Effect of cross-platform gene-expression, computational methods on breast cancer subtyping in PALOMA-2 and PALLET studies.

Intrinsic breast cancer molecular subtyping (IBCMS) provides significant prognostic information for patients with breast cancer and helps determine treatment. This study compared IBCMS methods on various gene-expression platforms in PALOMA-2 and PALLET trials. PALOMA-2 tumor samples were profiled using EdgeSeq and nanostring and subtyped with AIMS, PAM50, and research-use-only (ruo)Prosigna. PALLET tumor biopsies were profiled using mRNA sequencing and subtyped with AIMS and PAM50. In PALOMA-2 (n = 222), a 54% agreement was observed between results from AIMS and gold-standard ruoProsigna, with AIMS assigning 67% basal-like to HER2-enriched. In PALLET (n = 224), a 69% agreement was observed between results from PAM50 and AIMS. Different IBCMS methods may lead to different results and could misguide treatment selection; hence, a standardized clinical PAM50 assay and computational approach should be used.Trial number: NCT01740427.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Simultaneous mass spectrometric quantification of trace amines, their precursors and metabolites.

OBJECTIVES: Trace amines are powerful neuromodulators influencing the release and reuptake of catecholamines. These low concentrated endogenous amines impact mood, cognition, and hormone regulation. Dysregulation of trace amines have been associated with a variety of diseases, such as schizophrenia, Parkinson's disease, migraine, depression and more. Succesfull simultaneous quantification of trace amines, their precursors and metabolites would benefit both research and patient care. Since these compounds have various functional groups and are present in biological matrices with large concentration difference, their simultaneous quantification is an analytical challenge. Our goal was to develop a highly sensitive LC-MS/MS assay to simultaneously quantify trace amines, their precursors and metabolites in plasma. METHODS: Our method is based on a simple two-step in-matrix derivatization protocol: propionic anhydride (PA) and 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide (EDC) in combination with 2,2,2-trifluoroethylamine (TFEA) followed by online solid phase extraction combined with LC-MS/MS. Fifteen metabolites can be measured simultaneously, three precursors, eight trace amines and four metabolites. Validation of this method was performed according to international validation guidelines. The pre-analytical stability of trace amines was assessed. RESULTS: This novel method was successful in quantifying trace amines, their precursors, and metabolites in plasma. Using just 50 µl human plasma, we were able to accomplish limit of quantification for 2-phenylethylamine and N-methyl-phenylethylamine of 0.2 nmol/L and 0.1 nmol/L for tyramine and n-methyltyramine. Inter-and intra-assay imprecision was < 15 % for all analytes. Stability assessment showed susceptibility of certain trace amines e.g. 2-phenylethylamine and N-methyl-phenylethylamine to enzymatic degradation in plasma. The addition of the monoamine oxidase inhibitor pargyline to plasma prevented this enzymatic degradation. CONCLUSIONS: We developed a novel LC-MS/MS method that1) uses a new double derivatization technique, 2) is automated with online SPE, 3) uses far less sample volume then previous methods and 4) detects more components in the same sample (eight trace amines, three precursors, and four metabolites) with high specificity and selectivity. Furthermore, addition of MAO A/B inhibitor prevents degradation and guarantees more accurate quantification of trace amines.

Conserved structures and dynamics in 5'-proximal regions of Betacoronavirus RNA genomes.

Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5'-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5'-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.

Comparative Analysis of the Functions Work Groups and Informal Subgroups Carry out in Relation to their Members: The Essence, Conditions of Implementation, Effects, and Dysfunctions.

In this article, we attempt to integrate and further develop conceptual ideas about functions of small groups and the informal subgroups that arise within them in relation to their respective members, namely, the functions of: (1) creating possibilities for realizing individual goals and meeting individual needs; (2) providing protection from external and intragroup social threats; (3) providing information to members; (4) educating members; (5) providing adaptive capacities to members; and (6) providing control and regulation. First, drawing on a functional analysis, we defined the concept of "function." Next, we touched upon such issues as: the essence of each function; conditions for implementing the functions; the difference between an informal subgroup and a small group in how they implement the functions for their respective members; the effects of implementing the functions; and the related dysfunctions. This versatile account of the functions that small groups and informal subgroups implement in relation to their members allowed us to expand our understanding of these functions and their effects on attitudes, activities and the development of group (subgroup) members, as well as of some aspects of group and subgroup processes and performance. We conclude by presenting both theoretical and practical applications of the analysis of the functions of groups and subgroups and, accordingly, posed some important questions for further research and discussion.

Determination of Soil Contamination at the Wildland-Urban Interface after the 2021 Marshall Fire in Colorado, USA.

