225 research outputs found
Unraveling the Mechanism of Luteinizing Hormone Receptor Activation : Hinge Region as a Key Player
No full textGPCRs, influencing myriads of cellular functions, are the members of the largest family of the membrane proteins. However, their structures and the signaling mechanisms still remain enigmatic. In case of the Glycoprotein Hormone Receptor (GpHR) family the structure-function relationship is less understood because of a large extra-cellular domain (ECD). This large ECD, consisting of Leucine Rich Repeats (LRRs) and membrane-proximal hinge region, is sufficient for specific binding to the hormone (Ascoli, Fanelli, & Segaloff, 2002), but for receptor activation, hormone binding is translated via a conformation wave starting at hinge region and relayed to the transmembrane domain. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure-function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005; Fox, Dias, & Van Roey, 2001; Wu, Lustbader, Liu, Canfield, & Hendrickson, 1994). However, the complete understanding of the hormone‐receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action or regulating receptor activation in a hormone independent manner.
The crystal structure of FSHR ECD (amino acids 17-366) revealed that LRRs form a semicircular palm shaped structure with the C terminus region, designated as the hinge region, protruding out like a thumb. The hinge region, rather than being a separate functional unit, was found to be an integral part of the LRR domain, having two such repeats (LRR11 &12). LRR 11 is connected to LRR12 through a hairpin loop (amino acids 280-344) harboring the invariant sulfated tyrosine residue (sTyr) in YD/EY motif (X. Jiang et al., 2012). The heterodimeric hormones consisting of a common subunit and a hormone specific subunit, bind to the primary hormone binding site at LRR 4-6 as reported in the FSHR-FSH co crystal (Fan & Hendrickson, 2005). This primary binding of the hormone at LRR 4-6 creates a pocket (comprising of the residues P16α, L17α, F18α, F74α, L37β, Y39β, and P45β) in the hormone for secondary binding at sTyr residue. This interaction is proposed to initiate conformation change in the hinge region which further leads to FSHR activation (X. Jiang et al., 2012). Thus, the role of hinge region in GpHR activation got evolved from a linker to a switch, which decides the fate of the receptor activity (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). sTyr residue being conserved, presents itself as a potential player in activation mechanism of all the three receptors of the family (Bonomi, Busnelli, Persani, Vassart, & Costagliola, 2006; Kreuchwig, Kleinau, & Krause, 2013). Precise involvement of sTyr in GpHR activation is yet to be explored. The previous studies from the laboratory using the hinge region specific polyclonal and monoclonal antibodies established the unequivocal role of the hinge region in FSHR and TSHR activation (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). However, its function in LHR activation has not been conclusively established. Due to the unavailability of the structural information of LHR ECD/hinge, it is more difficult to study and explain the role of hinge region in LHR activation. The hormone independent signaling by point mutants of LHR also remains poorly understood.
In the present study an attempt has been made to understand the role of the hinge region in LHR signaling and modulating role of LRRs in hinge mediated LHR activation. The present study was initiated with an overall objective of understanding the molecular details of LHR activation mechanism keeping hinge at the centre of the picture. To have clarity of this picture with a holistic view of the mechanism, multi-pronged approach was adopted. Initially, ScFvs against LHR hinge region were employed as tools to probe into the hormone‐receptor interactions. Antibodies against glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone‐receptor interactions and signal transduction (Agrawal & Dighe, 2009; Dighe & Moudgal, 1983; Gadkari, Sandhya, Sowdhamini, & Dighe, 2007; Gadkari et al., 2007; Kene, Nalavadi, Dighe, Iyer, & Mahale, 2004; Majumdar, Railkar, & Dighe, 2012a, 2012b). In this study, Single chain Fragment variables (ScFvs) against the hinge region of LH receptor have been employed to understand the mechanism of receptor activation. The effects of LHR ScFvs on hCG-LHR interactions have been investigated and three of the ScFvs, JE10, JE4 and JG1 could bypass the hormone and activate the receptor directly, with JE10 being the most potent one. The effect on the signaling was specific for LHR as no increase in cAMP response was observed for TSHR/FSHR in presence of these ScFvs. JE10 surprisingly was unique and could alter the hCG-LHR interaction by decreasing hormone affinity and simultaneously increasing the Bmax for the hormone. JE10 binding was decreased to the pre-formed hormone receptor complex suggesting that hCG and the stimulatory antibody show stearic hindrance at the binding sites on hinge or hormone binding induces conformational change in the epitope of JE10. The change in affinity and Bmax of the hormone by JE10 could be due to unmasking of new binding sites for hormones or an allosteric effect on the protomer interaction like explained
in case of a small TMD specific allosteric modulator of FSHR (Xuliang Jiang et al., 2014). JE10 could also potentiate hCG signaling at sub-saturating concentrations of hCG, the precise mechanism of which is not clear. Through TSHR-LHR chimeric mutants, a stretch from amino acids 313-349, within the hinge region, was identified as the site recognized by JE10.
