Search results for: chaperone assisted protein folding

Authors: Yuting Wu, Faraz Choudhury, Daniel Benjamin, James Whalin, Joshua Blatz, Leon Shohet, Michael Sussman, Mark Richards

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Plasma-Induced Modification of Biomolecules (PLIMB) has been developed as a technology, which, together with mass spectrometry, measures three-dimensional structural characteristics of proteins. This technique uses hydroxyl radicals generated by atmospheric-pressure plasma discharge to react with the solvent-accessible side chains of protein in an aqueous solution. In this work, we investigate the three-dimensional structure of hemoglobin and myoglobin using PLIMB. Additional modifications to these proteins, such as oxidation, fragmentations, and conformational changes caused by PLIMB are also explored. These results show that PLIMB, coupled with mass spectrometry, is an effective way to determine solvent access to hemoproteins. Furthermore, we show that many factors, including pH and the electrical parameters used to generate the plasma, have a significant influence on solvent accessibility.

Keywords: plasma, hemoglobin, myoglobin, solvent access

Procedia PDF Downloads 194

Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Sadeq Al Yaari, Ayman Al Yaari, Adham Al Yaari, Montaha Al Yaari, Aayah Al Yaari, Sajedah Al Yaari

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Background & Objectives: Audio-visual aids and computer-assisted language instruction (CALI) effects are strong in teaching language components (sound system, grammatical structures and vocabulary) to students of special needs. To explore the effects of the audio-visual aids and CALI in teaching sound system to this class of students by speech language therapists (SLTs), an experiment has been undertaken to evaluate their performance during their study of the sound system course. Methods: Forty students (males and females) of special needs at al-Malādh school for teaching students of special needs in Dhamar (Yemen) range between 8 and 18 years old underwent this experimental study while they were studying language sound system course. Pre-and-posttests have been administered at the begging and end of the semester. Students' treatment was compared to a similar group (control group) of the same number under the same environment. Whereas the first group was taught using audio-visual aids and CALI, the second was not. Students' performances were linguistically and statistically evaluated. Results & conclusions: Compared with the control group, the treatment group showed significantly higher scores in the posttest (72.32% vs. 31%). Compared with females, males scored higher marks (1421 vs. 1472). Thus, we should take the audio-visual aids and CALI into consideration in teaching sound system to students of special needs.

Keywords: language components, sound system, audio-visual aids, CALI, students, special needs, SLTs

Procedia PDF Downloads 48

Authors: Akhil Raj Pushparajan, Ranjit Ramachandran, Jijimole Gopi Reji, Ajay Kumar Ramakrishnan

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death by an infectious disease. In spite of the availability of effective drugs and a vaccine, TB is a major health concern and was declared a global emergency by the World Health Organization (WHO). The success of intracellular pathogens like Mtb depends on its ability to overcome the challenging environment in the host. Gene regulation controlled by transcriptional regulators (TRs) plays a crucial role for the bacteria to adapt to the host environment. In vitro studies on gene regulatory mechanisms during dormancy and reactivation have provided insights into the adaptations employed by Mtb to survive in the host. Here we present our efforts to functionally characterize Rv1019, a putative TR of Mtb H37Rv which was found to be present at significantly varying levels during dormancy and reactivation in vitro. The expression of this protein in the dormancy-reactivation model was validated by qRT-PCR and western blot. By DNA- protein interaction studies and reporter assays we found that under normal laboratory conditions of growth this protein behaves as an auto-repressor and tetracycline was found to abrogate this repression by interfering with its ability to bind DNA. Further, by cDNA analysis, we found that this TR is co-transcribed with its downstream genes Rv1020 (mfd) and Rv1021 (mazG) which are involved in DNA damage response in Mtb. Constitutive expression of this regulator in the surrogate host M. smegmatis showed downregulation of the orthologues of downstream genes suggested that Rv1019 could negatively regulate these genes. Our finds also show that M. smegmatis expressing Rv1019 is sensitive to DNA damage suggests the role of this protein in regulating DNA damage response induced by oxidative stress. Because of its role in regulating DNA damage response which may help in the persistence of Mtb, Rv1019 could be used as a prospective target for therapeutic intervention to fight TB.

Keywords: auto-repressor, DNA repair, mycobacterium smegmatis, mycobacterium tuberculosis, tuberculosis

Procedia PDF Downloads 139

Authors: Wael El-Deeb

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The present study aimed to investigate the diagnostic importance of acute phase proteins in urinary tract infection (UTI) in cattle. We describe the clinical, bacteriological and biochemical findings in 99 lactating cows. Blood and urine samples from diseased (n=84) and control healthy cows (n=15) were submitted to laboratory investigations. The urine analysis revealed hematuria and pyuria in UTI group. The isolated bacteria were E.coli (43/84) Corynebacterium spp, (31/84), Proteus spp. (6/84) and Streptococcus spp (4/84). The concentrations of Haptoglobin (Hp), serum amyloid A (SAA), α1-Acid glycoprotein (AGP), fibrinogen (Fb), total protein, albumen, and globulin were higher in cows with UTI when compared to healthy ones. Fifty-one of 84 cows with UTI were successfully treated. The levels of Hp, SAA, AGP, total protein, and globulin were associated with the odds of treatment failure. Conclusively, acute phase proteins could be used as diagnostic and prognostic biomarkers in cows with UTI.