Wildfires at the wildland-urban interface (WUI) are increasingly common. The impacts of such events are likely distinct from those that occur strictly in wildland areas, as we would expect an elevated likelihood of soil contamination due to the combustion of anthropogenic materials. We evaluated the impacts of a wildfire at the WUI on soil contamination, sampling soils from residential and nonresidential areas located inside and outside the perimeter of the 2021 Marshall Fire in Colorado, USA. We found that fire-affected residential properties had elevated concentrations of some heavy metals (including Zn, Cu, Cr, and Pb), but the concentrations were still below levels of likely concern, and we observed no corresponding increases in concentrations of polycyclic aromatic hydrocarbons (PAHs). The postfire increases in metal concentrations were not generally observed in the nonresidential soils, highlighting the importance of combustion of anthropogenic materials for potential soil contamination from wildfires at the WUI. While soil contamination from the 2021 Marshall Fire was lower than expected, and likely below the threshold of concern for human health, our study highlights some of the challenges that need to be considered when assessing soil contamination after such fires.

The colitis may be microscopic, but the diarrhea is not: update on the treatment of microscopic colitis and immune checkpoint inhibitor colitis.

PURPOSE OF REVIEW: Microscopic colitis is an inflammatory disease of the colon that presents as watery diarrhea with minimal to normal endoscopic changes on colonoscopy. It encompasses two common subtypes, lymphocytic colitis and collagenous colitis, which are both treated similarly.Immune checkpoint inhibitor colitis is among the most common immune-related adverse events. Endoscopic and histological findings range from normal colonic mucosa to inflammatory bowel like changes. This review article provides update in treatment and management of microscopic colitis and immune checkpoint inhibitor colitis (ICPi colitis). RECENT FINDINGS: Recent studies on microscopic colitis have focused on the successful use of immunomodulators such as biologics for treatment of budesonide refractory microscopic colitis cases. Microscopic colitis does not confer an added risk for colorectal cancer.With the increasing usage of immunotherapy agents, immune checkpoint inhibitor colitis is becoming more common. ICPi colitis can be successfully managed with steroids, with treatment stepped up to biologics for moderate to severe cases or for mild cases that do not respond to steroids. Immunotherapy agents can be carefully re-introduced in mild cases, after treatment of ICPi colitis. SUMMARY: Biologics can be used to treat budesonide refractory microscopic colitis. ICPi colitis can be managed with steroids and biologics in moderate to severe cases.

Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase.

Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66' homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain. Target sites at subdomain interfaces were identified and computational analysis used to obtain an initial set of ligands for screening. Chromatographic evaluations of the p51 homodimer/monomer ratio support the feasibility of this approach. Ligands that bind near the interfaces and a ligand that binds directly to a region of the fingers subdomain involved in subunit interface formation were identified, and the interactions were further characterized by NMR spectroscopy and X-ray crystallography. Although these ligands were found to reduce dimer formation, further efforts will be required to obtain ligands with higher binding affinity. In contrast with previous ligand identification studies performed on the RT heterodimer, subunit interface surfaces are solvent-accessible in the p51 and p66 monomers, making these constructs preferable for identification of ligands that directly interfere with dimerization.

Multiple Non-Canonical Base-Stacking Interactions as One of the Major Determinants of RNA Tertiary Structure Organization.

Stacking interactions of heterocyclic bases of ribonucleotides are one of the most important factors in the organization of RNA secondary and tertiary structure. Most of these (canonical) interactions are formed between adjacent residues in RNA polynucleotide chains. However, with the accumulation of data on the atomic tertiary structures of various RNAs and their complexes with proteins, it has become clear that nucleotide residues that are not adjacent in the polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of nucleotides can interact via (non-canonical) base stacking. This paper presents an exhaustive database of such nonadjacent base-stacking elements (NA-BSEs) and their environment in the macromolecules of natural and synthetic RNAs. Analysis of these data showed that NA-BSE-forming nucleotides, on average, account for about a quarter of all nucleotides in a particular RNA and, therefore, should be considered as bona fide motifs of the RNA tertiary structure. We also classified NA-BSEs by their location in RNA macromolecules. It was shown that the structure-forming role of NA-BSEs involves compact folding of single-stranded RNA loops, transformation of double-stranded bulges into imperfect helices, and binding of RNA regions distant in the primary and secondary RNA structure.

RNA tertiary structure prediction in CASP15 by the GeneSilico group: Folding simulations based on statistical potentials and spatial restraints.

