Sustainable and Efficient Recovery of Polyhydroxyalkanoate Polymer from Cupriavidus necator using Environment Friendly Solvents
An imprudent use of environmentally hazardous petrochemical-based plastics and limited availability of fossil fuels have provoked research interests towards production of biodegradable plastics - polyhydroxyalkanoate (PHAs). However, the industrial application of PHAs based products is primarily restricted by their high cost of recovery and extraction protocols. Moreover, solvents used for the extraction and purification are toxic and volatile which causes adverse environmental hazards. Development of efficient downstream recovery strategies along with utilization of non-toxic solvents will accelerate their commercialization. In this study, various extraction strategies were designed for sustainable and cost-effective recovery of PHAs from Cupriavidus necator using non-toxic environment friendly solvents viz. 1,2-propylene carbonate, ethyl acetate, isoamyl alcohol, butyl acetate. The effect of incubation time i.e. 10, 30 and 50 min and temperature i.e. 60, 80, 100, 120°C was tested to identify the most suitable solvent. PHAs extraction using a recyclable solvent, 1,2 propylene carbonate, showed the highest recovery yield (90%) and purity (93%) at 120°C and 30 min incubation. Ethyl acetate showed the better capacity to recover PHAs from cells than butyl acetate. Extraction with ethyl acetate exhibited high recovery yield and purity of 96% and 92%, respectively at 100°C. Effect of non-toxic surfactant such as linear alkylbenzene sulfonic acid (LAS) was also studied at 40, 60 and 80°C, and detergent pH range of 3.0, 5.0, 7.0 and 9.0 for the extraction of PHAs from the cells. LAS gave highest yield of 86% and purity of 88% at temperature 80°C and 5.0 pH.
An Automated Sensor System for Cochlear Implants Electrode Array Insertion
A cochlear implant, referred to as a CI, is a small electronic device that can provide direct electrical stimulation to the auditory nerve. During cochlear implant surgery, atraumatic electrode array insertion is considered to be a crucial step. However, during implantation, the mechanical behaviour of an electrode array inside the cochlea is not known. The behaviour of an electrode array inside of the cochlea is hardly identified by regular methods. In this study, a CI electrode array capacitive sensor system is proposed. It is able to automatically determine the array state as a result of the capacitance variations. Instead of applying sensors to the electrode array, the capacitance information from the electrodes will be gathered and analysed. Results reveal that this sensing method is capable of recognising different states when fed into a pre-shaped model.
Influence of Infrared Radiation on the Growth Rate of Microalgae Chlorella sorokiniana
Nowadays, with dramatic increasing of global energy demand along with progressively decreasing of raw materials, development of bioeconomy concept and close-loop technologies allowing achieving resource efficiency and sustainability have to become one of the main priorities worldwide. At this point, a potential of microalgae as a substrate for value-added products and CO₂-neutral energy production is becoming more and more discussed topic over the last few decades. In comparison to conventional biomass, microalgae can be cultivated in wide range of conditions without compromising food and feed production thus addressing issues associated with negative social and the environmental impacts. However, one of the most challenging tasks is to undergo seasonal variations and to achieve optimal growing conditions for indoor closed systems that can cover further demand for material and energetic utilization of microalgae. For instance, outdoor cultivation in St. Petersburg (Russia) is only suitable within rather narrow time frame (from mid-May to mid-September). At earlier and later periods insufficient sunlight and heat for the growth of microalgae were detected. On the other hand, without additional physical effects the biomass increment in summer is 3-5 times per week, depending on the solar radiation and the ambient temperature. In order increase biomass production, scientists from all over the world have proposed various technical solutions for cultivators and have been studying the influence of various physical factors affecting the biomass growth namely: magnetic field, radiation impact, electric field, etc. In this paper, the influence of infrared (IR) radiation and fluorescent light on the growth rate of microalgae Chlorella sorokiniana have been studied. The cultivation of Chlorella sorokiniana was carried out in 500 ml cylindrical glass vessels, which were constantly aerated. To accelerate the cultivation process, the mixture was stirred for 15 minutes at 500 rpm following 120 minutes of the rest time. At the same time, metabolic needs in nutrients were provided by addition of micro- and macronutrients in the microalgae growing medium. Lighting was carried out by fluorescent lamps with the intensity of 2500 ± 300 Lx. The influence of infrared radiation was determined using IR lamps with a voltage of 220 V, power of 250 W in order to achieve the intensity of 13 600 ± 500 Lx. Obtained results showed that under the influence of IR radiation along with combined effect of active aeration and variable mixing, the biomass increment on the 2nd day was 3 times compared to the blind sample, and on the 7th day, it was eight-fold. On the other hand, application of IR lamps for the biomass growth allows maintaining the optimal temperature of microalgae suspension at approximately 25…28 °C, which might especially be beneficial during the cold season in extreme climate zones.
Designing Next Generation Platforms for Recombinant Protein Production by Genome Engineering of Escherichia coli
We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.