Keywords: cows, urinary, infections, haptoglobin, serum Amyloid A

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Authors: A. Alsoqeer

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The establishment, dry matter production and feeding value of six perennial grasses were measured over two growing seasons in a field experiments. The experiments were conducted at the Agricultural and Veterinary Medicine Research Station, Faculty of Agriculture and Veterinary Medicine, Qassim University, Kingdom of Saudi Arabia in 2009 and 2010 seasons. The six perennial grasses were: creeping bluegrass (Bothriochloa insculpta cv. Bisset), digit grass (Digitaria smutsi), Jarra digit grass (Digitaria milanjiana), panic (Panicum coloratum cv. Bambatsii), Sabi grass (Urochloa mosambicensis) and setaria (Setaria sphacelata cv. Kazungula). The experimental design used was a completely randomized block design with four replications. The results revealed significant differences among plant species of all agronomic characters and quality traits in the first year, while in the second year, plant species differed significantly for quality traits only. D. smutsi had a superior performance for all agronomic characters, however, it had the lowest values in protein content in the two years comparing with other genotypes. D. milanjiana and U. mosambicensis showed high values in dry matter yield and protein content in the first year, but showed a very poor performance in the second year because most of plants were die due to the low temperatures in the winter. These two species appear to be suitable for annual cultivation. The other species tolerate the cold winter and were a highly productive in the second year.

Keywords: dry mater yield, grass species, cuts, quality traits, crude protein content

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Authors: Mauricio Quintero Angel, Alexander Pereira, Selene Alarcón

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One of the elements of greatest importance in agricultural production is the harvesting; an activity associated to different occupational health risks such as harvesting in high altitudes, the transport of heavy materials and the application of excessive muscle strain that leads to muscular-bone disorders. Therefore, there is an urgent necessity to improve and validate interventions to reduce exposition and risk to harvesters. This article has the objective of describing the co-development under the ergonomic analysis framework of an assisted manual harvesting tool for peach palm oriented to reduce the risk of death and accidents as it avoid the harvest in heights. The peach palm is a palm tree that is cultivated in Colombia, Perú, Brasil, Costa Rica, among others and that reaches heights of over 20 m, with stipes covered with spines. The fruits are drupes of variable size. For the harvesting of peach palm, in Colombia farmers use the “Marota” or “Climber”, a tool in a closed X shape built in wood, that has two supports adjusted at the stipe, that elevate alternately until reaching a point high enough to grab the bunch that is brought down using a rope. An activity of high risk since it is done at a high altitude without any type of protection and safety measures. The Marota is alternated with a rod, which as variable height between 5 and 12 Meters with a harness system at one end to hold the bunch that is lowered with the whole system (bamboo bunch). The rod is used from the ground or from the Marota in height. As an alternative to traditional tools, the Bajachonta was co-developed with farmers, a tool that employs a traditional bamboo hook system with modifications, to be able to hold it with a rope that passes through a pulley. Once the bunch is hitched, the hook system is detached and this stays attached to the peduncle of the palm tree, afterwards through a pulling force being exerted towards the ground by tensioning the rope, the bunch comes loose to be taken down using a rope and the pulley system to the ground, reducing the risk and efforts in the operation. The bajachonta was evaluated in tree productive zones of Colombia, with innovative farmers, were the adoption is highly probable, with some modifications to improve its efficiency and effectiveness, keeping in mind that the farmers perceive in it an advantage in the reduction of death and accidents by not having to harvest in heights.

Keywords: assisted harvesting, ergonomics, harvesting in high altitudes, participative design, peach palm

Procedia PDF Downloads 408

Authors: P. M. Ikonić, T. A. Tasić, L. S. Petrović, S. B. Škaljac, M. R. Jokanović, V. M. Tomović, B. V. Šojić, N. R. Džinić, A. M. Torbica, B. B. Ikonić

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The aim of the study was to determine how different ripening processes (traditional vs. industrial) influenced the proteolysis in traditional Serbian dry-fermented sausage Petrovská klobása. The obtained results indicated more intensive pH decline (0.7 units after 9 days) in industrially ripened products (I), what had a positive impact on drying process and proteolytic changes in these samples. Thus, moisture content in I sausages was lower at each sampling time, amounting 24.7% at the end of production period (90 days). Likewise, the process of proteolysis was more pronounced in I samples, resulting in higher contents of non-protein nitrogen (NPN) and free amino acids nitrogen (FAAN), as well as in faster and more intensive degradation of myosin (≈220 kDa), actin (≈45 kDa) and other polypeptides during processing. Consequently, the appearance and accumulation of several protein fragments were registered.

Keywords: dry-fermented sausage, Petrovská klobása, proteolysis, ripening process

Procedia PDF Downloads 333

Authors: Hien Thi Thu Le, Nuno Rafael Candeias, Olli Yli-Harja, Meenakshisundaram Kandhavelu

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Purinergic receptor 1 (P2Y1R) is the potential therapeutic target for inducing prostate cancer (PCa) cell death. Recently, 1-indolinoalkyl 2-phenolic derivative, HIC, was identified as a P2Y1R agonist that increases apoptosis and inhibits cell proliferation of PCa. However, the biological effects of HIC have not been extensively studied at the molecular level. In the present study, we have investigated the anticancer effects of HIC and the molecular mechanisms underlying in PCa cells. Half maximal inhibitory concentration (IC₅₀) of HIC was measured as 15.98 μM and 15.64 μM for DU145 and PC3 cells, respectively. In addition, we found that HIC inhibited cell growth and metastasis of PC3 and DU145 cells colonies, spheroid areas, and migrated cells. RNA seq analysis revealed significant changes of over 3000 genes (p value < 0.05) upon HIC treatment in PC3 and DU145 cells. Genes involved in DNA damage, apoptosis, cell cycle arrest at G1/S phase were modulated by HIC treatment. MAPK and NF-κB protein array revealed the increased expression of ERK1/2, JNK1/2, p53 phosphorylation, and p53 protein. ERK1/2 and JNK1/2 activations are known to increase the stabilization of p53, a tumor suppressor protein, which is required to arrest the cell cycle at G1/S phase and cause cell death of PCa cells. Overall, our results suggest that HIC can serve as a multi-dimensional chemotherapeutic agent possessing strong cytotoxic, anti-cancer, and anti-metastasis against PCa growth.

