Implementation of Enhanced Recovery Protocols for Gastrointestinal Surgery in Children: Practical Tools From Key Stakeholders.

INTRODUCTION: We explored patient, caregiver, and provider recommendations for development of a tool kit to implement enhanced recovery protocols (ERPs) for pediatric patients undergoing gastrointestinal surgery. ERPs are widely used for adults to decrease hospital length of stay, hospital costs, and complications while hastening patient recovery after surgery. With limited data available for ERPs among pediatric populations informed modification of adult ERPs is needed to facilitate successful implementation for pediatric surgery. METHODS: Using a qualitative research design, semistructured interviews were conducted with hospital-based teams including surgeons, anesthesiologists, gastroenterologists, nursing, and physician assistants. Four in-person focus groups were held at two pediatric hospitals with patients and caregivers. Codes were developed and applied to interview and focus groups transcripts for structural content analysis. Thematic analysis guided by the Active Implementation Framework, included recommendations that informed ERP implementation tool kit development. RESULTS: Key components of the ERP tool kit included the need for a structured and systematic approach, leadership support from key champions, and buy-in from surgical partners and hospital management. Providers identified the need for multimodal educational materials on ERP elements for staff and patients; use of uniform checklists, care sets and an electronic repository to collect outcome data for quality assurance assessment. Patients and caregivers endorsed expansion of the team to include child-life specialists, nutritionists, and patient-parent supporters to help navigate the surgical experience. CONCLUSIONS: This study is the first to leverage key input from patients, caregivers, and providers to identify practical components for an ERP implementation tool kit for children undergoing gastrointestinal surgery.

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection.

Low-dose human interleukin-2 (hIL-2) treatment is used clinically to treat autoimmune disorders due to the cytokine's preferential expansion of immunosuppressive regulatory T cells (Tregs). However, off-target immune cell activation and short serum half-life limit the clinical potential of IL-2 treatment. Recent work showed that complexes comprising hIL-2 and the anti-hIL-2 antibody F5111 overcome these limitations by preferentially stimulating Tregs over immune effector cells. Although promising, therapeutic translation of this approach is complicated by the need to optimize dosing ratios and by the instability of the cytokine/antibody complex. We leverage structural insights to engineer a single-chain hIL-2/F5111 antibody fusion protein, termed F5111 immunocytokine (IC), which potently and selectively activates and expands Tregs. F5111 IC confers protection in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus. These results provide a roadmap for IC design and establish a Treg-biased immunotherapy that could be clinically translated for autoimmune disease treatment.

cDC1 coordinate innate and adaptive responses in the omentum required for T cell priming and memory.

In the peritoneal cavity, the omentum contains fat-associated lymphoid clusters (FALCs) whose role in response to infection is poorly understood. After intraperitoneal immunization with Toxoplasma gondii, conventional type 1 dendritic cells (cDC1s) were critical to induce innate sources of IFN-γ and cellular changes in the FALCs. Unexpectedly, infected peritoneal macrophages that migrated into the FALCs primed CD8+ T cells. Although T cell priming was cDC1 independent, these DCs were required for maximal CD8+ T cell expansion. An agent-based computational model and experimental data highlighted that cDC1s affected the magnitude of the proliferative burst and promoted CD8+ T cell expression of nutrient uptake receptors and cell survival. Thus, although FALCs lack the organization of secondary lymphoid organs, cDC1s resident in this tissue coordinate innate responses to microbial challenge and provide secondary signals required for T cell expansion and memory formation.

A qualitative examination of barriers and facilitators of pediatric enhanced recovery protocol implementation among 18 pediatric surgery services.

