They all rock: A systematic comparison of conformational movements in LeuT-fold transporters.

Many membrane transporters share the LeuT fold-two five-helix repeats inverted across the membrane plane. Despite hundreds of structures, whether distinct conformational mechanisms are supported by the LeuT fold has not been systematically determined. After annotating published LeuT-fold structures, we analyzed distance difference matrices (DDMs) for nine proteins with multiple available conformations. We identified rigid bodies and relative movements of transmembrane helices (TMs) during distinct steps of the transport cycle. In all transporters the bundle (first two TMs of each repeat) rotates relative to the hash (third and fourth TMs). Motions of the arms (fifth TM) to close or open the intracellular and outer vestibules are common, as is a TM1a swing, with notable variations in the opening-closing motions of the outer vestibule. Our analyses suggest that LeuT-fold transporters layer distinct motions on a common bundle-hash rock and demonstrate that systematic analyses can provide new insights into large structural datasets.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Scene-selectivity in CA1/subicular complex: Multivoxel pattern analysis at 7T.

Prior univariate functional magnetic resonance imaging (fMRI) studies in humans suggest that the anteromedial subicular complex of the hippocampus is a hub for scene-based cognition. However, it is possible that univariate approaches were not sufficiently sensitive to detect scene-related activity in other subfields that have been implicated in spatial processing (e.g., CA1). Further, as connectivity-based functional gradients in the hippocampus do not respect classical subfield boundary definitions, category selectivity may be distributed across anatomical subfields. Region-of-interest approaches, therefore, may limit our ability to observe category selectivity across discrete subfield boundaries. To address these issues, we applied searchlight multivariate pattern analysis to 7T fMRI data of healthy adults who undertook a simultaneous visual odd-one-out discrimination task for scene and non-scene (including face) visual stimuli, hypothesising that scene classification would be possible in multiple hippocampal regions within, but not constrained to, anteromedial subicular complex and CA1. Indeed, we found that the scene-selective searchlight map overlapped not only with anteromedial subicular complex (distal subiculum, pre/para subiculum), but also inferior CA1, alongside posteromedial (including retrosplenial) and parahippocampal cortices. Probabilistic overlap maps revealed gradients of scene category selectivity, with the strongest overlap located in the medial hippocampus, converging with searchlight findings. This was contrasted with gradients of face category selectivity, which had stronger overlap in more lateral hippocampus, supporting ideas of parallel processing streams for these two categories. Our work helps to map the scene, in contrast to, face processing networks within, and connected to, the human hippocampus.

Disruption of Aldehyde Dehydrogenase 2 protects against bacterial infection.

The ALDH2*2 (rs671) allele is one of the most common genetic mutations in humans, yet the positive evolutionary selective pressure to maintain this mutation is unknown, despite its association with adverse health outcomes. ALDH2 is responsible for the detoxification of metabolically produced aldehydes, including lipid-peroxidation end products derived from inflammation. Here, we demonstrate that host-derived aldehydes 4-hydroxynonenal (4HNE), malondialdehyde (MDA), and formaldehyde (FA), all of which are metabolized by ALDH2, are directly toxic to the bacterial pathogens Mycobacterium tuberculosis and Francisella tularensis at physiological levels. We find that Aldh2 expression in macrophages is decreased upon immune stimulation, and that bone marrow-derived macrophages from Aldh2 -/- mice contain elevated aldehydes relative to wild-type mice. Macrophages deficient for Aldh2 exhibited enhanced control of Francisella infection. Finally , mice lacking Aldh2 demonstrated increased resistance to pulmonary infection by M. tuberculosis , including in a hypersusceptible model of tuberculosis, and were also resistant to Francisella infection. We hypothesize that the absence of ALDH2 contributes to the host's ability to control infection by pathogens such as M. tuberculosis and F. tularensis , and that host-derived aldehydes act as antimicrobial factors during intracellular bacterial infections. One sentence summary: Aldehydes produced by host cells contribute to the control of bacterial infections.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter.

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.

Coordination of bacterial cell wall and outer membrane biosynthesis.

Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.

Mutation-related magnetization-transfer, not axon density, drives white matter differences in premanifest Huntington disease: Evidence from in vivo ultra-strong gradient MRI.

