Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3+ bacteria were present within single-membrane phagosomes that are LAMP1+. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3+ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection

Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish

Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible cre(ERt2)/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa(+) cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models

Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread

Efferocytosis, the process by which dying/dead cells are removed by phagocytosis, plays an important role in development, tissue homeostasis and innate immunity1. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes (Lm), can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells using the pore-forming toxin Listeriolysin O (LLO) and two phospholipases C2. Expression of the cell surface protein ActA allows Lm to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. We show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 contributes to efficient cell-to-cell spread by Lm in macrophages in vitro and growth of these bacteria is impaired in TIM-4−/− mice. Thus, Lm promotes its dissemination in a host by exploiting efferocytosis. Our study suggests that PS-targeted therapeutics may be useful in the fight against infections by Lm and other bacteria that utilize similar strategies of cell-to-cell spread during infection

Bit-Vector Model Counting using Statistical Estimation

Approximate model counting for bit-vector SMT formulas (generalizing \#SAT) has many applications such as probabilistic inference and quantitative information-flow security, but it is computationally difficult. Adding random parity constraints (XOR streamlining) and then checking satisfiability is an effective approximation technique, but it requires a prior hypothesis about the model count to produce useful results. We propose an approach inspired by statistical estimation to continually refine a probabilistic estimate of the model count for a formula, so that each XOR-streamlined query yields as much information as possible. We implement this approach, with an approximate probability model, as a wrapper around an off-the-shelf SMT solver or SAT solver. Experimental results show that the implementation is faster than the most similar previous approaches which used simpler refinement strategies. The technique also lets us model count formulas over floating-point constraints, which we demonstrate with an application to a vulnerability in differential privacy mechanisms

Rehabilitation Treatment Specification System for Voice Therapy: Application to Everyday Clinical Care

PURPOSE: Rehabilitation intervention descriptions often do not explicitly identify active ingredients or how those ingredients lead to changes in patient functioning. The Rehabilitation Treatment Specification System (RTSS) provides guidance to identify the critical aspects of any rehabilitation therapy and supported the development of standardly named ingredients and targets in voice therapy (Rehabilitation Treatment Specification System for Voice Therapy [RTSS-Voice]). This study sought to test the content validity of the RTSS-Voice and determine if the RTSS-Voice can be used to identify commonalities and differences in treatment (criterion validity) across clinicians in everyday clinical practice. METHOD: Five speech-language pathologists from different institutions videotaped one therapy session for 59 patients diagnosed with a voice or upper airway disorder. Specifications were created for each video, and iterative rounds of revisions were completed with the treating clinician and two RTSS experts until consensus was reached on each specification. RESULTS: All 59 sessions were specified without the addition of any targets or ingredients. There were two frequent targets: (a) increased volition and (b) decreased strained voice quality. There were three frequent ingredients: (a) information regarding the patient\u27s capability and motivation to perform a therapeutic behavior, (b) knowledge of results feedback, and (c) opportunities to practice voicing with improved resonance and mean airflow. Across sessions treating vocal hyperfunction, there was large variability across clinicians regarding the types and number of treatment components introduced, types of feedback provided, and vocal practice within spontaneous speech and negative practice. CONCLUSIONS: The RTSS and the RTSS-Voice demonstrated strong content validity, as they comprehensively characterized 59 therapy sessions. They also demonstrated strong criterion validity, as commonalities and differences were identified in everyday voice therapy for vocal hyperfunction across multiple clinicians. Future work to translate RTSS principles and RTSS-Voice terms into clinical documentation can help to understand how clinician and patient variability impacts outcomes and bridge the research-practice gap. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.24796875

Understanding the links between perceiving gratitude and romantic relationship satisfaction using an accuracy and bias framework

Perceiving a partner’s gratitude has several benefits for romantic relationships. We aimed to better understand these associations by decomposing perceptions into accuracy and bias. Specifically, we examined whether accuracy and bias in perceiving a partner’s experience (Study 1: Ndyads= 205) and expression (Study 2: Ndyads= 309) of gratitude were associated with romantic relationship satisfaction. Using the Truth and Bias Model of Judgment, we found that perceivers generally underestimated their partner’s gratitude, and lower perceptions of gratitude were related to lower perceiver satisfaction. Perceivers reported greater satisfaction when they assumed their partner’s gratitude was similar to their own. Partners reported greater satisfaction when perceivers accurately gauged their partners’ gratitude experience (but not expression) and lower satisfaction when perceivers underestimated their gratitude expression (but not experience). Overall, by decomposing gratitude perceptions into accuracy and bias, we provide insight into how these components differentially relate to relationship satisfaction