Ribonucleic acid (RNA) molecules serve as master regulators of cells by encoding their biological function in the ribonucleotide sequence, particularly their ability to interact with other molecules. To understand how RNA molecules perform their biological tasks and to design new sequences with specific functions, it is of great benefit to be able to computationally predict how RNA folds and interacts in the cellular environment. Our workflow for computational modeling of the 3D structures of RNA and its interactions with other molecules uses a set of methods developed in our laboratory, including MeSSPredRNA for predicting canonical and non-canonical base pairs, PARNASSUS for detecting remote homology based on comparisons of sequences and secondary structures, ModeRNA for comparative modeling, the SimRNA family of programs for modeling RNA 3D structure and its complexes with other molecules, and QRNAS for model refinement. In this study, we present the results of testing this workflow in predicting RNA 3D structures in the CASP15 experiment. The overall high score of the computational models predicted by our group demonstrates the robustness of our workflow and its individual components in terms of predicting RNA 3D structures of acceptable quality that are close to the target structures. However, the variance in prediction quality is still quite high, and the results are still too far from the level of protein 3D structure predictions. This exercise led us to consider several improvements, especially to better predict and enforce stacking interactions and non-canonical base pairs.

The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos.

The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. Here we determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. We probed interactions of Glo qRRMs with TCEI_III RNA using NMR paramagnetic relaxation experiments. Our in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.

A comprehensive survey of long-range tertiary interactions and motifs in non-coding RNA structures.

Understanding the 3D structure of RNA is key to understanding RNA function. RNA 3D structure is modular and can be seen as a composition of building blocks of various sizes called tertiary motifs. Currently, long-range motifs formed between distant loops and helical regions are largely less studied than the local motifs determined by the RNA secondary structure. We surveyed long-range tertiary interactions and motifs in a non-redundant set of non-coding RNA 3D structures. A new dataset of annotated LOng-RAnge RNA 3D modules (LORA) was built using an approach that does not rely on the automatic annotations of non-canonical interactions. An original algorithm, ARTEM, was developed for annotation-, sequence- and topology-independent superposition of two arbitrary RNA 3D modules. The proposed methods allowed us to identify and describe the most common long-range RNA tertiary motifs. Along with the prevalent canonical A-minor interactions, a large number of previously undescribed staple interactions were observed. The most frequent long-range motifs were found to belong to three main motif families: planar staples, tilted staples, and helical packing motifs.

Molecular profiling of aromatase inhibitor sensitive and resistant ER+HER2- postmenopausal breast cancers.

Aromatase inhibitors (AIs) reduce recurrences and mortality in postmenopausal patients with oestrogen receptor positive (ER+) breast cancer (BC), but >20% of patients will eventually relapse. Given the limited understanding of intrinsic resistance in these tumours, here we conduct a large-scale molecular analysis to identify features that impact on the response of ER + HER2- BC to AI. We compare the 15% of poorest responders (PRs, n = 177) as measured by proportional Ki67 changes after 2 weeks of neoadjuvant AI to good responders (GRs, n = 190) selected from the top 50% responders in the POETIC trial and matched for baseline Ki67 categories. In this work, low ESR1 levels are associated with poor response, high proliferation, high expression of growth factor pathways and non-luminal subtypes. PRs having high ESR1 expression have similar proportions of luminal subtypes to GRs but lower plasma estradiol levels, lower expression of estrogen response genes, higher levels of tumor infiltrating lymphocytes and immune markers, and more TP53 mutations.

Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer.

Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated ß2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of ß2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer.

The proteomic landscape of soft tissue sarcomas.

Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research.

A Transcriptome-Based Precision Oncology Platform for Patient-Therapy Alignment in a Diverse Set of Treatment-Resistant Malignancies.

Predicting in vivo response to antineoplastics remains an elusive challenge. We performed a first-of-kind evaluation of two transcriptome-based precision cancer medicine methodologies to predict tumor sensitivity to a comprehensive repertoire of clinically relevant oncology drugs, whose mechanism of action we experimentally assessed in cognate cell lines. We enrolled patients with histologically distinct, poor-prognosis malignancies who had progressed on multiple therapies, and developed low-passage, patient-derived xenograft models that were used to validate 35 patient-specific drug predictions. Both OncoTarget, which identifies high-affinity inhibitors of individual master regulator (MR) proteins, and OncoTreat, which identifies drugs that invert the transcriptional activity of hyperconnected MR modules, produced highly significant 30-day disease control rates (68% and 91%, respectively). Moreover, of 18 OncoTreat-predicted drugs, 15 induced the predicted MR-module activity inversion in vivo. Predicted drugs significantly outperformed antineoplastic drugs selected as unpredicted controls, suggesting these methods may substantively complement existing precision cancer medicine approaches, as also illustrated by a case study. SIGNIFICANCE: Complementary precision cancer medicine paradigms are needed to broaden the clinical benefit realized through genetic profiling and immunotherapy. In this first-in-class application, we introduce two transcriptome-based tumor-agnostic systems biology tools to predict drug response in vivo. OncoTarget and OncoTreat are scalable for the design of basket and umbrella clinical trials. This article is highlighted in the In This Issue feature, p. 1275.