A Case Study: Innovative Aquatic-Based Bioeconomy for Chlorella Sorokiniana
Over the last decade due to the climate change and a strategy of natural resources preservation the interest for the aquatic biomass has dramatically increased. Along with mitigation of the environmental pressure and connection of waste streams (including CO2 and heat emissions), microalgae bioeconomy can supply food, feed, pharmaceutical and power industry with number of value-added products. Furthermore, in comparison to conventional biomass, microalgae can be cultivated in wide range of conditions without compromising food and feed production thus addressing issues associated with negative social and the environmental impacts. This paper presents the state-of-the art technology for microalgae bioeconomy from cultivation process to production of valuable components and by-streams. Microalgae Chlorella sorokiniana were cultivated in the pilot-scale innovation concept in Hamburg (Germany) using different systems such as race way ponds (2 x 5000 L) and flat panel reactors (8 x 180 L). In order to achieve the optimum growth conditions along with suitable cellular composition for the further extraction of the value added components, process parameters such as light intensity, temperature and pH are continuously being monitored. On the other hand, metabolic need in nutrients is provided by addition of micro and macronutrients into a medium. After repeated-batch cultivation processes to biomass dry matter content of 4…5 g∙L -1 efficacious harvesting and cell disruption, determination of lipids, proteins and saccharides was carried out. Lipids extraction is conducted in repeated-batch semi-automatic mode using hot extraction method according to Randall. As solvents hexane and ethanol are used at different ratio of 9:1 and 1:9 respectively. Depending on cell disruption method along with solvents ratio, the total lipids content showed significant variations between 8,1 and 13,9 %. The highest percentage of extracted biomass was reached with a sample pretreated with microwave digestion using 90 % of hexane and 10 % of ethanol as solvents. Proteins content in microalgae was determined by 2 different methods, namely: Total Kejadahl Nitrogen (TKN), which further was converted to protein content, as well as Bradford method using Brilliant Blue G-250 dye. Obtained results, showed a good correlation between both methods with protein content being in the range of 39,8 – 47,1 %. Characterization of neutral and acid saccharides from microalgae was conducted by phenol-sulfuric acid method at two wavelengths of 480 and 490 nm. The average concentration of neutral and acid saccharides under the optimal cultivation conditions was 19,5 and 26,1 % respectively. Subsequently, biomass residues are used as substrate for anaerobic digestion on the pilot-scale (2.1 m3 ) with an aim to supply the whole process with the heat and electricity. The methane concentration, which was measured on the daily bases, showed some variations over the cultivation season and was in the range between 48 and 55 %. CO2 which is formed during the fermentation process and after the combustion in the Combined Heat and Power unit is used within the cultivation process as a carbon source for the photoautotrophic synthesis of biomass.
Orthosis and Finite Elements: A Study for Development of New Designs through Additive Manufacturing
The gait pattern in people that present motor limitations foment the demand for auxiliary locomotion devices. These artifacts for movement assistance vary according to its shape, size, and functional features, following the clinical applications desired. Among the ortheses of lower limbs, the ankle-foot orthesis aims to improve the ability to walk in people with different neuromuscular limitations, although they do not always answer patients' expectations for their aesthetic and functional characteristics. The purpose of this study is to explore the possibility of using new design in additive manufacturer to reproduce the shape and functional features of an ankle-foot orthesis in an efficient and modern way. Therefore, this work presents a study about the performance of the mechanical forces through the analysis of finite elements in an ankle-foot orthesis. It will be demonstrated a study of distribution of the stress on the orthopedic device in orthostatism and during the movement in the course of patient's walk.
Biosorption of Nickel by Penicillium simplicissimum SAU203 Isolated from Indian Metalliferous Mining Overburden
Nickel, an industrially important metal is not mined in India, due to the lack of its primary mining resources. But, the chromite deposits occurring in the Sukinda and Baula-Nuasahi region of Odhisa, India, is reported to contain around 0.99% of nickel entrapped in the goethite matrix of the lateritic iron rich ore. Weathering of the dumped chromite mining overburden often leads to the contamination of the ground as well as the surface water with toxic nickel. Microbes inherent to this metal contaminated environment are reported to be capable of removal as well as detoxification of various metals including nickel. Nickel resistant fungal isolates obtained in pure form from the metal rich overburden were evaluated for their potential to biosorb nickel by using their dried biomass. Penicillium simplicissimum SAU203 was the best nickel biosorbant among the 20 fungi tested and was capable to sorbing 16.85 mg Ni/g biomass from a solution containing 50 mg/l of Ni. The identity of the isolate was confirmed using 18S rRNA gene analysis. The sorption capacity of the isolate was further standardized following Langmuir and Freundlich adsorption isotherm models and the results reflected energy efficient sorption. Fourier-transform infrared spectroscopy studies of the nickel loaded and control biomass in a comparative basis revealed the involvement of hydroxyl, amine and carboxylic groups in Ni binding. The sorption process was also optimized for several standard parameters like initial metal ion concentration, initial sorbet concentration, incubation temperature and pH, presence of additional cations and pre-treatment of the biomass by different chemicals. Optimisation leads to significant improvements in the process of nickel biosorption on to the fungal biomass. P. simplicissimum SAU203 could sorb 54.73 mg Ni/g biomass with an initial Ni concentration of 200 mg/l in solution and 21.8 mg Ni/g biomass with an initial biomass concentration of 1g/l solution. Optimum temperature and pH for biosorption was recorded to be 30°C and pH 6.5 respectively. Presence of Zn and Fe ions improved the sorption of Ni(II), whereas, cobalt had a negative impact. Pre-treatment of biomass with various chemical and physical agents has affected the proficiency of Ni sorption by P. simplicissimum SAU203 biomass, autoclaving as well as treatment of biomass with 0.5 M sulfuric acid and acetic acid reduced the sorption as compared to the untreated biomass, whereas, NaOH and Na₂CO₃ and Twin 80 (0.5 M) treated biomass resulted in augmented metal sorption. Hence, on the basis of the present study, it can be concluded that P. simplicissimum SAU203 has the potential for the removal as well as detoxification of nickel from contaminated environments in general and particularly from the chromite mining areas of Odhisa, India.