White matter (WM) alterations have been observed in Huntington disease (HD) but their role in the disease-pathophysiology remains unknown. We assessed WM changes in premanifest HD by exploiting ultra-strong-gradient magnetic resonance imaging (MRI). This allowed to separately quantify magnetization transfer ratio (MTR) and hindered and restricted diffusion-weighted signal fractions, and assess how they drove WM microstructure differences between patients and controls. We used tractometry to investigate region-specific alterations across callosal segments with well-characterized early- and late-myelinating axon populations, while brain-wise differences were explored with tract-based cluster analysis (TBCA). Behavioral measures were included to explore disease-associated brain-function relationships. We detected lower MTR in patients' callosal rostrum (tractometry: p = .03; TBCA: p = .03), but higher MTR in their splenium (tractometry: p = .02). Importantly, patients' mutation-size and MTR were positively correlated (all p-values < .01), indicating that MTR alterations may directly result from the mutation. Further, MTR was higher in younger, but lower in older patients relative to controls (p = .003), suggesting that MTR increases are detrimental later in the disease. Finally, patients showed higher restricted diffusion signal fraction (FR) from the composite hindered and restricted model of diffusion (CHARMED) in the cortico-spinal tract (p = .03), which correlated positively with MTR in the posterior callosum (p = .033), potentially reflecting compensatory mechanisms. In summary, this first comprehensive, ultra-strong gradient MRI study in HD provides novel evidence of mutation-driven MTR alterations at the premanifest disease stage which may reflect neurodevelopmental changes in iron, myelin, or a combination of these.

Subiculum-BNST structural connectivity in humans and macaques.

Invasive tract-tracing studies in rodents implicate a direct connection between the subiculum and bed nucleus of the stria terminalis (BNST) as a key component of neural pathways mediating hippocampal regulation of the Hypothalamic-Pituitary-Adrenal (HPA) axis. A clear characterisation of the connections linking the subiculum and BNST in humans and non-human primates is lacking. To address this, we first delineated the projections from the subiculum to the BNST using anterograde tracers injected into macaque monkeys, revealing evidence for a monosynaptic subiculum-BNST projection involving the fornix. Second, we used in vivo diffusion MRI tractography in macaques and humans to demonstrate substantial subiculum complex connectivity to the BNST in both species. This connection was primarily carried by the fornix, with additional connectivity via the amygdala, consistent with rodent anatomy. Third, utilising the twin-based nature of our human sample, we found that microstructural properties of these tracts were moderately heritable (h2 ∼ 0.5). In a final analysis, we found no evidence of any significant association between subiculum complex-BNST tract microstructure and indices of perceived stress/dispositional negativity and alcohol use, derived from principal component analysis decomposition of self-report data. Our findings address a key translational gap in our knowledge of the neurocircuitry regulating stress.

Mucosal Vaccination with Cyclic Dinucleotide Adjuvants Induces Effective T Cell Homing and IL-17-Dependent Protection against Mycobacterium tuberculosis Infection.

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines.

Extended-amygdala intrinsic functional connectivity networks: A population study.

Pre-clinical and human neuroimaging research implicates the extended-amygdala (ExtA) (including the bed nucleus of the stria terminalis [BST] and central nucleus of the amygdala [CeA]) in networks mediating negative emotional states associated with stress and substance-use behaviours. The extent to which individual ExtA structures form a functionally integrated unit is controversial. We utilised a large sample (n > 1,000 healthy young adult humans) to compare the intrinsic functional connectivity networks (ICNs) of the BST and CeA using task-free functional magnetic resonance imaging (fMRI) data from the Human Connectome Project. We assessed whether inter-individual differences within these ICNs were related to two principal components representing negative disposition and alcohol use. Building on recent primate evidence, we tested whether within BST-CeA intrinsic functional connectivity (iFC) was heritable and further examined co-heritability with our principal components. We demonstrate the BST and CeA to have discrete, but largely overlapping ICNs similar to previous findings. We found no evidence that within BST-CeA iFC was heritable; however, post hoc analyses found significant BST iFC heritability with the broader superficial and centromedial amygdala regions. There were no significant correlations or co-heritability associations with our principal components either across the ICNs or for specific BST-Amygdala iFC. Possible differences in phenotype associations across task-free, task-based, and clinical fMRI are discussed, along with suggestions for more causal investigative paradigms that make use of the now well-established ExtA ICNs.

Host and Pathogen Communication in the Respiratory Tract: Mechanisms and Models of a Complex Signaling Microenvironment.

Chronic lung diseases are a leading cause of morbidity and mortality across the globe, encompassing a diverse range of conditions from infections with pathogenic microorganisms to underlying genetic disorders. The respiratory tract represents an active interface with the external environment having the primary immune function of resisting pathogen intrusion and maintaining homeostasis in response to the myriad of stimuli encountered within its microenvironment. To perform these vital functions and prevent lung disorders, a chemical and biological cross-talk occurs in the complex milieu of the lung that mediates and regulates the numerous cellular processes contributing to lung health. In this review, we will focus on the role of cross-talk in chronic lung infections, and discuss how different cell types and signaling pathways contribute to the chronicity of infection(s) and prevent effective immune clearance of pathogens. In the lung microenvironment, pathogens have developed the capacity to evade mucosal immunity using different mechanisms or virulence factors, leading to colonization and infection of the host; such mechanisms include the release of soluble and volatile factors, as well as contact dependent (juxtracrine) interactions. We explore the diverse modes of communication between the host and pathogen in the lung tissue milieu in the context of chronic lung infections. Lastly, we review current methods and approaches used to model and study these host-pathogen interactions in vitro, and the role of these technological platforms in advancing our knowledge about chronic lung diseases.

Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease.

Cholesterol-dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC-1) on specific immune cells thereby evoking an anti-inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC-1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC-1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC-induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY-induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre-treatment of human DCs with peptides blocked bacterial uptake via MRC-1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co-administration of peptide-loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC-1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs.

Localized Cell-Surface Sampling of a Secreted Factor Using Cell-Targeting Beads.

Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques (p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.

A Modular Microscale Granuloma Model for Immune-Microenvironment Signaling Studies in vitro.

Tuberculosis (TB) is one of the most potent infectious diseases in the world, causing more deaths than any other single infectious agent. TB infection is caused by inhalation of Mycobacterium tuberculosis (Mtb) and subsequent phagocytosis and migration into the lung tissue by innate immune cells (e.g., alveolar macrophages, neutrophils, and dendritic cells), resulting in the formation of a fused mass of immune cells known as the granuloma. Considered the pathological hallmark of TB, the granuloma is a complex microenvironment that is crucial for pathogen containment as well as pathogen survival. Disruption of the delicate granuloma microenvironment via numerous stimuli, such as variations in cytokine secretions, nutrient availability, and the makeup of immune cell population, can lead to an active infection. Herein, we present a novel in vitro model to examine the soluble factor signaling between a mycobacterial infection and its surrounding environment. Adapting a newly developed suspended microfluidic platform, known as Stacks, we established a modular microscale infection model containing human immune cells and a model mycobacterial strain that can easily integrate with different microenvironmental cues through simple spatial and temporal "stacking" of each module of the platform. We validate the establishment of suspended microscale (4 µL) infection cultures that secrete increased levels of proinflammatory factors IL-6, VEGF, and TNFα upon infection and form 3D aggregates (granuloma model) encapsulating the mycobacteria. As a proof of concept to demonstrate the capability of our platform to examine soluble factor signaling, we cocultured an in vitro angiogenesis model with the granuloma model and quantified morphology changes in endothelial structures as a result of culture conditions (P < 0.05 when comparing infected vs. uninfected coculture systems). We envision our modular in vitro granuloma model can be further expanded and adapted for studies focusing on the complex interplay between granulomatous structures and their surrounding microenvironment, as well as a complementary tool to augment in vivo signaling and mechanistic studies.

Healthy Hearts via Live Videoconferencing: An Exercise and Diet Intervention in Pediatric Heart Transplant Recipients.

Background Pediatric heart transplant recipients have high-risk cardiovascular profiles that can affect their long-term outcomes; however, promoting exercise and healthy diet has not been a major focus in the field. The objective of this study was to test the feasibility and impact of a supervised exercise and diet intervention delivered via live videoconferencing in this population. Methods and Results Patients 8 to 19 years of age at least 1 year post heart transplantation were enrolled. The 12- to 16-week intervention phase included live video-supervised exercise (×3/week) and nutrition (×1/week) sessions. The 12- to 16-week maintenance phase included ×1/week live video-supervised exercise and nutrition sessions and ×2/week self-directed exercise sessions. Cardiac, vascular, nutritional, and functional health indices were obtained at baseline, after intervention, and after maintenance. Fourteen patients (median age, 15.2; interquartile range, 14.3-16.7 years) at a median of 3.3 (interquartile range, 1.5-9.7) years after heart transplant completed the intervention. Patients attended 89.6±11% of exercise and 88.4±10% of nutrition sessions during the intervention and 93.4±11% of exercise and 92.3±11% of nutrition sessions during maintenance. After intervention, body mass index percentile (median, -27%; P=0.02), endothelial function (median, +0.29; P=0.04), maximum oxygen consumption (median, +2 mL/kg per minute; P=0.002). Functional Movement Screening total score (median, +2.5; P=0.002) and daily consumption of saturated fat (median, -6 g; P=0.02) improved significantly. After maintenance, improvements in maximum oxygen consumption (median, +3.2 mL/kg per minute; P=0.02) and Functional Movement Screening total score (median, +5; P=0.002) were sustained. Conclusions In pediatric heart transplant recipients, a live video-supervised exercise and diet intervention is feasible. Our results demonstrate excellent adherence with significant improvements in cardiovascular and functional health. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02519946.

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins.

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.

Droplet Incubation and Splitting in Open Microfluidic Channels.