Activity and Interconnections of Individual and Collective Actors: An Integrative Approach to Small Group Research.

In this article, we attempted to integrate and further develop theoretical ideas in the area of the small group research about all group activity levels (types of actors) - individual, informal subgroup, and group - and about connections among them. We have touched upon such issues as (a) modes of group activity represented by activities of each type of the actors; (b) structural and functional associations among the actors; (c) functions that each type of actors carries out with respect to another type of actors; (d) direct and indirect links among actors; (e) the influence of links between some actors on links among other actors; and (f) processes of integration and disintegration as the main mechanism for changing connections among actors. Special attention is paid to direct (immediate) personalized and depersonalized connections among actors, as well as to connections mediated by actors' connections with another actor or some object. Discussion of these issues leads to formulation of some specific propositions. Simultaneous research coverage of all three types of actors and various connections among them should allow for creating a more complete picture of small group activities and various psychological phenomena within it, including multifaceted and complex ones. It should also enable considering group structure and the essence of group dynamics differently. We conclude this article by presenting both theoretical and practical implications of the proposed integrative perspective and by posing some important questions in line with it for further discussion.

A gut microbiome metabolite paradoxically depresses contractile function while activating mitochondrial respiration.

Trimethylamine-N-oxide (TMAO) is an end-product of gut microbiome metabolism linked to cardiovascular disease (CVD). However, precise cardiovascular influences of the TMAO concentrations reported in early or severe disease remain to be detailed. We investigated acute effects of TMAO on cardiac contractile, coronary and mitochondrial function. Male C57Bl/6 mouse hearts were Langendorff perfused to assess concentration-dependent effects of TMAO (1-300 µM) on left ventricular (LV) function, coronary flow and select protein expression. Effects of 10 µM and 100 µM TMAO on LV mitochondrial function were examined via respirometry. TMAO at 10-300 µM concentration-dependently depressed LV contractile function, with coronary flow paralleling changes in isovolumic pressure development. Direct coronary effects were evident at >30 µM TMAO in hearts performing minimal isovolumic work, although this response was reduced by >65%. In contrast, exposure to 10 µM or 100 µM TMAO increased mitochondrial complex I, II and maximal respiratory fluxes while appearing to reduce outer membrane integrity. Expression of phosphorylated AMPKα and total GSK-3ß declined. Thus, acute exposure of mouse hearts to TMAO levels reported in advanced CVD significantly inhibits cardiac contractility and induces modest coronary constriction while paradoxically overactivating mitochondrial respiration.

Nematically Templated Vortex Lattices in Superconducting FeSe.

New pathways to controlling the morphology of superconducting vortex lattices─and their subsequent dynamics─are required to guide and scale vortex world-lines into a computing platform. We have found that the nematic twin boundaries align superconducting vortices in the adjacent terraces due to the incommensurate potential between vortices surrounding twin boundaries and those trapped within them. With the varying density and morphology of twin boundaries, the vortex lattice assumes several distinct structural phases, including square, regular, and irregular one-dimensional lattices. Through concomitant analysis of vortex lattice models, we have inferred the characteristic energetics of the twin boundary potential and furthermore predicted the existence of geometric size effects as a function of increasing confinement by the twin boundaries. These findings extend the ideas of directed control over vortex lattices to intrinsic topological defects and their self-organized networks, which have direct implications for the future design and control of strain-based topological quantum computing architectures.

Structural and ligand binding analysis of the pet allergens Can f 1 and Fel d 7.

Introduction: Pet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands. Methods: To provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7. Results: Structural comparisons with reported lipocalins revealed that Can f 1 and Fel d 7 calyxes are open and positively charged while other dog lipocalin allergens are closed and negatively charged. We screened fatty acids as surrogate ligands, and found that Can f 1 and Fel d 7 bind multiple ligands with preferences for palmitic acid (16:0) among saturated fatty acids and oleic acid (18:1 cis-9) among unsaturated ones. NMR analysis of methyl probes reveals that conformational changes occur upon binding of pinolenic acid inside the calyx. Molecular dynamics simulation shows that the carboxylic group of fatty acids shuttles between two positively charged amino acids inside the Can f 1 and Fel d 7 calyx. Consistent with simulations, the stoichiometry of oleic acid-binding is 2:1 (fatty acid: protein) for Can f 1 and Fel d 7. Discussion: The results provide valuable insights into the determinants of selectivity and candidate ligands for pet lipocalin allergens Can f 1 and Fel d 7.