In order to study structural features of the JE10 epitope, LHR ECD was modeled on the basis of FSHRED crystal structure. With most of the motifs being structurally conserved (CF3 and YPSHCCAFF); the major portion of the hinge region was found to be unstructured. This unstructured region harbored the JE10 epitope as well as the functionally important conserved sTyr residue. The CD spectra of LHR hinge in presence of ScFv JE10 suggested a ScFv induced helical conformation and stabilization of the hinge loop region, which was constrained in the homology model into helices. As loop was now constrained in the Mode 2, so was the interaction of sTyr, which was now in contact with positively charged residues, probably stabilizing its charge. The YEY motif mutants further confirmed the indirect essential role of Y331 in activation of LHR by JE10.
Another approach followed to study hCG-LHR interactions was use of a series of LHR N-terminal truncation mutants and truncation mutants along with one of the LHR CAM (S277Q/D578Y). The effect of these truncations on hormone binding and receptor activation was investigated. The deletion of Cysteine box (Cb-1) of LHR (present at N-terminus of ECD) leads to abrogation of hCG binding, indicating importance of this region in maintaining ECD conformation required for hormone binding. This is the most unexplored region of the ECD. Though Cb-1 does not bind to the hormone directly (as is evident from the crystal structure) but it is indirectly essential for hormone binding. The basal activity of these truncated mutants was as low as that of the wild type LHR, reconfirming that no region of LHR ECD acts as an inverse agonist for the TMD (Karges, Gidenne, Aumas, Kelly, & Milgrom, 2005). Truncation mutants with CAM (double mutants) also showed low basal activity, suggesting that intact ECD is prerequisite for keeping LHR in a conformation, best suited for hormone binding and binding of G protein for activation. That best conformation still needs to be explored. Truncation mutants did not get stimulated by JE10 also. This observation is opposite to the previous studies in which FSHR/TSHR truncated mutants could be stimulated by hinge specific antibodies (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). This difference points out to the variations in which LHR hinge-TMD interactions prevail and lead to the receptor activation. This variation was also
confirmed with a previous report in which the binding of TSHR-ECL specific antisera to wild type LHR and TSHR-LHR 6 chimeric mutant suggested that hinge of LHR does not seem to be constraining the TMD (Majumdar et al., 2012b). Thus the LHR TMD itself possesses all the inhibitory interactions, also indicated by the presence of most of the activating mutations in LHR TMD (Piersma, Verhoef-post, Berns, & Themmen, 2007).
Protomer interaction is the newest aspect of GpHR activation mechanism and has not reached any conclusive, physiologically relevant explanations yet. By co-transfection of wild type LHR and ECD truncated mutants, this study suggests the LHR protomer interaction and proposes the involvement of allosteric effect of ECD on LHR protomer interaction.
