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.
Selective Recovery and Molecular Identification of Laccase-Producing Bacteria from Selected Terrestrial and Aquatic Milieu in the Eastern Cape, South Africa: Toward the Production of Environmentally Relevant Biocatalysts
Laccase is constantly gaining status as important biocatalyst in biotechnology. The illimitable potential of its industrial applications and the corresponding aggressive need for phenomenal volumes of extracellularly secreted laccases have called for its interminable production from sources which are able to meet this demand within a relatively short period of time, preferably bacteria. In response to this call, this study was designed to source for laccase-producing bacteria from different environmental matrices. Three sampling environments were chosen such as wastewater treatment plants, University of Fort Hare vicinity and the Hogback woodland, all within the Eastern Cape, South Africa. Samples such as effluents, sediments, leaf litters, degrading wood and rock scrapings were selectively enriched with some model aromatic compounds and were further screened qualitatively and quantitatively on five phenolic substrates ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), Guaiacol, 1-Naphthol, Potassium Ferric Cyanide and Syringaldazine). Basis for selection was their ability to elicit a colour change on at least three of the above mentioned agar based assay substrates. The choice isolates were further identified based on 16S rRNA molecular identification techniques. 33 isolates were screened out of the 40 representative distinct colonies during the qualitative plate screens, while quantitative screens selected out 11 bacterial isolates. They were, based on molecular identification, desginated as members of the genera Pseudomonas, Stenotrophomonas and Citrobacter of the gammaproteobacteria and Bordetalla and Achromobacter of the betaproteobacteria respectively. We therefore conclude based on our outcomes that we may have isolated efficient laccase-producing bacteria, which might be of beneficial significance in catalysis and biotechnology.
De Novo Assembly and Characterization of the Transcriptome during Seed Development, and Generation of Genic-SSR Markers in Pomegranate (Punica granatum L.)
Pomegranate (Punica granatum L.) is known to be one of the oldest edible fruit tree species, with a wide geographical global distribution. Fruits from the two defined varieties (Hicaznar and 33N26) were taken at intervals after pollination and fertilization at different sizes. Seed samples were used for transcriptome sequencing. Primary sequencing was produced by Illumina Hi-Seq™ 2000. Firstly, we had raw reads, and it was subjected to quality control (QC). Raw reads were filtered into clean reads and aligned to the reference sequences. De novo analysis was performed to detect genes expressed in seeds of pomegranate varieties. We performed downstream analysis to determine differentially expressed genes. We generated about 27.09 gb bases in total after Illumina Hi-Seq sequencing. All samples were assembled together, we got 59,264 Unigenes, the total length, average length, N50, and GC content of Unigenes are 84.547.276 bp, 1.426 bp, 2,137 bp, and 46.20 %, respectively. Unigenes were annotated with 7 functional databases, finally, 42.681(NR: 72.02%), 39.660 (NT: 66.92%), 30.790 (Swissprot: 51.95%), 20.212 (COG: 34.11%), 27.689 (KEGG: 46.72%), 12.328 (GO: 20.80%), and 33,833 (Interpro: 57.09%) Unigenes were annotated. With functional annotation results, we detected 42.376 CDS, and 4.999 SSR distribute on 16.143 Unigenes.
Production of Antimicrobial Agents against Multidrug-Resistant Staphylococcus Aureus through the Biocatalysis of Vegetable Oils
Structural modification of natural lipids via chemical reaction or microbial bioconversion can change their properties or even create novel functionalities. Enzymatic oxidation of lipids leading to formation of oxylipin is one of those modifications. Hydroxy fatty acids, one of those oxylipins have gained important attentions because of their structural and functional properties compared with other non-hydroxy fatty acids. Recently 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from lipid-containing oleic acid by microbial conversion, and the further study confirmed that DOD contained strong antimicrobial activities against a broad range of microorganisms. In this study, we tried to modify DOD molecules by the enzymatic or physical reaction to create new functionality or to enhance the antimicrobial activity of DOD. After modification of DOD molecules by different ways, we confirmed that the antimicrobial activity of DOD was highly enhanced and presented strong antimicrobial activities against multidrug-resistant Staphylococcus aureus, suggesting that DOD and its derivatives can be used as efficient antimicrobial agents for medical and industrial applications.
Cr³⁺/SiO₄⁴⁻ Codoped Hydroxyapatite Nanorods: Fabrication and Microstructure Analysis
In this study, nanorods of Cr³⁺/SiO₄⁴⁻ codoped hydroxyapatite (Cr³⁺/SiO₄⁴⁻-HA) were synthesized successfully and rapidly through microwave irradiation technique, using (Ca(NO₃)₂•4H₂O), ((NH₄)₂HPO₄), (SiC₈H₂₀O₄) and (Cr(NO₃)₃.9H₂O) as source materials for Ca²⁺, PO₄³⁻, SiO₄⁴⁻ and Cr³⁺ ions, respectively. The impact of dopants on the phase formation and microstructure of the powders were investigated by means of X-ray diffraction (XRD), Fourier transform infrared spectrum analysis (FT-IR) and Field emission electron microscopy (FESEM) techniques. XRD analysis showed that with an incorporation of Cr³⁺/SiO₄⁴⁻ ions into HA structure resulted in peak broadening and reduced peak height due to the amorphous nature and reduced crystallinity of the resulting HA powder. FTIR spectroscopy revealed the existence of the different vibrational modes matching to phosphates and hydroxyl groups. The FESEM analysis showed a change in the crystal shape from spherical to rod shaped particles upon Cr³⁺ doping into the crystal structure. Acknowledgments: This study was supported by Karabük University (Project no. KBÜBAP-17-YD-144). The authors would like to thank for support.
