The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment.

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

Where's Whaledo: A software toolkit for array localization of animal vocalizations.

Where's Whaledo is a software toolkit that uses a combination of automated processes and user interfaces to greatly accelerate the process of reconstructing animal tracks from arrays of passive acoustic recording devices. Passive acoustic localization is a non-invasive yet powerful way to contribute to species conservation. By tracking animals through their acoustic signals, important information on diving patterns, movement behavior, habitat use, and feeding dynamics can be obtained. This method is useful for helping to understand habitat use, observe behavioral responses to noise, and develop potential mitigation strategies. Animal tracking using passive acoustic localization requires an acoustic array to detect signals of interest, associate detections on various receivers, and estimate the most likely source location by using the time difference of arrival (TDOA) of sounds on multiple receivers. Where's Whaledo combines data from two small-aperture volumetric arrays and a variable number of individual receivers. In a case study conducted in the Tanner Basin off Southern California, we demonstrate the effectiveness of Where's Whaledo in localizing groups of Ziphius cavirostris. We reconstruct the tracks of six individual animals vocalizing concurrently and identify Ziphius cavirostris tracks despite being obscured by a large pod of vocalizing dolphins.

Distinct Coordination Chemistry of Fe(III)-Based MRI Probes.

Contrast agents are used in approximately 40% of all magnetic resonance imaging (MRI) procedures to improve the quality of the images based on the distribution and dynamic clearance of the agent. To date, all clinically approved contrast agents are Gd(III) coordination complexes that serve to shorten the longitudinal (T1) and transverse (T2) proton relaxation times of water. Recent interest in replacing Gd with biologically relevant metal ions such as Mn or Fe has led to increased interest in the aqueous coordination chemistry of their complexes. In this Account, we focus on high-spin Fe(III) complexes that have been recently reported as MRI contrast agents or probes in our laboratory.The highly Lewis acidic Fe(III) center has distinct coordination chemistry in aqueous solutions, facilitating alternative strategies in the design of MRI probes. To illustrate this, we describe different classes of Fe(III) MRI probes with a focus on macrocyclic complexes and multinuclear complexes such as self-assembled metal organic polyhedra (MOP). Our initial efforts focused on macrocyclic complexes of Fe(III) in order to tune spin and oxidation states with the goal of stabilizing high-spin Fe(III) in reducing biological environments. Our probes feature six-coordinate Fe(III) complexes of 1,4,7-triazacyclononane with hydroxypropyl, phosphonate, or carboxylate pendant groups to produce Fe(III) complexes that shorten proton T1 times predominantly from second-sphere or outer-sphere interactions at neutral pH. Analogues with pentadentate macrocyclic ligands have an inner-sphere water that does not exchange rapidly on the NMR time scale, yet these complexes are effective relaxation agents. Fe(III) macrocyclic complexes in this class can be modified to modulate their biodistribution and pharmacokinetic clearance in mice. The goal of these studies is for the Fe(III) agents to clear as extracellular fluid agents and produce profiles similar to those of Gd agents. Finally, studies of multimeric Fe(III) complexes are of interest to produce probes that give large proton relaxivity. In this approach the two Fe(III) centers are connected through aryl linkers as demonstrated for several macrocyclic complexes. Even more tightly connected Fe(III) centers are produced in a Fe(III) self-assembled cage with relaxivity of 21 mM-1 s-1 at 4.7 T, 37 °C in the presence of serum albumin to which it is tightly bound. This cage enhances contrast of the vasculature as a blood pool agent and accumulates in tumors. Finally, we present our perspectives on the further development of Fe(III) complexes for various applications in MRI.

Transcriptional Circuitry of NKX2-1 and SOX1 Defines an Unrecognized Lineage Subtype of Small-Cell Lung Cancer.

Rationale: The current molecular classification of small-cell lung cancer (SCLC) on the basis of the expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. Objectives: To refine the current SCLC classification with epigenomic profiles and to identify features of the redefined SCLC subtypes. Methods: We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance of NKX2-1 (NK2 homeobox 1) was evaluated by cell growth, apoptosis, and xenograft using clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-associated protein 9)-mediated deletion. NKX2-1-specific cistromic profiles were determined using chromatin immunoprecipitation followed by sequencing, and its functional transcriptional partners were determined using coimmunoprecipitation followed by mass spectrometry. Rb1flox/flox; Trp53flox/flox and Rb1flox/flox; Trp53flox/flox; Nkx2-1flox/flox mouse models were engineered to explore the function of Nkx2-1 in SCLC tumorigenesis. Epigenomic landscapes of six human SCLC specimens and 20 tumors from two mouse models were characterized. Measurements and Main Results: We identified two epigenomic subclusters of the major SCLC-A subtype: SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found that NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. In addition, we found that maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor, SOX1 (SRY-box transcription factor 1). Conclusions: We comprehensively describe additional epigenomic heterogeneity of the major SCLC-A subtype and define the SCLC-Aα subtype by the core regulatory circuitry of NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

Bayesian detection and tracking of odontocetes in 3-D from their echolocation clicks.