Mutation spectrum of NOD2 reveals recessive inheritance as a main driver of Early Onset Crohn’s Disease

Inflammatory bowel disease (IBD), clinically defined as Crohn’s disease (CD), ulcerative colitis (UC), or IBD-unclassified, results in chronic inflammation of the gastrointestinal tract in genetically susceptible hosts. Pediatric onset IBD represents ≥ 25% of all IBD diagnoses and often presents with intestinal stricturing, perianal disease, and failed response to conventional treatments. NOD2 was the first and is the most replicated locus associated with adult IBD, to date. However, its role in pediatric onset IBD is not well understood. We performed whole-exome sequencing on a cohort of 1,183 patients with pediatric onset IBD (ages 0–18.5 years). We identified 92 probands with biallelic rare and low frequency NOD2 variants accounting for approximately 8% of our cohort, suggesting a Mendelian inheritance pattern of disease. Additionally, we investigated the contribution of recessive inheritance of NOD2 alleles in adult IBD patients from a large clinical population cohort. We found that recessive inheritance of NOD2 variants explains ~ 7% of cases in this adult IBD cohort, including ~ 10% of CD cases, confirming the observations from our pediatric IBD cohort. Exploration of EHR data showed that several of these adult IBD patients obtained their initial IBD diagnosis before 18 years of age, consistent with early onset disease. While it has been previously reported that carriers of more than one NOD2 risk alleles have increased susceptibility to Crohn’s Disease (CD), our data formally demonstrate that recessive inheritance of NOD2 alleles is a mechanistic driver of early onset IBD, specifically CD, likely due to loss of NOD2 protein function. Collectively, our findings show that recessive inheritance of rare and low frequency deleterious NOD2 variants account for 7–10% of CD cases and implicate NOD2 as a Mendelian disease gene for early onset Crohn’s Disease

Proangiogenic contribution of adiponectin toward mammary tumor growth in vivo

PURPOSE: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. EXPERIMENTAL DESIGN: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. RESULTS: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. CONCLUSIONS: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic micro-environment

Diagnostic Delay Is Associated with Complicated Disease and Growth Impairment in Paediatric Crohn\u27s Disease

Background: Paediatric data on the association between diagnostic delay and inflammatory bowel disease [IBD] complications are lacking. We aimed to determine the effect of diagnostic delay on stricturing/fistulising complications, surgery, and growth impairment in a large paediatric cohort, and to identify predictors of diagnostic delay. Methods: We conducted a national, prospective, multicentre IBD inception cohort study including 1399 children. Diagnostic delay was defined as time from symptom onset to diagnosis \u3e75th percentile. Multivariable proportional hazards [PH] regression was used to examine the association between diagnostic delay and stricturing/fistulising complications and surgery, and multivariable linear regression to examine the association between diagnostic delay and growth. Predictors of diagnostic delay were identified using Cox PH regression. Results: Overall (64% Crohn\u27s disease [CD]; 36% ulcerative colitis/IBD unclassified [UC/IBD-U]; 57% male]), median time to diagnosis was 4.2 (interquartile range [IQR] 2.0-9.2) months. For the overall cohort, diagnostic delay was \u3e9.2 months; in CD, \u3e10.8 months and in UC/IBD-U, \u3e6.6 months. In CD, diagnostic delay was associated with a 2.5-fold higher rate of strictures/internal fistulae (hazard ratio [HR] 2.53, 95% confidence interval [CI] 1.41-4.56). Every additional month of diagnostic delay was associated with a decrease in height-for-age z-score of 0.013 standard deviations [95% CI 0.005-0.021]. Associations persisted after adjusting for disease location and therapy. No independent association was observed between diagnostic delay and surgery in CD or UC/IBD-U. Diagnostic delay was more common in CD, particularly small bowel CD. Abdominal pain, including isolated abdominal pain in CD, was associated with diagnostic delay. Conclusions: Diagnostic delay represents a risk factor for stricturing/internal fistulising complications and growth impairment in paediatric CD