Characterization of Articular Cartilage Based on the Response of Cartilage Surface to Loading/Unloading
Articular cartilage is a fluid-swollen tissue of synovial joints that functions by providing a lubricated surface for articulation and to facilitate the load transmission. The biomechanical function of this tissue is highly dependent on the integrity of its ultrastructural matrix. Any alteration of articular cartilage matrix, either by injury or degenerative conditions such as osteoarthritis (OA), compromises its functional behaviour. Therefore, the assessment of articular cartilage is important in early stages of degenerative process to prevent or reduce further joint damage with associated socio-economic impact. Therefore, there has been increasing research interest into the functional assessment of articular cartilage. This study developed a characterization parameter for articular cartilage assessment based on the response of cartilage surface to loading/unloading. This is because the response of articular cartilage to compressive loading is significantly depth-dependent, where the superficial zone and underlying matrix respond differently to deformation. In addition, the alteration of cartilage matrix in the early stages of degeneration is often characterized by PG loss in the superficial layer. In this study, it is hypothesized that the response of superficial layer is different in normal and proteoglycan depleted tissue. To establish the viability of this hypothesis, samples of visually intact and artificially proteoglycan-depleted bovine cartilage were subjected to compression at a constant rate to 30 percent strain using a ring-shaped indenter with an integrated ultrasound probe and then unloaded. The response of articular surface which was indirectly loaded was monitored using ultrasound during the time of loading/unloading (deformation/recovery). It was observed that the rate of cartilage surface response to loading/unloading was different for normal and PG-depleted cartilage samples. Principal Component Analysis was performed to identify the capability of the cartilage surface response to loading/unloading, to distinguish between normal and artificially degenerated cartilage samples. The classification analysis of this parameter showed an overlap between normal and degenerated samples during loading. While there was a clear distinction between normal and degenerated samples during unloading. This study showed that the cartilage surface response to loading/unloading has the potential to be used as a parameter for cartilage assessment.
In vitro Plant Regeneration of Gonystylus Bancanus (Miq) Kurz. Through Direct Organogenesis
Plant regeneration was achieved from shoot tip and nodal segment of Gonystylus bancanus (Miq) Kurz. cultured in Murashige and Skoog’s medium supplemented with various concentrations of 6-benzylaminopurine (BAP). The most optimum concentration of BAP for shoot initiation is 10.0 mgl⁻¹ with approximately 10% of shoot tip and 15% of nodal segment produced single shoot after 28 and 15 days of culture incubation respectively. Rooting was achieved when shoots were transferred into MS medium supplemented with 5.0 mgl⁻¹ Naphthalene acetic acid (NAA). Synthesizing results developed through this research can be a starting point for the upscalling and optimization process in future.
Milk Yield and Fingerprinting of Beta-Casein Precursor (CSN2) Gene in Some Saudi Camel Breeds
Camels are substantial providers of transport, milk, sport, meat, shelter, fuel, security and capital in many countries, particularly Saudi Arabia. Identification of animal breeds has progressed rapidly during the last decade. Advanced molecular techniques are playing a significant role in breeding or strain protection laws. On the other hand, fingerprinting of some molecular markers related to some productive traits in farm animals represents most important studies to our knowledge, which aim to conserve these local genetic resources, and to the genetic improvement of such local breeds by selective programs depending on gene markers. Milk records were taken two days in each week from female camels of Majahem, Safara, Wathaha, and Hamara breeds, respectively from different private farms in northern Jeddah, Riyadh and Alwagh governorates and average weekly yields were calculated. DNA sequencing for CSN2 gene was used for evaluating the genetic variations and calculating the genetic distance values among four Saudi camel populations which are Hamra(R), Safra(Y), Wadha(W) and Majaheim(M). In addition, this marker was analyzed for reconstructing the Neighbor joining tree among evaluating camel breeds. In respect to milk yield during winter season, result indicated that average weekly milk yield of Safara camel breed (30.05 Kg/week) is significantly (p < 0.05) lower than the other 3 breeds which ranged from 39.68 for Hamara to 42.42 Kg/week for Majahem, while there are not significant differences between these three breeds. The Neighbor Joining analysis that re-constructed based on DNA variations showed that samples are clustered into two unique clades. The first clade includes Y (from Y4 to Y18) and M (from M1, to M9). On the other hand, the second cluster is including all R (from R1 to R6) and W (from W1 to W6). The genetic distance values were equal 0.0068 (between the groups M&Y and R&W) and equal 0 (within each group).
