Search results for: in vitro folding

Authors: Siddharth Deshpande, Nihar Masurkar, Vallerinteavide Mavelli Girish, Malan Desai, Chester Drum

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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.

Keywords: thermostable shell, in vitro folding, stability, functional yield

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Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

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Authors: Ahmed H. Amer, A. Amar, Sh. Hamada, I. I. Bondouk, F. A. El-Hussiny

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Elastic scattering of α-particles from 9Be and 11B nuclei at different alpha energies have been analyzed. Optical model parameters (OMPs) of α-particles elastic scattering by these nuclei at different energies have been obtained. In the present calculations, the real part of the optical potential are derived by folding of nucleon-nucleon (NN) interaction into nuclear matter density distribution of the projectile and target nuclei using computer code FRESCO. A density-dependent version of the M3Y interaction (CDM3Y6), which is based on the G-matrix elements of the Paris NN potential, has been used. Volumetric integrals of the real and imaginary potential depth (JR, JW) have been calculated and found to be energy dependent. Good agreement between the experimental data and the theoretical predictions in the whole angular range. In double folding (DF) calculations, the obtained normalization coefficient Nr is in the range 0.70–1.32.

Keywords: elastic scattering, optical model, double folding model, density distribution

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Authors: Abd Elrahman Elgamala, N. Darwish, I. Bondouk, Sh. Hamada

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Available experimental angular distributions for ⁶Li elastically scattered from ¹⁶O nucleus in the energy range 13.0–50.0 MeV are investigated and reanalyzed using optical model of the conventional phenomenological potential and also using double folding optical model of different interaction models: DDM3Y1, CDM3Y1, CDM3Y2, and CDM3Y3. All the involved models of interaction are of M3Y Paris except DDM3Y1 which is of M3Y Reid and the main difference between them lies in the different values for the parameters of the incorporated density distribution function F(ρ). We have extracted the renormalization factor N_R for ⁶Li+¹⁶O nuclear system in the energy range 13.0–50.0 MeV using the aforementioned interaction models.

Keywords: elastic scattering, optical model, folding potential, density distribution

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Authors: Do-Jin Jang, Sung-Ah Kim

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In designing a kinetic façade, it is hard for the designer to make digital models due to its complex geometry with motion. This paper aims to present a methodology of converting a point cloud of a physical model into a single digital model with a certain topology and motion. The method uses a Microsoft Kinect sensor, and color markers were defined and applied to three paper folding-inspired designs. Although the resulted digital model cannot represent the whole folding range of the physical model, the method supports the designer to conduct a performance-oriented design process with the rough physical model in the reduced folding range.

Keywords: design media, kinetic facades, tangible user interface, 3D scanning

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Authors: Sh. Hamada

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The current work aims to study the rainbow like-structure observed in the elastic scattering of alpha particles on both ¹²C and ¹⁶O nuclei. We reanalyzed the experimental elastic scattering angular distributions data for α+¹²C and α+¹⁶O nuclear systems at different energies using both optical model and double folding potential of different interaction models such as: CDM3Y1, DDM3Y1, CDM3Y6 and BDM3Y1. Potential created by BDM3Y1 interaction model has the shallowest depth which reflects the necessity to use higher renormalization factor (N_r). Both optical model and double folding potential of different interaction models fairly reproduce the experimental data.

Keywords: density distribution, double folding, elastic scattering, nuclear rainbow, optical model

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Authors: Rajnarayan Damor, Gayatri Swarnakar

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Fasciola gigantica are present in the biliary ducts of liver and gall bladder of domestic buffaloes. They are very harmful and causes significant lose to live stock owners, on account of poor growth and lower productivity of domestic buffaloes. Synthetic veterinary drugs have been used to eliminate parasites from cattle but these drugs are unaffordable and inaccessible for poor cattle farmers. The in vitro anthelmintic effect of Citrullus colocynthis fruit extract against Fasciola gigantica parasites were observed by light and scanning electron microscopy. Fruit extracts of C. colocynthis exhibit highest mortality 100% at 50 mg/ml in 15th hour of exposure. The oral and ventral sucker appeared to be slightly more swollen than control and synthetic drug albendazole. The tegument showed submerged spines by the swollen tegument around them. The tegument of the middle region showed deep furrows, folding and submerged spines which either lied very flat against the surface or had become submerged in the tegument by the swollen tegument around them leaving deep furrows. Posterior region showed with deep folding in the tegument, completely disappearance of spines and swelling of the tegument led to completely submerged spines leaving spine socket. The present study revealed that fruit extracts of Citrullus colocynthis found to be potential sources for novel anthelmintic and justify their ethno-veterinary use.