Evaluation of Antimicrobial Properties of Lactic Acid Bacteria of Enterococcus Genus
The ability of the lactic acid bacteria (LAB) to prevent and cure a variety of diseases, their protective role against infections and colonization of pathogenic microorganisms in the digestive tract, has lead to the coining of the term probiotics or pro-life. LAB inhibiting the growth of pathogenic and food spoilage microorganisms, maintaining the nutritive quality and improving the shelf life of foods. They have also been used as flavor and texture producers. Enterococcus strains have been used for treatment of diseases such as diarrhea or antibiotic associated diarrhea, inflammatory pathologies that affect colon such as irritable bowel syndrome, or immune regulation, diarrhea caused by antibiotic treatments. The obtaining and investigation of biological properties of proteinoceous antibiotics, on the basis of probiotic LAB shown, that bacteriocins, metabiotics, and peptides of LAB represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics. The genotyping by 16S rRNA sequencing for LAB were used. Cell free culture broth (CFC) broth was purified by the Gel filtration method on the Sephadex Superfine G 25 resin. Antimicrobial activity was determined by spot-on-lawn method and expressed in arbitrary units (AU/ml). The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republics of Armenia and Nagorno Karabakh were examined. It was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. The influences of partially purified antimicrobial preparations (AMP), obtained from the different strains of Enterococcus genus on the growth of MDR pathogenic bacteria were investigated. It was shown, that bacteriocin containing partially purified preparations, obtained from different strains of Enterococcus faecium and durans species, possess bactericidal or bacteriostatic activity against antibiotic resistant intestinal, spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella. Endemic strains of LAB, isolated from Matsoni made from donkey, buffalo and goat milk, shown broad spectrum of activity against food spoiling microorganisms, moulds and fungi, such as Salmonella sp., Esherichia coli, Aspergillus and Penicillium species. Highest activity against MDR pathogens shown bacteria, isolated from goat milk products. High stability of the investigated strains of the genus Enerococcus, isolated from samples of matsun from different regions of Nagorno-Karabakh (NKR) to the antibiotics was shown. The obtained data show high stability of the investigated different strains of the genus Enerococcus. The high genetic diversity in Enterococcus group suggests adaptations for specific mutations in different environments. Thus, endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms isolated from different sources and belong to different taxonomic group. Prospect of the use of certain antimicrobial preparations against pathogenic strains is obvious. These AMP can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.
Use of Microbial Fuel Cell for Metal Recovery from Wastewater
Metal containing wastewater is generated in large quintiles due to rapid industrialization. Generally, the metal present in wastewater is not biodegradable and can be accumulated in living animals, humans and plant tissue, causing disorder and diseases. The conventional metal recovery methods include chemical, physical and biological methods, but these are chemical and energy intensive. The recent development in microbial fuel cell (MFC) technology provides a new approach for metal recovery; this technology offers a flexible platform for both reduction and oxidation reaction oriented process. The use of MFCs will be a new platform for more efficient and low energy approach for metal recovery from the wastewater. So far metal recover was extensively studied using chemical, physical and biological methods. The MFCs present a new and efficient approach for removing and recovering metals from different wastewater, suggesting the use of different electrode for metal recovery can be a new efficient and effective approach.
Magnetophotonics 3D MEMS/NEMS System for Quantitative Mitochondrial DNA Defect Profiling
Mitochondrial defects have a significant impact in many human diseases and aging associated phenotypes. The pathogenic mitochondrial DNA (mtDNA) mutations are diverse and usually present as heteroplasmic. mtDNA 4977bps deletion is one of the common mtDNA defects, and the ratio of mutated versus normal copy is significantly associated with clinical symptoms thus their quantitative detection has become an important unmet needs for advanced disease diagnosis and therapeutic guidelines. This study revealed a Micro-electro-mechanical-system (MEMS) enabled automatic microfluidic chip that only required minimal sample. The system integrated multiple laboratory operation steps into a Lab-on-a-Chip for high-sensitive and prompt measurement. The entire process including magnetic nanoparticle based mtDNA extraction in chip, mutation selective photonic DNA cleavage, and nanoparticle accelerated photonic quantitative polymerase chain reaction (qPCR). All subsystems were packed inside a miniature three-dimensional micro structured system and operated in an automatic manner. Integration of magnetic beads with microfluidic transportation could promptly extract and enrich the specific mtDNA. The near infrared responsive magnetic nanoparticles enabled micro-PCR to be operated by pulse-width-modulation controlled laser pulsing to amplify the desired mtDNA while quantified by fluorescence intensity captured by a complementary metal oxide system array detector. The proportions of pathogenic mtDNA in total DNA were thus obtained. Micro capillary electrophoresis module was used to analyze the amplicone products. In conclusion, this study demonstrated a new magnetophotonic based qPCR MEMS system that successfully detects and quantify specific disease related DNA mutations thus provides a promising future for rapid diagnosis of mitochondria diseases.