Validation of a Fluid-Structure Interaction Model of an Aortic Dissection versus a Bench Top Model
The aim of this investigation was to validate the fluid-structure interaction model of type B aortic dissection with our experimental results from a bench-top-model. Another objective was to study the relationship between the size of a septectomy that increases the outflow of the false lumen and its effect on the values of the differential of pressure between true lumen and false lumen. Fluid-structure interaction analysis based on Galerkin’s formulation was used in this investigation to study flow pattern and hemodynamics within a flexible type B aortic dissection model using boundary conditions from our experimental data. The numerical results of our model were verified against the experimental data for various tear size and location. Thus, CFD tools have a potential role in evaluating different scenarios and aortic dissection configurations.
Construction of the Large Scale Biological Networks from Microarrays
One of the sustainable goals of the system biology is understanding gene-gene interactions. Hence, gene regulatory networks (GRN) need to be constructed for understanding the disease ontology and to reduce the cost of drug development. To construct gene regulatory from gene expression we need to overcome many challenges such as data denoising and dimensionality. In this paper, we develop an integrated system to reduce data dimension and remove the noise. The generated network from our system was validated via available interaction databases and was compared to previous methods. The result revealed the performance of our proposed method.
Hemodynamics of a Cerebral Aneurysm under Rest and Exercise Conditions
Physiological flow under rest and exercise conditions in patient-specific cerebral aneurysm models is numerically investigated. A finite-volume based code with BiCGStab as the linear equation solver is used to simulate unsteady three-dimensional flow field through the incompressible Navier-Stokes equations. Flow characteristics are first established in a healthy cerebral artery for both physiological conditions. The effect of saccular aneurysm on cerebral hemodynamics is then explored through a comparative analysis of the velocity distribution, nature of flow patterns, wall pressure and wall shear stress (WSS) against the reference configuration. The efficacy of coil embolization as a potential strategy of surgical intervention is also examined by modelling coil as a homogeneous and isotropic porous medium where the extended Darcy’s law, including Forchheimer and Brinkman terms, is applicable. The Carreau-Yasuda non-Newtonian blood model is incorporated to capture the shear thinning behavior of blood. Rest and exercise conditions correspond to normotensive and hypertensive blood pressures respectively. The results indicate that the fluid impingement on the outer wall of the arterial bend leads to abnormality in the distribution of wall pressure and WSS, which is expected to be the primary cause of the localized aneurysm. Exercise correlates with elevated flow velocity, vortex strength, wall pressure and WSS inside the aneurysm sac. With the insertion of coils in the aneurysm cavity, the flow bypasses the dilatation, leading to a decline in flow velocities and WSS. Particle residence time is observed to be lower under exercise conditions, a factor favorable for arresting plaque deposition and combating atherosclerosis.
Metagenomics Analysis of Bacteria in Sorghum Using next Generation Sequencing
Sorghum is an important cereal crop in the world. In particular, it has attracted breeders due to capacity to serve as food, feed, fiber and bioenergy crop. Like any other plant, sorghum hosts a variety of microbes, which can either, have a neutral, negative and positive influence on the plant. In the current study, regions (V3/V4) of 16 S rRNA were targeted to extensively assess bacterial multitrophic interactions in the phyllosphere of sorghum. The results demonstrated that the presence of a pathogen has a significant effect on the endophytic bacterial community. Understanding these interactions is key to develop new strategies for plant protection.
Europium Chelates as a Platform for Biosensing
Rare earth nanotechnology has gained a considerable amount of interest in the field of biosensing due to the unique luminescence properties of lanthanides. Chelating rare earth ions plays a significant role in biological labelling applications including medical diagnostics, due to their different excitation and emission wavelengths, variety of their spectral properties, sharp emission peaks and long fluorescence lifetimes. We aimed to develop a platform for biosensors based on Europium (Eu³⁺) chelates against biomarkers of cardiac injury (heart-type fatty acid binding protein; H-FABP3) and stroke (glial fibrillary acidic protein; GFAP). Additional novelty in this project is the use of synthetic binding proteins (Affimers), which could offer an excellent alternative targeting strategy to the existing antibodies. Anti-GFAP and anti-HFABP3 Affimer binders were modified to increase the number of carboxy functionalities. Europium nitrate then incubated with the modified Affimer. The luminescence characteristics of the Eu³⁺ complex with modified Affimers and antibodies against anti-GFAP and anti-HFABP3 were measured against different concentrations of the respective analytes on excitation wavelength of 395nm. Bovine serum albumin (BSA) was used as a control against the IgG/Affimer Eu³⁺ complexes. The emission spectrum of Eu³⁺ complex resulted in 5 emission peaks ranging between 550-750 nm with the highest intensity peaks were at 592 and 698 nm. The fluorescence intensity of Eu³⁺ chelates with the modified Affimer or antibodies increased significantly by 4-7 folder compared to the emission spectrum of Eu³⁺ complex. The fluorescence intensity of the Affimer complex was quenched proportionally with increased analyte concentration, but this did not occur with antibody complex. In contrast, the fluorescence intensity for Eu³⁺ complex increased slightly against increased concentration of BSA. These data demonstrate that modified Affimers Eu³⁺ complexes can function as nanobiosensors with potential diagnostic and analytical applications.