The effect of JE10 on activating and inactivating mutants of LHR were quite interesting. The ScFv could bind to the activating mutant D578Y (associated with precocious puberty). This mutant exhibited higher basal cAMP production, but was activated even further by the ScFv. The inactivating mutant A593P is a completely inactive receptor associated with (associated with pseudo-hermaphroditism. It does not respond to the hormone at all. The ScFv JE10 binds to this receptor and stimulates cAMP production. This observation is rather striking, as it is possible to activate a completely inactive mutant that could not be stimulated by the hormone by a binder specific for the hinge region. It is not clear how the binder that interacts with the hinge region affects the function of the inactive TMD thus providing an interesting tool to investigate the interactions between the hinge region and TMD that are probably key to understand the activation of GpHR. which has been shown to be central to the GpHR activation mechanism, (Agrawal & Dighe, 2009; Majumdar et al., 2012b; Schaarschmidt, Huth, Meier, Paschke, & Jaeschke, 2014). As per the recently suggested model by Deupi et. al., that each mutation and agonist can take a different pathway during activation (Kobilka & Deupi, 2007). The activated state induced by JE10 in D578Y and A593P seems to be different from the wild type LHR, with each activated receptor state having different capacity to bind to the G protein. The difference in G protein capacity in itself reflects the different receptor turnover or different Gs uncouplings or different Gs binding affinities, which needs to be further investigated, opening up another avenue for exploration. There is a lacuna in understanding the signal relay from the hinge to TMD. However, JE10 seems to be activating the wild type LHR and the mutants directly or indirectly by modulating the 6th helix of the TMD, known to be important for hormone independent
activation of LHR (Fanelli, 2000; Latronico & Segaloff, 2007; Majumdar et al., 2012b).
As evident from the absence of any hinge mediated constrain on LHR TMD and absence of uncharged residues present in LHR LRRD-TMD interface (LHR ECD Model 1), LHR hinge does not seem to be maintaining significant interactions with the TMD in absence of a ligand or in its basal state. Hormone/ agonist binding or activating mutations act as a positive regulator (inducing conformation change in hinge), required to bridge the interactions between LHR hinge and the TMD, which is supported by various studies in the past (Karges et al., 2005; Majumdar et al., 2012b; Nishi, Nakabayashi, Kobilka, & Hsueh, 2002; Osuga et al., 1997; Ryu, Gilchrist, Tung, Ji, & Ji, 1998; Zeng, Phang, Song, Ji, & Ji, 2001). This interaction bridged by the conformational change in the hinge region, seems to isomerize the closed state of LHR into an activated state. The present study supports the conformational induction model for receptor activation in which intramolecular interactions between the two domains (hinge-TMD) lead to the receptor activation.
In conclusion, this study presents a possible mechanism of activation of LHR by a partial agonist ScFv, which induces the conformation change in the disordered loop region (a.a.313-349) of the hinge and stabilizes it into helical state. This conformation change is predicted to be important for relaying the activation signal to the TMD. The study also demonstrates the activation of a completely inactive mutant A593P by JE10, suggesting a distinct possibility of its use as a therapeutic tool in treating infertility caused by inactivating mutations in LHR.
On a second note, the study extends the role of LRRs, apart from direct hormone binding, to an indirect allosteric role in hormone binding, LHR activation and functional stability. This functional stability does not seem to be restricted to a single LHR but also depends on its interaction with nearby protomers. Though there are evidences for and against each of the above discussed possibilities, as yet there is no accepted model that explains the precise steps of receptor activation, hence, the molecular details of these interactions needs to be investigated in future
The effects of Wnt5a and Wnt3a and PCP signaling on Schwann cell biology and myelination
No full textPlanar cell polarity (PCP) is known as the polarization of cells within the plane of the tissue layer. This form of polarization controls several epithelial and non-epithelial morphological processes, such as the orientation of primary cilia in the inner ear, convergent extension (CE) and directed migration. A three tiered model of PCP regulation has been proposed which consists of the global, core, and effector modules. However there is one addition level of modulation through non-canonical Wnt signaling pathway. Of the many Wnt proteins a few have been identified to signal primarily through this pathway. One such protein is Wnt5a, which has been shown to modulate PCP during directed cell migration. In this study we gather preliminary data for the presence of PCP signaling components in Schwann cells and investigate the effect of Wnt5a and its antagonist Wnt3a on Schwann cell proliferation, migration and myelination.M.S.Includes bibliographical referencesby Neha Jan
The effects of immediate versus delayed feedback after multiple-choice questions on subsequent exam performance