Keywords: anthelmintic, buffalo, Citrullus colocynthis, Fasciola gigantica, mortality, tegument

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Authors: Vineeta Kaushik, Manisha Goel

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Chaperones are proteins that help other proteins fold correctly, and are found in all domains of life i.e., prokaryotes, eukaryotes and archaea. Various comparative genomic studies have suggested that the archaeal protein folding machinery appears to be highly similar to that found in eukaryotes. In case of protein folding; slow rotation of peptide prolyl-imide bond is often the rate limiting step. Formation of the prolyl-imide bond during the folding of a protein requires the assistance of other proteins, termed as peptide prolyl cis-trans isomerases (PPIases). Cyclophilins constitute the class of peptide prolyl isomerases with a wide range of biological function like protein folding, signaling and chaperoning. Most of the cyclophilins exhibit PPIase enzymatic activity and play active role in substrate protein folding which classifies them as a category of molecular chaperones. Till date, there is not very much data available in the literature on archaeal cyclophilins. We aim to compare the structural and biochemical features of the cyclophilin protein from within the three domains to elucidate the features affecting their stability and enzyme activity. In the present study, we carry out in-silico analysis of the cyclophilin proteins to predict their conserved residues, sites under positive selection and compare these proteins to their bacterial and eukaryotic counterparts to predict functional divergence. We also aim to clone and express these proteins in heterologous system and study their biophysical characteristics in detail using techniques like CD and fluorescence spectroscopy. Overall we aim to understand the features contributing to the folding, stability and dynamics of the archaeal cyclophilin proteins.

Keywords: biophysical characterization, x-ray crystallography, chaperone-like activity, cyclophilin, PPIase activity

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Authors: N. Burtebayev, Sh. Hamada, Zh. Kerimkulov, D. K. Alimov, A. V. Yushkov, N. Amangeldi, A. N. Bakhtibaev

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The main aim of the current work is to examine if 14N is candidate to be clusterized nuclei or not. In order to check this attendance, we have measured the angular distributions for 14N ion beam elastically scattered on 12C target nuclei at different low energies; 17.5, 21, and 24.5MeV which are close to the Coulomb barrier energy for 14N+12C nuclear system. Study of various transfer reactions could provide us with useful information about the attendance of nuclei to be in a composite form (core + valence). The experimental data were analyzed using two approaches; Phenomenological (Optical Potential) and semi-microscopic (Double Folding Potential). The agreement between the experimental data and the theoretical predictions is fairly good in the whole angular range.

Keywords: deuteron transfer, elastic scattering, optical model, double folding, density distribution

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Authors: Ravinder Singh, C Rajesh, Saroj Badhan, Shailendra Mishra, Ranjit Singh Kataria

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Heat shock protein 60 and 70 are crucial chaperones that guide appropriate folding of denatured proteins under heat stress conditions. HSP60 and HSP70 provide assistance in correct folding of a multitude of denatured proteins. The heat shock factors are the family of some transcription factors which controls the regulation of gene expression of proteins involved in folding of damaged or improper folded proteins during stress conditions. Under normal condition heat shock proteins bind with HSF-1 and act as its repressor as well as aids in maintaining the HSF-1’s nonactive and monomeric confirmation. The experimental protein structure for all these proteins in Bubalus bubalis is not known till date. Therefore computational approach was explored to identify three-dimensional structure analysis of all these proteins. In this study, an extensive in silico analysis has been performed including sequence comparison among species to comparative modeling of Bubalus bubalis HSP60, HSP70 and HSF-1 protein. The stereochemical properties of proteins were assessed by utilizing several scrutiny bioinformatics tools to ensure model accuracy. Further docking approach was used to study interactions between Heat shock proteins and HSF-1.