Insights into Archaeological Human Sample Microbiome Using 16S rRNA Gene Sequencing
Human body is inhabited by a vast number of microorganisms, collectively known as the human microbiome, and there is a tremendous interest in evolutionary changes in human microbial ecology, diversity and function. The field of paleomicrobiology, study of ancient human microbiome, is powered by modern techniques of Next Generation Sequencing (NGS), which allows extracting microbial genomic data directly from archaeological sample of interest. One of the major techniques is 16S rRNA gene sequencing, by which certain 16S rRNA gene hypervariable regions are being amplified and sequenced. However, some limitations of this method exist including the taxonomic precision and efficacy of different regions used. The aim of this study was to evaluate the phylogenetic sensitivity of different 16S rRNA gene hypervariable regions for microbiome studies in the archaeological samples. Towards this aim, archaeological bone samples and corresponding soil samples from each burial environment were collected in Medieval cemeteries in Latvia. The Ion 16S™ Metagenomics Kit targeting different 16S rRNA gene hypervariable regions was used for library construction (Ion Torrent technologies). Sequenced data were analysed by using appropriate bioinformatic techniques; alignment and taxonomic representation was done using Mothur program. Sequences of most abundant genus were further aligned to E. coli 16S rRNA gene reference sequence using MEGA7 in order to identify the hypervariable region of the segment of interest. Our results showed that different hypervariable regions had different discriminatory power depending on the groups of microbes, as well as the nature of samples. On the basis of our results, we suggest that wider range of primers used can provide more accurate recapitulation of microbial communities in archaeological samples. Acknowledgements. This work was supported by the ERAF grant Nr. 22.214.171.124/16/A/101.
Study of Microbial Diversity Associated with Tarballs and Their Exploitation in Crude Oil Degradation
Tarballs are crude oil remnants found in oceans after long term weathering process and are a global concern since several decades as potential marine pollutant. Being complicated in structure microbial remediation of tarballs in natural environment is a slow process. They are rich in high molecular weight alkanes and poly aromatic hydrocarbons which are resistant to microbial attack and other environmental factors, therefore remain in environment for long time. However, it has been found that many bacteria and fungi inhabit on tarballs for nutrients and shelter. Many of them are supposed to be oil degraders, while others are supposed to be getting benefited by byproducts formed during hydrocarbon metabolism. Thus tarballs are forming special interesting ecological niche of microbes. This work aimed to study diversity of bacteria and fungi from tarballs and to see their potential application in crude oil degradation. The samples of tarballs were collected from Betul beach of south Goa (India). Different methods were used to isolate culturable fraction of bacteria and fungi from it. Those were sequenced for 16S rRNA gene and ITS for molecular level identification. The 16S rRNA gene sequence analysis revealed the presence of 13 bacterial genera/clades (Alcanivorax, Brevibacterium, Bacillus, Cellulomonas, Enterobacter, Klebsiella, Marinobacter, Nitratireductor, Pantoea, Pseudomonas, Pseudoxanthomonas, Tistrella and Vibrio), while the ITS sequence analysis placed the fungi in 8 diverse genera/ clades (Aspergillus, Byssochlamys, Monascus, Paecilomyces, Penicillium, Scytalidium/ Xylogone, Talaromyces and Trichoderma). All bacterial isolates were screened for oil degradation capacity. Potential strains were subjected to crude oil degradation experiment for quantification. Results were analyzed by GC-MS-MS.
Membrane Spanning DNA Origami Nanopores for Protein Translocation
Single-molecule sensing via protein nanopores has achieved a step-change in portable and label-free DNA sequencing. However, protein pores of both natural or engineered origin are not able to produce the tunable diameters needed for effective protein sensing. Here, we describe a generic strategy to build synthetic DNA nanopores that are wide enough to accommodate folded protein. The pores are composed of interlinked DNA duplexes and carry lipid anchors to achieve the required membrane insertion. Our demonstrator pore has a contiguous cross-sectional channel area of 50 nm2 which is 6-times larger than the largest protein pore. Consequently, transport of folded protein across bilayers is possible. The modular design is amenable for different pore dimensions and can be adapted for protein sensing or to create molecular gates in synthetic biology.
Effect of Low Temperature on Structure and RNA Binding of E.coli CspA: A Molecular Dynamics Based Study
Cold shock protein A (CspA) is major cold inducible protein present in Escherichia coli. The protein is involved in stabilizing secondary structure of RNA by working as chaperone during cold temperature. Two RNA binding motifs play key role in the stabilizing activity. This study aimed to investigate implications of low temperature on structure and RNA binding activity of E. coli CspA. Molecular dynamics simulations were performed to compare the stability of the protein at 37°C and 10 °C. The protein was mutated at RNA binding motifs and docked with RNA to assess the stability of both complexes. Results suggest that CspA as well as CspA-RNA complex is more stable at low temperature. It was also confirmed that RNP1 and RNP2 play key role in RNA binding.
7,10-Epoxy Octadeca 7,9-Dienoic Acid: Potential Candidate of Antibacterial Agent against Multidrug-Resistant Staphylococcus aureus
Structural modification of natural lipids by biocatalysis can change their properties or even create novel functionalities. Hydroxy fatty acid, one of those oxylipins, can be produced from microbial bioconversion of the natural vegetable oils. Recently 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from olive oil by bacterial strain Pseudomonas aeruginosa PR3, and further study confirmed that DOD contained strong antimicrobial activities against broad range of microorganisms. In this study, we tried to modify DOD molecules by physical reaction to create new functionality and/or to enhance the antimicrobial activity of DOD. After the harsh heat-treatment, a novel furan fatty acid, 7,10-epoxy octadeca 7,9-dienoic acid (EODA) was produced from DOD. We confirmed that EODA presented strong antibacterial activity against multidrug-resistant Staphylococcus aureus and also EODA showed a recuperative effect of the beta-lactam antibiotics activity against methicillin-resistant Staphylococcus aureus.