Statistical Studies on Lead Bioremediation by Marine Halomonas ES015: Optimization of Lead Bioremediation by Marine Halomonas ES015 Using Statistical Experimental Designs
Bioremediation technology is now used for treatment instead of traditional metal removal methods. A strain was isolated from Marsa Alam, Red sea, Egypt showed high resistance to high lead concentration and was identified by the 16S rRNA gene sequencing technique as Halomonas sp ES015. Medium optimization was carried out using Plackett-Burman design, and the most significant factors were yeast extract, casamino acid and inoculums size. The optimized media obtained by the statistical design raised the removal efficiency from 84% to 99% from initial concentration 250 ppm of lead. Moreover, Box-Behnken experimental design was applied to study the relationship between yeast extract concentration, casamino acid concentration and inoculums size. The optimized medium increased removal efficiency to 97% from initial concentration 500 ppm of lead. Immobilized Halomonas sp. ES015 cells on sponge cubes using optimized medium in loop bioremediation column, showed relatively constant lead removal efficiency when reused for six successive cycles till the 6th run over the range of time interval. Also metal removal efficiency was not affected by flow rate changes. Finally, the results of this research refer to the possibility of lead bioremediation by free or immobilized cells of Halomonas sp ES015. Also, bioremediation can be done in batch cultures and semicontinuous cultures using column technology.
Association of Lipoprotein Lipase Gene (HindIII rs320) Polymorphisms with Moderate Hypertriglyceridemia Secondary to Metabolic Syndrome
Lipoprotein Lipase (LPL) is a key enzyme for lipid metabolism; its genetic polymorphism can be a candidate for modulating lipids parameters in metabolic syndrome. The objective of the present study was to determine whether lipoproteins lipase polymorphisMetS (LPL-HindIII) could be associated with moderate hypertriglyceridemia (secondary to metabolism syndrome). The polymorphism Hind III (rs320) was assessed by PCR-RFLP in 51 MetS patients and 17 healthy controls from the hospital in Tlemcen. The logistic regression analyses showed no significant association with Hind III genotype and hypertriglyceridemia (TG ≥ 1,5g/l or TG lower treatment) (P=0,455), metabolic syndrome (P=0,455), hypertension (P=0,802) and type 2 diabetes (P=0,144). In terms of plasma biomarkers, although not statistically significant, there was a difference in TG levels (P > 0,05), which was lowest among carriers of the homogenous mutant allele (H-). In this study, there was no association between the rare allele (H-) and disease protection, and between the frequent allele (H+) and disease prevalence (hypertriglyceridemia, metabolic syndrome, hypertension, type 2 diabetes).
Some Extreme Halophilic Microorganisms Produce Extracellular Proteases with Long Lasting Tolerance to Ethanol Exposition
Extremophiles constitute a potentially valuable source of proteases for the development of biotechnological processes; however, the number of available studies in the literature is limited compared to mesophilic counterparts. Therefore, in this study, Peruvian halophilic microorganisms were characterized to select suitable proteolytic strains that produce active proteases under exigent conditions. Proteolysis was screened using the streak plate method with gelatin or skim milk as substrates. After that, proteolytic microorganisms were selected for phenotypic characterization and screened by a semi-quantitative proteolytic test using a modified method of diffusion agar. Finally, proteolysis was evaluated using partially purified extracts by ice-cold ethanol precipitation and dialysis. All analyses were carried out over a wide range of NaCl concentrations, pH, temperature and substrates. Of a total of 60 strains, 21 proteolytic strains were selected, of these 19 were extreme halophiles and 2 were moderates. Most proteolytic strains demonstrated differences in their biochemical patterns, particularly in sugar fermentation. A total of 14 microorganisms produced extracellular proteases, 13 were neutral, and one was alkaline showing activity up to pH 9.0. Proteases hydrolyzed gelatin as the most specific substrate. In general, catalytic activity was efficient under a wide range of NaCl (1 to 4 M NaCl), temperature (37 to 55 °C) and after an ethanol exposition performed at -20 °C for 24 hours. In conclusion, this study reported 14 candidates extremely halophiles producing extracellular proteases capable of being stable and active on a wide range of NaCl, temperature and even long lasting ethanol exposition.
Treatment of Histopathological Symptoms in N-Nitrosopyrrolidine Induced Changes in Lung Tissue by Isolated Flavonoid from Indigofera tinctoria
N-nitrosopyrollidine or NPYR is a tobacco-specific nitrosamine which upon intoxicated causes abnormal production of Reactive Oxygen Species disrupt the endogenous antioxidant system. The study was designed to evaluate the histological changes in lung tissue of Mus musculus in NPYR administered lungs and effect of isolated flavonoid 3,6-dihydroxy-(3’,4’,7’-trimethoxyphenyl)-chromen-4-one-7-glucoside (ITC) from experimental plant Indigofera tinctorial. Post treatment with isolated compound significantly restored the abnormal symptoms and changes in pulmonary tissue. Transverse section of mouse lung in control animals appeared as a thin lace. Histologically, most of the lung was arranged as alveoli which were thin walled structures made up of single layered squamous epithelial cells. In the transverse section of lung at 100 X will clearly show the component of alveoli, surround by a thin layer of connective tissue and blood vessels. Smaller bronchioles were lined by cuboidal epithelial cells while larger bronchioles were lined by ciliated columnar epithelium layer while in NPYR intoxicated lungs signs of vast pulmonary damages and carcinogenesis as alveolar damage, necrosis, DADs or defused alveolar damages hyperplasia, metaplasia, dysplasia and next stage of carcinogenesis were revealed. Treatment with ITC showed the significant positive changes in the lung tissue due to the side hydroxyl and methoxy groups in its structure which help in combating oxidative injuries and give protection from the free radicals generated during the metabolism of NPYR in body. Thus, histopathological analysis confirms the development of the cancerous conditions in the lung tissue in mice model and the protective effects of ITC.
