324,325 research outputs found
Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance
No full textProteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.Proteorhodopsin (PR) wurde ursprünglich aus nicht kultivierten γ-Proteobakterium isoliert und ist in großen Mengen in den Ozeanen enthalten. PR ist wie sein homolog Bakteriorhodopsin (BR) ein TypI Retinal Bindeprotein und die Sequenzen sind zu 26% identisch. Innerhalb der PR Familie haben die Mitglieder eine Sequenzhomologie zu ungefähr 78% und zeigen einen Unterschied von 40 nm im absorptions spektrum. Diese Eigenschaft bietet ein gutes Modelsystem um zu verstehen durch welchen Mechanismus das Absorptionsspektrum moduliert wird. PR ist ein photoaktive Protonenpumpe und es wird angenommen, dass die Richtung des Protonentransfers vom pH-wert abhängt, was auf eine Rolle als ein pH abhängiger Regulator hindeutet. Da PR sowohl in der Zelle in hoher Zahl, als auch in den Ozeanen in unterschiedlichen Tiefen weit verbreitet ist, wird angenommen, dass PR bei der Verwertung von Sonnenlicht, im Energiestoffwechsel und beim Kohlenstoffumsatz beteiligt ist. Im Gegensatz zu BR, welches bekannterweise 2D Kristalle bildet, ist etwas vergleichbares für PR bis heute nicht bekannt. Weder der Mechanismus von PR noch seine 3D Struktur sind bisher gelöst. Die vorliegende Doktorarbeit versucht offene Punkte zum Mechanismus und zur Struktur von PR zu klären. Für die Charakterisierung werden 2D Kristallographie, "Atomic Force Microscopy" und Festkörper NMR verwendet. Für die Bildung von 2D Kristallen konnte eine große Auswahl an Kristallisationbedingungen ermittelt werden, was auf deutliche Protein Protein Wechselwirkungen hindeutet. Zieht man die hohe Zahl an PR Molekülen pro zelle in betracht, ist es wahrscheinlich, dass durch diese Interaktionen auch in der natürlichen Membran eine dichte Packung der Proteine auftritt. Elektronenmikroskopische Aufnahmen mit geringer Auflösung deuten auf eine ringförmige Anordnung der Proteine in einem hexagonalen Gitter mit einer Einheitszelle von 87Å * 87Å. Vorläufige FTIR Messungen deuten darauf hin, dass diese Anordnung den Photozyklus nicht behindert und sowohl K als auch M Zustand konnten identifiziert werden. Um weitere Informationen über den Oligomerisierungszustand der 2D Kristalle zu gewinnen wurden Einzelmolekül - und Rasterkraft Mikroskopie durchgeführt. Hierbei zeigte sich, dass das Protein in kristallinen und dicht rekonstituierten Regionen überwiegend als Hexamer vorliegt. Daneben kann zu einem geringen Anteil auch ein pentamerer Zustand beobachtet werden. Der Mechanismus der Proteinentfaltung war vergleichbar zu anderen, besser untersuchten Mitgliedern der Rhodopsinfamilie. Zwischen den Daten aus der "Atomic Force Microscopy" und der Elektronenmikroskopie zeigt sich eine gute Korrelation. Festkörper NMR an vollständig und selektiv markierten 2D Kristallen ergaben Spektren mit einer typischen 15N Linienbreite von 0,6 bis 1,2 ppm. Die 15N chemische Verschiebung der Schiffschen Base hat im Kristall den gleichen Wert wie funktional aktiv rekonstitutierte Proben, was indirekt die Funktionalität und die korrekte Faltung bestätigt. Die Zuordnung der 15N Signale für Tryptophan wurde durch "Rotational Echo Double Resonance" Experimente vorgenommen. 2D kreuzpolarisation Lee Goldburg Messungen zeigen den dynamischen Zustand des Proteins trotz der eingeschränkten Mobilität im kristallinen Zustand. Das Verhalten der Lipide wurde mit 31P messungen der Lipidkopfgruppe untersucht und zeigt, dass diese nicht fest gebunden sind, sondern sich mehr wie in einer Lipiddoppelschicht verhalten. Für 13C-13C homonukleare korrelations Experimente wurde die Mischzeit durch die Analyse von Aufbaukurven optimiert. Diese Versuche deuten darauf hin, dass es möglich ist einzelne Resonanzen aufzulösen, wie im Fall des Glutamat gezeigt mit einem gutem Signal zu Rauschen Verhältnis. Selektives "unlabeling" ist eine effizente Methode um die Ueberlappung der Signal zu reduzieren. Darüberhinaus sind für eine weitere Chrakterisisierung effizentere Markierungsschemata notwendig. Die bisherige spektrale Auflösung ist gut genug für die Untersuchung einzelner Aminosäuren, für vollständig markierte Proben sind weitere Verbesserungen notwendig
mRNA translation from an antigen presentation perspective: A tribute to the works of Nilabh Shastri