Transcriptomic Response of Calmodulin Encoding Gene (CaM) in Pesticide Utilizing Talaromyces Fungal Strains
Calmodulin is one of the intracellular calcium proteins that regulates large spectrum of enzymes and cellular functions including metabolism of cyclic nucleotides and glycogen. The potentials of calmodulin gene in fungi necessitates their genetic response and their strong cassette of enzyme secretions for pesticide degradation. Therefore, this study was carried out to investigate the ‘Transcriptomic’ response of calmodulin encoding genes in Talaromyces fungi in response to 2, 2-dichlorovinyl dimethyl phosphate (DDVP or Dichlorvos) an organophosphate pesticide and γ-Hexachlorocyclohexane (Lindane) an organochlorine pesticide. Fungi strains isolated from rhizosphere from grasses rhizosphere in pesticide polluted sites were subjected to percentage incidence test. Two most frequent fungi were further characterized using ITS gene amplification (ITS1 and ITS4 combinations), they were thereafter subjected to In-vitro DDVP and lindane tolerance tests at different concentrations. They were also screened for presence and expression of calmodulin gene (caM) using RT-PCR technique. The two Talaromyces strains had the highest incidence of 50-72% in pesticide polluted site, they were both identified as Talaromyces astroroseus asemoG and Talaromyces purpurogenum asemoN submitted in NCBI gene-bank with accession numbers KY488464 and KY488468 respectively. T. astroroseus KY488464 tolerated DDVP (1.23±0.023 cm) and lindane (1.11±0.018 cm) at 25 % concentration while T. purpurogenum KY488468 tolerated DDVP (1.33±0.061 cm) and lindane (1.54±0.077 cm) at this concentration. Calmodulin gene was detected in both strains, but RT-PCR expression of caM gene revealed at 900-1000 bp showed an under-expression of caM in T. astrorosues KY488464 but overexpressed in T. purpurogenum KY488464. Thus, the calmodulin gene response of these fungal strains to both pesticides could be considered in monitoring the potentials of fungal strains to pesticide tolerance and bioremediation of pesticide in polluted soil.
Detection and Expression of Peroxidase Genes in Trichoderma harzianum KY488466 and its Response to Crude Oil Degradation
Fungi have potentials for degrading hydrocarbons through the secretion of different enzymes. Crude oil tolerance and degradation by Trichoderma harzianum was investigated in this study with its ability to produce peroxidase enzymes (LiP and MnP). Many fungal strains were isolated from rhizosphere of grasses growing on a crude oil spilled site, and the most frequent strain based on percentage incidence was further characterized using morphological and molecular characteristics. Molecular characterization was done through the amplification of Ribosomal-RNA regions of 18s (1609-1627) and 28s (287-266) using ITS1 and ITS4 combinations and it was identified using NCBI BLAST tool. The selected fungus was also subjected to an in-vitro tolerance test at crude oil concentrations of 5, 10, 15, 20 and 25% while 0% served as control. In addition, lignin peroxidase genes (lig1-6) and manganese peroxidase gene (mnp) were detected and expressed in this strain using RT-PCR technique, its peroxidase producing activities was also studied in aliquots (U/ml). This strain had highest incidence of 80%, it was registered in NCBI as Trichoderma harzianum asemoJ KY488466. The strain KY488466 responded to crude oil concentrations as it increase, the dose inhibition response percentage (DIRP) increased from 41.67 to 95.41 at 5 to 25 % crude oil concentrations. All the peroxidase genes are present in KY488466, and expressed with amplified 900-1000 bp through RT-PCR technique. In this strain, lig2, lig4 and mnp genes were over-expressed, lig 6 was moderately expressed, while none of the genes was under-expressed. The strain also produced 90±0.87 U/ml lignin peroxidase and 120±1.23 U/mil manganese peroxidase enzymes in aliquots. These results imply that KY488466 can tolerate and survive high crude oil concentration and could be exploited for bioremediation of oil-spilled soils, the produced peroxidase enzymes could also be exploited for other biotechnological experiments.
Influence of Iron Content in Carbon Nanotubes on the Intensity of Hyperthermia in the Cancer Treatment
The term ‘cancer’ is given to a collection of related diseases that may affect any part of the human body. It is a pathological behaviour of cells with the potential to undergo abnormal breakdown in the processes that control cell proliferation, differentiation, and death of particular cells. Although cancer is commonly considered as modern disease, there are beliefs that drastically growing number of new cases can be linked to the extensively prolonged life expectancy and enhanced techniques for cancer diagnosis. Magnetic hyperthermia therapy is a novel approach to cancer treatment, which may greatly contribute to higher efficiency of the therapy. Employing carbon nanotubes as nanocarriers for magnetic particles, it is possible to decrease toxicity and invasiveness of the treatment by surface functionalisation. Despite appearing in recent years, magnetic particle hyperthermia has already become of the highest interest in the scientific and medical environment. The reason why hyperthermia therapy brings so much hope for future treatment of cancer lays in the effect that it produces in malignant cells. Subjecting them to thermal shock results in activation of numerous degradation processes inside and outside the cell. The heating process initiates mechanisms of DNA destruction, protein denaturation and induction of cell apoptosis, which may lead to tumour shrinkage, and in some cases, it may even cause complete disappearance of cancer. The factors which have the major impact on the final efficiency of the treatment include temperatures generated inside the tissues, time of exposure to the heating process, and the character of an individual cancer cell type. The vast majority of cancer cells is characterised by lower pH, persistent hypoxia and lack of nutrients, which can be associated to abnormal microvasculature. Since in healthy tissues we cannot observe presence of these conditions, they should not be seriously affected by elevation of the temperature. The aim of this work is to investigate the influence of iron content in iron filled Carbon Nanotubes on the desired nanoparticles for cancer therapy. In the article, the development and demonstration of the method and the model device for hyperthermic selective destruction of cancer cells are presented. This method was based on the synthesis and functionalization of carbon nanotubes serving as ferromagnetic material nanocontainers. The methodology of the production carbon- ferromagnetic nanocontainers (FNCs) includes the synthesis of carbon nanotubes, chemical, and physical characterization, increasing the content of a ferromagnetic material and biochemical functionalization involving the attachment of the key addresses. The ferromagnetic nanocontainers were synthesised in CVD and microwave plasma system. The research work has been financed from the budget of science as a research project No. PBS2/A5/31/2013.
