CaSSiDI: novel single-cell "Cluster Similarity Scoring and Distinction Index" reveals critical functions for PirB and context-dependent Cebpb repression.

PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called "Cluster Similarity Scoring and Distinction Index" (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB's effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ "neuronal-primed" subset and an CD68hiFtl1+ "iron-loaded" subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.

Chemical carcinogens: implications for cancer treatment.

Carcinogen exposure has been associated with enhanced cancer immunogenicity that is often attributed to neoantigen generation. However, the broader, neoantigen-independent impact of carcinogens on immune responses to cancer cells remains underexplored. In this issue of the JCI, Huang et al. uncover a mechanism wherein carcinogen-treated cancer cells exhibit an inability to establish an immunosuppressive tumor microenvironment (TME) due to reduced M-CSF expression. Intriguingly, the so-called carcinogen-induced tumor-associated macrophages (TAMs) within this TME exhibited anti-tumor properties instead of the conventional immunosuppressive phenotype. This phenomenon extended to human lung cancers, as evidenced by TAM reprogramming in smokers versus nonsmokers. This study substantially advances our understanding of carcinogen-mediated effects on cancer immunogenicity, potentially redirecting approaches to cancer immunotherapy.

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

Cholinergic control of Th17 cell pathogenicity in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFß, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.

Antimicrobial stewardship intervention bundle decreases outpatient fluoroquinolone prescribing for urinary tract infections.

An antimicrobial stewardship bundle was implemented in 23 community health system urgent care and primary care clinics to reduce fluoroquinolone prescribing in urinary tract infections. The percentage of urinary tract infection (UTI) visits prescribed a fluoroquinolone subsequently decreased from 17.6% to 3% in urgent care and from 23.8% to 6.8% in primary care.

Serious Bacterial Infections in Young Febrile Infants With Positive Urinalysis Results.

It is unknown whether febrile infants 29 to 60 days old with positive urinalysis results require routine lumbar punctures for evaluation of bacterial meningitis. OBJECTIVE: To determine the prevalence of bacteremia and/or bacterial meningitis in febrile infants ≤60 days of age with positive urinalysis (UA) results. METHODS: Secondary analysis of a prospective observational study of noncritical febrile infants ≤60 days between 2011 and 2019 conducted in the Pediatric Emergency Care Applied Research Network emergency departments. Participants had temperatures ≥38°C and were evaluated with blood cultures and had UAs available for analysis. We report the prevalence of bacteremia and bacterial meningitis in those with and without positive UA results. RESULTS: Among 7180 infants, 1090 (15.2%) had positive UA results. The risk of bacteremia was higher in those with positive versus negative UA results (63/1090 [5.8%] vs 69/6090 [1.1%], difference 4.7% [3.3% to 6.1%]). There was no difference in the prevalence of bacterial meningitis in infants ≤28 days of age with positive versus negative UA results (∼1% in both groups). However, among 697 infants aged 29 to 60 days with positive UA results, there were no cases of bacterial meningitis in comparison to 9 of 4153 with negative UA results (0.2%, difference -0.2% [-0.4% to -0.1%]). In addition, there were no cases of bacteremia and/or bacterial meningitis in the 148 infants ≤60 days of age with positive UA results who had the Pediatric Emergency Care Applied Research Network low-risk blood thresholds of absolute neutrophil count <4 × 103 cells/mm3 and procalcitonin <0.5 ng/mL. CONCLUSIONS: Among noncritical febrile infants ≤60 days of age with positive UA results, there were no cases of bacterial meningitis in those aged 29 to 60 days and no cases of bacteremia and/or bacterial meningitis in any low-risk infants based on low-risk blood thresholds in both months of life. These findings can guide lumbar puncture use and other clinical decision making.

Child Abuse Recognition Training for Prehospital Providers Using Deliberate Practice.