Keywords: prostate cancer, P2Y1 receptor, apoptosis, metastasis

Procedia PDF Downloads 133

Authors: Rabitah Zakaria, Chan M. Luan, Nor Hakimah Ramly

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The extraction of vegetable oil using aqueous extraction process assisted by ionic extended surfactant has been investigated as an alternative to hexane extraction. However, the ionic extended surfactant has not been commercialised and its safety with respect to food processing is uncertain. Hence, food-grade non-ionic surfactants (Tween 20, Span 20, and Span 80) were proposed for the extraction of residual oil from palm-pressed mesocarp fibre. Palm-pressed mesocarp fibre contains a significant amount of residual oil ( 5-10 wt %) and its recovery is beneficial as the oil contains much higher content of vitamin E, carotenoids, and sterols compared to crude palm oil. In this study, the formulation of food-grade surfactants using a combination of high hydrophilic-lipophilic balance (HLB) surfactants and low HLB surfactants to produce micro-emulsion with very low interfacial tension (IFT) was investigated. The suitable surfactant formulation was used in the oil extraction process and the efficiency of the extraction was correlated with the IFT, droplet size and viscosity. It was found that a ternary surfactant mixture with a HLB value of 15 (82% Tween 20, 12% Span 20 and 6% Span 80) was able to produce micro-emulsion with very low IFT compared to other HLB combinations. Results suggested that the IFT and droplet size highly affect the oil recovery efficiency. Finally, optimization of the operating parameters shows that the highest extraction efficiency of 78% was achieved at 1:31 solid to liquid ratio, 2 wt % surfactant solution, temperature of 50˚C, and 50 minutes contact time.

Keywords: food-grade surfactants, aqueous extraction of residual oil, palm-pressed mesocarp fibre, interfacial tension

Procedia PDF Downloads 390

Authors: Manal Kamel, Sara Maher, Hanan El Baz, Faten Salah, Omar Sayyouh, Zeinab Demerdash

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In the recent COVID 19 pandemic, experts of public health have emphasized testing, tracking infected people, and tracing their contacts as an effective strategy to reduce the spread of the virus. Development of rapid and sensitive diagnostic assays to replace reverse transcription polymerase chain reaction (RT-PCR) is mandatory..Our innovative test strip relying on the application of nanoparticles conjugated to recombinant nanobodies for SARS-COV-2 spike protein (S1) & angiotensin-converting enzyme 2 (that is responsible for the virus entry into host cells) for rapid detection of SARS-COV-2 spike protein (S1) in saliva or sputum specimens. Comparative tests with RT-PCR will be held to estimate the significant effect of using COVID 19 nanobodies for the first time in the development of lateral flow test strip. The SARS-CoV-2 S1 (3 ng of recombinant proteins) was detected by our developed LFIA in saliva specimen of COVID-19 Patients No cross-reaction was detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS- CoV antigens..Our developed system revealed 96 % sensitivity and 100% specificity for saliva samples compared to 89 % and 100% sensitivity and specificity for nasopharyngeal swabs. providing a reliable alternative for the painful and uncomfortable nasopharyngeal swab process and the complexes, time consuming PCR test. An increase in testing compliances to be expected.

Keywords: COVID 19, diagnosis, LFIA, nanobodies, ACE2

Procedia PDF Downloads 137

Authors: Rana Bilal, Faiz-Ul-Hassan, Moazzam Jameel

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There is a dire need to replace high-cost protein with more economical protein to overcome animal protein shortage in developing nations especially countries like Pakistan. In conjunction with these efforts, the current study was planned to evaluate the effects of various dried distillery yeast sludge (DYS) levels on the immune level, egg quality, and performance of layers by replacing soybean meal. The study was designed with two hundred layers of Hy-Line variety. Distillery yeast sludge was dried and ground for 2 mm mesh size and after this proximate and mineral analysis was determined. Five isocaloric and isonitrogeneous feeds were given containing C (control), 5, 10, 15, 20% distillery yeast sludge by replacing soybean meal. The trial was performed in the completely randomized design with five treatments, 4 replicates and 10 hen per replicate. Results demonstrated that feed intake, egg production, feed conversion ratio decreased (P < 0.05) with the increased dietary DYS. However, statistically significant decrease (P < 0.05) was found in hens having DYS20 diet than control. Layers on Diets C, DYS5 and DYS10 exerted a higher immune level than DYS15 and DYS20 diets. Egg weight, eggshell weight, eggshell thickness, egg albumen height as well as haugh unit score were affected significantly by the increased level of DYS. In general, results of this study demonstrated that inclusion of DYS up to 10% showed no adverse effects on health and performance of layers.

