Maximizing the effectiveness of 1.5 mg levonorgestrel for emergency contraception: The case for precoital use.

Objectives: U.S. and World Health Organization Selected Practice Recommendations for Contraceptive Use state people may have an advanced supply of emergency contraception (EC) to minimize treatment delays. We sought to characterize the potential improvement in effectiveness of 1.5 mg levonorgestrel (LNG-EC) if it were taken up to a few hours before unprotected sex. Study design: We expanded on an existing mathematical model for the maximum attainable effectiveness of LNG-EC, assuming it exclusively works to disrupt ovulation, and compared results with point estimates from nine studies when it was taken up to 72 hours after sex. We then modelled how effectiveness might have improved if subjects had taken LNG-EC up to 3 hours before sex. Results: Taking LNG-EC immediately after sex could potentially reduce the risk of unintended pregnancy by 91%. However, population-average maximum attainable effectiveness levels ranged from just 49% to 67% when accounting for the distributions of postcoital treatment delays in the example studies. If half the subjects had taken it 3 hours before sex, then maximum effectiveness levels would have ranged from 70% to 81%. Conclusions: At the individual level, taking LNG-EC a few hours before sex is a logical extension of Selected Practice Recommendations regarding an advanced supply of EC and, based on our modeling, should be advocated for people who can reasonably anticipate an unprotected sex act. In the absence of more clinical data, however, people should not routinely rely on precoital use of LNG-EC to prevent pregnancy unless modern, effective contraceptives are inaccessible to them. Implications: Based on mathematical modeling, individuals who anticipate needing to take LNG-EC for an impending unprotected act of sex could further reduce their chance of an undesired pregnancy by taking it a few hours in advance.

Anxiety and curiosity in hierarchical models of neural emotion processing-A mini review.

Traditionally, two fundamentally different theoretical approaches have been used in emotion research to model (human) emotions: discrete emotion theories and dimensional approaches. More recent neurophysiological models like the hierarchical emotion theory suggest that both should be integrated. The aim of this review is to provide neurocognitive evidence for this perspective with a particular focus on experimental studies manipulating anxiety and/or curiosity. We searched for evidence that the neuronal correlates of discrete and dimensional emotional systems are tightly connected. Our review suggests that the ACC (anterior cingulate cortex) responds to both, anxiety, and curiosity. While amygdala activation has been primarily observed for anxiety, at least the NAcc (nucleus accumbens) responds to both, anxiety and curiosity. When these two areas closely collaborate, as indicated by strong connectivity, this may indicate emotion regulation, particularly when the situation is not predictable.

Irinotecan and temozolomide in combination with dasatinib and rapamycin versus irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma (RIST-rNB-2011): a multicentre, open-label, randomised, controlled, phase 2 trial.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. METHODS: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. FINDINGS: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). INTERPRETATION: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting. FUNDING: Deutsche Krebshilfe.

Changes to Secondary School Physical Activity Programs and Policy after Emerging from COVID-19 Lockdowns.

The purpose of this study was to explore the adaptations that schools made to physical activity programs and facilities, and disparities by area urbanicity and income, during the first school year after the emergence of the COVID-19 pandemic. In a convenience sample of 132 secondary schools in Canada, school contacts responded to an annual survey in the 2020-2021 school year on changes to physical activity programs and facilities, and related staff training. Content analysis categorized open-ended text responses, and schools were compared based on area urbanicity and median income. Most schools canceled all interschool sports (88.9%) and intramurals (65.9%). New programs were added by 12.6% of schools, and about half (49%) of schools reported some continuing programs, most of which were sports programs, followed by facility and equipment access. Physical activity facilities were closed in 18.1% of schools, while 15.7% had new facilities added, and 11% temporarily converted facilities into learning spaces. Large/medium urban schools were at greater odds of having made any change to their facilities compared to schools in rural/small urban areas (odds ratio (95% confidence interval): 2.3 (1.1, 4.8)). The results demonstrate the considerable scale and nature of the restrictions in school provisions of physical activity opportunities during this period, as well as the resourcefulness of some schools in adding new programs and facilities.

The brain microvasculature is a primary mediator of interferon-α neurotoxicity in human cerebral interferonopathies.

Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.

Transient Differentiation-State Plasticity Occurs During Acute Lymphoblastic Leukemia Initiation.