BACKGROUND: Enhanced recovery protocols (ERPs) are an evidence-based intervention to optimize post-surgical recovery. Several studies have demonstrated that the use of an ERP for gastrointestinal surgery results in decreased length of stay, shortened time to a regular diet, and fewer administered opioids, while also trending toward lower complication and 30-day readmission rates. Yet, implementation of ERPs in pediatric surgery is lagging compared to adult surgery. The study's purpose was to conduct a theory-guided evaluation of barriers and facilitators to ERP implementation at US hospitals with a pediatric surgery service. METHODS: We conducted semi-structured interviews at 18 hospitals with 48 participants, including pediatric surgeons, anesthesiologists, gastroenterologists, nurses, and physician assistants. Interviews were conducted online, audio-recorded, and transcribed verbatim. To identify barriers and facilitators to ERP implementation, we conducted an analysis using deductive logics based on the five Active Implementation Frameworks (AIFs). RESULTS: Effective practices (usable innovations) were challenged by a lack of compliance to ERP elements, and facilitators were having standardized protocols in place and organization support for implementation. Effective implementation (stages of implementation and implementation drivers) had widespread barriers to implementation across the stages from exploration to full implementation. Barriers included needing dedicated teams for ERP implementation and buy-in from hospital leadership. These items, when present, were strong facilitators of effective implementation, in addition to on-site, checklists, protected time to oversee ERP implementation, and order sets for ERP elements built into the electronic medical record. The enabling context (teams) focused on teams' engagement in ERP implementation and how they collaborated to implement ERPs. Barriers included having surgical team members resistant to change or who were not bought into ERPs in pediatric practice. Facilitators included engaging a multi-disciplinary team and engaging patients and families early in the implementation process. CONCLUSIONS: Barriers to ERP implementation in pediatric surgery highlighted can be addressed through providing guidelines to ERP implementation, team-based support for change management, and protocols for developing an ERP implementation team. Future steps are to apply and evaluate these strategies in a stepped-wedge, cluster randomized trial to increase the implementation of ERPs at these 18 hospitals.

Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis.

Initial TCR engagement (priming) of naive CD8+ T cells results in T cell expansion, and these early events influence the generation of diverse effector and memory populations. During infection, activated T cells can re-encounter cognate antigen, but how these events influence local effector responses or formation of memory populations is unclear. To address this issue, OT-I T cells which express the Nur77-GFP reporter of TCR activation were paired with the parasite Toxoplasma gondii that expresses OVA to assess how secondary encounter with antigen influences CD8+ T cell responses. During acute infection, TCR stimulation in affected tissues correlated with parasite burden and was associated with markers of effector cells while Nur77-GFP- OT-I showed signs of effector memory potential. However, both Nur77-GFP- and Nur77-GFP+ OT-I from acutely infected mice formed similar memory populations when transferred into naive mice. During the chronic stage of infection in the CNS, TCR activation was associated with large scale transcriptional changes and the acquisition of an effector T cell phenotype as well as the generation of a population of CD103+ CD69+ Trm like cells. While inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight that recent TCR activation contributes to the phenotypic heterogeneity of the CD8+ T cell response but suggest that this process has a limited impact on memory populations at acute and chronic stages of infection.

PD-L1-PD-1 interactions limit effector regulatory T cell populations at homeostasis and during infection.

Phenotypic and transcriptional profiling of regulatory T (Treg) cells at homeostasis reveals that T cell receptor activation promotes Treg cells with an effector phenotype (eTreg) characterized by the production of interleukin-10 and expression of the inhibitory receptor PD-1. At homeostasis, blockade of the PD-1 pathway results in enhanced eTreg cell activity, whereas during infection with Toxoplasma gondii, early interferon-γ upregulates myeloid cell expression of PD-L1 associated with reduced Treg cell populations. In infected mice, blockade of PD-L1, complete deletion of PD-1 or lineage-specific deletion of PD-1 in Treg cells prevents loss of eTreg cells. These interventions resulted in a reduced ratio of pathogen-specific effector T cells: eTreg cells and increased levels of interleukin-10 that mitigated the development of immunopathology, but which could compromise parasite control. Thus, eTreg cell expression of PD-1 acts as a sensor to rapidly tune the pool of eTreg cells at homeostasis and during inflammatory processes.

Age- and Sex-Specific Needs for Children Undergoing Inflammatory Bowel Disease Surgery: A Qualitative Study.

INTRODUCTION: The use of enhanced recovery protocols (ERP) is extending to pediatric surgical populations, such as patients with inflammatory bowel diseases (IBDs). Given the variation in age- and sex-specific characteristics of pediatric IBD patients, it is important to understand the unique needs of subgroups, such as male versus female or preadolescent versus older patients, when implementing ERPs. We gathered clinician, patient, and caregiver perspectives on age- and sex-specific needs for children undergoing IBD surgery. METHODS: We used semistructured interviews and focus groups to assess ERP needs and perceived differences in needs between preadolescent (10-13 y), older (14-19 y), male, and female IBD patients. Participants included clinicians, patients who had recent IBD surgery, and patients' caregivers. RESULTS: Forty-eight clinicians, six patients, and eight caregivers participated. Three broad categories of themes emerged: concerns, needs, and experiences related to the (1) surgical care process; (2) continuum of IBD care; and (3) suggestions to make surgical care more patient centered. With regard to surgical care processes, stakeholders reported different communication needs for preadolescent and older children. Key themes about the continuum of IBD care were the need (1) for support from child life specialists and (b) to address young women's health issues. Suggestions to make surgical care more patient centered included providing older children with patient experiences that reflect their perspective as young adults. CONCLUSIONS: The findings highlight the need to adopt a patient-centered approach for ERP use that actively addresses age- and sex-specific factors while engaging patients and caregivers as partners with clinicians to improve surgical care for children with IBD.