Keywords: egg quality, immunity, layers, performance

Procedia PDF Downloads 233

Authors: R. S. Yadav, J. Havlica, I. Kuřitka, Z. Kozakova, J. Masilko, L. Kalina, M. Hajdúchová, V. Enev, J. Wasserbauer

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In this work, copper ferrite CuFe2O4 spinel ferrite nanoparticles with different particle size at different annealing temperature were synthesized using the starch-assisted sol-gel auto-combustion method. The synthesized nanoparticles were characterized by conventional powder X-ray diffraction (XRD) spectroscopy, Raman Spectroscopy, Fourier Transform Infrared Spectroscopy, Field-Emission Scanning Electron Microscopy, X-ray Photoelectron Spectroscopy, and Vibrating Sample Magnetometer. The XRD patterns confirmed the formation of CuFe2O4 spinel ferrite nanoparticles. Field-Emission Scanning Electron Microscopy revealed that particles are of spherical morphology with particle size 5-20 nm at lower annealing temperature. An infrared spectroscopy study showed the presence of two principal absorption bands in the frequency range around 530 cm-1 (ν1) and around 360 cm-1 (ν2); which indicate the presence of tetrahedral and octahedral group complexes, respectively, within the spinel ferrite nanoparticles. Raman spectroscopy study also indicated the change in octahedral and tetrahedral site related Raman modes in copper ferrite nanoparticles with change of particle size. This change in magnetic behavior with change of particle size of CuFe2O4 nanoparticles was also observed. The change in magnetic properties with change of particle size is due to cation redistribution, which was confirmed by X-Ray photoelectron study.

Keywords: copper ferrite, nanoparticles, magnetic property, CuFe2O4

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Authors: Shalini Singh, Sanjay K. Srivastava, Govind, Mukhtar. A. Khan, P. K. Singh

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Nanotechnology is revolutionizing the development of biosensors. Nanomaterials and nanofabrication technologies are increasingly being used to design novel biosensors. Sensitivity and other attributes of biosensors can be improved by using nanomaterials with unique chemical, physical, and mechanical properties in their construction. Silicon is a promising biomaterial that is non-toxic and biodegradable and can be exploited in chemical and biological sensing. Present study demonstrated the streptavidin–biotin interaction on silicon surfaces with different topographies such as flat and nanostructured silicon (nanowires) surfaces. Silicon nanowires with wide range of surface to volume ratio were prepared by electrochemical etching of silicon wafer. The large specific surface of silicon nanowires can be chemically modified to link different molecular probes (DNA strands, enzymes, proteins and so on), which recognize the target analytes, in order to enhance the selectivity and specificity of the sensor device. The interaction of streptavidin with biotin was carried out on 3-aminopropyltriethoxysilane (APTS) functionalized silicon surfaces. Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS) studies have been performed to characterize the surface characteristics to ensure the protein attachment. Silicon nanowires showed the enhance protein attachment, as compared to flat silicon surface due to its large surface area and good molecular penetration to its surface. The methodology developed herein could be generalized to a wide range of protein-ligand interactions, since it is relatively easy to conjugate biotin with diverse biomolecules such as antibodies, enzymes, peptides, and nucleotides.

Keywords: FTIR, silicon nanowires, streptavidin-biotin, XPS

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Authors: M. Dehestani, F. Kamali, M. Klantari Pour, L. Zeidabadi-Nejad

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Chemical bonding between polyethylene glycol (PEG) with pharmaceutical proteins called pegylation is one of the most effective methods of improving the pharmacological properties. The covalent attachment of polyethylene glycol (PEG) to proteins will increase their pharmacologic properties. For the formation of a combination of pegylated protein should first be activated PEG and connected to the protein. Interferons(IFNs) are a family of cytokines which show antiviral effects in front of the biological and are responsible for setting safety system. In this study, the nature and properties of the interaction between active positions of IFNs and polyethylene glycol have been investigated using molecular dynamics simulation. The main aspect of this theoretical work focuses on the achievement of valuable data on the reaction pathways of PEG-IFNs and the transition state energy. Our results provide a new perspective on the interactions, chemical properties and reaction pathways between IFNs and PEG.

Keywords: interaction, interferons, molecular dynamics simulation, polyethylene glycol

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Authors: Eric Granholm, Christophe Delay, Jason Holden, Peter Link

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Negative symptoms are an important unmet treatment needed for schizophrenia. We conducted an open trial of a novel blended intervention called mobile-assisted cognitive behavior therapy for negative symptoms (mCBTn). mCBTn is a weekly group therapy intervention combining in-person and smartphone-based CBT (CBT2go app) to improve experiential negative symptoms in people with schizophrenia. Both the therapy group and CBT2go app included recovery goal setting, thought challenging, scheduling of pleasurable activities and social interactions, and pleasure savoring interventions to modify defeatist attitudes, a target mechanism associated with negative symptoms, and improve experiential negative symptoms. We tested whether participants with schizophrenia or schizoaffective disorder (N=31) who met prospective criteria for persistent negative symptoms showed improvement in experiential negative symptoms. Retention was excellent (87% at 18 weeks) and severity of defeatist attitudes and motivation and pleasure negative symptoms declined significantly in mCBTn with large effect sizes. We also tested whether pupillary responses, a measure of cognitive effort, predicted improvement in negative symptoms mCBTn. Pupillary responses were recorded at baseline using a Tobii pupillometer during the digit span task with 3-, 6- and 9-digit spans. Mixed models showed that greater dilation during the task at baseline significantly predicted a greater reduction in experiential negative symptoms. Pupillary responses may provide a much-needed prognostic biomarker of which patients are most likely to benefit from CBT. Greater pupil dilation during a cognitive task predicted greater improvement in experiential negative symptoms. Pupil dilation has been linked to motivation and engagement of executive control, so these factors may contribute to benefits in interventions that train cognitive skills to manage negative thoughts and emotions. The findings suggest mCBTn is a feasible and effective treatment for experiential negative symptoms and justify a larger randomized controlled clinical trial. The findings also provide support for the defeatist attitude model of experiential negative symptoms and suggest that mobile-assisted interventions like mCBTn can strengthen and shorten intensive psychosocial interventions for schizophrenia.