Leukemia is characterized by oncogenic lesions that result in a block of differentiation while phenotypic plasticity is retained. A better understanding of how these two phenomena arise during leukemogenesis in humans could help inform diagnosis and treatment strategies. Here, we leveraged the well-defined differentiation states during T-cell development to pinpoint the initiation of T-cell acute lymphoblastic leukemia (T-ALL), an aggressive form of childhood leukemia, and study the emergence of phenotypic plasticity. Single-cell whole genome sequencing of leukemic blasts was combined with multiparameter flow cytometry to couple cell identity and clonal lineages. Irrespective of genetic events, leukemia-initiating cells altered their phenotypes by differentiation and de-differentiation. Construction of phylogenies of individual leukemias using somatic mutations revealed that phenotypic diversity is reflected by the clonal structure of the cancer. The analysis also indicated that the acquired phenotypes are heritable and stable. Together, these results demonstrate a transient period of plasticity during leukemia initiation where phenotypic switches appear unidirectional.

Morphological changes and their associations with clinical parameters in children with nephropathic cystinosis and chronic kidney disease prior to kidney replacement therapy over 25 years.

BACKGROUND: Infantile nephropathic cystinosis (INC) is a rare lysosomal storage disorder, mostly and often firstly affecting the kidneys, together with impaired disharmonious growth and rickets, eventually resulting in progressive chronic kidney disease (CKD). With the introduction of cysteamine therapy, most pediatric patients reach adulthood with no need for kidney replacement therapy. Still, detailed changes in INC patients' clinical and morphological presentation over the past decades have not yet been thoroughly investigated. METHODS: Two groups with a respective total of 64 children with INC and 302 children with CKD, both treated conservatively and aged 2 to 18 years, were prospectively observed in the time span from 1998 to 2022 with 1186 combined annual clinical and morphological examinations clustered into two measurement periods (1998 to 2015 and ≥ 2016). RESULTS: In INC patients, thoracic proportion indices remained markedly increased, whereas body fat stores remained decreased over the past 25 years (+ 1 vs. below ± 0 z-score, respectively). Their CKD peers presented with overall improved growth, general harmonization of body proportions, and improved body fat stores, while INC patients only presented with an isolated significant increase in leg length over time (∆0.36 z-score). eGFR adjusted for age did not significantly change over the past 25 years in both groups. Alkaline phosphatase (ALP) showed a significant decrease in CKD patients over time, while remaining above normal levels in INC patients. CONCLUSIONS: Disproportionate thoracic shape and impaired body fat stores remain the most characteristic morphological traits in INC patients over the past 25 years, while causal mechanisms remain unclear.

Health-related quality of life of children with X-linked hypophosphatemia in Germany.

BACKGROUND: X-linked hypophosphatemia (XLH) is a rare inherited phosphate-wasting disorder associated with bone and dental complications. Health-related quality of life (HRQoL) is reduced in XLH patients on conventional treatment with phosphate supplements and active vitamin D, while information on patients treated with burosumab is rare. METHODS: HRQoL was assessed in 63 pediatric XLH patients participating in a prospective, observational study and patient registry in Germany using the KIDSCREEN-52 survey instrument and standardized qualitative interviews. RESULTS: The median age of the XLH patients was 13.2 years (interquartile range 10.6 - 14.6). At the time of the survey, 55 (87%) patients received burosumab and 8 (13%) conventional treatment. Forty-six patients (84%) currently being treated with burosumab previously received conventional treatment. Overall, HRQoL was average compared to German reference values (mean ± SD: self-report, 53.36 ± 6.47; caregivers' proxy, 51.33 ± 7.15) and even slightly above average in some dimensions, including physical, mental, and social well-being. In general, XLH patients rated their own HRQoL higher than their caregivers. In qualitative interviews, patients and caregivers reported that, compared with conventional therapy, treatment with burosumab reduced stress, bone pain, and fatigue, improved physical health, and increased social acceptance by peers and the school environment. CONCLUSIONS: In this real-world study in pediatric XLH patients, HRQoL was average or even slightly above that of the general population, likely due to the fact that the vast majority of patients had their treatment modality switched from conventional treatment to burosumab resulting in improved physical health and well-being.

An Evolutionary Psychological Approach Toward BDSM Interest and Behavior.