Keywords: Bubalus bubalis, comparative modelling, docking, heat shock protein

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Authors: Ana Maria Reis D'Azevedo Breda, Catarina Maria Neto da Cruz

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The Japanese art of origami provides a rich context for designing exploratory mathematical activities for children and young people. By folding a simple sheet of paper, fascinating and surprising planar and spatial configurations emerge. Equally surprising is the unfolding process, which also produces striking patterns. The procedure of folding, unfolding, and folding again allows the exploration of interesting geometric patterns. When adequately and systematically done, we may deduce some of the mathematical rules ruling origami. As the child/youth folds the sheet of paper repeatedly, he can physically observe how the forms he obtains are transformed and how they relate to the pattern of the corresponding unfolding, creating space for the understanding/discovery of mathematical principles regulating the folding-unfolding process. As part of a 2023 Summer Academy organized by a Portuguese university, a session entitled “Folding, Thinking and Generalizing” took place. Twenty-three students attended the session, all enrolled in the 2nd cycle of Portuguese Basic Education and aged between 10 and 12 years old. The main focus of this session was to foster the development of critical cognitive and socio-emotional skills among these young learners using origami. These skills included creativity, critical analysis, mathematical reasoning, collaboration, and communication. Employing a qualitative, descriptive, and interpretative analysis of data collected during the session through field notes and students’ written productions, our findings reveal that structured origami-based activities not only promote student engagement with mathematical concepts in a playful and interactive but also facilitate the development of socio-emotional skills, which include collaboration and effective communication between participants. This research highlights the value of integrating origami into educational practices, highlighting its role in supporting comprehensive cognitive and emotional learning experiences.

Keywords: skills, origami rules, active learning, hands-on activities

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Authors: Danny Barash

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Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is that many are found in prokaryotes but only a small percentage of known riboswitches have been found in certain eukaryotic organisms. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods that include some slight structural considerations. These pattern-matching methods were the first ones to be applied for the purpose of riboswitch detection and they can also be programmed very efficiently using a data structure called affix arrays, making them suitable for genome-wide searches of riboswitch patterns. However, they are limited by their ability to detect harder to find riboswitches that deviate from the known patterns. Several methods have been developed since then to tackle this problem. The most commonly used by practitioners is Infernal that relies on Hidden Markov Models (HMMs) and Covariance Models (CMs). Profile Hidden Markov Models were also carried out in the pHMM Riboswitch Scanner web application, independently from Infernal. Other computational approaches that have been developed include RMDetect by the use of 3D structural modules and RNAbor that utilizes Boltzmann probability of structural neighbors. We have tried to incorporate more sophisticated secondary structure considerations based on RNA folding prediction using several strategies. The first idea was to utilize window-based methods in conjunction with folding predictions by energy minimization. The moving window approach is heavily geared towards secondary structure consideration relative to sequence that is treated as a constraint. However, the method cannot be used genome-wide due to its high cost because each folding prediction by energy minimization in the moving window is computationally expensive, enabling to scan only at the vicinity of genes of interest. The second idea was to remedy the inefficiency of the previous approach by constructing a pipeline that consists of inverse RNA folding considering RNA secondary structure, followed by a BLAST search that is sequence-based and highly efficient. This approach, which relies on inverse RNA folding in general and our own in-house fragment-based inverse RNA folding program called RNAfbinv in particular, shows capability to find attractive candidates that are missed by Infernal and other standard methods being used for riboswitch detection. We demonstrate attractive candidates found by both the moving-window approach and the inverse RNA folding approach performed together with BLAST. We conclude that structure-based methods like the two strategies outlined above hold considerable promise in detecting riboswitches and other conserved RNAs of functional importance in a variety of organisms.

Keywords: riboswitches, RNA folding prediction, RNA structure, structure-based methods

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Authors: O. Kahraman, M. S. Alatas, O. B. Citil

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In determination of values of feeds which, are used in ruminant nutrition, different methods are used like in vivo, in vitro, in situ or in sacco. Generally, the most reliable results are taken from the in vivo studies. But because of the disadvantages like being hard, laborious and expensive, time consuming, being hard to keep the experiment conditions under control and too much samples are needed, the in vitro techniques are more preferred. The most widely used in vitro techniques are two-staged digestion technique and gas production technique. In vitro gas production technique is based on the measurement of the CO2 which is released as a result of microbial fermentation of the feeds. In this review, the factors affecting the results obtained from in vitro gas production technique (Hohenheim Feed Test) were discussed. Some factors must be taken into consideration when interpreting the findings obtained in these studies and also comparing the findings reported by different researchers for the same feeds. These factors were discussed in 3 groups: factors related to animal, factors related to feeds and factors related with differences in the application of method. These factors and their effects on the results were explained. Also it can be concluded that the use of in vitro gas production technique in feed evaluation routinely can be contributed to the comprehensive feed evaluation, but standardization is needed in this technique to attain more reliable results.