Keywords: cognitive-behavioral therapy, mobile interventions, negative symptoms, pupillometry schizophrenia

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Authors: Yunha Ryu, Kyoungsik Kim

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Raman spectroscopy is one of the most powerful techniques for chemical detection, but the low sensitivity originated from the extremely small cross-section of the Raman scattering limits the practical use of Raman spectroscopy. To overcome this problem, Surface Enhanced Raman Scattering (SERS) has been intensively studied for several decades. Because the SERS effect is mainly induced from strong electromagnetic near-field enhancement as a result of localized surface plasmon resonance of metallic nanostructures, it is important to design the plasmonic structures with high density of electromagnetic hot spots for SERS substrate. One of the useful fabrication methods is using porous nanomaterial as a template for metallic structure. Internal pores on a scale of tens of nanometers can be strong EM hotspots by confining the incident light. Also, porous structures can capture more target molecules than non-porous structures in a same detection spot thanks to the large surface area. Herein we report the facile fabrication method of porous SERS substrate by integrating solvent-assisted nanoimprint lithography and selective etching of block copolymer. We obtained nanostructures with high porosity via simple selective etching of the one microdomain of the diblock copolymer. Furthermore, we imprinted of the nanocone patterns into the spin-coated flat block copolymer film to make three-dimensional SERS substrate for the high density of SERS hot spots as well as large surface area. We used solvent-assisted nanoimprint lithography (SAIL) to reduce the fabrication time and cost for patterning BCP film by taking advantage of a solvent which dissolves both polystyrenre and poly(methyl methacrylate) domain of the block copolymer, and thus block copolymer film was molded under the low temperature and atmospheric pressure in a short time. After Ag deposition, we measured Raman intensity of dye molecules adsorbed on the fabricated structure. Compared to the Raman signals of Ag coated solid nanocone, porous nanocone showed 10 times higher Raman intensity at 1510 cm(-1) band. In conclusion, we fabricated porous metallic nanocone arrays with high density electromagnetic hotspots by templating nanoimprinted diblock copolymer with selective etching and demonstrated its capability as an effective SERS substrate.

Keywords: block copolymer, porous nanostructure, solvent-assisted nanoimprint, surface-enhanced Raman spectroscopy

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Authors: Sujata Sharma, Avinash Singh, Lovely Gautam, Pradeep Sharma, Mau Sinha, Asha Bhushan, Punit Kaur, Tej P. Singh

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Peptidyl-tRNA hydrolase (Pth) Pth is an essential bacterial enzyme that catalyzes the release of free tRNA and peptide moeities from peptidyl tRNAs during stalling of protein synthesis. In order to design inhibitors of Pth from Pseudomonas aeruginosa (PaPth), we have determined the structures of PaPth in its native state and in the bound states with two compounds, amino acylate-tRNA analogue (AAtA) and 5-azacytidine (AZAC). The peptidyl-tRNA hydrolase gene from Pseudomonas aeruginosa was amplified by Phusion High-Fidelity DNA Polymerase using forward and reverse primers, respectively. The E. coliBL21 (λDE3) strain was used for expression of the recombinant peptidyl-tRNA hydrolase from Pseudomonas aeruginosa. The protein was purified using a Ni-NTA superflow column. The crystallization experiments were carried out using hanging drop vapour diffusion method. The crystals diffracted to 1.50 Å resolution. The data were processed using HKL-2000. The polypeptide chain of PaPth consists of 194 amino acid residues from Met1 to Ala194. The centrally located β-structure is surrounded by α-helices from all sides except the side that has entrance to the substrate binding site. The structures of the complexes of PaPth with AAtA and AZAC showed the ligands bound to PaPth in the substrate binding cleft and interacted with protein atoms extensively. The residues that formed intermolecular hydrogen bonds with the atoms of AAtA included Asn12, His22, Asn70, Gly113, Asn116, Ser148, and Glu161 of the symmetry related molecule. The amino acids that were involved in hydrogen bonded interactions in case of AZAC included, His22, Gly113, Asn116, and Ser148. As indicated by fittings of two ligands and the number of interactions made by them with protein atoms, AAtA appears to be a more compatible with the structure of the substrate binding cleft. However, there is a further scope to achieve a better stacking than that of O-tyrosyl moiety because it is not still ideally stacked. These observations about the interactions between the protein and ligands have provided the information about the mode of binding of ligands, nature and number of interactions. This information may be useful for the design of tight inhibitors of Pth enzymes.

Keywords: peptidyl tRNA hydrolase, Acinetobacter baumannii, Pth enzymes, O-tyrosyl

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Authors: M. Mortada, A. Seleem, M. El-Morsi, M. Younan

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The present study introduces the folding technology to be utilized for the first time in vertical diffusion stills. This work represents a model of the distillation process by utilizing chevron pattern of folded structure. An experimental setup has been constructed, to investigate the performance of the folded sheets in the vertical effect diffusion still for a specific range of operating conditions. An experimental comparison between the folded type and the flat type sheets has been carried out. The folded pattern showed a higher performance and there is an increase in the condensate to feed ratio that ranges from 20-30 % through the operating hot plate temperature that ranges through 60-90°C. In addition, a parametric analysis of the system using Design of Experiments statistical technique, has been developed using the experimental results to determine the effect of operating conditions on the system's performance and the best operating conditions of the system has been evaluated.