BACKGROUND: In most states, prehospital professionals (PHPs) are mandated reporters of suspected abuse but cite a lack of training as a challenge to recognizing and reporting physical abuse. We developed a learning platform for the visual diagnosis of pediatric abusive versus non-abusive burn and bruise injuries and examined the amount and rate of skill acquisition. METHODS: This was a prospective cross-sectional study of PHPs participating in an online educational intervention containing 114 case vignettes. PHPs indicated whether they believed a case was concerning for abuse and would report a case to child protection services. Participants received feedback after submitting a response, permitting deliberate practice of the cases. We describe learning curves, overall accuracy, sensitivity (diagnosis of abusive injuries) and specificity (diagnosis of non-abusive injuries) to determine the amount of learning. We performed multivariable regression analysis to identify specific demographic and case variables associated with a correct case interpretation. After completing the educational intervention, PHPs completed a self-efficacy survey on perceived gains in their ability to recognize cutaneous signs of abuse and report to social services. RESULTS: We enrolled 253 PHPs who completed all the cases; 158 (63.6%) emergency medical technicians (EMT), 95 (36.4%) advanced EMT and paramedics. Learning curves demonstrated that, with one exception, there was an increase in learning for participants throughout the educational intervention. Mean diagnostic accuracy increased by 4.9% (95% CI 3.2, 6.7), and the mean final diagnostic accuracy, sensitivity, and specificity were 82.1%, 75.4%, and 85.2%, respectively. There was an increased odds of getting a case correct for bruise versus burn cases (OR = 1.4; 95% CI 1.3, 1.5); if the PHP was an Advanced EMT/Paramedic (OR = 1.3; 95% CI 1.1, 1.4) ; and, if the learner indicated prior training in child abuse (OR = 1.2; 95% CI 1.0, 1.3). Learners indicated increased comfort in knowing which cases should be reported and interpreting exams in children with cutaneous injuries with a median Likert score of 5 out of 6 (IQR 5, 6). CONCLUSION: An online module utilizing deliberate practice led to measurable skill improvement among PHPs for differentiating abusive from non-abusive burn and bruise injuries.

IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis.

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Sierpinski Pyramids by Molecular Entanglement.

Planar, terpyridine-based metal complexes with the Sierpinski triangular motif and alkylated corners undergo a second self-assembly event to give megastructural Sierpinski pyramids; assembly is driven by the facile lipophilic-lipophilic association of the alkyl moieties and complementary perfect fit of the triangular building blocks. Confirmation of the 3D, pyramidal structures was verified and supported by a combination of TEM, AFM, and multiscale simulation techniques.

A Clinical Prediction Rule to Identify Febrile Infants 60 Days and Younger at Low Risk for Serious Bacterial Infections.

Importance: In young febrile infants, serious bacterial infections (SBIs), including urinary tract infections, bacteremia, and meningitis, may lead to dangerous complications. However, lumbar punctures and hospitalizations involve risks and costs. Clinical prediction rules using biomarkers beyond the white blood cell count (WBC) may accurately identify febrile infants at low risk for SBIs. Objective: To derive and validate a prediction rule to identify febrile infants 60 days and younger at low risk for SBIs. Design, Setting, and Participants: Prospective, observational study between March 2011 and May 2013 at 26 emergency departments. Convenience sample of previously healthy febrile infants 60 days and younger who were evaluated for SBIs. Data were analyzed between April 2014 and April 2018. Exposures: Clinical and laboratory data (blood and urine) including patient demographics, fever height and duration, clinical appearance, WBC, absolute neutrophil count (ANC), serum procalcitonin, and urinalysis. We derived and validated a prediction rule based on these variables using binary recursive partitioning analysis. Main Outcomes and Measures: Serious bacterial infection, defined as urinary tract infection, bacteremia, or bacterial meningitis. Results: We derived the prediction rule on a random sample of 908 infants and validated it on 913 infants (mean age was 36 days, 765 were girls [42%], 781 were white and non-Hispanic [43%], 366 were black [20%], and 535 were Hispanic [29%]). Serious bacterial infections were present in 170 of 1821 infants (9.3%), including 26 (1.4%) with bacteremia, 151 (8.3%) with urinary tract infections, and 10 (0.5%) with bacterial meningitis; 16 (0.9%) had concurrent SBIs. The prediction rule identified infants at low risk of SBI using a negative urinalysis result, an ANC of 4090/µL or less (to convert to ×109 per liter, multiply by 0.001), and serum procalcitonin of 1.71 ng/mL or less. In the validation cohort, the rule sensitivity was 97.7% (95% CI, 91.3-99.6), specificity was 60.0% (95% CI, 56.6-63.3), negative predictive value was 99.6% (95% CI, 98.4-99.9), and negative likelihood ratio was 0.04 (95% CI, 0.01-0.15). One infant with bacteremia and 2 infants with urinary tract infections were misclassified. No patients with bacterial meningitis were missed by the rule. The rule performance was nearly identical when the outcome was restricted to bacteremia and/or bacterial meningitis, missing the same infant with bacteremia. Conclusions and Relevance: We derived and validated an accurate prediction rule to identify febrile infants 60 days and younger at low risk for SBIs using the urinalysis, ANC, and procalcitonin levels. Once further validated on an independent cohort, clinical application of the rule has the potential to decrease unnecessary lumbar punctures, antibiotic administration, and hospitalizations.