Keywords: In vitro, gas production technique, Hohenheim feed test, standardization

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Authors: Mir Jalil Razavi, Tianming Liu, Xianqiao Wang

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Cortical folding, characterized by convex gyri and concave sulci, has an intrinsic relationship to the brain’s functional organization. Understanding the mechanism of the brain’s convoluted patterns can provide useful clues into normal and pathological brain function. During the development, the cerebral cortex experiences a noticeable expansion in volume and surface area accompanied by tremendous tissue folding which may be attributed to many possible factors. Despite decades of endeavors, the fundamental mechanism and key regulators of this crucial process remain incompletely understood. Therefore, to taking even a small role in unraveling of brain folding mystery, we present a mechanical model to find mechanism of 3-hinges formation in a growing brain that it has not been addressed before. A 3-hinge is defined as a gyral region where three gyral crests (hinge-lines) join. The reasons that how and why brain prefers to develop 3-hinges have not been answered very well. Therefore, we offer a theoretical and computational explanation to mechanism of 3-hinges formation in a growing brain and validate it by experimental observations. In theoretical approach, the dynamic behavior of brain tissue is examined and described with the aid of a large strain and nonlinear constitutive model. Derived constitute model is used in the computational model to define material behavior. Since the theoretical approach cannot predict the evolution of cortical complex convolution after instability, non-linear finite element models are employed to study the 3-hinges formation and secondary morphological folds of the developing brain. Three-dimensional (3D) finite element analyses on a multi-layer soft tissue model which mimics a small piece of the brain are performed to investigate the fundamental mechanism of consistent hinge formation in the cortical folding. Results show that after certain amount growth of cortex, mechanical model starts to be unstable and then by formation of creases enters to a new configuration with lower strain energy. By further growth of the model, formed shallow creases start to form convoluted patterns and then develop 3-hinge patterns. Simulation results related to 3-hinges in models show good agreement with experimental observations from macaque, chimpanzee and human brain images. These results have great potential to reveal fundamental principles of brain architecture and to produce a unified theoretical framework that convincingly explains the intrinsic relationship between cortical folding and 3-hinges formation. This achieved fundamental understanding of the intrinsic relationship between cortical folding and 3-hinges formation would potentially shed new insights into the diagnosis of many brain disorders such as schizophrenia, autism, lissencephaly and polymicrogyria.

Keywords: brain, cortical folding, finite element, three hinge

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Authors: Hosamuddin Hamza

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The major concept of 4D printing is self-folding under thermal and humidity changes. This concept relies on understanding how the microstructures of 3D-printed models can undergo spontaneous shape transformation under thermal and moisture changes. The transformation mechanism could be achieved by mixing, in a controllable pattern, a number of materials within the printed model, each with known strain/shrinkage properties. 4D printing has a strong potential to be applied in dentistry as the technology could produce dynamic and adaptable materials to be used as functional objects in the oral environment under the continuously changing thermal and humidity conditions. The motion criteria could override the undesired dimensional changes, thermal instability, polymerization shrinkage and microleakage. 4D printing could produce restorative materials being self-adjusted spontaneously without further intervention from the dentist or patient; that is, the materials could be capable of fixing its failed portions, compensating for some lost tooth structure, while avoiding microleakage or overhangs at the margins. In prosthetic dentistry, 4D printing could provide an option to manage the influence of bone and soft tissue imbalance during mastication (and at rest) with high predictability of the type/direction of forces. It can also produce materials with better fitting and retention characteristics than conventional or 3D-printed materials. Nevertheless, it is important to highlight that 4D-printed objects, having dynamic properties, could provide some cushion as they undergo self-folding compensating for any thermal changes or mechanical forces such as traumatic forces.

Keywords: functional material, self-folding material, 3D printing, 4D printing

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Authors: Hamza Javar Magnier, Robin Curtis