Localization and tracking of marine animals can reveal key insights into their behaviors underwater that would otherwise remain unexplored. A promising nonintrusive approach to obtaining location information of marine animals is to process their bioacoustic signals, which are passively recorded using multiple hydrophones. In this paper, a data processing chain that automatically detects and tracks multiple odontocetes (toothed whales) in three dimensions (3-D) from their echolocation clicks recorded with volumetric hydrophone arrays is proposed. First, the time-difference-of-arrival (TDOA) measurements are extracted with a generalized cross-correlation that whitens the received acoustic signals based on the instrument noise statistics. Subsequently, odontocetes are tracked in the TDOA domain using a graph-based multi-target tracking (MTT) method to reject false TDOA measurements and close gaps of missed detections. The resulting TDOA estimates are then used by another graph-based MTT stage that estimates odontocete tracks in 3-D. The tracking capability of the proposed data processing chain is demonstrated on real acoustic data provided by two volumetric hydrophone arrays that recorded echolocation clicks from Cuvier's beaked whales (Ziphius cavirostris). Simulation results show that the presented MTT method using 3-D can outperform an existing approach that relies on manual annotation.

Foxa2 and Cdx2 cooperate with Nkx2-1 to inhibit lung adenocarcinoma metastasis.

Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5long, the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program.

Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: classical and basal-like. The classical subtype is characterised by a more favourable prognosis and better response to chemotherapy than the basal-like subtype. The classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor hepatocyte nuclear factor 4 α (HNF4α). The objective of this study is to evaluate the role of HNF4α, SIX4 and SIX1 in regulating the growth and molecular subtype of PDAC. DESIGN: We manipulate the expression of HNF4α, SIX4 and SIX1 in multiple in vitro and in vivo PDAC models. We determine the consequences of manipulating these genes on PDAC growth, differentiation and molecular subtype using functional assays, gene expression analysis and cross-species comparisons with human datasets. RESULTS: We show that HNF4α restrains tumour growth and drives tumour cells toward an epithelial identity. Gene expression analysis of murine models and human tumours shows that HNF4α activates expression of genes associated with the classical subtype. HNF4α also directly represses SIX4 and SIX1, two mesodermal/neuronal lineage specifiers expressed in the basal-like subtype. Finally, SIX4 and SIX1 drive proliferation and regulate differentiation in HNF4α-negative PDAC. CONCLUSION: Our data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the classical gene expression programme and repression of SIX4 and SIX1, which may represent novel dependencies of the basal-like subtype.

Nkx2-1 represses a latent gastric differentiation program in lung adenocarcinoma.

Tissue-specific differentiation programs become dysregulated during cancer evolution. The transcription factor Nkx2-1 is a master regulator of pulmonary differentiation that is downregulated in poorly differentiated lung adenocarcinoma. Here we use conditional murine genetics to determine how the identity of lung epithelial cells changes upon loss of their master cell-fate regulator. Nkx2-1 deletion in normal and neoplastic lungs causes not only loss of pulmonary identity but also conversion to a gastric lineage. Nkx2-1 is likely to maintain pulmonary identity by recruiting transcription factors Foxa1 and Foxa2 to lung-specific loci, thus preventing them from binding gastrointestinal targets. Nkx2-1-negative murine lung tumors mimic mucinous human lung adenocarcinomas, which express gastric markers. Loss of the gastrointestinal transcription factor Hnf4α leads to derepression of the embryonal proto-oncogene Hmga2 in Nkx2-1-negative tumors. These observations suggest that loss of both active and latent differentiation programs is required for tumors to reach a primitive, poorly differentiated state.

Fetal Origins of Asthma: A Longitudinal Study from Birth to Age 36 Years.