IL-33 promotes innate lymphoid cell-dependent IFN-γ production required for innate immunity to Toxoplasma gondii.

IL-33 is an alarmin required for resistance to the parasite Toxoplasma gondii, but its role in innate resistance to this organism is unclear. Infection with T. gondii promotes increased stromal cell expression of IL-33, and levels of parasite replication correlate with release of IL-33 in affected tissues. In response to infection, a subset of innate lymphoid cells (ILC) emerges composed of IL-33R+ NK cells and ILC1s. In Rag1-/-mice, where NK cells and ILC1 production of IFN-γ mediate innate resistance to T. gondii, the loss of the IL-33R resulted in reduced ILC responses and increased parasite replication. Furthermore, administration of IL-33 to Rag1-/- mice resulted in a marked decrease in parasite burden, increased production of IFN-γ, and the recruitment and expansion of inflammatory monocytes associated with parasite control. These protective effects of exogenous IL-33 were dependent on endogenous IL-12p40 and the ability of IL-33 to enhance ILC production of IFN-γ. These results highlight that IL-33 synergizes with IL-12 to promote ILC-mediated resistance to T. gondii.

Animal-derived medications: cultural considerations and available alternatives.

CONTEXT: Cultural competency is a cornerstone of patient-centered health care. Religious doctrines may define appropriate consumption or use of certain animals and forbid use of others. Many medications contain ingredients that are animal-derived; these medications may be unacceptable to individual patients within the context of their religious beliefs and lifestyle choices. Knowledge of animal-derived medications as a component of cultural competency can facilitate a dialogue that shifts focus from the group to the individual, away from cultural competency toward cultural humility, and away from a paternalistic provider/patient dynamic toward one of partnership. OBJECTIVES: To explore how animal-derived drug components may impact medication selection and acceptability from the perspective of patients, physicians, and religious leaders as evidenced by studies that explore the question via survey or questionnaire. A secondary objective is to use the context of animal-derived drug products as a component of cultural competency to build a framework supporting the development of cultural humility. METHODS: A systematic search was performed in the PubMed, CINAHL, Cochrane, and ProQuest databases using combinations of the following terms: "medication selection," "medication," "adherence," "pharmaceutical preparations," "religion and medicine," "religion," "animal," "dietary," "porcine," and "bovine." Studies that reported using surveys or questionnaires to examine patient, physician, or religious leader perspective on animal-derived medications published in English between 1990 and 2020 were included. Review articles, opinion pieces, case reports, surveys of persons other than patients, religious leaders, or physicians, and studies published in languages other than English were excluded. Three authors independently reviewed articles to extract information pertaining to perspectives on animal-based medication ingredients. RESULTS: Eight studies meeting the described criteria were found that queried beliefs or knowledge of patients, religious leaders, or physicians regarding medications and medical products of biologic origin. Those studies are described in full in this review. CONCLUSIONS: Knowledge of animal-derived ingredients may help open conversations with patients around spiritual history and cultural competency, particularly for those patients belonging to religious sects with doctrines that define appropriate use of human- or animal-derived products. Further formal study is needed to explore more fully the extent to which religious beliefs may impact selection of animal- or human-derived medications. Guidelines developed from this knowledge may aid in identifying individual patients with whom the discussion may be particularly relevant. More studies are needed to quantify and qualify beliefs regarding animal-derived medication constituents.

Impact of Ion-Pairing Effects on Linear and Nonlinear Photophysical Properties of Polymethine Dyes*.

The two-photon absorption (2PA) and photophysics of heptamethine dyes featuring cyanine or dipolar electronic structures have been compared for the first time. The perfectly delocalized cyanine system is classically characterized by a two-photon transition matching the vibronic component of its lower energy absorption band. The dipolar species is generated by ion-pairing with a hard counterion in a non-dissociating solvent and displays significant modifications oft he optical properties, including a significant hypochromic shift of absorption, weaker emission and 2PA matching the lower energy transition, thus revealing symmetry breaking within the polymethine electronic structure.

Highly Conjugated, Fused-Ring, Quadrupolar Organic Chromophores with Large Two-Photon Absorption Cross-Sections in the Near-Infrared.

