Toolset for incorporation of unnatural amino acids into proteins expressed in mammalian cells

Proteins are biological nanomachines that perform almost all the biological processes. The survival of a living organism depends heavily on proper coordination and functioning of these biomolecules. Naturally, malfunctioning proteins may stunt the growth and proper development of an organism and might even lead to death. Because of their impact on human health, proteins are among the most studied biomolecules. For such studies, proteins are usually produced using protein expression systems (e.g., mammalian cells, insect cells, yeast cells or bacterial cells). For studying human proteins, mammalian systems or human cell- based systems are usually the best because they can most accurately mimic the production and maturation processes of the target-protein. To understand the biophysical properties and working mechanisms of proteins, many biophysical methods are available. Such methods often rely on molecules known as probes, that can sense a change in their local environment and generate measurable signals corresponding to these changes. To study a protein molecule, these probes, must be covalently attached to the protein via one of its chemically reactive sites. Attachment of biophysical probe to a specific site on the protein requires a unique reactive site on the protein molecule. However, site-specific attachment of a biophysical probe is either not feasible or not practical when many reactive sites are present on the protein or if the reactive sites are functionally relevant for the protein. In such a scenario, presence of a bioorthogonal reactive handle on the protein of interest may allow the introduction of the biophysical probe without interfering with the reactive sites or functionally relevant amino acids of proteins. Such bioorthogonal reactive handles can be introduced in proteins via unnatural amino acids. Using protein expression systems, unnatural amino acids can be genetically (or cotranslationally) incorporated into the proteins during the polypeptide biosynthesis. The most widely used strategy for doing this is genetic code expansion (G.C.E.) via stop codon suppression, where, by introducing an orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase pair in the protein expression system, one of the three stop-codons (Opal, Amber, or Ochre) is repurposed as a signal-codon to incorporate unnatural amino acid(s) in proteins. Several genetic code expansion systems have been developed to incorporate more than 100 different unnatural amino acids into the proteins expressed in mammalian systems. These unnatural amino acids provide a gamut of unique biophysical and biochemical characteristics for characterizing proteins and polypeptides. Among other applications, unnatural amino acids can introduce bioorthogonal reaction handles, cross-linking handles, post-translational modifications or fluorescent side chains into proteins. This work is dedicated for site-specific incorporation of unnatural amino acids in proteins expressed in mammalian cells. For doing so, we have developed mammalian expression vectors and, in this thesis, have demonstrated their potential for assimilating the existing G.C.E. systems. We have also developed a cell-based screening assay for quantification of the UAA incorporation efficiency of the different G.C.E. systems. We have demonstrated that this screening assay provides a holistic picture about UAA incorporation conditions. We have also demonstrated that this assay is compatible with high- throughput screening experiments. Finally, we have demonstrated a systematic strategy for efficiently incorporating two UAAs in proteins expressed in mammalian cells. Using this strategy, we have incorporated a fluorescent amino acid as well as a bioorthogonal handle in our test protein eGFP. The introduced biorthogonal handle can be used for site-specific incorporation of a fluorophore to perform FRET based experiments

Pessimistic Worldview and Existential Nihilism in Philip Larkin’s Poetry

 Philip Larkin is one of the most respected men of letters of the twentieth century. A major characteristic of his works was his preoccupation with the overshadowing gloom and despair that resulted from the Second World War and the loss of the golden era of the British Empire. Unsociable by temperament, Larkin was fully alive to the harsh realities of the modernized world that post-War generation was living in. As was natural to his nature, he developed a deep-rooted pessimism and even a nihilistic tendency towards life and human existence in general. This paper attempts to evaluate four of Larkin’s so-called pessimistic poems, namely, “Mr. Bleaney”, “Next, Please”, “Afternoons” and “Days” from his different collections of poetry in order to analyse his preoccupation with death and a general pessimistic attitude towards the worth of human life

(Re)Reading Animal Imagery in the Poetry of Ted Hughes

Ted Hughes has published several volumes of verse and his poems have a singular approach because of the themes they explore, as well as, the manner in which they explore them. The poets before Hughes were thoroughly consumed with the harrowing backwash of the Second World War and their poetry reflected the macabre front of a stony world where the question of human existence and the absurdity of life were the engrossing concerns. Hughes was a revolutionary poet in the sense that he revolted against this post-war tendency of being evasive to the harsh realities of the times. He painted bare the nightmarish world and emotional stagnation that the Great War had resulted into. Another distinctive feature of Hughes’ poetry is that he is a captivating poet of the animal world. His use of animals as a metaphor for the human world is not only intriguing but innovative. He presents not only the unruffled beauty, but also the violence and ruthlessness of the natural world. The symbolic reference to the modern mechanical world is staggering and impinges on our mind. The portrayal of this modern “waste land” is closely allied with his metaphorical usage of the faunae. It is this aspect of his poetry which the paper attempts to analyze

Implementation of an efficient Fuzzy Logic based Information Retrieval System

This paper exemplifies the implementation of an efficient Information Retrieval (IR) System to compute the similarity between a dataset and a query using Fuzzy Logic. TREC dataset has been used for the same purpose. The dataset is parsed to generate keywords index which is used for the similarity comparison with the user query. Each query is assigned a score value based on its fuzzy similarity with the index keywords. The relevant documents are retrieved based on the score value. The performance and accuracy of the proposed fuzzy similarity model is compared with Cosine similarity model using Precision-Recall curves. The results prove the dominance of Fuzzy Similarity based IR system.Comment: arXiv admin note: substantial text overlap with http://ntz-develop.blogspot.in/ , http://www.micsymposium.org/mics2012/submissions/mics2012_submission_8.pdf , http://www.slideshare.net/JeffreyStricklandPhD/predictive-modeling-and-analytics-selectchapters-41304405 by other author

Big Data Application for Traffic Estimation on a Website: Big Daddy Case

While living under rapidly changing conditions innovation, flexibility and readiness to change are grounding prosperity of the firm. But any changes for companies should be reasoned and made on the basis of analytical approach. Big data usually could help in this situation without spending time and money on expensive research activities. Therefore, this paper is focused on big data application on customers’ behavior switching from one product to new its look. Modeling is based on few monthsˈ daily data with application of regression analysis and the least squares method. The major finding comes up with the estimation output that the new webpage is more popular among IOS and WEB users, although Android systems showing negative impact on switching to new websit

Investigation of Thermoelectric properties of Magnetic Insulator FeRuTiSi Using First Principle Calculation

In this work, we have investigated the electronic structure and thermoelectric properties of quaternary heusler alloy, FeRuTiSi, using first principle DFT tools implemented in WIEN2k and BoltzTraP code. Electronic structure calculations using TB-mBJ potential shows appearance of flat band at the conduction band edge, thus electron in conduction band have the large effective mass (me*), and therefore mainly contribute for negatively large value of Seebeck coefficient (S). This alloy has indirect band gap of 0.59 eV, and shows the n-type transport behavior. Under the constant relaxation time approximation (tau = 10 -14 s), temperature dependent Seebeck coefficient, electrical conductivity (sigma), and electronic thermal conductivity (ke) were also estimated. The maximum figure-of-merit (ZT), for the FeRuTiSi compound is found to be ~0.86 at 840 K, with n-type doping, which suggests that this quaternary alloy can be a good candidate among the n-type material for thermoelectric applications in high-temperature regComment: 4 Pages, 4 figure