In silico Analysis of Differentially Expressed Genes in High-Grade Squamous Intraepithelial Lesion and Squamous Cell Carcinomas Stages of Cervical Cancer
Cervical cancer is one of the women related cancers which starts from the pre-cancerous cells and a fraction of women with pre-cancers of the cervix will develop cervical cancer. Cervical pre-cancers if treated in pre-invasive stage can prevent almost all true cervical squamous cell carcinoma. The present study investigates the genes and pathways that are involved in the progression of cervical cancer and are responsible in transition from pre-invasive stage to other advanced invasive stages. The study used GDS3292 microarray data to identify the stage specific genes in cervical cancer and further to generate the network of the significant genes. The microarray data GDS3292 consists of the expression profiling of 10 normal cervices, 7 HSILs and 21 SCCs samples. The study identifies 70 upregulated and 37 downregulated genes in HSIL stage while 95 upregulated and 60 downregulated genes in SCC stages. Biological process including cell communication, signal transduction are highly enriched in both HSIL and SCC stages of cervical cancer. Further, the ppi interaction of genes involved in HSIL and SCC stages helps in identifying the interacting partners. This work may lead to the identification of potential diagnostic biomarker which can be utilized for early stage detection.
miCoRe: Colorectal Cancer miRNAs Database
Colorectal cancer (CRC) also refers as bowel cancer or colon cancer. It involves the development of abnormal growth of cells in colon or rectum part of the body. This work leads to the development of a miRNA database in colorectal cancer. We named this database- miCoRe. This database comprises of all validated colon-rectal cancer miRNAs information from various published literature with an effectual knowledge based information retrieval system. miRNAs have been collected from various published literature reports. MySQL is used for main-framework of miCoRe while the front-end was developed in PHP script. The aim of developing miCoRe is to create a comprehensive central repository of colorectal carcinoma miRNAs with all germane information of miRNAs and their target genes. The current version of miCoRe consists of 238 miRNAs which are known to be implicated in malignancy of CRC. Alongside with miRNA information, miCoRe also contains the information related to the target genes of these miRNA. miCoRe furnishes the information about the mechanism of incidence and progression of the disease, which would further help the researchers to look for colorectal specific miRNAs therapies and CRC specific targeted drug designing. Moreover, it will also help in development of biomarkers for the better and early detection of CRC and will help in better clinical management of the disease.
Fluorescing Aptamer-Gold Nanoparticle Complex for the Sensitive Detection of Bisphenol A
Bisphenol A (BPA) is one of the endocrine disruptors (EDCs), which have been suspected to be associated with reproductive dysfunction and physiological abnormality in human. Since the BPA has been widely used to make plastics and epoxy resins, the leach of BPA from the lining of plastic products has been of major concern, due to its environmental or human exposure issues. The simple detection of BPA based on the self-assembly of aptamer-mediated gold nanoparticles (AuNPs) has been reported elsewhere, yet the detection sensitivity still remains challenging. Here we demonstrate an improved AuNP-based sensor of BPA by using fluorescence-combined AuNP colorimetry in order to overcome the drawback of traditional AuNP sensors. While the anti-BPA aptamer (full length or truncated ssDNA) triggered the self-assembly of unmodified AuNP (citrate-stabilized AuNP) in the presence of BPA at high salt concentrations, no fluorescence signal was observed by the subsequent addition of SYBR Green, due to a small amount of free anti-BPA aptamer. In contrast, the absence of BPA did not cause the self-assembly of AuNPs (no color change by salt-bridged surface stabilization) and high fluorescence signal by SYBP Green, which was due to a large amount of free anti-BPA aptamer. As a result, the quantitative analysis of BPA was achieved using the combination of absorption of AuNP with fluorescence intensity of SYBR green as a function of BPA concentration, which represented more improved detection sensitivity (as low as 1 ppb) than did in the AuNP colorimetric analysis. This method also enabled to detect high BPA in water-soluble extracts from thermal papers with high specificity against BPS and BPF. We suggest that this approach will be alternative for traditional AuNP colorimetric assays in the field of aptamer-based molecular diagnosis.
Visualizing Matrix Metalloproteinase-2 Activity Using Extracellular Matrix-Immobilized Fluorescence Resonance Energy Bioprobe in Cancer Cells
Visualizing matrix metalloproteinases (MMPs) activity is necessary for understanding cancer metastasis because they are implicated in cell migration and invasion by degrading the extracellular matrix (ECM). While much effort has been made to sense the MMP activity, but extracellularly long-term monitoring of MMP activity still remains challenging. Here, we report a collagen-bound fluorescent bioprobe for the detection of MMP-2 activity in the extracellular environment. This bioprobe consists of ECM-immobilized part (including collagen-bound protein) and MMP-sensing part (including peptide substrate linked with fluorescence resonance energy transfer (FRET) coupler between donor green fluorescent protein (GFP) and acceptor TAMRA dye), which was constructed through intein-mediated self-splicing conjugation. Upon being immobilized on the collagen-coated surface, this bioprobe enabled efficient long-lasting observation of MMP-2 activity in the cultured cells without affecting cell growth and viability. As a result, the FRET ratio (acceptor/donor) decreased as the MMP2 activity increased in cultured cancer cells. Furthermore, unlike wild-type MMP-2, mutated MMP-2 expression (Y580A in the hemopexin region) gave rise to lowering the secretion of MMP-2 in HeLa. Conclusively, our method is anticipated to find applications for tracing and visualizing enzyme activity.