The two-photon absorption (2PA) properties are investigated for two series of organic, π-conjugated, fused-ring, quadrupolar A-π-D-π-A chromophores of the type originally developed as nonfullerene acceptors for organic photovoltaics. These molecules are found to exhibit large nondegenerate two-photon absorption (ND2PA) cross-sections (ca. 6-27 × 103 GM) in the near-infrared (NIR). In the first series, involving molecules of varying core size, ND2PA spectra and cross-sections characterized by femtosecond ND2PA spectroscopy in chloroform solutions reveal that increases in core size, and thus conjugation length, leads to substantially red-shifted and enhanced 2PA. In a second series, variation of the strength of the terminal acceptor (A) with constant core size (seven rings, indacene-based) led to less dramatic variation in the 2PA properties. Among the two core types studied, compounds in which the donor has a thieno[3,2-b]thiophene center demonstrate larger 2PA cross-sections than their indacene-centered counterparts, due to the greater electron-richness of their cores amplifying intramolecular charge transfer. Excited-state absorption (ESA) contributions to nonlinear absorption measured by open-aperture Z-scans are deduced for some of the compounds by analyzing the spectral overlap between 2PA bands and NIR ESA transitions obtained by ND2PA and transient absorption measurements, respectively. ESA cross-sections extracted from transient absorption and irradiance-dependent open-aperture Z-scans are in reasonable agreement, and their moderate magnitudes (ca. 10-21 m2) suggest that, although ESA contributions are non-negligible, the effective response is predominantly instantaneous 2PA.

TeoNAM: A Nested Association Mapping Population for Domestication and Agronomic Trait Analysis in Maize.

Recombinant inbred lines (RILs) are an important resource for mapping genes controlling complex traits in many species. While RIL populations have been developed for maize, a maize RIL population with multiple teosinte inbred lines as parents has been lacking. Here, we report a teosinte nested association mapping (TeoNAM) population, derived from crossing five teosinte inbreds to the maize inbred line W22. The resulting 1257 BC1S4 RILs were genotyped with 51,544 SNPs, providing a high-density genetic map with a length of 1540 cM. On average, each RIL is 15% homozygous teosinte and 8% heterozygous. We performed joint linkage mapping (JLM) and a genome-wide association study (GWAS) for 22 domestication and agronomic traits. A total of 255 QTL from JLM were identified, with many of these mapping near known genes or novel candidate genes. TeoNAM is a useful resource for QTL mapping for the discovery of novel allelic variation from teosinte. TeoNAM provides the first report that PROSTRATE GROWTH1, a rice domestication gene, is also a QTL associated with tillering in teosinte and maize. We detected multiple QTL for flowering time and other traits for which the teosinte allele contributes to a more maize-like phenotype. Such QTL could be valuable in maize improvement.

IL-27 and TCR Stimulation Promote T Cell Expression of Multiple Inhibitory Receptors.

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR.

Tyrosine, cysteine, and proton coupled electron transfer in a ribonucleotide reductase-inspired beta hairpin maquette.

Tyrosine residues act as intermediates in proton coupled electron transfer reactions (PCET) in proteins. For example, in ribonucleotide reductase (RNR), a tyrosyl radical oxidizes an active site cysteine via a 35 Å pathway that contains multiple aromatic groups. When singlet tyrosine is oxidized, the radical becomes a strong acid, and proton transfer reactions, which are coupled with the redox reaction, may be used to control reaction rate. Here, we characterize a tyrosine-containing beta hairpin, Peptide O, which has a cross-strand, noncovalent interaction between its single tyrosine, Y5, and a cysteine (C14). Circular dichroism provides evidence for a thermostable beta-turn. EPR spectroscopy shows that Peptide O forms a neutral tyrosyl radical after UV photolysis at 160 K. Molecular dynamics simulations support a phenolic/SH interaction in the tyrosine singlet and radical states. Differential pulse voltammetry exhibits pH dependence consistent with the formation of a neutral tyrosyl radical and a pKa change in two other residues. A redox-coupled decrease in cysteine pKa from 9 (singlet) to 6.9 (radical) is assigned. At pD 11, picosecond transient absorption spectroscopy after UV photolysis monitors tyrosyl radical recombination via electron transfer (ET). The ET rate in Peptide O is indistinguishable from the ET rates observed in peptides containing a histidine and a cyclohexylalanine (Cha) at position 14. However, at pD 9, the tyrosyl radical decays via PCET, and the decay rate is slowed, when compared to the histidine 14 variant. Notably, the decay rate is accelerated, when compared to the Cha 14 variant. We conclude that redox coupling between tyrosine and cysteine can act as a PCET control mechanism in proteins.

Nonlinear refraction and absorption measurements of thin films by the dual-arm Z-scan method.