Rationale: Deficits in infant lung function-including the ratio of the time to reach peak tidal expiratory flow to the total expiratory time (tptef/te) and maximal expiratory flow at FRC (V̇maxFRC)-have been linked to increased risk for childhood asthma.Objectives: To examine the individual and combined effects of tptef/te and V̇maxFRC in infancy on risk for asthma and abnormalities of airway structure into mid-adult life.Methods: One hundred eighty participants in the Tucson Children's Respiratory Study birth cohort had lung function measured by the chest-compression technique in infancy (mean age ± SD: 2.0 ± 1.2 mo). Active asthma was assessed in up to 12 questionnaires between ages 6 and 36 years. Spirometry and chest high-resolution computed tomographic (HRCT) imaging were completed in a subset of participants at age 26. The relations of infant tptef/te and V̇maxFRC to active asthma and airway structural abnormalities into adult life were tested in multivariable mixed models.Measurements and Main Results: After adjustment for covariates, a 1-SD decrease in infant tptef/te and V̇maxFRC was associated with a 70% (P = 0.001) and 55% (P = 0.005) increased risk of active asthma, respectively. These effects were partly independent, and two out of three infants who were in the lowest tertile for both tptef/te and V̇maxFRC developed active asthma by mid-adult life. Infant V̇maxFRC predicted reduced airflow and infant tptef/te reduced HRCT airway caliber at age 26.Conclusions: These findings underscore the long-lasting effects of the fetal origins of asthma, support independent contributions by infant tptef/te and V̇maxFRC to development of asthma, and link deficits at birth in tptef/te with HRCT-assessed structural airway abnormalities in adult life.

Alveolar to arterial gas exchange during constant-load exercise in healthy active men and women.

Inadequate hyperventilation and inefficient alveolar to arterial gas exchange are gas exchange challenges that can limit capacity and cause exercise-induced arterial hypoxaemia (EIAH). This work evaluated if the prevalence of gas exchange inefficiencies, defined as AaDO2>25 mmHg, PaCO2>38 mmHg, and/or ΔPaO2>-10 mmHg at any point during constant-load exercise in healthy, active, but not highly trained, individuals suggested an innate sex difference that would make females more susceptible to EIAH. Sixty-four healthy, active males and females completed 18-min of cycling exercise (moderate and vigorous intensity, 9 min/stage). Arterial blood gases were measured at rest and every 3-min during exercise, while constantly assessing gas exchange. Both sexes demonstrated similar levels of AaDO2 widening until the final 3 min of vigorous exercise, where females demonstrated a trend for greater widening than males (16.3±6.2 mmHg vs. 19.1±6.0 mmHg, p=0.07). Males demonstrated a blunted ventilatory response to moderate exercise with higher PaCO2 (38.5±2.6 vs. 36.5±2.4, p=0.002) and a lower ventilation when corrected for workload (0.42±0.1 vs. 0.48±0.1, p=0.002). No significant arterial hypoxaemia occurred, but in 6 M and 5 F SaO2 dropped by ≥2%. There was no difference in prevalence of pulmonary gas exchange inefficiencies between sexes, but the type of inefficiency was influenced by sex.Abbreviations: AaDO2: alveolar-arterial oxygen difference; BP: blood pressure; EIAH: exercise-induced arterial hypoxaemia; F: females; HR: heart rate; M: males; Q: cardiac output; PaCO2: arterial partial pressure of carbon dioxide; PaO2: arterial partial pressure of oxygen; ΔPaO2: change in arterial partial pressure of oxygen; PAO2: alveolar partial pressure of oxygen; RPE: rating of perceived exertion; SaO2: arterial oxygen saturation; VE: ventilation; VE/VCO2: ventilatory equivalent for carbon dioxide; VO2PEAK: peak oxygen consumption; WMAX: workload maximum.

Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis.

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

Unwarranted philosophical assumptions in research on ANS.

Clarke and Beck import certain assumptions about the nature of numbers. Although these are widespread within research on number cognition, they are highly contentious among philosophers of mathematics. In this commentary, we isolate and critically evaluate one core assumption: the identity thesis.

Comparing life review writing with active control groups: Results of a feasibility study.

INTRODUCTION: Life review writing can be used to improve depressive symptoms in well older adults, but it is unclear whether the reminiscing, writing, or socialising is associated with the improvements. This study assessed the feasibility of a controlled trial comparing life review writing with two active control groups designed to isolate the acts of (a) engaging in a meaningful leisure activity and (b) socialising. METHODS: Twenty-four participants from a 100-bed assisted living facility were enrolled in the study. Participants ranged in age from 70 to 98 years (M = 83.9, SD = 7.6). Eight 1-hour weekly sessions were held, with participants assigned to one of three groups: (a) life review writing, (b) painting, or (c) coffee from around the world. At baseline, participants completed a demographic and health questionnaire, the Mini-Cog, and the Geriatric Depression Scale Short Form (GDS-15). The GDS-15 was administered again at weeks 4 and 8, along with the health questionnaire at post-test. RESULTS: Nearly 25% (N = 24) of the facility's residents attended at least one session, with an average attendance rate of 66.4%, or 5.3 sessions. However, to achieve this participation rate, our original plan to employ random assignment had to be abandoned. The groups were a partial success insofar as we successfully isolated engaging in a meaningful activity in the painting group, but the writing group engaged in less reminiscing than hoped. At post-test, seven participants reported health, mood, and social life benefits. GDS-15 scores fluctuated over time, with no significant improvement from pre- to post-test. CONCLUSION: The residents' low willingness to be randomised creates a serious selection bias, but randomising volunteers into different activities runs counter to client-centred therapy. To scrutinise the mechanisms of change of life review writing, we recommend comparing two writing groups: one that reminisces and one that does not.