Keywords: chevron pattern, fold structure, solar distillation, vertical diffusion still

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Authors: Lebogang L. R. Mphahlele, Bruce B. Sithole

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Chicken meat is the highest consumed meat in south Africa, with a per capita consumption of >33 kg yearly. Hence, South Africa produces over 250 million kg of waste chicken feathers each year, the majority of which is landfilled or incinerated. The discarded feathers have caused environmental pollution and natural protein resource waste. Therefore, the valorisation of waste chicken feathers is measured as a more environmentally friendly and cost-effective treatment. Feather contains 91% protein, the main component being beta-keratin, a fibrous and insoluble structural protein extensively cross linked by disulfide bonds. Keratin is usually converted it into nanofibers via electrospinning for a variety of applications. keratin nanofiber composites have many potential biomedical applications for their attractive features, such as high surface-to-volume ratio and very high porosity. The application of nanofibers in the biomedical wound dressing requires antimicrobial properties for materials. One approach is incorporating inorganic nanoparticles, among which silver nanoparticles played an important alternative antibacterial agent and have been studied against many types of microbes. The objective of this study is to combine synthetic polymer, chicken feather keratin, and antibacterial nanoparticles to develop novel electrospun antibacterial nanofibrous composites for possible wound dressing application. Furthermore, this study will converting a two-dimensional electrospun nanofiber membrane to three-dimensional fiber networks that resemble the structure of the extracellular matrix (ECM)

Keywords: chicken feather keratin, nanofibers, nanoparticles, nanocomposites, wound dressing

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Authors: Hanaa A. S. Oraby, Amal A. M. Hassan, Mahmoud M. Sakr, Atef A. A. Haiba

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Applying the profiling approach for the assessment of proteomic variations due to genetic modification of the Egyptian tomato cultivar "Edkawy", three complementary approaches were used. These methods are amino acids analysis, gel electrophoresis, and Gas chromatography coupled with mass spectrometry (GC/MS). The results of the present study Show evidence of proteomic variations between both modified tomato and its non-modified counterpart. Amino acids concentrations, and the protein patterns separation on the 1D SDS-PAGE were not similar in the case of transformed tomato compared to that of the non-transformed counterpart. These detected differences are most likely derived from the process of transformation. Results also revealed that the efficiency of GC/MS approach to identify a mixture of unknown proteins is limited. GC/MS analysis was only able to identify few number of protein molecules. Therefore, more advanced and specific technologies like MALDI-TOF-MS are recommended to be employed.

Keywords: GMOs, unintended effects, proteomic variations, 1D SDS-PAGE, GC/MS

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Authors: M. Cherki, I. Taouam, A. Amiri, F. Hmimid, T. Ould Bellahcen

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The Moroccan coasts represent an important wealth of red algae which have an economic interest. Among these algae, the Gelidium sesquipedale, which is exploited industrially for its richness in agar. The aim of this study is to establish a general overview of the macronutrient composition of Gelidium sesquipedale and to compare this composition according to three factors: the harvest site (El Jadida, Casablanca and Mohammadia), the harvest depth (coast and depth) and the drying process (open air and oven). Proteins, lipids, and carbohydrates are measured by different methods. The analysis of results show that the protein concentrations of the El Jadida and Mohammadia samples are significantly higher than that of Casablanca (0.026 ± 0.0007 µg/µg DW 0.024 ± 0.001 µg/µg DW and 0.006 ± 0.0007 µg/µg DW, p < 0.05 respectively). However, Casablanca samples are significantly richer in total sugars (0.023 ± 0.002 µg/µg DW, p < 0.05) and less rich in reducing sugars (0.0001 ± 0.00001 µg/µg DW, p < 0.05) compared to other samples. The lipid concentrations of the samples from the three harvest sites do not show any significant difference. With respect to depth, only total protein and total sugar concentrations were significantly higher in the coast versus depth samples (0.035 ± 0.004 µg/µg DW vs. 0.026 ± 0.0007 µg/µg DW and 0.035 ± 0.006 µg/µg DW vs. 0.012 ± 0.005 µg/µg DW p < 0.05 respectively). For the drying process, protein, total sugars and lipid concentrations were significantly higher in open air samples compared to oven samples (0.006 ± 0.0007 µg/µg DW). vs 0.004 ± 0.0003 µg/µg DW, 0.023 ± 0.002 µg/µg DW vs 0.007 ± 0.002 µg/µg DW and 8% vs 4% p < 0.05 respectively). Our results demonstrate that the chemical composition of Gelidium sesquipedale varies according to the harvest region. In addition, samples harvested on the coast and dried in the open air are the richest in macronutrients.

Keywords: biochemical composition, drying, depth, Gelidium sesquipedale, red algae, region

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Authors: Romeo A. Botes, Imelda Smit

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The demands of an ever changing and complex higher education environment, along with the profile of modern learners challenge current approaches to assessment and feedback. More learners enter the education system every year. The younger generation expects immediate feedback. At the same time, feedback should be meaningful. The assessment of practical activities in programming poses a particular problem, since both lecturers and learners in the information and computer science discipline acknowledge that paper-based assessment for programming subjects lacks meaningful real-life testing. At the same time, feedback lacks promptness, consistency, comprehensiveness and individualisation. Most of these aspects may be addressed by modern, technology-assisted assessment. The focus of this paper is the continuous development of an artefact that is used to assist the lecturer in the assessment and feedback of practical programming activities in a senior database programming class. The artefact was developed using three Design Science Research cycles. The first implementation allowed one programming activity submission per assessment intervention. This pilot provided valuable insight into the obstacles regarding the implementation of this type of assessment tool. A second implementation improved the initial version to allow multiple programming activity submissions per assessment. The focus of this version is on providing scaffold feedback to the learner – allowing improvement with each subsequent submission. It also has a built-in capability to provide the lecturer with information regarding the key problem areas of each assessment intervention.