Tackling the workforce crisis in district nursing: can the Dutch Buurtzorg model offer a solution and a better patient experience? A mixed methods case study.

Despite policy intentions for more healthcare out of hospital, district nursing services face multiple funding and staffing challenges, which compromise the care delivered and policy objectives. OBJECTIVES: What is the impact of the adapted Buurtzorg model on feasibility, acceptability and effective outcomes in an English district nursing service? DESIGN: Mixed methods case study. SETTING: Primary care. PARTICIPANTS: Neighbourhood nursing team (Buurtzorg model), patients and carers, general practitioners (GPs), other health professionals, managers and conventional district nurses. RESULTS: The adapted Buurtzorg model of community nursing demonstrated feasibility and acceptability to patients, carers, GPs and other health professionals. For many patients, it was preferable to previous experiences of district nursing in terms of continuity in care, improved support of multiple long-term conditions (encompassing physical, mental and social factors) and proactive care. For the neighbourhood nurses, the ability to make operational and clinical decisions at team level meant adopting practices that made the service more responsive, accessible and efficient and offered a more attractive working environment. Challenges were reported by nurses and managers in relation to the recognition and support of the concept of self-managing teams within a large bureaucratic healthcare organisation. While there were some reports of clinical effectiveness and efficiency, this was not possible to quantify, cost or compare with the standard district nursing service. CONCLUSIONS: The adapted Buurtzorg model of neighbourhood nursing holds potential for addressing issues of concern to patients, carers and staff in the community. The two interacting innovations, that is, a renewed focus on patient and carer-centred care and the self-managing team, were implemented in ways that patients, carers, other health professionals and nurses could identify difference for both the nursing care and also the nurses' working lives. It now requires longer term investigation to understand both the mechanism for change and also the sustainability.

Supramolecular arrays by the self-assembly of terpyridine-based monomers with transition metal ions.

Hierarchical construction of a highly ordered supramolecular array has been, in general, a challenge due to the complexation of building blocks and the hard-to-control weak interactions. Herein, we present a type of well-ordered nanoribbon, which was self-assembled via shape complimentary and hydrophobic effects from the bowl-shaped supramolecular components, which were synthesized by combining designer terpyridine-based monomers and two different metal ions (Ru2+, Zn2+). Interestingly, switching counter ions or changing monomer concentrations, a transformation between a uniform nanosphere and nanoribbon occurred. This opens a door to fabricate readily tailorable, large-scale, supramacromolecular materials.

The use of acuity and frailty measures for district nursing workforce plans.