No full textThis thesis investigates the effects of immediate versus delayed feedback following multiple-choice questions on subsequent performance on multiple-choice and recall questions. In three experiments, students in a college psychology lecture course received immediate or delayed feedback following multiple-choice questions on an initial unit exam which was followed up with exam(s) including both multiple-choice and short-answer questions. In the first experiment, the kind of feedback did not affect performance on the same multiple-choice questions when they were repeated on the final. In the second experiment, two subsequent follow-up exams included first a short-answer version of the multiple-choice question and then the same multiple-choice question. Performance on the short-answer questions was better following delayed feedback than following immediate feedback. However, the kind of feedback had no effect on the performance of the repeated multiple-choice questions. Also, the interval between the initial exam and the follow-up exam had no effect on performance. The third experiment examined whether delayed feedback increased confidence more than immediate feedback and whether the increase in confidence mediated the improved performance on subsequent short-answer questions. The delayed feedback had no effect on confidence for the subsequent short-answer and multiple-choice responses. Together, these results demonstrate that delayed feedback improves performance on the short-answer questions by increasing the subsequent generation of the correct response but does not influence recognition of it.M.S.Includes bibliographical referencesIncludes vitaby Neha Sinh
Moral Panic, Social Exclusion and The Human Rights of Same-Sex Partners in Ghana-RETRACTED
No full textThis article is retracted :
The retraction is based on the request of the author, Dr. Neha Jain, as it contains some exclusive and private data of a community out of India, that should not be released online. https://doi.org/10.55938/ijgasr.v1i3.20
Sincerely,Editorial Team, IJGASR
Announcement: https://journals.icapsr.com/index.php/ijgasr/announcement/view/17
Correction to: A cell-cycle signature classifier for pan-cancer analysis
No full textIn the original published version, the list of authors was incomplete. Theodora A. Constantin was missing, and Neha Tabassum and Theodora A. Constantin share first authorship. The correct author list is given above. The original article has been corrected. DOI to original article: https://doi.org/10.1038/s41388-020-01426-
Less-institutionalized social structures: a theoretical, methodological, and empirical analysis of how networks and culture matter for emergence
No full textUsing a networks and culture lens, I investigate the micro-level processes underlying the production of order in social contexts or locations that are relationally-defined and meaningful, but lack cultural cues to action and interpretation or are in the early stages of acquiring such meaning. Drawing on neoinstitutionalist theory, I refer to such social structures as less-institutionalized. To explain order in the absence of situational cultural cues literature in social network analysis has traditionally attributed regularities to situational structural tendencies that preclude shared understandings and/or subjective engagement. Recent literature in the sociology of culture that revives overarching moral intuitions as a basis for action similarly rejects the explanatory value of situational cultural cues. Arguing that culture is neither irrelevant nor implicated in an overarching way in culturally less-institutionalized situations, I posit that order can be linked to individuals’ tacit and discursive use of cultural repertoires acquired over the life-course through involvements in multiple networks of interaction and domains of shared meanings or ‘netdoms.’ I analytically distinguish between three categories of less-institutionalized situations of the basis of the degree of uncertainty in interpretation and action they impose upon their occupants: high, intermediate, and absence/low. I demonstrate my argument using three examples of less-institutionalized situations/positions from distinct sociological fields: (1) rapid labor-force feminization in South Asia (high-uncertainty); (2) an emergent area of knowledge production (intermediate-uncertainty); and (3) falling average sibship-size implicated in worldwide fertility decline (low/absent uncertainty). Elaborating upon three cross-netdom mechanisms - analogizing, contrasting, and spillovers – and using a mixture of interpretive techniques, multilevel statistical models, and exponential random graph models, I show that occupants use cultural repertoires discursively in high-uncertainty less-institutionalized positions, tacitly in low-uncertainty situations, and in a combination of tacit and deliberative ways under conditions of intermediate uncertainty. I also develop a mathematical model to show how less-institutionalized practices/interpretations can come to be institutionalized over time through management of uncertainty within homophilous networks. Lastly, positing a duality between the cultural repertoires of individuals and those of social locations, I conclude with a discussion on how less-institutionalized positions offer a unique window into investigating processes of emergence and social change.Ph. D.Includes bibliographical referencesIncludes vitaby Neha Gonda
Competing bimetallic ratios: Amsterdam, London and bullion arbitrage in the 18th century
No full textThis article analyses the stability of bimetallism in the mid-18th century for the case of two large centres that had different legal ratios and only one international market ratio. A new theoretical framework is articulated for the situation of international independence to set legal bimetallic ratios by monetary authorities in different countries. Then, using new data handcollected from archival sources and relevant to the two main bullion markets in the 18th century, Amsterdam and London, this theoretical framework is utilised to identify the regimes that actually prevailed during that period, in which Amsterdam was effectively on the bimetallic standard while London was on the gold standard de facto.Bimetallism, Bimetallic stability, Bullion markets, Arbitrage, Specie-point mechanism, Melting-minting points
Regional empty marine container management
No full textEmpty container repositioning is one of the longstanding and ongoing issues in the containerized maritime trade. Even though it is a non-revenue generating, expensive and undesirable exercise, it is an integral part of an overall efficient global transportation system, which balances demand and supply of empty containers between regions. Empty containers are repositioned at three levels - global, inter-regional and regional-level. The focus of this dissertation is at the regional level of empty container repositioning.