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There have been rapid advances in the upstream processing of protein therapeutics, which has shifted the bottleneck to downstream purification and formulation. Finding liquid formulations with shelf lives of up to two years is increasingly difficult for some of the newer therapeutics, which have been engineered for activity, but their formulations are often viscous, can phase separate, and have a high propensity for irreversible aggregation1. We explore means to develop improved predictive ability from a better understanding of how protein-protein interactions on formulation conditions (pH, ionic strength, buffer type, presence of excipients) and how these impact upon the initial steps in protein self-association and aggregation. In this work, we study the initial steps in the aggregation pathways using a minimal protein model based on square-well potentials and discontinuous molecular dynamics. The effect of model parameters, including range of interaction, stiffness, chain length, and chain sequence, implies that protein models fold according to various pathways. By reducing the range of interactions, the folding- and collapse- transition come together, and follow a single-step folding pathway from the denatured to the native state2. After parameterizing the model interaction-parameters, we developed an understanding of low-temperature conformational properties and fluctuations, and the correlation to the folding transition of proteins in isolation. The model fluctuations increase with temperature. We observe a low-temperature point, below which large fluctuations are frozen out. This implies that fluctuations at low-temperature can be correlated to the folding transition at the melting temperature. Because proteins “breath” at low temperatures, defining a native-state as a single structure with conserved contacts and a fixed three-dimensional structure is misleading. Rather, we introduce a new definition of a native-state ensemble based on our understanding of the core conservation, which takes into account the native fluctuations at low temperatures. This approach permits the study of a large range of length and time scales needed to link the molecular interactions to the macroscopically observed behaviour. In addition, these models studied are parameterized by fitting to experimentally observed protein-protein interactions characterized in terms of osmotic second virial coefficients.

Keywords: protein folding, native-ensemble, conformational fluctuation, aggregation

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Authors: R. Devicharan

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In a short span 3D Printing is expected to play a vital role in our life. The possibility of creativity and speed in manufacturing through various 3D printing processes is infinite. This study is performed on the FDM (Fused Deposition Modelling) method of 3D printing, which is one of the pre-dominant methods of 3D printing technologies. This study focuses on physical properties of the objects produced by 3D printing which determine the applications of the 3D printed objects. This paper specifically aims at the study of the tensile strength and the folding endurance of the 3D printed objects through the FDM (Fused Deposition Modelling) method using the ABS (Acronitirile Butadiene Styrene) and PLA (Poly Lactic Acid) plastic materials. The study is performed on a controlled environment and the specific machine settings. Appropriate tables, graphs are plotted and research analysis techniques will be utilized to analyse, verify and validate the experiment results.

Keywords: FDM process, 3D printing, ABS for 3D printing, PLA for 3D printing, rapid prototyping

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Authors: Kevin Wang, Jian Ge, Yinan Zhao, Kevin Willis

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Kepler has discovered over 4000 exoplanets and candidates. However, current transit planet detection techniques based on the wavelet analysis and the Box Least Squares (BLS) algorithm have limited sensitivity in detecting minor planets with a low signal-to-noise ratio (SNR) and long periods with only 3-4 repeated signals over the mission lifetime of 4 years. This paper presents a novel precise-period transit signal detection methodology based on a new Graphics Processing Unit (GPU) Fast Folding algorithm in conjunction with a Convolutional Neural Network (CNN) to detect low SNR and/or long-period transit planet signals. A comparison with BLS is conducted on both simulated light curves and real data, demonstrating that the new method has higher speed, sensitivity, and reliability. For instance, the new system can detect transits with SNR as low as three while the performance of BLS drops off quickly around SNR of 7. Meanwhile, the GPU Fast Folding method folds light curves 25 times faster than BLS, a significant gain that allows exoplanet detection to occur at unprecedented period precision. This new method has been tested with all known transit signals with 100% confirmation. In addition, this new method has been successfully applied to the Kepler of Interest (KOI) data and identified a few new Earth-sized Ultra-short period (USP) exoplanet candidates and habitable planet candidates. The results highlight the promise for GPU Fast Folding as a replacement to the traditional BLS algorithm for finding small and/or long-period habitable and Earth-sized planet candidates in-transit data taken with Kepler and other space transit missions such as TESS(Transiting Exoplanet Survey Satellite) and PLATO(PLAnetary Transits and Oscillations of stars).

Keywords: algorithms, astronomy data analysis, deep learning, exoplanet detection methods, small planets, habitable planets, transit photometry