We extend the recently developed dual-arm Z-scan to increase the signal-to-noise ratio (SNR) for measuring the nonlinear refraction (NLR) of thin films on thick substrates. Similar to the case of solutes in solution, the phase shift due to NLR in a thin film can often be dominated by the phase shift due to NLR in the much thicker substrate. SNR enhancement is accomplished by simultaneously scanning a bare substrate and the film plus substrate in two separate but identical Z-scan arms. The subtraction of these signals taken simultaneously effectively cancels the nonlinear signal from the substrate, leaving only the signal from the film. More importantly, the SNR is increased since the correlated noise from effects such as beam-pointing instabilities cancels. To show the versatility of the dual-arm Z-scan method, we perform measurements on semiconductor and organic thin films, some less than 100 nm thick and with thicknesses up to 4 orders of magnitude less than the substrate.

Structure and Function of Tryptophan-Tyrosine Dyads in Biomimetic ß Hairpins.

Tyrosine-tryptophan (YW) dyads are ubiquitous structural motifs in enzymes and play roles in proton-coupled electron transfer (PCET) and, possibly, protection from oxidative stress. Here, we describe the function of YW dyads in de novo designed 18-mer, ß hairpins. In Peptide M, a YW dyad is formed between W14 and Y5. A UV hypochromic effect and an excitonic Cotton signal are observed, in addition to singlet, excited state (W*) and fluorescence emission spectral shifts. In a second Peptide, Peptide MW, a Y5-W13 dyad is formed diagonally across the strand and distorts the backbone. On a picosecond timescale, the W* excited-state decay kinetics are similar in all peptides but are accelerated relative to amino acids in solution. In Peptide MW, the W* spectrum is consistent with increased conformational flexibility. In Peptide M and MW, the electron paramagnetic resonance spectra obtained after UV photolysis are characteristic of tyrosine and tryptophan radicals at 160 K. Notably, at pH 9, the radical photolysis yield is decreased in Peptide M and MW, compared to that in a tyrosine and tryptophan mixture. This protective effect is not observed at pH 11 and is not observed in peptides containing a tryptophan-histidine dyad or tryptophan alone. The YW dyad protective effect is attributed to an increase in the radical recombination rate. This increase in rate can be facilitated by hydrogen-bonding interactions, which lower the barrier for the PCET reaction at pH 9. These results suggest that the YW dyad structural motif promotes radical quenching under conditions of reactive oxygen stress.

Nonlinear optical components for all-optical probabilistic graphical model.

The probabilistic graphical models (PGMs) are tools that are used to compute probability distributions over large and complex interacting variables. They have applications in social networks, speech recognition, artificial intelligence, machine learning, and many more areas. Here, we present an all-optical implementation of a PGM through the sum-product message passing algorithm (SPMPA) governed by a wavelength multiplexing architecture. As a proof-of-concept, we demonstrate the use of optics to solve a two node graphical model governed by SPMPA and successfully map the message passing algorithm onto photonics operations. The essential mathematical functions required for this algorithm, including multiplication and division, are implemented using nonlinear optics in thin film materials. The multiplication and division are demonstrated through a logarithm-summation-exponentiation operation and a pump-probe saturation process, respectively. The fundamental bottlenecks for the scalability of the presented scheme are discussed as well.

Individually Dispersed Gold Nanoshell-Bearing Cellulose Nanocrystals with Tailorable Plasmon Resonance.

Cellulose nanocrystals (CNCs) can be attractive templates for the generation of functional inorganic/organic nanoparticles, given their fine sizes, aspect ratios, and sustainable worldwide availability in abundant quantities. Here, we present for the first time a scalable, surfactant-free, tailorable wet chemical process for converting commercially available CNCs into individual aspected gold nanoshell-bearing particles with tunable surface plasmon resonance bands. Using a rational cellulose functionalization approach, stable suspensions of positively charged CNCs have been generated. Continuous, conductive, nanocrystalline gold coatings were then applied to the individual, electrostatically stabilized CNCs via decoration with 1-3 nm diameter gold particles followed by electroless gold deposition. Optical analyses indicated that these core-shell nanoparticles exhibited two surface plasmon absorbance bands, with one located in the visible range (near 550 nm) and the other at near infrared (NIR) wavelengths. The NIR band possessed a peak maximum wavelength that could be tuned over a wide range (1000-1300 nm) by adjusting the gold coating thickness. The bandwidth and wavelength of the peak maximum of the NIR band were also sensitive to the particle size distribution and could be further refined by fractionation using viscosity gradient centrifugation.

Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes.

Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor-acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a Kd of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.