Regional repositioning of empty containers involves empty container movement between regional importers, marine terminals, empty container depots, and export customers. This chain movement generates excessive unproductive empty vehicle miles in a region. The problem of empty vehicle miles travelled becomes more prominent when empty container depots are located close to the port and import and export customers are inland. Stakeholders incur large system costs in repositioning empty containers between the regional import-export business locations and the port/depots. Regions with high import activity are concerned with the increase in containerized trade volumes and the persistent trade imbalance because of the capacity shortfall at their existing depots.
This thesis addresses the above two regional concerns of excessive empty vehicle miles and empty container storage capacity shortfall by proposing an 'Inland-Depots-for-Empty-Containers (IDEC)' system. It recommends opening new empty container depots inland in the region, closer to high volume import-export customer clusters, in addition to the depots currently being located near the ports. The dissertation discusses the feasibility, viability, and effectiveness of the proposed system.
It develops mathematical models for the IDEC system to determine the optimal number and location of inland depots in a given region under deterministic and stochastic demand patterns. Exploiting the structure of the NP-hard problem, it develops a heuristic based on the randomized rounding algorithm to solve large scale, realistic depot-location problems. To implement a successful and sustainable IDEC system, it explicitly considers the varied perspectives of different maritime stakeholders involved in the container movement. Based on the models and quantitative analyses, it demonstrates that an IDEC system has great potential in improving regional empty moves, increasing both business profitability and social welfare simultaneously.Ph.D.Includes bibliographical references (p. 133-144)
Generative modeling for relational databases
No full textThesis: M. Eng. in Computer Science and Engineering, Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (pages 79-80).The goal of this thesis is to build a system that automatically creates synthetic data for enabling data science endeavors. To meet this goal, we present the Synthetic Data Vault (SDV), a system that builds generative models of relational databases. We are able to sample from the model and create synthetic data, hence the name SDV. When implementing the SDV, we developed an algorithm that computes statistics at the intersection of related database tables. We then use a state-of-the-art multivariate modeling approach to model this data. The SDV iterates through all possible relations, ultimately creating a model for the entire database. Once this model is computed, the same relational information allows the SDV to synthesize data by sampling from any part of the database. After building the SDV, we used it to generate synthetic data for five different publicly available datasets. We then published the datasets and asked data scientists to develop predictive models for them as part of a crowdsourced experiment. On May 18, 2016, preliminary analysis from the ongoing experiment provided evidence that the synthetic data can successfully replace original data for data science. Our analysis indicates that there is no significant difference in the work produced by data scientists who used synthetic data as opposed to real data. We conclude that the SDV is a viable solution for synthetic data generation. Our primary contribution is that we designed and implemented the first generative modeling system for relational databases that demonstratively synthesizes realistic data.by Neha Patki.M. Eng. in Computer Science and Engineerin
After Columbus: Explaining the Global Trade Boom 1500-1800
No full textThis paper documents the size and timing of the world inter-continental trade boom following the greate voyages in the 1490s of Columbus, da Gama and their followers. Indeed, a trade boom followed over the subsequent three centuries. But what was its cause? The conventionnal wisdom in the world history literature offers globalization as the answer: it alleges that declining trade barriers falling transport costs and overseas "discovery" explains the boom. In contrast, this paper reports the evidence that confirms unambiguously that there was no commodity price convergence between continents, something that would have emerged had globalization been a force that mattered. Thus, the trade boom must have been caused by some combination of European import demand and foreign export supply from Asia and the Americas. Furthermore, the behavior of the relative price of foreign importables in European cities should tell us which mattered most and when. We offer detailed evidence on the relative prices of such importables in European markets over the five centuries 1350-1850. We then offer a model which is used to decompose the sources of the trade boom 1500-1800.
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