Distinct Properties of Ca2+Ca^{2+}-Calmodulin Binding to N- and C-Terminal Regulatory Regions of the TRPV1 Channel

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM $(Ca^{2+}–CaM)$ in complex with the TRPV1-CT segment, determined to $1.95-\mathring{A}$ resolution. The two lobes of $Ca^{2+}–CaM$ wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of $Ca^{2+}–CaM$, respectively. This structure is similar to canonical $Ca^{2+}–CaM$-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity $Ca^{2+}–CaM$-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the $Ca^{2+}–CaM$-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the $Ca^{2+}–CaM$-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.Molecular and Cellular Biolog

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.Molecular and Cellular Biolog

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection.

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens

Glia Delimit Shape Changes Of Sensory Neuron Receptive Endings in C. elegans

Neuronal receptive endings such as dendritic spines and sensory protrusions are structurally remodeled by experience. How receptive endings acquire their remodeled shapes is not well understood. In response to environmental stressors, including starvation, crowding and high temperature, the nematode Caenorhabditis elegans enters a diapause state, termed dauer, which is accompanied by remodeling of sensory neuron receptive endings. Here, we demonstrate that sensory receptive endings of the AWC amphid neurons in dauer animals remodel in the confines of a compartment defined by the amphid sheath glial cells that envelop these endings. The glia remodel concomitantly with and independently of AWC receptive endings to delimit AWC receptive ending growth. Remodeling of the glia requires the Otd/Otx transcription factor TTX-1, the C2H2 zinc finger transcription factor ZTF-16, the fusogen AFF-1, and likely the VEGFR-related protein VER-1, all acting within the glial cell. ver-1 expression is induced by dauer entry and by cultivation at high temperature, and requires direct binding of TTX-1 and perhaps also ZTF-16 to ver-1 regulatory sequences. Our results demonstrate that experience-induced changes in glial compartment size provide spatial constraints on neuronal receptive ending growth

On the Provenance of Software Systems: Automating Software Traceability with Knowledge Graph and Large Language Model Synergy

The present dissertation delineates a system that enables those engaged in software development to automatically generate and maintain project life cycle provenance. All projects are implemented and made manifest with the development of artifacts, e.g., papers, code files, etc. Tools exist to accelerate artifact creation, but little focus is paid to the processes that produce them. In terms of Ontology, or, from Ancient Greek, the study of being, the two most basic entities in reality are Continuant and Occurrent, or, roughly, “Artifact” and “Process”. This dissertation posits that for any created artifact, its process of creation, i.e., its life cycle provenance, must also be captured and maintained. Artifacts are often delivered without an explicit trace of their evolution. This is particularly unacceptable for critical systems, where requirements documents, codebases, and other meta-artifacts are revisited without a corresponding history of how or why they came to be, leading to confusion and rework. While the software development life cycle (SDLC) incorporates meta-artifacts like traceability matrices to improve artifact provenance, these are typically informal, heavy with natural language, and lack structured explainability. This work proposes that each artifact should be attended by a machine-readable, human-interpretable, extensible provenance record, implemented in the form of a knowledge graph, backed by well-established ontologies. The developed system, ProvTracer, leverages structured knowledge via PROV-O and the Basic Formal Ontology alongside generative natural language capabilities via the Generative Pretrained Transformer (GPT) series of Multimodal Large Language Models (MLLMs), to create real-time, traceable, and explainable links between development activities and their resulting artifacts. By capturing these provenance trace links automatically through multimodal signals, e.g., screenshots, peripheral device input, etc., ProvTracer aims to bridge the gap between implicit processes and explicit traces, enabling developers to understand, query, maintain, integrate, and trust the evolution of their projects and systems. The synergy between knowledge graphs and MLLMs enables a novel form of interactive, explainable software development. Natural language queries of provenance trace link knowledge graphs can reduce information overload, extract developer rationale and decision histories, support task assignment, and a range of project management activities. This aligns with a burgeoning trend in research demonstrating that structured knowledge improves machine learning trust, transparency and reproducibility. The present dissertation addresses the challenges of traceability and explainability in the SDLC by presenting a system that automatically captures artifact provenance and operationalizes it for practical use in real-world software development

Functionalization of C-aryl glycals and studies toward the total synthesis of 5-hydroxyaloin A

textIn the context of ongoing efforts toward C-aryl glycoside synthesis, a recently developed approach to form C-aryl glycals from 2-deoxysugar lactones was expanded to form novel substrates. This approach has been applied to the synthesis of various furyl glycals, allowing access to C-aryl glycals via a benzyne furan (4+2) cycloaddition methodology. The hydroboration-oxidation of said C-aryl glycals has allowed access to C(2)-oxygenated C-aryl glycosides via the benzyne cycloaddition approach. An approach to the total synthesis of 5-hydoxyaloin A is detailed, in which regioselective benzyne furan (4+2) cycloadditions were achieved via the use of a silicon tether. Two approaches to the anthrone core have been applied; one in which an unsymmetrically-substituted aryl ring is first constructed by means of a silicon tether, and one in which the unsymmetrically-substituted ring is formed last, also utilizing a silicon tether. The latter approach has allowed access to the anthrone core of 5-hydroxyaloin A, and only a final desulfurization remains in order to access the natural product.Chemistr

Towards Agile Academia: An Approach to Scientific Paper Writing Inspired by Software Engineering

