Bisphenol F affects neurodevelopmental gene expression, mushroom body development, and behavior in Drosophila melanogaster.

Bisphenol F (BPF) is a potential neurotoxicant used as a replacement for bisphenol A (BPA) in polycarbonate plastics and epoxy resins. We investigated the neurodevelopmental impacts of BPF exposure using Drosophila melanogaster as a model. Our transcriptomic analysis indicated that developmental exposure to BPF caused the downregulation of neurodevelopmentally relevant genes, including those associated with synapse formation and neuronal projection. To investigate the functional outcome of BPF exposure, we evaluated neurodevelopmental impacts across two genetic strains of Drosophila- w1118 (control) and the Fragile X Syndrome (FXS) model-by examining both behavioral and neuronal phenotypes. We found that BPF exposure in w1118 Drosophila caused hypoactive larval locomotor activity, decreased time spent grooming by adults, reduced courtship activity, and increased the severity but not frequency of ß-lobe midline crossing defects by axons in the mushroom body. In contrast, although BPF reduced peristaltic contractions in FXS larvae, it had no impact on other larval locomotor phenotypes, grooming activity, or courtship activity. Strikingly, BPF exposure reduced both the severity and frequency of ß-lobe midline crossing defects in the mushroom body of FXS flies, a phenotype previously observed in FXS flies exposed to BPA. This data indicates that BPF can affect neurodevelopment and its impacts vary depending on genetic background. Further, BPF may elicit a gene-environment interaction with Drosophila fragile X messenger ribonucleoprotein 1 (dFmr1)-the ortholog of human FMR1, which causes fragile X syndrome and is the most common monogenetic cause of intellectual disability and autism spectrum disorder.

Overexpression of T3SS translocation signals in Salmonella causes delayed attenuation.

Engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We previously engineered Salmonella enterica serovar Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. Here, we show that after 48 hours, the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFN-γ-deficient mice, this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell's physiology, which makes host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Apoptotic signaling clears engineered Salmonella in an organ-specific manner.

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. When a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we engineered S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. To clear an infection, cells may have specific tasks that they must complete before they die; different modes of cell death could initiate these 'bucket lists' in either convergent or divergent ways.

Although alive and healthy cells are essential for survival, in certain circumstances ­ such as when a cell becomes infected ­ it is beneficial for cells to deliberately die through a process known as regulated cell death. There are several types of regulated cell death, each with distinct pathways and mechanisms. However, if the initial pathway is blocked, cells can use an alternative one, suggesting that they can compensate for one other. Two forms of regulated cell death ­ named pyroptosis and apoptosis ­ can be used by infected cells to limit the spread of pathogens. However, it was not clear if these two forms or additional 'back-up' apoptosis pathways ­ which are induced when pyroptosis fails ­ are equally efficient at clearing infections and how they might vary in different cell types. To address this, Abele et al. investigated cell death in live mice infected with the bacterium Salmonella. Different organs in which the bacterium infects distinct cell types were examined. Experiments showed that pyroptosis could eliminate bacteria from both intestinal cells as well as immune cells found throughout the body, called macrophages. In contrast, apoptosis was only able to clear infection from intestinal cells. The findings can be explained by prior studies showing both apoptosis and pyroptosis lead to the same outcome in intestinal cells ­ dead cells are expelled from the body through a process called extrusion to maintain the barrier function of the intestine. However, in macrophages, the different pathways lead to different outcomes, indicating they are not entirely interchangeable. Overall, the findings of Abele et al. underscore the complexity of cellular responses to infection and the nuanced roles of different cell death pathways. This provides further evidence that cells might have specific tasks they need to complete before death in order to effectively clear an infection. These tasks may differ depending on cell type and the form of regulated cell death, and may not be equally efficient at clearing an infection.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover an innate granuloma model in mice with an environmental bacterium called Chromobacterium violaceum. Granuloma formation not only successfully walls off, but also clears, the infection. The infected lesion can arise from a single bacterium that replicates despite the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum-induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. This innate granuloma successfully eradicates C. violaceum infection. Therefore, this C. violaceum-induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby resolve infection by an environmental pathogen.