Experimental Design in Extraction of Pseudomonas sp. Protease from Fermented Broth by Polyethylene Glycol/Citrate Aqueous Two-Phase System
Aqueous two-phase system (ATPS) is an interesting alternative for separating industrial enzymes due to it is easy to scale-up and low cost. Polyethylene glycol (PEG) mixed with potassium phosphate or magnesium sulfate is one of the most frequently polymer/salt ATPS used, but the consequences of its use is a high concentration of phosphates and sulfates in wastewater causing environmental issues. Citrate could replace these inorganic salts due to it is biodegradable and does not produce toxic compounds. On the other hand, statistical design of experiments is widely used for ATPS optimization and it allows to study the effects of the involved variables in the purification, and to estimate their significant effects on selected responses and interactions. The 24 factorial design with four central points (20 experiments) was employed to study the partition and purification of proteases produced by Pseudomonas sp. in PEG/citrate ATPS system. ATPS was prepared with different sodium citrate concentrations [14, 16 and 18% (w/w)], pH values (7, 8 and 9), PEG molecular weight (2,000; 4,000 and 6,000 g/mol) and PEG concentrations [18, 20 and 22 % (w/w)]. All system components were mixed with 15% (w/w) of the fermented broth and deionized water was added to a final weight of 12.5 g. Then, the systems were mixed and kept at room temperature until to reach two-phases separation. Volumes of the top and bottom phases were measured, and aliquots from both phases were collected for subsequent proteolytic activity and total protein determination. Influence of variables such as PEG molar mass (MPEG), PEG concentration (CPEG), citrate concentration (CSal) and pH were evaluated on the following responses: purification factor (PF), activity yield (Y), partition coefficient (K) and selectivity (S). STATISTICA program version 10 was used for the analysis. According to the obtained results, higher levels of CPEG and MPEG had a positive effect on extraction, while pH did not influence on the process. On the other hand, the CSal could be related with low values of Y because of the citrate ions have a negative effect on solubility and enzymatic structure. The optimum values of Y (66.4 %), PF (1.8), K (5.5) and S (4.3) were obtained at CSal (18%), MPEG (6,000 g/mol), CPEG (22%) and pH 9. These results indicated that the PEG/citrate system is accurate to purify these Pseudomonas sp. proteases from fermented broth as a first purification step.
In vitro Protein Folding and Stability Using Thermostable Exoshells
Folding and stabilization of recombinant proteins remain a consistent challenge for industrial and therapeutic applications. Proteins derived from thermophilic bacteria often have superior expression and stability qualities. To develop a generalizable approach to protein folding and stabilization, we tested the hypothesis that wrapping a thermostable exoshell around a protein substrate would aid folding and impart thermostable qualities to the internalized substrate. To test the effect of internalizing a protein within a thermostable exoshell (tES), we tested in vitro folding and stability using green fluorescent protein (GFPuv), horseradish peroxidase (HRP) and renilla luciferase (rLuc). The 8nm interior volume of a thermostable ferritin assembly was engineered to accommodate foreign proteins and either present a positive, neutral or negative interior charge environment. We further engineered the tES complex to reversibly assemble and disassemble with pH titration. Template proteins were expressed as inclusion bodies and an in vitro folding protocol was developed that forced proteins to fold inside a single tES. Functional yield was improved 100-fold, 100-fold and 150-fold with use of tES for GFPuv, HRP and rLuc respectively and was highly dependent on the internal charge environment of the tES. After folding, functional proteins could be released from the tES folding cavity using size exclusion chromatography at pH 5.8. Internalized proteins were tested for improved stability against thermal, organic, urea and guanidine denaturation. Our results demonstrated that thermostable exoshells can efficiently refold and stabilize inactive aggregates into functional proteins.
Growth Performance Of fresh Water Microalgae Chlorella sp. Exposed to Carbon Dioxide
It is generally recognized, that algae could be an interesting option for reducing CO₂ emissions. Based on light and CO₂, algae can be used for the production various economically interesting products. Current algae cultivation techniques, however, still present a number of limitations. Efficient feeding of CO₂, especially on a large scale, is one of them. Current methods for CO₂ feeding to algae cultures rely on the sparging pure CO₂ or directly from flue gas. The limiting factor in this system is the solubility of CO₂ in water, which demands a considerable amount of energy for an effective gas to liquid transfer and leads to losses to the atmosphere. Due to the current ineffective methods for CO₂ introduction into algae ponds very large surface areas would be required for enough ponds to capture a considerable amount of the CO₂. The purpose of this study is to assess technology to capture carbon dioxide (CO₂) emissions generated by industry by utilizing of microalgae Chlorella sp. The microalgae were cultivated in a bioreactor culture pond raceway type. The result is expected to be useful in mitigating the effects of greenhouse gases in reducing the CO₂ emissions. The research activities include: (1) Characterization of boiler flue gas, (2) Operation of culture pond, (3) Sampling and sample analysis. The results of this study showed that the initial assessment absorption of the flue gas by microalgae using 1000 L raceway pond completed by heat exchanger were quite promising. The transfer of CO₂ into the pond culture system was run well. This identified from the success of cooling the boiler flue gas from the temperature of about 200 °C to below ambient temperature. Except for the temperature, the gas bubbles into the culture media were quite fine. Therefore, the contact between the gas and the media was well performed. The efficiency of CO₂ absorption by Chlorella sp reached 6.68 % with an average CO₂ loading of 0.29 g/L/day.