Droplet-based microfluidics enables compartmentalization and controlled manipulation of small volumes. Open microfluidics provides increased accessibility, adaptability, and ease of manufacturing compared to closed microfluidic platforms. Here, we begin to build a toolbox for the emerging field of open channel droplet-based microfluidics, combining the ease of use associated with open microfluidic platforms with the benefits of compartmentalization afforded by droplet-based microfluidics. We develop fundamental microfluidic features to control droplets flowing in an immiscible carrier fluid within open microfluidic systems. Our systems use capillary flow to move droplets and carrier fluid through open channels and are easily fabricated through 3D printing, micromilling, or injection molding; further, droplet generation can be accomplished by simply pipetting an aqueous droplet into an empty open channel. We demonstrate on-chip incubation of multiple droplets within an open channel and subsequent transport (using an immiscible carrier phase) for downstream experimentation. We also present a method for tunable droplet splitting in open channels driven by capillary flow. Additional future applications of our toolbox for droplet manipulation in open channels include cell culture and analysis, on-chip microscale reactions, and reagent delivery.

Healthy hearts in pediatric heart transplant patients with an exercise and diet intervention via live video conferencing-Design and rationale.

BACKGROUND: Pediatric heart transplant (PedHtx) patients have increased cardiovascular risk profiles that affect their long-term outcomes and quality of life. We designed a 12- to 16-week diet and exercise intervention delivered via live video conferencing to improve cardiovascular health. Our methodology and baseline assessment of the first 13 enrolled patients are reported. METHODS: Inclusion criteria are as follows: (a) 8-19 years old; (b) heart transplant >12 months; (c) ability to fast overnight; (d) cardiac clearance by cardiologist; and (e) presence of an adult at home during exercise sessions for patients <14 years old. Exclusion criteria are as follows: (a) acute illness; (b) latex allergy; (c) transplant rejection <3 months ago; and (d) multi-organ transplantation. The intervention consists of one diet and three exercise sessions weekly via live video conferencing. Study visits are conducted at baseline, intervention completion, and end of maintenance period. RESULTS: A total of 13 participants (15.2 [2.3] years) have been enrolled. Median percent-predicted VO2 max was 56.8 [20.7]% (10 patients <70%). Ten patients had abnormal endothelial function (reactive hyperemia index <1.9; 1.4 [0.325]) and 11 patients had stiff arteries (pulse wave velocity ≧5.5 m/s for 15-19 years, ≧4.5 m/s for 8-14 years; 5.6 [0.7] m/s). Patients had suboptimal diets (saturated fat: 22.7 [23.8] g/d, sodium: 2771 [1557] mg/d) and were sedentary at a median of 67.5 [13.8]% of their time. CONCLUSIONS: Baseline assessment confirms that PedHtx patients have abnormal cardiac, vascular, and functional health indices, poor dietary habits, and are sedentary. These results support the rationale to test the feasibility and impact of a non-pharmacologic lifestyle intervention in this patient population.

Upgrading well plates using open microfluidic patterning.

Cellular communication between multiple cell types is a ubiquitous process that is responsible for vital physiological responses observed in vivo (e.g., immune response, organ function). Many in vitro coculture strategies have been developed, both in traditional culture and microscale systems, and have shown the potential to recreate some of the physiological behaviors of organs or groups of cells. A fundamental limitation of current systems is the difficulty of reconciling the additional engineering requirements for creating soluble factor signaling systems (e.g., segregated cell culture) with the use of well-characterized materials and platforms that have demonstrated successful results and biocompatibility in assays. We present a new open-microfluidic platform, the Monorail Device, that is placed in any existing well plate or Petri dish and enables patterning of segregated coculture regions, thereby allowing the direct upgrade of monoculture experiments into multiculture assays. Our platform patterns biocompatible hydrogel walls via microfluidic spontaneous capillary flow (SCF) along a rail insert set inside commercially available cultureware, creating customized pipette-accessible cell culture chambers that require fewer cells than standard macroscale culture. Importantly, the device allows the use of native surfaces without additional modification or treatments, while creating permeable dividers for the diffusion of soluble factors. Additionally, the ease of patterning afforded by our platform makes reconfiguration of the culture region as simple as changing the rail insert. We demonstrate the ability of the device to pattern flows on a variety of cell culture surfaces and create hydrogel walls in complex and precise shapes. We characterize the physical parameters that enable a reproducible SCF-driven flow and highlight specialized design features that increase the ease of use of the device and control of the open microfluidic flow. Further, we present the performance of our platform according to useful coculture criteria, including permeability and integrity of our hydrogel walls and surface-sensitive cell culture. Lastly, we show the potential of this type of platform to create modular multikingdom culture systems that can be used to study soluble factor signaling between mammalian cells, bacteria, and fungi, as well as the potential for adaptation of this technology by researchers across multiple fields.