Radio Frequency Heating of Iron-Filled Carbon Nanotubes for Cancer Treatment
There exist more than one hundred different types of cancer, and therefore no particular treatment is offered to people struggling with this disease. The character of treatment proposed to a patient will depend on a variety of factors such as type of the cancer diagnosed, advancement of the disease, its location in the body, as well as personal preferences of a patient. None of the commonly known methods of cancer-fighting is recognised as a perfect cure, however great advances in this field have been made over last few decades. Once a patient is diagnosed with cancer, he is in need of medical care and professional treatment for upcoming months, and in most cases even for years. Among the principal modes of treatment offered by medical centres, one can find radiotherapy, chemotherapy, and surgery. All of them can be applied separately or in combination, and the relative contribution of each is usually determined by medical specialist in agreement with a patient. In addition to the conventional treatment option, every day more complementary and alternative therapies are integrated into mainstream care. There is one promising cancer modality - hyperthermia therapy which is based on exposing body tissues to high temperatures. This treatment is still being investigated and is not widely available in hospitals and oncological centres. There are two kinds of hyperthermia therapies with direct and indirect heating. The first is not commonly used due to low efficiency and invasiveness, while the second is deeply investigated and a variety of methods have been developed, including ultrasounds, infrared sauna, induction heating and magnetic hyperthermia. The aim of this work was to examine possibilities of heating magnetic nanoparticles under the influence of electromagnetic field for cancer treatment. For this purpose, multiwalled carbon nanotubes used as nanocarriers for iron particles were investigated for its heating properties. The samples were subjected to an alternating electromagnetic field with frequency range between 110-619 kHz. Moreover, samples with various concentrations of carbon nanotubes were examined. The lowest frequency of 110 kHz and sample containing 10 wt% of carbon nanotubes occurred to influence the most effective heating process. Description of hyperthermia therapy aiming at enhancing currently available cancer treatment was also presented in this paper. Most widely applied conventional cancer modalities such as radiation or chemotherapy were also described. Methods for overcoming the most common obstacles in conventional cancer modalities, such as invasiveness and lack of selectivity, has been presented in magnetic hyperthermia characteristics, which explained the increasing interest of the treatment.
Designing Active Sites on Amicyanin Using Histidine S Plus Cobalt, and Measuring Their Functional Activity
There is a growing interest in introducing a desired functional group on enzymes in the field of protein engineering. In here, various redox centers were newly created using histidine tag, which is widely used for protein purification, plus cobalt in one of cupredoxins, amicyanin. The coordination of Cobalt-His tag and reactivity of the Co²⁺ loaded His-tag also were characterized. 3xHis-tag, 6xHis-tag, and 9xHis-tag were introduced on amicyanin by site-directed mutagenesis, and then Co²⁺ was loaded on each His-tagged amicyanin. The spectral changes at 330 nm corresponding to cobalt binding on His-tag site indicated the binding ratio of 3xHis-tag, 6xHis-tag, and 9xHis-tag to cobalt as 1:1, 1:2, 1:3 respectively. Based on kinetic studies of binding cobalt to 3xHis-tag, 6xHis-tag, and 9xHis-tagged amicyanin, the nature of the sites was elucidated. In addition, internal electron transfer properties between Cu¹⁺ site and engineered site of amicyanin were determined. These results provide insight into improvement of metal coordination and alternation of the redox properties of metal as a new catalytic site on proteins.
Effects of Cassia tora Seeds Extract on Type 2 Diabetes Induced Mice
Type 2 diabetes (T2D) is characterized by insulin resistance, the inability of β-cell and the dysfunction of mitochondria. To characterize effects of Cassia tora extract on mitochondrial dysfunction related T2D, the reduced glutathione level, amount of mitochondrial complexes and activities of mitochondrial complexes were measured. Three groups of mice were modeled; a control group was fed a normal diet, a diabetic group was fed a diabetic diet high in fat and carbohydrates, and a third group was fed a diabetic diet + 70% ethanol extracted Cassia tora seeds for 12 weeks. The amount of mitochondria was determined by Bradford assay after isolation of mitochondria in liver from each group. During isolation of mitochondria, cytosolic fractions of the tissue were collected to measure the reduced glutathione level. Interestingly, high level of the reduced glutathione was observed in Cassia tora treated group and decreased activities of mitochondrial complexes in Cassia tora treated group compared to the diabetic diet group. It indicates that Cassia tora has the potential to increase the reduced form of glutathione functioned as an important antioxidant in cells, and to reduce mitochondrial metabolic compensatory mechanism.