Influence of Pseudomonas japonica on Growth and Metal Tolerance of Celosia cristata L.
Heavy metals are one of the priority pollutants as they pose serious health and environmental threats. They can be removed by various physiochemical methods but are costly and responsible for additional environmental problems. Bioremediation that exploits plants and their associated microbes have been referred as cost effective and environmental friendly technique. In this study, a pot experiment was conducted in a greenhouse to evaluate the potential of Celosia cristata and effects of bacteria, Pseudomonas japonica, and organic amendment moss/compost on tolerating/accumulating heavy metals. Two weeks old seedlings were transferred to soil in pots, and after four weeks they were inoculated with bacterial strain, while after growth of six weeks they were watered with a metal containing synthetic wastewater and were harvested after a growth period of nine weeks. After harvesting, morphological and physiological parameters and metal content of plants were measured. The results showed highest plant growth and biomass production in case of organic amendments while highest metal uptake has been found in non-amended pots. Positive controls have shown highest Pb uptake of 2900 mg/kg DW, while P. japonica amended pots have shown highest Cd, Cr, Ni and Cu uptake of 963.53, 1481.17, 1022.01 and 602.17 mg/kg DW, respectively. In conclusion organic amendments have strong impacts on growth enhancement while P. japonica enhances metal translocation and accumulation to aerial parts with little significant involvement in plant growth.
Bulk Modification of Poly(Dimethylsiloxane) for Biomedical Applications
In the last decade microfabrication processes including rapid prototyping techniques have advanced rapidly and achieved a fairly matured stage. These advances encouraged and enabled the use of microfluidic devices by a wider range of users with applications in biological separations, and cell and organoid cultures. Accordingly, a significant current challenge in the field is controlling biomolecular interactions at interfaces and the development of novel biomaterials to satisfy the unique needs of the biomedical applications. Poly(dimethylsiloxane) (PDMS) is by far the most preferred material in the fabrication of microfluidic devices. This can be attributed its favorable properties, including: (1) simple fabrication by replica molding, (2) good mechanical properties, (3) excellent optical transparency from 240 to 1100 nm, (4) biocompatibility and non-toxicity, and (5) high gas permeability. However, high hydrophobicity (water contact angle ~108°±7°) of PDMS often limits its applications where solutions containing biological samples are concerned. In our study, we created a simple, easy method for modifying the surface chemistry of PDMS microfluidic devices through the addition of surface-segregating additives during manufacture. In this method, a surface segregating copolymer is added to precursors for silicone and the desired device is manufactured following the usual methods. When the device surface is in contact with an aqueous solution, the copolymer self-organizes to expose its hydrophilic segments to the surface, making the surface of the silicone device more hydrophilic. This can lead to several improved performance criteria including lower fouling, lower non-specific adsorption, and better wettability. Specifically, this approach is expected to be useful for the manufacture of microfluidic devices. It is also likely to be useful for manufacturing silicone tubing and other materials, biomaterial applications, and surface coatings.
Potentiality of Biohythane Process for the Gaseous Energy Recovery from Organic Wastes
A two-phase anaerobic process combining biohydrogen followed by biomethane (biohythane technology) serves as an environment-friendly and economically sustainable approach for the improved valorization of organic wastes. Suitability of the pure cultures like Klebsiela pneumonia, C. freundii, B. coagulan, etc. and mixed acidogenic cultures for the biohydrogen production was already studied. The characteristics of organic wastes play a critical role in biohydrogen production. The choice of an appropriate combination of complementary organic wastes can vastly improve the bioenergy generation besides achieving the significant cost reduction. Suitability and economic viability of using the groundnut deoiled cake (GDOC), mustard deoiled cake (MDOC), distillers’ dried grain with soluble (DDGS) and algal biomass (AB) as a co-substrate were studied for a biohythane production. Results show that maximum gaseous energy of 20.7, 9.3, 16.7 and 15.6 % was recovered using GDOC, MDOC, DDGS and AB in the two stage biohythane production, respectively. Both GDOC and DDGS were found to be better co-substrates as compared to MDOC and AB in terms of hythane production, respectively. The maximum cumulative hydrogen and methane production of 150 and 64 mmol/L were achieved using GDOC. Further, 98 % reduction in substrate input cost (SIC) was achieved using the co-supplementation procedure.
