors and interactions between them. We have previously used response surface methodology (RSM) for the optimization of soluble expression of TNF-α and RANKL. In this work, we initially applied RSM for the optimization of recombinant HO-1 and a 91% increase of protein production was achieved. Subsequently, we slightly modified a published incomplete factorial approach (called IF1) in order to evaluate the effect of three expression variables (bacterial strains, induction temperatures and culture media) on soluble expression levels of the three tested proteins. However, soluble expression yields of TNF-α and RANKL obtained by the IF1 method were significantly lower (<50%) than those obtained by RSM. We further modified the IF1 approach by replacing the culture media with induction times and the resulted method called IF-STT (Incomplete Factorial-Stain/Temperature/Time) was validated using the three proteins. Interestingly, soluble expression levels of the three proteins obtained by IF-STT were only 1.2-fold lower than those obtained by RSM. Although RSM is probably the best approach for optimization of biological processes, the IF-STT is faster, it examines the most important factors (bacterial strain, temperature and time) influencing protein soluble expression in a single experiment, and can be used in any recombinant protein expression project as a starting point. © 2015 Elsevier Inc. All rights reserved

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

Statistical approaches to maximize recombinant protein expression in Escherichia coli: A general review

The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth. (C) 2013 Elsevier Inc. All rights reserved

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

Optimization of TNF-alpha, overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies

Tumor necrosis factor-alpha (TNF-alpha) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-alpha is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-alpha inhibitors. In this work, induction conditions for the production of human TNF-alpha fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD600nm), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-alpha. An 11% increase of TNF-alpha production was achieved after determination of the optimum induction conditions: OD600nm prior induction 0.55, a post induction temperature of 25 degrees C, an IPTG concentration of 1 mM and a post-induction time of 4 h. We have also studied TNF-alpha oligomerization, the major property of this protein, and a K-d value of 0.26 nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable Kd value for protein trimerization probably due to the complexibility of our model. (C) 2012 Elsevier Inc. All rights reserved