Visual representations of single- and multi-sport participation in a youth swimming sample: Implications for definitions and discussions of early specialization.

Academic literature and sport policy documents have cited concerns about an increasing prevalence of early sport specialization, with associated burnout, dropout, and injury. However, evidence to support such statements is limited. Definitions of early specialization vary, but a common criterion is continued participation in a single sport, prior to adolescence. We explored the prevalence of single-sport participation and other patterns of sport involvement from ages 6-12 in a Canadian swimming sample using retrospective longitudinal methods. Parents of 236 competitive swimmers (ages 12-17) completed surveys on their children's sport backgrounds, including the number of sports participated in annually from age 6-12. A cluster heat map elucidated single- and multi-sport patterns over time. Mixed analyses of variance tested for differences by gender and club type. Fourteen percent of our sample showed stable participation in either one sport or multiple sports per year over time, 25% decreased their annual number of sports, and 60% increased. This trend of increasing, rather than decreasing the number of sports in their annual activity roster when approaching age 12 was particularly pronounced for girls. Only 10 participants (4% of the sample) consistently engaged in a single sport each year from age 6-12. Summer (seasonal) swimmers consistently did more sports than year-round swimmers. Overall, our findings showed highly idiosyncratic longitudinal patterns of sport participation that did not easily conform to current sport activity guidelines. We also found similar idiosyncrasy in an ad-hoc analysis of participants who had dropped out of swimming a year later. If single-sport participation is considered a key criterion for defining early specialization, our findings suggest the prevailing narrative around early specialization may be overstated in relation to the number of single-sport athletes. Alternatively, other components of early specialization may be more prevalent and deserving of attention due to possible associations with harmful outcomes.

Apoptotic signaling clears engineered Salmonella in an organ-specific manner.

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria in the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular 'bucket lists' to be accomplished before a cell dies.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover a novel granuloma model in mice. Chromobacterium violaceum is an environmental bacterium that stimulates granuloma formation that not only successfully walls off but also clears the infection. The infected lesion can arise from a single bacterium that replicates in the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum -induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. These innate granulomas successfully eradicate C. violaceum infection. Therefore, this new C. violaceum -induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby eradicate infection by an environmental pathogen.

Role of Caspases and Gasdermin A during HSV-1 Infection in Mice.

Herpes simplex virus type 1 (HSV-1) infection can manifest locally as mucocutaneous lesions or keratitis and can also spread to the central nervous system to cause encephalitis. HSV-1 establishes a lifelong latent infection and neither cure nor vaccine is currently available. The innate immune response is the first line of defense against infection. Caspases and gasdermins are important components of innate immunity. Caspases are a family of cysteine proteases, most of which mediate regulated cell death. Gasdermins are a family of pore-forming proteins that trigger lytic cell death. To determine whether caspases or gasdermins contribute to innate immune defenses against HSV-1, we screened mice deficient in specific cell death genes. Our results indicate a modest role for caspase-6 in defense against HSV-1. Further, Asc-/-Casp1/11-/- mice also had a modest increased susceptibility to HSV-1 infection. Caspase-7, -8, and -14 did not have a notable role in controlling HSV-1 infection. We generated Gsdma1-Gsdma2-Gsdma3 triple knockout mice, which also had normal susceptibility to HSV-1. We confirmed that the previously published importance of RIPK3 during systemic HSV-1 infection also holds true during skin infection. Overall, our data highlight that as a successful pathogen, HSV-1 has multiple ways to evade host innate immune responses.

Caspase-7 activates ASM to repair gasdermin and perforin pores.

Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons1. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)2,3. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.

Standardized Neurodevelopmental Surveillance of High-risk Infants Using Telehealth: Implementation Study during COVID-19.