Differential Expression Analysis of Busseola fusca Larval Transcriptome in Response to Cry1Ab Toxin Challenge
Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stem borer, is a major pest in sub-Saharan Africa. It causes economic damage to maize and sorghum crops and has evolved non-recessive resistance to genetically modified (GM) maize expressing the Cry1Ab insecticidal toxin. Since B. fusca is a non-model organism, very little genomic information is publicly available, and is limited to some cytochrome c oxidase I, cytochrome b, and microsatellite data. The biology of B. fusca is well-described, but still poorly understood. This, in combination with its larval-specific behavior, may pose problems for limiting the spread of current resistant B. fusca populations or preventing resistance evolution in other susceptible populations. As part of on-going research into resistance evolution, B. fusca larvae were collected from Bt and non-Bt maize in South Africa, followed by RNA isolation (15 specimens) and sequencing on the Illumina HiSeq 2500 platform. Quality of reads was assessed with FastQC, after which Trimmomatic was used to trim adapters and remove low quality, short reads. Trinity was used for the de novo assembly, whereas TransRate was used for assembly quality assessment. Transcript identification employed BLAST (BLASTn, BLASTp, and tBLASTx comparisons), for which two libraries (nucleotide and protein) were created from 3.27 million lepidopteran sequences. Several transcripts that have previously been implicated in Cry toxin resistance was identified for B. fusca. These included aminopeptidase N, cadherin, alkaline phosphatase, ATP-binding cassette transporter proteins, and mitogen-activated protein kinase. MEGA7 was used to align these transcripts to reference sequences from Lepidoptera to detect mutations that might potentially be contributing to Cry toxin resistance in this pest. RSEM and Bioconductor were used to perform differential gene expression analysis on groups of B. fusca larvae challenged and unchallenged with the Cry1Ab toxin. Pairwise expression comparisons of transcripts that were at least 16-fold expressed at a false-discovery corrected statistical significance (p) ≤ 0.001 were extracted and visualized in a hierarchically clustered heatmap using R. A total of 329,194 transcripts with an N50 of 1,019 bp were generated from the over 167.5 million high-quality paired-end reads. Furthermore, 110 transcripts were over 10 kbp long, of which the largest one was 29,395 bp. BLAST comparisons resulted in identification of 157,099 (47.72%) transcripts, among which only 3,718 (2.37%) were identified as Cry toxin receptors from lepidopteran insects. According to transcript expression profiles, transcripts were grouped into three subclusters according to the similarity of their expression patterns. Several immune-related transcripts (pathogen recognition receptors, antimicrobial peptides, and inhibitors) were up-regulated in the larvae feeding on Bt maize, indicating an enhanced immune status in response to toxin exposure. Above all, extremely up-regulated arylphorin genes suggest that enhanced epithelial healing is one of the resistance mechanisms employed by B. fusca larvae against the Cry1Ab toxin. This study is the first to provide a resource base and some insights into a potential mechanism of Cry1Ab toxin resistance in B. fusca. Transcriptomic data generated in this study allows identification of genes that can be targeted by biotechnological improvements of GM crops.
Effect of Span 60, Labrasol and Cholesterol on Labisia pumila Loaded Niosomes Quality
Labisia pumila (LP) plant extract has the potential to be applied in cosmeceutical products due to its anti-photoaging properties. The main purpose of this study was to improve transdermal delivery of Labisia pumila by encapsulating LP in niosomes. Niosomes loaded LP were prepared by coacervation phase separation method using non-ionic surfactant (Span 60), labrasol and cholesterol. The optimum formula obtained were Span 60, Labrasol and Cholesterol at the mole ratio of 6:1:4. At the optimum formulation, the niosome obtained significantly improved the quality of transdermal penetration of LP compared to free LP.
Studies of Carbohydrate, Antioxidant, Nutrient and Genomic DNA Characterization of Fresh Olive Treated with Alkaline and Acidic Solvent: An Innovation
Fresh ripen olive cannot be consumed immediately after harvest due to the excessive bitterness having polyphenol as antioxidant. Industrial processing needs to be edible the fruit. The laboratory processing technique has been used to make it edible by using acid (vinegar, 5% acetic acid) and alkaline solvent (NaOH). Based on the treatment and consequence, innovative data have been found in this regard. The experiment was conducted to investigate biochemical content, nutritional and DNA characterization of olive fruit treated with alkaline (Sodium chloride anhydrous) and acidic solvent (5% acetic acid, vinegar). The treatments were used as control (no water), water control, 10% sodium chloride anhydrous (NaOH), vinegar (5% acetic acid), vinegar + NaOH and vinegar + NaOH + hot water treatment. Our results showed that inverted sugar and glucose content were higher in the vinegar and NaOH treated olive than in other treatments. Fructose content was the highest in vinegar + NaOH treated fruit. Nutrient contents NO3 K, Ca and Na were found higher in the treated fruit than the control fruit. Moreover, maximum K content was observed in the case of all treatments compared to the other nutrient content. The highest acidic (lower pH) condition (sour) was found in treated fruit. DNA yield was found higher in water control than acid and alkaline treated olives. DNA band was wider in the olive treated water control compared to the NaOH, vinegar, vinegar + NaOH and vinegar + NaOH + Hot water treatment. Finally, results suggest that vinegar + NaOH treated olive fruit was the best for fresh olive homemade processing after harvesting for edible purpose.