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Authors: Isaac Samuel

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A study was conducted to determine the in-vitro degradation of selected Nigerian browse plants consumed by small ruminants on free range in northern guinea savannah region of Nigeria using in vitro gas production, proximate composition, fibre components, methane gas production and dry matter degradation as tools. The leaves samples of the selected browse plants were collected, processed and incubated using in vitro gas dry matter degradation techniques. Results obtained showed variation in the rate of degradation. The result obtained from chemical analysis showed that the CP content of A. occidentale (26.49%) was higher than F. thonningi (23.58%), M. indica (20.58%) and T. catappa (18.61%). Both ADF and NDF of A. occidentale (40.00 and 50.00) were as well higher than F. thonningi (20.00 and 40.00), M. indica (20.00 and 40.00) and T.catappa (20.00 and 42.00). Results from in vitro gas production however showed that T. catappa (23.67ml/DM) has a significantly higher (p<0.05) value than F.thonningi (20.67ml/DM), A. occidentale (16.67ml/DM), and M. indica(14.00ml/DM) at 72 hours of incubation. Methane gas production and in vitro gas production can be used to predict dry matter degradation and nutritive value of feedstuff for small ruminants. A. occidentale with the least methane gas production and highest crude protein (CP) content might have the most nutritive value among the browse plants investigated.

Keywords: in vitro, degradation, browse, gas production

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Authors: Sarita Puri, Tapan K. Chaudhuri

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Chaperonin GroEL is a tetradecameric Escherichia coli protein having identical subunits of 57 kDa. The elucidation of thermodynamic parameters related to stability for the native GroEL is not feasible as it undergoes irreversible unfolding because of its large size (800kDa) and multimeric nature. Nevertheless, it is important to determine the thermodynamic stability parameters for the highly stable GroEL protein as it helps in folding and holding of many substrate proteins during many cellular stresses. Properly folded monomers work as building-block for the formation of native tetradecameric GroEL. Spontaneous refolding behavior of monomeric GroEL makes it suitable for protein-denaturant interactions and thermodynamic stability based studies. The urea mediated unfolding is a three state process which means there is the formation of one intermediate state along with native and unfolded states. The heat mediated denaturation is a two-state process. The unfolding process is reversible as observed by the spontaneous refolding of denatured protein in both urea and head mediated refolding processes. Analysis of folding/unfolding data provides a measure of various thermodynamic stability parameters for the monomeric GroEL. The proposed mechanism of unfolding of monomeric GroEL is a three state process which involves formation of one stable intermediate having folded apical domain and unfolded equatorial, intermediate domains. Research in progress is to demonstrate the importance of specific residues in stability and oligomerization of GroEL protein. Several mutant versions of GroEL are under investigation to resolve the above mentioned issue.

Keywords: equilibrium unfolding, monomeric GroEl, spontaneous refolding, thermodynamic stability

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Authors: Justo Rodriguez, Daming Chen, Amador M. Guzman

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The detection, treatment, and control of rapidly propagating, deadly viruses such as COVID-19, require the development of inexpensive, fast, and accurate devices to address the urgent needs of the population. Microfluidics-based sensors are amongst the different methods and techniques for detection that are easy to use. A micro analyzer is defined as a microfluidics-based sensor, composed of a network of microchannels with varying functions. Given their size, portability, and accuracy, they are proving to be more effective and convenient than other solutions. A micro analyzer based on the concept of “Lab on a Chip” presents advantages concerning other non-micro devices due to its smaller size, and it is having a better ratio between useful area and volume. The integration of multiple processes in a single microdevice reduces both the number of necessary samples and the analysis time, leading the next generation of analyzers for the health-sciences. In some applications, the flow of solution within the microchannels is originated by a pressure gradient, which can produce adverse effects on biological samples. A more efficient and less dangerous way of controlling the flow in a microchannel-based analyzer is applying an electric field to induce the fluid motion and either enhance or suppress the mixing process. Electrokinetic flows are characterized by no less than two non-dimensional parameters: the electric Rayleigh number and its geometrical aspect ratio. In this research, stable and unstable flows have been studied numerically (and when possible, will be experimental) in a T-shaped microchannel. Additionally, unstable electrokinetic flows for Rayleigh numbers higher than critical have been characterized. The flow mixing enhancement was quantified in relation to the stretching and folding that fluid particles undergo when they are subjected to supercritical electrokinetic flows. Computational simulations were carried out using a finite element-based program while working with the flow mixing concepts developed by Gollub and collaborators. Hundreds of seeded massless particles were tracked along the microchannel from the entrance to exit for both stable and unstable flows. After post-processing, their trajectories, the folding and stretching values for the different flows were found. Numerical results show that for supercritical electrokinetic flows, the enhancement effects of the folding and stretching processes become more apparent. Consequently, there is an improvement in the mixing process, ultimately leading to a more homogenous mixture.