No full textThe field of mRNA translation has witnessed an impressive expansion in the last decade. The once standard model of translation initiation has undergone, and is still undergoing, a major overhaul, partly due to more recent technical advancements detailing, for example, initiation at non-AUG codons. However, some of the pioneering works in this area have come from immunology and more precisely from the field of antigen presentation to the major histocompatibility class I (MHC-I) pathway. Despite early innovative studies from the lab of Nilabh Shastri demonstrating alternative mRNA translation initiation as a source for MHC-I peptide substrates, the mRNA translation field did not include these into their models. It was not until the introduction of the ribo-sequence technique that the extent of non-canonical translation initiation became widely acknowledged. The detection of peptides on MHC-I molecules by CD8 + T cells is extremely sensitive, making this a superior model system for studying alternative mRNA translation initiation from specific mRNAs. In view of this, we give a brief history on alternative initiation from an immunology perspective and its fundamental role in allowing the immune system to distinguish self from non-self and at the same time pay tribute to the works of Nilabh Shastri
Molecular heterogeneity and immunosuppressive microenvironment in Glioblastoma
No full textCopyright © 2020 DeCordova, Shastri, Tsolaki, Yasmin, Klein, Singh and Kishore. Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a poor prognosis, despite surgical resection combined with radio- and chemotherapy. The major clinical obstacles contributing to poor GBM prognosis are late diagnosis, diffuse infiltration, pseudo-palisading necrosis, microvascular proliferation, and resistance to conventional therapy. These challenges are further compounded by extensive inter- and intra-tumor heterogeneity and the dynamic plasticity of GBM cells. The complex heterogeneous nature of GBM cells is facilitated by the local inflammatory tumor microenvironment, which mostly induces tumor aggressiveness and drug resistance. An immunosuppressive tumor microenvironment of GBM provides multiple pathways for tumor immune evasion. Infiltrating immune cells, mostly tumor-associated macrophages, comprise much of the non-neoplastic population in GBM. Further understanding of the immune microenvironment of GBM is essential to make advances in the development of immunotherapeutics. Recently, whole-genome sequencing, epigenomics and transcriptional profiling have significantly helped improve the prognostic and therapeutic outcomes of GBM patients. Here, we discuss recent genomic advances, the role of innate and adaptive immune mechanisms, and the presence of an established immunosuppressive GBM microenvironment that suppresses and/or prevents the anti-tumor host response
Diffusive author(s), cohesive author: Analysis of S/N (1994)
No full textThis study indicates the ways in which various aspects of the author(s) are brought forth in Dumb type’s performance art, the S/N production. Previous research has suggested a non-hierarchical organization of Dumb type and the absence of a “privileged author” in Dumb type’s collaborative work, S/N. However, the results that I have investigated from member’s interviews on the creative process of S/N along with my analysis of the recorded images of S/N, indicate a different aspect of the author(s). First, S/N was created through, so to speak, the collective ideas of the members of Dumb type. Further, S/N has at least nine quotations from previous performances, installations, and printed writings, besides the work-in-progress technique. Explicating one of the “author functions” as given by Michel Foucault, each text has plural subjects of the author. However, it has been revealed from members’ interviews that Teiji Furuhashi had a decision-making role in selecting the members’ ideas within the performance. Since then, S/N has had plural subjects of creation; however, Furuhashi is one of the subjects of creation along with the “privileged author.” S/N has plural authors (diffusive authors) yet at the same time, it has a “privileged author,” Teiji Furuhashi (cohesive author)
Innate immunity and neuroinflammation
No full textCopyright © 2013 Abhishek Shastri et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Inflammation of central nervous system (CNS) is usually associated with trauma and infection. Neuroinflammation occurs in close relation to trauma, infection, and neurodegenerative diseases. Low-level neuroinflammation is considered to have beneficial effects whereas chronic neuroinflammation can be harmful. Innate immune system consisting of pattern-recognition receptors, macrophages, and complement system plays a key role in CNS homeostasis following injury and infection. Here, we discuss how innate immune components can also contribute to neuroinflammation and neurodegeneration
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Estimation of perceptible water vapor of atmosphere using artificial neural network, support vector machine and multiple linear regression algorithm and their comparative study
No full text
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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