Bondage/discipline, Dominance/submission, and Sadism/Masochism (BDSM) have gained increased attention and discussion in recent years. This prevalence is accompanied by a shift in perceptions of BDSM, including the declassification of sadomasochism as a paraphilic disorder. Evolutionary psychology offers a unique perspective of why some individuals are interested in BDSM and why some prefer certain elements of BDSM over others (e.g., dominance versus submission). In this paper, we examine BDSM from an evolutionary standpoint, examining biopsychosocial factors that underlie the BDSM interests and practice. We articulate this perspective via an exploration of: proximate processes, such as the role of childhood experiences, sexual conditioning, and physiological factors; as well as ultimate explanations for power play and pain play dimensions of BDSM, highlighting the potential adaptive advantages of each. While BDSM may not be adaptive in itself, we examine the literature of sex differences in BDSM role preferences and argue that these preferences may stem from the extreme forms of behaviors which enhance reproductive success. In the realm of pain play, we explore the intersection of pain and pleasure from both physiological and psychological perspectives, highlighting the crucial role of psychological and play partner factors in modulating the experience of pain. Finally, we encourage future research in social sciences to utilize evolutionary frameworks to further explore the subject and help alleviate the mystification surrounding BDSM. This multifaceted exploration of BDSM provides valuable insights for clinicians, kink-identified individuals, and scholars seeking to understand the evolutionary perspectives of human sexual behavior and preferences.

Identification of the Campanian Ignimbrite in the Dead Sea and consequent time-transgressive hydroclimatic shifts in the Eastern Mediterranean.

Robust chronologies and time equivalent tephra markers are essential to better understand spatial palaeoenvironmental response to past abrupt climatic changes. Identification of well-dated and widely dispersed volcanic ash by tephra and cryptotephra (microscopic volcanic ash) provides time synchronous tie-points and strongly reduces chronological uncertainties. Here, we present the major, minor and trace element analyses of cryptotephra shards in the Dead Sea Deep Drilling sedimentary record (DSDDP 5017-1A) matching the Campanian Ignimbrite (CI). This geochemical identification expands the known dispersal range of the CI to the southeastern Mediterranean, over 2300 km from the volcanic source. Due to the CI eruption occurring near-synchronous with North Atlantic ice surge of Heinrich Event 4 (HE4), this tephra provides insights into regional responses to large-scale climatic change in the Mediterranean. In the Dead Sea, the CI layer is associated with wetter climatic conditions. This contrasts with the contemporaneous occurrence of the CI deposition and dry conditions in the central and eastern Mediterranean suggesting a possible climate time-transgressive expansion of HE4. Our finding underscores the temporal and spatial complexity of regional climate responses and emphasises the importance of tephra as a time marker for studying large-scale climatic changes verses regional variations.

The brain insulin receptor gene network and associations with frailty index.

OBJECTIVE: To investigate longitudinal associations between variations in the co-expression-based brain insulin receptor polygenic risk score and frailty, as well as change in frailty across follow-up. METHODS: This longitudinal study included 1605 participants from the Helsinki Birth Cohort Study. Biologically informed expression-based polygenic risk scores for the insulin receptor gene network, which measure genetic variation in the function of the insulin receptor, were calculated for the hippocampal (hePRS-IR) and the mesocorticolimbic (mePRS-IR) regions. Frailty was assessed in at baseline in 2001-2004, 2011-2013 and 2017-2018 by applying a deficit accumulation-based frailty index. Analyses were carried out by applying linear mixed models and logistical regression models adjusted for adult socioeconomic status, birthweight, smoking and their interactions with age. RESULTS: The FI levels of women were 1.19%-points (95% CI 0.12-2.26, P = 0.029) higher than in men. Both categorical and continuous hePRS-IR in women were associated with higher FI levels than in men at baseline (P < 0.05). In women with high hePRS-IR, the rate of change was steeper with increasing age compared to those with low or moderate hePRS-IR (P < 0.05). No associations were detected between mePRS-IR and frailty at baseline, nor between mePRS-IR and the increase in mean FI levels per year in either sex (P > 0.43). CONCLUSIONS: Higher variation in the function of the insulin receptor gene network in the hippocampus is associated with increasing frailty in women. This could potentially offer novel targets for future drug development aimed at frailty and ageing.

Frailty in Midlife as a Predictor of Changes in Body Composition from Midlife into Old Age: A Longitudinal Birth Cohort Study.

INTRODUCTION: Few studies have investigated the association between frailty and subsequent body composition. METHODS: We performed separate linear mixed model analyses to study the associations between changes in the participant frailty status assessed by a frailty index (FI) and subsequent body mass index (BMI), lean mass index (LMI), fat mass index (FMI), and FMI to LMI ratio values assessed on three occasions over 17 years. The analyses were carried out among 996 participants spanning from age 57 to 84 years. RESULTS: With advancing age, LMI and BMI decreased, whereas FMI and FMI to LMI ratio increased. Participants with "stable frailty," followed by those with "increasing frailty" experienced faster decreases in LMI and faster increases in FMI and FMI to LMI ratio values from midlife into old age relative to those in the group "stable not frail." Contrastingly, those in the highest third of absolute annual increase in FMI and FMI to LMI ratio became more frail faster from midlife into old age relative to those in the lowest third. CONCLUSIONS: We found evidence of an adverse health outcome of frailty where lean indices declined faster and fat indices and fat-to-lean ratios increased faster from midlife into old age. The changes resembled those that occurred with aging, but at a faster pace. The relationship between body composition and frailty is likely bidirectional, where high or increasing levels of fat are associated with the risk of becoming more frail earlier, but where a longer duration of frailty may increase the risk of faster age-related changes to body composition.