INTRODUCTION: Neurodevelopmental surveillance is critical for high-risk infants following neonatal intensive care discharge and is traditionally performed in-person. COVID-19 interruption of regular surveillance necessitated a rapid development of telehealth models for effective and standardized care. METHODS: We used implementation science and lean methodologies to develop an effective telehealth neurodevelopmental surveillance program for high-risk infants. Interventions included reorganization of visit flow processes and a telehealth toolkit for standardized neurological and developmental assessments. We tested and improved our intervention through plan-do-study-act cycles, value-added analysis, and parent- and provider-satisfaction questionnaires. Process metrics (standard elements, subspecialty referrals, diagnostic tests, and prescriptions ordered) were compared in group-level analyses between telehealth patients (N = 97) March 16, 2020-July 1, 2020 and a matched in-person cohort at the same period the previous year. Run charts examined shifts in balancing measures (provider efficiency and missed visits) over 8 weeks before and after implementation. RESULTS: Primary outcomes were visit completion (100%), patient parent satisfaction (>90% strongly agreed or agreed telehealth procedures were valuable and easy to use) and ability to accurately diagnose cerebral palsy (no statistical difference with comparison visits). Providers (N = 6) rated telehealth experiences favorably. Process metrics indicated no differences between telehealth and in-person visits (all P > 0.05). Following telehealth implementation, provider efficiency increased to near baseline (median 88.9% versus 91.7%) and median missed visits decreased to 0% from 20% (in-person). CONCLUSIONS: Implementation of telehealth for neurodevelopmental surveillance in a tertiary high-risk infant follow-up clinic successfully provided standardized and timely care during stay-at-home orders; broader telehealth applications may overcome access barriers in this field.

Photogrammetric Outcomes of Primary Nasal Correction in Unilateral Cleft Lip Patients: Early Childhood Results From a Single Surgeon's Experience.

BACKGROUND: Concerns of nonlasting results and potential nasal growth damage precluded cleft nasal correction at the time of initial cleft lip repair. Our goal was to evaluate the outcome of primary cleft nasal correction in our patients with unilateral cleft lip. METHODS: A retrospective review of patients with complete and incomplete unilateral cleft lip who underwent primary cleft nasal correction from 2010 to 2017 by the same surgeon was performed. The cleft-to-noncleft nostril height, width, one-fourth medial part of nostril height, nasal sill height, and nostril area ratios, as well as inner nostril height-to-width ratios were determined from standard basilar view photographs taken in different time points (T1, <3 months; T2, 3-12 months; T3, 12-36 months; and T4, >36 months after surgery). A 5-point visual analog scale (1 = worst, 5 = best) was used to assess each patient's nose appearance. RESULTS: Seventy-two patients were identified (66.7% male, 51.3% with a complete cleft lip). Average visual analog scale scores T1-T4 were 3.88 ± 0.85, 3.72 ± 0.93, 3.54 ± 0.99, and 3.40 ± 0.71, respectively. Intraclass correlation ranged from 0.61 to 0.94. A significant decrease [mean difference (SD)] was found for cleft-to-noncleft nostril width ratio [0.15 (0.18)] from T1 to T2, and an increase for one-fourth medial height ratio [-0.09 (0.07)] and for inner nostril height-to-width ratio in the noncleft side [-0.23 (0.25)] from T1 to T3. Thirteen patients required secondary surgical revision. CONCLUSION: Based on photogrammetry, primary cleft nasal correction in our patients with unilateral cleft lip achieved acceptable and stable outcomes during early childhood.

Markers of Early Specialization and Their Relationships With Burnout and Dropout in Swimming.

Empirical evidence directly associating early sport specialization with burnout and dropout is lacking, although a relationship is theorized. Research in this area relies on time-intensive retrospective interviews or questionnaires that generate large amounts of data. The optimal use of these data for assessing early specialization (ES) and its relationship with key criterion variables is unclear. The purpose of this study was to add empirical evidence to the literature regarding ES, burnout, and dropout. This involved examining a large number of hypothesized markers of ES and reducing them to a smaller set useful for predicting burnout and dropout. Survey data were collected from 137 swimmers, age 12-13 years, and their parents, including descriptions of swimmers' sport backgrounds from age 6 until present. Contrary to what was expected, the ES items were not positively related to burnout and dropout. The authors present several possible explanations, including key motivational considerations.

Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487.

The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2(*)2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k(cat) for these processes and increases the K(m) for NAD(+), as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2(*)2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2(*)2 complexed with coenzyme to 2.5A(.) We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2(*)2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2(*)2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2(*)2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme-binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant.

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.