Keywords: programming, computer-aided assessment, technology-assisted assessment, programming assessment software, design science research, mixed-method

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Authors: G. C. Jadeja, M. A. Desai, D. R. Bhatt, J. K. Parikh

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Green extraction techniques are fast paving ways into various industrial sectors due to the stringent governmental regulations leading to the banning of toxic chemicals’ usage and also due to the increasing health/environmental awareness. The present work describes the ionic liquids based sonication method for selectively extracting patchoulol from the leaves of patchouli. 1-Butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) and N,N,N,N’,N’,N’-Hexaethyl-butane-1,4-diammonium dibromide (dicationic ionic liquid - DIL) were selected for extraction. Ultrasound assisted ionic liquid extraction was employed considering concentration of ionic liquid (4–8 %, w/w), ultrasound power (50–150 W for [Bmim]BF4 and 20–80 W for DIL), temperature (30–50 oC) and extraction time (30–50 min) as major parameters influencing the yield of patchoulol. Using the Taguchi method, the parameters were optimized and analysis of variance (ANOVA) was performed to find the most influential factor in the selected extraction method. In case of [Bmim]BF4, the optimum conditions were found to be: 4 % (w/w) ionic liquid concentration, 50 W power, 30 oC temperature and extraction time of 30 min. The yield obtained under the optimum conditions was 3.99 mg/g. In case of DIL, the optimum conditions were obtained as 6 % (w/w) ionic liquid concentration, 80 W power, 30 oC temperature and extraction time of 40 min, for which the yield obtained was 4.03 mg/g. Temperature was found to be the most significant factor in both the cases. Extraction time was the insignificant parameter while extracting the product using [Bmim]BF4 and in case of DIL, power was found to be the least significant factor affecting the process. Thus, a green method of recovering patchoulol is proposed.

Keywords: green extraction, ultrasound, patchoulol, ionic liquids

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Authors: Kai Pai Huang

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Background: There are several types of plasma cell neoplasms including multiple myeloma, plasmacytoma, lymphoplasmacytic lymphoma, and monoclonal gammopathy of undetermined significance (MGUS) are found in our lab. Today, we want to compare with two cases using the method of capillary electrophoresis. Method: Serum is prepared and electrophoresis is performed at alkaline PH in a capillary using the Sebia® Capillary 2. Albumin and globulins are detected by the detector which is located in the cathode of the capillary and the signals are transformed to peaks. Serum was treated with beta-mercaptoethanol which reducing the polymerized immunoglobulin to monomer immunoglobulin to clarify two M-protein are secreted from the same plasma cell clone in bone marrow. Result: Case 1: A 78-year-old female presenting dysuria, oliguria and leg edema for several months. Laboratory data showed proteinuria, leukocytosis, results of high serum IgA and lambda light chain. A renal biopsy found amyloid fibrils in the glomerular mesangial area. Serum protein electrophoresis shows a major monoclonal peak in the β region and minor small peak in gamma region, and the immunotyping studies for serum showed two IgA/λ type. Case 2: A 55-year-old male presenting abdominal distension and low back pain for more than one month. Laboratory data showed T12 T8 compression fracture, results of high serum IgM and kappa light chain. Bone marrow aspiration showed the cells from the bone marrow are B cells with monotypic kappa chain expression. Bone marrow biopsy found this is lymphoplasmacytic lymphoma (Waldenstrom macroglobulin). Serum protein electrophoresis shows a monoclonal peak in the β region and the immunotyping studies for serum showed IgM/κ type. Conclusion: Plasma cell neoplasm can be diagnosed by many examinations. Among them, using capillary electrophoresis by a lab can separate several types of gammopathy and the quantification of a monoclonal peak can be used to evaluate the patients’ prognosis or treatment.

Keywords: plasma cell neoplasm, capillary electrophoresis, serum protein electrophoresis, immunotyping

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Authors: Sulhee Lee, Geon Kim, Young-Seo Park

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Unlike the traditional milk sterilization methods (LTLT, HTST, or UHT), pulsed electric field (PEF) technology is a non-thermal pasteurization process. This technology minimizes energy required for heat treatment in food processing, changes in sensory properties, and physical losses. In this study, structural changes of bovine milk proteins, the amount of immunoproteins such as IgG, and their storability by PEF treatment were examined. When the changes of protein content in PEF-treated milk were examined using HPLC, the amounts of α-casein and β-lactoglobulin were reduced over 40% each, whereas those of κ-casein and β-casein did not change. The amount of α-casein in HTST milk was reduced to 50%. When PEF was applied to milk at the energy level of 250 kJ, the amounts of IgG, IgA, β-lactoglobulin (β-LG), lactoferrin, and α-lactalbumin (α-LA) decreased by 43, 41, 35, 63, and 45%, respectively. When milk was sterilized by LTLT process followed by PEF process at the level of 150 kJ, the concentrations of IgG, IgA, β-LG, lactoferrin, and α-LA were 56.6, 10.6, 554, 2.8 and 660.1 μg/mL, respectively. When the bovine milk was sterilized by LTLT process followed by PEF process at the energy level of 180 kJ, storability of immunoproteins of milk was the highest and the concentrations of IgG, IgA, and β-LG decreased by 79.5, 6.5, and 134.5 μg/mL, respectively, when compared with the initial concentrations of those proteins. When bovine milk was stored at 4℃ after sterilization through HTST sterilizer followed by PEF process at the energy level of 200 kJ, the amount of lactoferrin decreased 7.3% after 36 days of storage, whereas that of lactoferrin of raw milk decreased 16.4%. Our results showed that PEF treatment did not change the protein structure nor induce protein denaturation in milk significantly when compared with LTLT or HTST sterilization. Also, LTLT or HTST process in combination with PEF were more effective than LTLT only or HTST only process in the conservation of immunoproteins in bovine milk.