A Class of FeIII Macrocyclic Complexes with Alcohol Donor Groups as Effective T1 MRI Contrast Agents.

Early studies suggested that FeIII complexes cannot compete with GdIII complexes as T1 MRI contrast agents. Now it is shown that one member of a class of high-spin macrocyclic FeIII complexes produces more intense contrast in mice kidneys and liver at 30 minutes post-injection than does a commercially used GdIII agent and also produces similar T1 relaxivity in serum phantoms at 4.7 T and 37 °C. Comparison of four different FeIII macrocyclic complexes elucidates the factors that contribute to relaxivity in vivo including solution speciation. Variable-temperature 17 O NMR studies suggest that none of the complexes has a single, integral inner-sphere water that exchanges rapidly on the NMR timescale. MRI studies in mice show large in vivo differences of three of the FeIII complexes that correspond, in part, to their r1 relaxivity in phantoms. Changes in overall charge of the complex modulate contrast enhancement, especially of the kidneys.

Influence of ß2 adrenergic receptor genotype on risk of nocturnal ventilation in patients with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease resulting in severe respiratory derangements. As such, DMD patients are at a high risk of nocturnal hypoventilation, thereby requiring nocturnal ventilation (NV). To this end, NV is an important clinical milestone in the management of DMD. Emerging evidence suggests that ß2 adrenergic receptors (ADRB2) may play a role in determining respiratory function, whereby more functional ADRB2 genotype variants (e.g., Gly16) are associated with improved pulmonary function and respiratory muscle strength. These findings suggest that the more functional ADRB2 genotype may help to preserve respiratory function in patients with DMD. The purpose of this study was to identify the influence of ADRB2 genotype on the risk of NV use in DMD. Data from the CINRG Duchenne Natural History Study including 175 DMD patients (3-25 yrs) were analyzed focusing on ADRB2 genotype variants. Time-to-event analyses were used to examine differences in the age at prescription of full-time NV use between genotypes. There were no differences between genotype groups in age, height, weight, corticosteroid use, proportion of ambulatory patients, or age at loss of ambulation. DMD patients expressing the Gly16 polymorphism had a significantly (P < 0.05) lower mean age at NV prescription compared with those patients expressing the Arg16 polymorphism (21.80 ± 0.59 yrs. vs 25.91 ± 1.31 yrs., respectively). In addition, a covariate-adjusted Cox model revealed that the Gly16 variant group possessed a 6.52-fold higher risk of full-time NV use at any given age compared with the Arg16 polymorphism group. These data suggest that genetic variations in the ADRB2 gene may influence the age at which DMD patients are first prescribed NV, whereby patients with the Gly16 polymorphism are more likely to require NV assistance at an earlier age than their Arg16 counterparts.

Nuclear factor I/B is an oncogene in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

Inner-Sphere and Outer-Sphere Water Interactions in Co(II) paraCEST Agents.

High-spin Co(II) complexes are promising for development as paraCEST agents (paraCEST = paramagnetic chemical exchange saturation transfer) for magnetic resonance imaging applications. The first examples of Co(II) paraCEST agents with bound water ligands are presented here. Four Co(II) macrocyclic complexes based on 1,4,7-triazacyclononane and containing either pendent alcohol or pendent amide groups were prepared. Two of the macrocycles encapsulate the Co(II) and contain no water ligands as shown by X-ray crystallographic studies, and two complexes have macrocycles with only five ligand donor groups to leave an open coordination site for bound water. The ionization of alcohol, water, or amide groups in the complexes was characterized by using pH potentiometry. These data show that one of the complexes has a readily deprotonated group with a pKa close to 6, which is assigned as an alcohol pendent. Amide pendents deprotonate at high pH (>8), and the water ligands of the Co(II) complexes are not deprotonated at neutral pH. All complexes produce CEST peaks through either alcohol OH or amide NH proton exchange. The water ligands exchange too rapidly to produce a CEST effect as shown by variable-temperature 17O NMR spectroscopy studies. The complexes with available coordination sites for inner-sphere water ligands produce large paramagnetic shifts and broadening of the 17O resonances of bulk water, whereas the encapsulated complexes show much less shifting and broadening of 17O resonances. All complexes produce substantial paramagnetic shifts of the 1H resonances of bulk water, which is promising for the development of supramolecular CEST agents.