This article discusses the use of Quest acuity and frailty measures for community nursing interventions to quantify and qualify the contributions of district nursing teams. It describes the use of a suite of acuity and frailty tools tested in 8 UK community service trusts over the past 5years. In addition, a competency assessment tool was used to gauge both capacity and capability of individual nurses. The consistency of the results obtained from the Quest audits offer significant evidence and potential for realigning community nursing services to offer improvements in efficiency and cost-effectiveness. The National Quality Board (NQB) improvement resource for the district nursing services ( NQB, 2017 ) recommends a robust method for classifying patient acuity/frailty/dependency. It is contended the Quest tools and their usage articulated here offer a suitable methodology.

Concentration dependent supramolecular interconversions of triptycene-based cubic, prismatic, and tetrahedral structures.

The quantitative, single step, self-assembly of a shape-persistent, three-dimensional C3v-symmetric, triptycene-based tris-terpyridinyl ligand initially gives a platonic-based cubic architecture, which was unequivocally characterized by 1D and 2D NMR spectroscopy, mass spectrometry, and single crystal X-ray structural analysis. The unique metal-ligand binding properties of the Cd2+ analogue of this construct give rise to a concentration-dependent dynamic equilibrium between cube, prism, and tetrahedron-shaped architectures. Dilution transforms this cube into two identical tetrahedra through a stable prism-shaped intermediate; increasing the concentration reverses the process.

Neuropeptide Y regulates a vascular gateway for hematopoietic stem and progenitor cells.

Endothelial cells (ECs) are components of the hematopoietic microenvironment and regulate hematopoietic stem and progenitor cell (HSPC) homeostasis. Cytokine treatments that cause HSPC trafficking to peripheral blood are associated with an increase in dipeptidylpeptidase 4/CD26 (DPP4/CD26), an enzyme that truncates the neurotransmitter neuropeptide Y (NPY). Here, we show that enzymatically altered NPY signaling in ECs caused reduced VE-cadherin and CD31 expression along EC junctions, resulting in increased vascular permeability and HSPC egress. Moreover, selective NPY2 and NPY5 receptor antagonists restored vascular integrity and limited HSPC mobilization, demonstrating that the enzymatically controlled vascular gateway specifically opens by cleavage of NPY by CD26 signaling via NPY2 and NPY5 receptors. Mice lacking CD26 or NPY exhibited impaired HSPC trafficking that was restored by treatment with truncated NPY. Thus, our results point to ECs as gatekeepers of HSPC trafficking and identify a CD26-mediated NPY axis that has potential as a pharmacologic target to regulate hematopoietic trafficking in homeostatic and stress conditions.

Supercharged, Precise, Megametallodendrimers via a Single-Step, Quantitative, Assembly Process.

Synthesis of giant unimolecular dendrimers is challenging due, in part, to difficulties encountered at higher generations, in both convergent and divergent protocols because of the multistep construction/purification process. Herein, we report a hybrid synthetic procedure in which the core is constructed last. This quantitative assembly generated a metallodendrimer that is supercharged (120+), large (11.3 nm diameter), and its core was previously established. The series of complexes has been unequivocally characterized by NMR, ESI-IM-MS, and TEM techniques.

Combinatorial Library Screening with Liposomes for Discovery of Membrane Active Peptides.

Membrane active peptides (MAPs) represent a class of short biomolecules that have shown great promise in facilitating intracellular delivery without disrupting cellular plasma membranes. Yet their clinical application has been stalled by numerous factors: off-target delivery, a requirement for high local concentration near cells of interest, degradation en route to the target site, and in the case of cell-penetrating peptides, eventual entrapment in endolysosomal compartments. The current method of deriving MAPs from naturally occurring proteins has restricted the discovery of new peptides that may overcome these limitations. Here, we describe a new branch of assays featuring high-throughput functional screening capable of discovering new peptides with tailored cell uptake and endosomal escape capabilities. The one-bead-one-compound (OBOC) combinatorial method is used to screen libraries containing millions of potential MAPs for binding to synthetic liposomes, which can be adapted to mimic various aspects of limiting membranes. By incorporating unnatural and d-amino acids in the library, in addition to varying buffer conditions and liposome compositions, we have identified several new highly potent MAPs that improve on current standards and introduce motifs that were previously unknown or considered unsuitable. Since small variations in pH and lipid composition can be controlled during screening, peptides discovered using this methodology could aid researchers building drug delivery platforms with unique requirements, such as targeted intracellular localization.