The construction of scientific papers is performed in service of the greater scientific community. This iterative process is, in effect, an academic economy, where all members benefit from well-written papers. However, many published scientific papers are poorly written; they often lack sufficient detail to allow replication, there is improper usage of citations or a lack of regard to relevant work, reporting is vague or without linked empirical data to allow verification, figures do not correspond to text or are non-sensical, literary elements, e.g., bulleted lists, are used ineffectively, formatting renders certain sections unreadable, and grammatical errors abound. The issues of paper quality are widespread and of varying concern. Similarly, the development of software systems is rife with many processual issues, from high-level architectural flaws to small developer errors, e.g., setting a Boolean value to true instead of false, which can be disastrous in large systems. As an answer to these longstanding concerns, software development methods have emerged over decades, most notably, the Waterfall and Agile approaches. These methods have established software engineering as a professional discipline backed by rigorous, empirical evaluation on many systems. A scientific paper is, conceptually, a system to be developed, much like a software system: it has a name, particular sections codified for different purposes, e.g., as the abstract summarizes and the conclusion concludes, it has an author or authors, it goes through several iterations of refinement, it may reference outside systems and it is eventually released to the public, and possibly maintained in future versions. It is posited that, due to the relatively small nature of most scientific papers (4-20 pages), the Agile method of software development can be used to produce more reliable scientific papers, in a more efficient manner and with better availability to readers, by employing the principles of open-source software, and a version control system, e.g., Git. Agile methods consistently provide deliverables of higher quality; this work intends to demonstrate that Agile can be adapted to streamline the scientific writing process and improve publication quality

Mitotic Exit: Thresholds and Targets

Cyclin dependent kinases (CDKs) are at the heart of the cell cycle. Throughout the cycle, these complexes modify many proteins, changing various aspects of their regulation (stability, localization, etc.). As cells exit mitosis, the CDK that has driven many of the cell cycle processes is inhibited and degraded, allowing many of the kinase substrates to return to their unphosphorylated state. This assures that each subsequent cell cycle is begun in the same naïve state, again ready for CDK-dependent regulation. The studies in this thesis focus on two mechanisms by which this restoration is accomplished in the budding yeast, Saccharomyces cerevisiae: (1) a transcriptional program that transcribes many of the genes required for physically dividing the mother and daughter cells and beginning the next round of cell division and (2) a phosphatase that specifically removes the phosphates from sites modified by CDK during exit from mitosis. Two transcription factors, Swi5 and Ace2, transcribe many of the genes required for physically dividing the mother and daughter cells and beginning the next round of cell division. Previously our lab has shown that locking mitotic cyclin levels, by inducing transcription of an undegradable form of the protein, causes dose-dependent delays in different cell cycle events. The first chapter addresses the contribution of the transcriptional program to this phenomenon. Interestingly, in these cells where mitotic cyclin levels were sustained, deletion of the transcription factor Swi5 increases the mitotic cyclin inhibition, specifically as it relates to budding and cytokinesis. Importantly, when phosphorylated by CDK, Swi5 is excluded from the nucleus, so in the second chapter, we investigate its localization when mitotic cyclin levels are locked. Swi5 still enters the nucleus. In fact in some cells, Swi5 enters the nucleus several times before the cell cycle advances. Given previous studies from our lab showing that the release of Cdc14 phosphatase also oscillates under these conditions, the reentry of Swi5 may support a model that a kinase/phosphatase balance allows cell cycle progression in these cells. All this suggests that Swi5 promotes the transcription of genes important for promoting cytokinesis and budding despite high mitotic cyclin levels. In the third chapter, we begin to assess the contribution of specific targets of the mitotic exit transcriptional program to the mitotic cyclin-dependent regulation of specific cell cycle events. Finally, Cdc14, a phosphatase that removes the phosphate groups added by CDKs, is sequestered for most of the cell cycle but released from the nucleolus during the end of mitosis. In the fourth chapter, we examine the physiological relevance of these dephosphorylation events on novel targets of the Cdc14 phosphatase

IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

Background: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC). Methodology/Principal Findings: Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1β and IL-6 ("conv. mix"). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC. Conclusion: Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy. © 2013 Schilling et al

Quantitative Conservation of the Gray Wolf (Canis lupus): Implications of Monitoring and Modeling the Yellowstone Wolves

The 1995 reintroduction of wolves to the Greater Yellowstone ecosystem had lasting effects on our understanding of reintroduction biology as a whole. However, continued study of a system as complex and intricately interwoven as this should be done with minimal human influence on the ecosystem. The states comprising the tri-state area surrounding Yellowstone National Park—Wyoming, Idaho, and Montana—have recently delisted wolves from the endangered species list. Here, I assess the greater implications in conservation that can be deduced from quantitative analysis of the Yellowstone wolves, and question whether the species is, in fact, stable enough for delisting in this area. I used multiple regressions on population growth as a function of population size to test the claim that the carrying capacity for wolves in Yellowstone is approximately 170 individuals. After finding that the estimate of 170 is much larger than my calculated values of carrying capacity, I move forward to design six distinct models that each enforce a predesignated carrying capacity on 100 different simulated wolf populations over the next 100 years. All of these simulations experienced zero probability of extinction. However, I discuss general trends that may still be extracted from the models, potential flaws in my modeling for this particular system, and a variety of different approaches to designing simulations that may more effectively predict the fate of the Yellowstone wolf population. I offer observations of how population dynamics change with respect to carrying capacities once the carrying capacity has been attained, and address differences between population dynamics in a pre-established population versus and those in a reintroduced population. Overall, I conclude by noting that the fate of the wolves is not certain, and should therefore be monitored and maintained with a more adaptive management strategy