Short-Term Exposing Effects of 4,4'-DDT on Mitochondrial Electron Transport Complexes in Eyes of Zebrafish
4,4’-Dichlorodiphenyltrichloroethane (4,4’-DDT) is colorless, odorless organochlorine and known as persistent toxic organic pollutant accumulated in organs. In this study, effects of 4,4’-DDT on activities of mitochondrial electron transport chain system was analyzed. 4,4’-DDT is directly treated to isolated mitochondria from eyes of zebrafish and then activities of mitochondrial complex I, II, III, IV were measured spectrophotometrically. The reaction was proceeded immediately after adding 4,4’-DDT to examine the short-term exposing effects of persistent organic pollutant. As a result, high concentration of 4,4’-DDT treated mitochondria exhibited slightly enhanced activity in all complexes than non-treated one except complex III in male. Particularly, 4,4’-DDT was more effective on enzymatic activity in mitochondria isolated from eyes of male zebrafish. These results represented that 4,4’-DDT might temporarily induce to open up ion channel on isolated mitochondria resulting in increasing the functional activity of mitochondrial electron transport chain system.
Compensatory Increased Activities of Mitochondrial Respiratory Chain Complexes from Eyes of Glucose-Immersed Zebrafish
Diabetes is a metabolic disease characterized by hyperglycemia, insulin resistant, mitochondrial dysfunction. Diabetes is associated with the development of diabetic retinopathy resulting in worsening vision and eventual blindness. In this study, eyes were enucleated from glucose-immersed zebrafish which is a good animal model to generate diabetes, and then mitochondria were isolated to evaluate activities of mitochondrial electron transfer complexes. Surprisingly, the amount of isolated mitochondria was increased in eyes from glucose-immersed zebrafish compared to those from non-glucose-immerged zebrafish. Spectrophotometric analysis for measuring activities of mitochondrial complex I, II, III, and IV revealed that mitochondria functions was even enhanced in eyes from glucose-immersed zebrafish. These results indicated that 3 days or 7 days glucose-immersion on zebrafish to induce diabetes might contribute metabolic compensatory mechanism to restore their mitochondrial homeostasis on the early stage of diabetes in eyes.
A Meridian Alignment Hypothesis for Binocular Extension of Listing's Law
Retinal image disparity exists between two eyes in binocular fixation due to horizontal separation of the eyeball. Cyclovergence rotates the eyes to reduce the cyclodisparity of the retinal image. It is known that human vergence following the binocular extension of Listing's Law in dark environments. This paper proposes a new hypothesis explaining the purpose of non-visually induced cyclovergence. The hypothesize states that the non-visually mediated cyclovergence minimizes the cyclodisparity by maximizing the alignment between two eyes’ horizontal and vertical meridians. Numerical simulations show that the binocular extension of Listing's Law can be explained by this hypothesis. The hypothesis will improve the understanding of human visual perception, which could potentially help the clinical diagnosis of visual disorder and the development of virtual reality tools.
Development of Bacterial Nanocellulose Bioink for 3D Bioprinting of Human Cartilage
Bacterial nanocellulose (BNC) consists of a 3D network of nanofibrils with excellent biocompatibility. These nanofibrils are tighly entangled, and in order to create a printable material for 3D bioprinting, the BNC network has to be disassembled into shorter fibrils. In this study, we evaluate two disassembly methods; acid hydrolysis, and mechanical defibrillation with the novel aqueous counter collision technique (ACC-BNC), to create bioinks with good shear thinning properties and desired printability. Post-printing, the ACC-BNC hydrogels were mixed with alginate to enable crosslinking, in order to retain the shape fidelity of the constructs during cultivation. The ACC-BNC bioink was also evaluated in an in vitro experiment with fibroblasts as well as by a clinically relevant setting in vivo, to study chondrogenesis. The cell proliferation was assessed by morphological and immunohistochemical analysis. The ACC-BNC bioink showed outstanding printability and post-printing mechanical stability. Furthermore, both the in vitro and in vivo results, in terms of cell proliferation of human fibroblasts and chondrocytes, respectively, were excellent. The human nasal chondrocyte ACC-BNC constructs had excellent structural integrity and a gradually increasing production of glycosaminoglycans and collagen II after 60 days of implantation in nude mice. The novel ACC disentanglement technique makes the BNC bioink highly suitable for 3D bioprinting in reconstructive surgery.