Controlled Interconversion of Superposed-Bistriangle, Octahedron, and Cuboctahedron Cages Constructed Using a Single, Terpyridinyl-Based Polyligand and Zn(2.).

Metallomacromolecular architectural conversion is expanded by the characterization of three different structures. A quantitative, single-step, self-assembly of a shape-persistent monomer, containing a flexible crown ether moiety, gives an initial Archimedean-based cuboctahedron that has been unequivocally characterized by 1D and 2D NMR spectroscopy, mass spectrometry, and collision cross section analysis. Both dilution and exchange of counterions, transforms this cuboctahedron into two identical octahedrons, which upon further dilution convert into four, superposed, bistrianglar complexes; increasing the concentration reverses the process. Ion binding studies using the cuboctahedral cage were undertaken.

Association of RNA Biosignatures With Bacterial Infections in Febrile Infants Aged 60 Days or Younger.

IMPORTANCE: Young febrile infants are at substantial risk of serious bacterial infections; however, the current culture-based diagnosis has limitations. Analysis of host expression patterns ("RNA biosignatures") in response to infections may provide an alternative diagnostic approach. OBJECTIVE: To assess whether RNA biosignatures can distinguish febrile infants aged 60 days or younger with and without serious bacterial infections. DESIGN, SETTING, AND PARTICIPANTS: Prospective observational study involving a convenience sample of febrile infants 60 days or younger evaluated for fever (temperature >38° C) in 22 emergency departments from December 2008 to December 2010 who underwent laboratory evaluations including blood cultures. A random sample of infants with and without bacterial infections was selected for RNA biosignature analysis. Afebrile healthy infants served as controls. Blood samples were collected for cultures and RNA biosignatures. Bioinformatics tools were applied to define RNA biosignatures to classify febrile infants by infection type. EXPOSURE: RNA biosignatures compared with cultures for discriminating febrile infants with and without bacterial infections and infants with bacteremia from those without bacterial infections. MAIN OUTCOMES AND MEASURES: Bacterial infection confirmed by culture. Performance of RNA biosignatures was compared with routine laboratory screening tests and Yale Observation Scale (YOS) scores. RESULTS: Of 1883 febrile infants (median age, 37 days; 55.7% boys), RNA biosignatures were measured in 279 randomly selected infants (89 with bacterial infections-including 32 with bacteremia and 15 with urinary tract infections-and 190 without bacterial infections), and 19 afebrile healthy infants. Sixty-six classifier genes were identified that distinguished infants with and without bacterial infections in the test set with 87% (95% CI, 73%-95%) sensitivity and 89% (95% CI, 81%-93%) specificity. Ten classifier genes distinguished infants with bacteremia from those without bacterial infections in the test set with 94% (95% CI, 70%-100%) sensitivity and 95% (95% CI, 88%-98%) specificity. The incremental C statistic for the RNA biosignatures over the YOS score was 0.37 (95% CI, 0.30-0.43). CONCLUSIONS AND RELEVANCE: In this preliminary study, RNA biosignatures were defined to distinguish febrile infants aged 60 days or younger with vs without bacterial infections. Further research with larger populations is needed to refine and validate the estimates of test accuracy and to assess the clinical utility of RNA biosignatures in practice.

Developing voluntary standards for district nurse education and practice.

This article charts the development of a project, funded by the Queen's Nursing Institute and Queen's Nursing Institute Scotland, to develop voluntary standards that reflect the contemporary and future practice of district nurses. The standards are designed to enhance, but not replace, the Nursing and Midwifery Council standards for district nurse specialist practice. The project encompassed the four UK countries and gathered data from a wide range of sources to inform the new standards that were launched in September 2015.