Limited capability of MRI radiomics to predict primary tumor histology of brain metastases in external validation.

Background: Growing research demonstrates the ability to predict histology or genetic information of various malignancies using radiomic features extracted from imaging data. This study aimed to investigate MRI-based radiomics in predicting the primary tumor of brain metastases through internal and external validation, using oversampling techniques to address the class imbalance. Methods: This IRB-approved retrospective multicenter study included brain metastases from lung cancer, melanoma, breast cancer, colorectal cancer, and a combined heterogenous group of other primary entities (5-class classification). Local data were acquired between 2003 and 2021 from 231 patients (545 metastases). External validation was performed with 82 patients (280 metastases) and 258 patients (809 metastases) from the publicly available Stanford BrainMetShare and the University of California San Francisco Brain Metastases Stereotactic Radiosurgery datasets, respectively. Preprocessing included brain extraction, bias correction, coregistration, intensity normalization, and semi-manual binary tumor segmentation. Two-thousand five hundred and twenty-eight radiomic features were extracted from T1w (±â€…contrast), fluid-attenuated inversion recovery (FLAIR), and wavelet transforms for each sequence (8 decompositions). Random forest classifiers were trained with selected features on original and oversampled data (5-fold cross-validation) and evaluated on internal/external holdout test sets using accuracy, precision, recall, F1 score, and area under the receiver-operating characteristic curve (AUC). Results: Oversampling did not improve the overall unsatisfactory performance on the internal and external test sets. Incorrect data partitioning (oversampling before train/validation/test split) leads to a massive overestimation of model performance. Conclusions: Radiomics models' capability to predict histologic or genomic data from imaging should be critically assessed; external validation is essential.

PSAURON: a tool for assessing protein annotation across a broad range of species.

Evaluating the accuracy of protein-coding sequences in genome annotations is a challenging problem for which there is no broadly applicable solution. In this manuscript we introduce PSAURON (Protein Sequence Assessment Using a Reference ORF Network), a novel software tool developed to assess the quality of protein-coding gene annotations. Utilizing a machine learning model trained on a diverse dataset from over 1000 plant and animal genomes, PSAURON assigns a score to coding DNA or protein sequence that reflects the likelihood that the sequence is a genuine protein coding region. PSAURON scores can be used for genome-wide protein annotation assessment as well as the rapid identification of potentially spurious annotated proteins. Validation against established benchmarks demonstrates PSAURON's effectiveness and correlation with recognized measures of protein quality, highlighting its potential use as a general-purpose method to evaluate gene annotation. PSAURON is open source and freely available at https://github.com/salzberg-lab/PSAURON . One-Sentence Summary: PSAURON is a machine learning-based tool for rapid assessment of protein coding gene annotation.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Cardiac function in a large animal model of myocardial infarction at 7 T: deep learning based automatic segmentation increases reproducibility.

Cardiac magnetic resonance (CMR) imaging allows precise non-invasive quantification of cardiac function. It requires reliable image segmentation for myocardial tissue. Clinically used software usually offers automatic approaches for this step. These are, however, designed for segmentation of human images obtained at clinical field strengths. They reach their limits when applied to preclinical data and ultrahigh field strength (such as CMR of pigs at 7 T). In our study, eleven animals (seven with myocardial infarction) underwent four CMR scans each. Short-axis cine stacks were acquired and used for functional cardiac analysis. End-systolic and end-diastolic images were labelled manually by two observers and inter- and intra-observer variability were assessed. Aiming to make the functional analysis faster and more reproducible, an established deep learning (DL) model for myocardial segmentation in humans was re-trained using our preclinical 7 T data (n = 772 images and labels). We then tested the model on n = 288 images. Excellent agreement in parameters of cardiac function was found between manual and DL segmentation: For ejection fraction (EF) we achieved a Pearson's r of 0.95, an Intraclass correlation coefficient (ICC) of 0.97, and a Coefficient of variability (CoV) of 6.6%. Dice scores were 0.88 for the left ventricle and 0.84 for the myocardium.

Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Heterogeneous and Cooperative Rupture of Histidine-Ni2+ Metal-Coordination Bonds on Rationally Designed Protein Templates.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.