Keywords: microchannel, stretching and folding, electro kinetic flow mixing, micro-analyzer

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Authors: Zelikha Labbani

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The in vitro isolated microspore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, microspore became a strategy to achieve various objectives particularly in genetic engineering. In this communication we would show the most recent advances in the producing haploid embryos via in vitro isolated microspore culture.

Keywords: in vitro isolated microspore culture, success, haploid cells, bioinformatics, biomedicine

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Authors: Adel S. El-Azab, Alaa A. M. Abdel-Aziz, Ibrahim A. Al-Suwaidan, Amer M. Alanazi

Abstract:

A novel series of 2,3,6-trisubstitute quinazolinone were designed, synthesized, and evaluated for their in-vitro antitumor activity. 3 (Benzylideneamino)-6-chloro-2-p-tolylquinazolin-4(3H)-One, 2-[(4-oxo-3-phenethyl-3,4-dihydroquinazolin-2-yl)thio]-N-(3,4;5-trimethoxyphenyl) acetamide and 3-(3-benzyl-6-methyl-4-oxo-3, 4-dihydroquinazolin-2-ylthio)-N-(3,4,5-trimethoxyphenyl) propanamide have shown amazing broad spectrum antitumor activity with mean GI50; 15.8, 3.16, and 7.4 μM respectively compared to known Quinazoline Derivatives antitumor drug 5-FU mean GI50=22.6 μM.

Keywords: quinazoline derivatives, in vitro antitumor, synthesis, 5-FU, NCI

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Authors: Babak Qasemi-Panahi, Gholamali Moghaddam, Seyed-Abbas Rafat, Hossein Daghigh Kia, Mansoureh Movahedin, Reza Hadavi

Abstract:

The objective of this study was to determine the effects of IGF-I on ovine oocytes maturation and subsequent development of embryos derived from in vitro fertilization (IVF). In vitro maturation (IVM) of oocytes and in vitro culture (IVC) of embryos was conducted with or without 100 ng/mL IGF-1. In the IGF-I treated group, mean percentage of oocyte maturation was significantly higher than the control group (57.67 ± 3.04 versus 49.81 ± 3.04%, respectively, P < 0.05). However, in comparison with control group, there was no significant effect of IGF-1 on rates of cleavage, morula, and blastocyst formation (85% versus 84%; 63% versus 65%, and 40% to 39%, respectively). These data demonstrate that IGF-I has a positive effect on ovine oocyte maturation rate, but it has not the significant outcome on embryo development.

Keywords: ovine, IGF-I, IVM, ICSI

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Authors: Zelikha Labbani

Abstract:

The In Vitro isolated micro spore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a micro spore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the micro spore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of micro spore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, micro spore became a strategy to achieve various objectives particularly in genetic engineering. In this study we would show the most recent advances in the producing haploid embryos via In Vitro isolated micro spore culture.

Keywords: haploid cells, In Vitro isolated microspore culture, success

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Authors: Rupesh K. Mishra, Gaëlle Catanante, Akhtar Hayat, Jean-Louis Marty

Abstract:

Ochratoxins (OTA) are secondary metabolites present in a wide variety of food stuff. They are dangerous by-products mainly produced by several species of storage fungi including the Aspergillus and Penicillium genera. OTA is known to have nephrotoxic, immunotoxic, teratogenic and carcinogenic effects. Thus, needs a special attention for a highly sensitive and selective detection system that can quantify these organic toxins in various matrices such as cocoa beans. This work presents a folding based aptasensors by employing an aptamer conjugated redox probe (methylene blue) specifically designed for OTA. The aptamers were covalently attached to the screen printed carbon electrodes using diazonium grafting. Upon sensing the OTA, it binds with the immobilized aptamer on the electrode surface, which induces the conformational changes of the aptamer, consequently increased in the signal. This conformational change of the aptamer before and after biosensing of target OTA could produce the distinguishable electrochemical signal. The obtained limit of detection was 0.01 ng/ml for OTA samples with recovery of up to 88% in contaminated cocoa samples.

Keywords: ochratoxin A, cocoa, DNA aptamer, labelled probe

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Authors: Zelikha Labbani

Abstract:

Since its creation in 1964 by Guha and Maheshwari in India on Datura innoxia Mill, in vitro androgenesis has become the method of choice in the production of doubled haploid in many species. However, in durum wheat, the Doubled haploid plant breeding programs remained limited due to the low production of androgenetic embryos and converting them into fertile green plants. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat.