Effect of Black Cumin (Nigella Sativa) Extract on Damaged Brain Cells
The nervous system is made up of complex delicate structures such as the spinal cord, peripheral nerves and the brain. These are prone to various types of injury ranging from neurodegenerative diseases to trauma leading to diseases like Parkinson's, Alzheimer's, multiple sclerosis, amyotrophic lateral sclerosis (ALS), multiple system atrophy etc. Unfortunately, because of the complicated structure of nervous system, spontaneous regeneration, repair and healing is seldom seen due to which brain damage, peripheral nerve damage and paralysis from spinal cord injury are often permanent and incapacitating. Hence, innovative and standardized approach is required for advance treatment of neurological injury. Nigella sativa (N. sativa), an annual flowering plant native to regions of southern Europe and Asia; has been suggested to have neuroprotective and anti-seizures properties. Neuroregeneration is found to occur in damaged cells when treated using extract of N. sativa. Due to its proven health benefits, lots of experiments are being conducted to extract all the benefits from the plant. The flowers are delicate and are usually pale blue and white in color with small black seeds. These seeds are the source of active components such as 30–40% fixed oils, 0.5–1.5% essential oils, pharmacologically active components containing thymoquinone (TQ), ditimoquinone (DTQ) and nigellin. In traditional medicine, this herb was identified to have healing properties and was extensively used Middle East and Far East for treating diseases such as head ache, back pain, asthma, infections, dysentery, hypertension, obesity and gastrointestinal problems. Literature studies have confirmed the extract of N. sativa seeds and TQ have inhibitory effects on inducible nitric oxide synthase and production of nitric oxide as well as anti-inflammatory and anticancer activities. Experimental investigation will be conducted to understand which ingredient of N. sativa causes neuroregeneration and roots to its healing property. An aqueous/ alcoholic extract of N. sativa will be made. Seed oil is also found to have used by researchers to prepare such extracts. For the alcoholic extracts, the seeds need to be powdered and soaked in alcohol for a period of time and the alcohol must be evaporated using rotary evaporator. For aqueous extracts, the powder must be dissolved in distilled water to obtain a pure extract. The mobile phase will be the extract while the suitable stationary phase (substance that is a good adsorbent e.g. silica gels, alumina, cellulose etc.) will be selected. Different ingredients of N. sativa will be separated using High Performance Liquid Chromatography (HPLC) for treating damaged cells. Damaged brain cells will be treated individually and in different combinations of 2 or 3 compounds for different intervals of time. The most suitable compound or a combination of compounds for the regeneration of cells will be determined using DOE methodology. Later the gene will also be determined and using Polymerase Chain Reaction (PCR) it will be replicated in a plasmid vector. This plasmid vector shall be inserted in the brain of the organism used and replicated within. The gene insertion can also be done by the gene gun method. The gene in question can be coated on a micro bullet of tungsten and bombarded in the area of interest and gene replication and coding shall be studied. Investigation on whether the gene replicates in the organism or not will be examined.
Chondrocytes and Stem Cells in 3D-Bioprinted Structures Create Human Cartilage in Vivo
Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glucosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.
Enhancement of Morphogenetic Potential to Obtain Elite Varities of Sauropus androgynous (L.) Merr. through Somatic Embryogenesis
Somatic embryogenesis is a remarkable illustration of the dictum of plant totipotency where developmental reconstruction of somatic cells takes place towards the embryogenic pathway. It recapitulates the morphological and developmental process that occurs in zygotic embryogenesis. S. androgynous commonly called as multivitamin plant. The leaves are consumed as green leafy vegetable by the Southeast Asian communities due to their rich nutritional profile. Despite being a good nutritional vegetable with proteins, vitamins, minerals, amino acids, it is warned for excessive intake due to the presence of alkoloid called papaverine. Papaverine at higher concentrations is toxic and leads to a syndrome called Bronchiolitis Obliterans. In the present study, morphogenetic potential of shoot tip, leaf and nodal explants of Sauropus androgynous was investigated to develop and enhance the reliable plant regeneration protocol via somatic embryogenesis. Somatic embryos were derived directly from the embryogenic callus derived from shoot tip, node and leaf cultures on Phillips and Collins (L2) medium supplemented with NAA at various concentrations ranging from 5.3 µM/l to 26.85 µM/l within two months of inoculation. Thus obtained embryos were sub cultured to modified L2 media supplemented with increased vitamin level for the further growth. Somatic embryos with well-developed cotyledons were transferred to normal and modified L2 basal medium for conversion. The plantlets thus obtained were subjected to brief acclimatization before transferring them to land. About 95% of survival rate was recorded. The augmentation process of culturing various explants through somatic embryogenesis using synthetic medium with various plant growth regulators under controlled conditions have aggrandized the commercial production of Sauropus making it easily available over the conventional propagation methods. In addition, regeneration process through somatic embryogenesis has ameliorated the development of desired character in Sauropus with low papaverine content thereby providing a valuable resource to the food and pharmaceutical industry. Based on this research, plant tissue culture techniques have shown promise for economical and convenient application in Sauropus androgynous breeding.
Evaluation of Anti-Inflammatory Activities in Wild Herb Urginea wightii
The present work focusses on anti-inflammatory action of Urginea wightii in-vitro. Urginea wightii is a member of Hyacinthaceae and considered to be wonder plant because of its varied important medicinal properties. The plant is endemic to India, Africa, and Mediterranian regions. Presence of alkaloids, flavonoid-glycosides especially flavonone derivatives are responsible for the strong anti-inflammatory activity of Urginea wightii. In present research work, anti-inflammatory activity of methanol extract of the bulb powder was tested on Male Wistar Rats. In these test animals, inflammation was induced by injecting carrageenan as the irritant to induce paw edema in Wistar rats. Inflammation of Paw edema was treated with both plant extract and Pyrox gel a known synthetic anti-inflammatory drug through external application. The result indicated that anti-inflammatory activity of Urginea wightii extract was almost similar to the synthetic Pyrox gel. This disproves the modern world's scepticism towards the herbal medicines and encourages to rely on natural plant extracts.
An Android Application for ECG Monitoring and Evaluation Using Pan-Tompkins Algorithm
Parallel to the fast worldwide increase of elderly population and spreading unhealthy life habits, there is a significant rise in the number of patients and health problems. The supervision of people who have health problems and oversight in detection of people who have potential risks, bring a considerable cost to health system and increase workload of physician. To provide an efficient solution to this problem, in the recent years mobile applications have shown their potential for wide usage in health monitoring. In this paper we present an Android mobile application that records and evaluates ECG signal using Pan-Tompkins algorithm for QRS detection. The application model includes an alarm mechanism that is proposed to be used for sending message including abnormality information and location information to health supervisor.