Keywords: pulsed electric field, bovine milk, immunoproteins, sterilization

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Authors: Jayaraman Muniyappan, Bachchan Kr Mishra, Gautam Salkar, Swetha Manian Sridhar

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Vacuum Assisted Resin Transfer Molding (VARTM), a cost effective method of Liquid Composite Molding (LCM), is a single step process where the resin, at atmospheric pressure, is infused through a preform that is maintained under vacuum. This hydrodynamic pressure gradient is responsible for the flow of resin through the dry fabric preform. The current study has a slight variation to traditional VARTM, wherein, the resin infuses through the fabric placed on a heated mold to reduce its viscosity. The saturated preform is subjected to a cure cycle where the resin hardens as it undergoes curing. During this cycle, an uneven temperature distribution through the thickness of the composite and excess exothermic heat released due to different cure rates result in non-uniform curing. Additionally, there is a difference in thermal expansion coefficient between fiber and resin in a given plane and between adjacent plies. All these effects coupled with orthotropic coefficient of thermal expansion of the composite give rise to process-induced stresses in the laminate. Such stresses lead to part deformation when the laminate tries to relieve them as the part is released off the mold. The current study looks at simulating resin infusion, cure kinetics and the structural response of composite laminate subject to process-induced stresses.

Keywords: cure kinetics, process-induced stresses, thermal expansion coefficient, vacuum assisted resin transfer molding

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Authors: S. D. N. K. Bathige, G. I. Godahewa, Navaneethaiyer Umasuthan, Jehee Lee

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Kunitz-type serine protease inhibitors (KTIs) are identified in various organisms including animals, plants and microbes. These proteins shared single or multiple Kunitz inhibitory domains link together or associated with other types of domains. Characteristic Kunitz type domain composed of around 60 amino acid residues with six conserved cysteine residues to stabilize by three disulfide bridges. KTIs are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis and inflammation. In this study, two Kunitz-type domain containing protein was identified from rock bream database and designated as RbKunitz. The coding sequence of RbKunitz encoded for 507 amino acids with 56.2 kDa theoretical molecular mass and 5.7 isoelectric point (pI). There are several functional domains including MANEC superfamily domain, PKD superfamily domain, and LDLa domain were predicted in addition to the two characteristic Kunitz domain. Moreover, trypsin interaction sites were also identified in Kunitz domain. Homology analysis revealed that RbKunitz shared highest identity (77.6%) with Takifugu rubripes. Completely conserved 28 cysteine residues were recognized, when comparison of RbKunitz with other orthologs from different taxonomical groups. These structural evidences indicate the rigidity of RbKunitz folding structure to achieve the proper function. The phylogenetic tree was constructed using neighbor-joining method and exhibited that the KTIs from fish and non-fish has been evolved in separately. Rock bream was clustered with Takifugu rubripes. The SYBR Green qPCR was performed to quantify the RbKunitz transcripts in different tissues and challenged tissues. The mRNA transcripts of RbKunitz were detected in all tissues (muscle, spleen, head kidney, blood, heart, skin, liver, intestine, kidney and gills) analyzed and highest transcripts level was detected in gill tissues. Temporal transcription profile of RbKunitz in rock bream blood tissues was analyzed upon LPS (lipopolysaccharide), Poly I:C (Polyinosinic:polycytidylic acid) and Edwardsiella tarda challenge to understand the immune responses of this gene. Compare to the unchallenged control RbKunitz exhibited strong up-regulation at 24 h post injection (p.i.) after LPS and E. tarda injection. Comparatively robust expression of RbKunits was observed at 3 h p.i. upon Poly I:C challenge. Taken together all these data indicate that RbKunitz may involve into to immune responses upon pathogenic stress, in order to protect the rock bream.

Keywords: Kunitz-type, rock bream, immune response, serine protease inhibitor

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Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

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Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast

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Authors: Hainan Chen, Jina Qing, Xiao Zhu, Ling Gao, Ampadu O. Jackson, Min Zhang, Kai Yin

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Objective: Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is highly associated with mitochondrial respiration. Recent studies have suggested that mitochondrial apolipoprotein A-1 binding protein (APOA1BP) was essential for the cellular metabolite NADHX repair to NADH, which is necessary for the mitochondrial function. The exact role of APOA1BP in the repolarization of M1 to M2, however, is uncertain. Material and method: THP-1-derived macrophages were incubated with LPS (10 ng/ml) or/and IL-4 (100 U/ml) for 24 hours. Biochemical parameters of oxidative phosphorylation and M1/M2 markers were analyzed after overexpression of APOA1BP in cells. Results: Compared with control and IL-4-exposed M2 cells, APOA1BP was downregulated in M1 macrophages. APOA1BP restored the decline in mitochondrial function to improve metabolic and phenotypic reprogramming of M1 to M2 macrophages. Blocking oxidative phosphorylation by oligomycin blunts the effects of APOA1BP on M1 to M2 repolarization. Mechanistically, LPS triggered the hydration of NADH and increased its hydrate NADHX which inhibit cellular NADH dehydrogenases, a key component of electron transport chain for oxidative phosphorylation. APOA1BP decreased the level of NADHX via converting R-NADHX to biologically useful S-NADHX. The mutant of APOA1BP aspartate188, the binding site of NADHX, fail to repair oxidative phosphorylation, thereby preventing repolarization. Conclusions: Restoring mitochondrial function by increasing mitochondrial APOA1BP might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control inflammatory diseases.

Keywords: inflammatory diseases, macrophage repolarization, mitochondrial respiration, apolipoprotein A-1 binding protein, NADHX, NADH

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