Keywords: durum wheat, haploid embryos, on in vitro, pretreatment

Procedia PDF Downloads 355

Authors: Gayatri Nahak, Satyajit Kanungo, Rajani Kanta Sahu

Abstract:

Holarrhena antidysenterica Linn. (Apocynaceae) is a typical Indian medicinal plant popularly known as “Indrajav”. Traditionally the plant has been considered a popular remedy for the treatment of dysentery, diarrhea, intestinal worms and the seeds of this plant are also used as an anti-diabetic remedy. In the present study axillary shoot multiplication, callus induction and shoot regeneration from callus culture were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. Then in vivo and in vitro grown healthy plants were selected for study of antioxidant activity through DPPH and OH methods. Significantly higher antioxidant activity and phenol contents were observed in vitro raised plant in comparison to in vivo plants. The findings indicated the greater amount of phenolic compounds leads to more potent radical scavenging effect as shown in in vitro raised plant in comparison to in vivo plants which showed the ability to utilize tissue culture techniques towards development of desired bioactive metabolites from in vitro culture as an alternative way to avoid using endangered plants in pharmaceutical purposes.

Keywords: Holarrhena antidysenterica, in vitro, in vivo, antioxidant activity

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Authors: Ibrahim A. H. Barakat, Ahmed R. Al-Himaidi

Abstract:

The present study was conducted to determine the role and optimum concentration of fenugreek seed extract during in-vitro maturation on in-vitro maturation and developmental competence of Neaimi sheep oocytes following in-vitro fertilization. The Cumulus Oocyte Complexes (COCs) collected from sheep slaughterhouse ovaries were randomly divided into three groups, and they were matured for 24 hrs. in maturation medium containing fenugreek seed extract (0, 1 and 10 µg ml-1). Oocytes of a control group were matured in a medium containing 1 µg ml-1 estradiol 17β. After maturation, half of oocytes were fixed and stained for evaluation of nuclear maturation. The rest of oocytes were fertilized in vitro with fresh semen, then cultured for 9 days for the assessment of the developmental capacity of the oocytes. The results showed that the mean values of oocytes with expanded cumulus cells percentage were not significantly different among all groups (P < 0.05). But nuclear maturation rate of oocytes matured with 10 µg ml-1 fenugreek seed extract was significantly higher than that of the control group. The maturation rate and development to morula and blastocyst stage for oocytes matured at 10 µg ml-1 fenugreek seed extract was significantly higher than those matured at 1µg ml-1 of fenugreek seed extract and the control group. In conclusion, better maturation and developmental capacity rate to morula and blastocyst stage were obtained by the addition of 10 µg ml-1 fenugreek seed extract to maturation medium than addition of 1 µg ml-1 estradiol-17β (P < 0.05).

Keywords: fenugreek seed extract, in vitro maturation, sheep oocytes, in vitro fertilization, embryo development

Procedia PDF Downloads 392

Authors: Bello Gonzalez, Setten Van M., Brouwer M.

Abstract:

The chicken small intestine represents a dynamic and complex organ in which the enzymatic digestion and absorption of nutrients take place. The development of an in vitro fermentation chicken small intestinal model could be used as an alternative to explore the interaction between the microbiota and nutrient metabolism and to enhance the efficacy of targeting interventions to improve animal health. In the present study we have developed an in vitro fermentation chicken ileum microbiota model for unrevealing the complex interaction of ileum microbial community under physiological conditions. A two-vessel continuous fermentation process simulating in real-time the physiological conditions of the ileum content (pH, temperature, microaerophilic/anoxic conditions, and peristaltic movements) has been standardized as a proof of concept. As inoculum, we use a pool of ileum microbial community obtained from chicken broilers at the age of day 14. The development and validation of the model provide insight into the initial characterization of the ileum microbial community and its dynamics over time-related to nutrient assimilation and fermentation. Samples can be collected at different time points and can be used to determine the microbial compositional structure, dynamics, and diversity over time. The results of studies using this in vitro model will serve as the foundation for the development of a whole small intestine in vitro fermentation chicken gastrointestinal model to complement our already established in vitro fermentation chicken caeca model. The insight gained from this model could provide us with some information about the nutritional strategies to restore and maintain chicken gut homeostasis. Moreover, the in vitro fermentation model will also allow us to study relationships between gut microbiota composition and its dynamics over time associated with nutrients, antimicrobial compounds, and disease modelling.

Keywords: broilers, in vitro model, ileum microbiota, fermentation

Procedia PDF Downloads 57