The insufficiency of ATG4A in macroautophagy.

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Moms know best?: Children's evaluations of mothers' unfair responses to peer conflicts.

Research on children's evaluations of parental discipline or parental responses to peer conflicts has focused on parents' responses to hypothetical or actual child behavior. These parent behaviors are typically depicted as fair, reasonable, and appropriate, but what if they are not? In daily life, parents do sometimes act unfairly, or children evaluate parents' responses as such. This study examined 90 4.5- to 10-year-old U.S. middle-class children's (Mage = 7.42 years, SD = 1.70) evaluations of four scenarios describing hypothetical mothers' unfair responses to peer conflicts (unjustified stealing; intentional harm; accidental harm; ambiguous harm). Across ages, children overwhelmingly judged mothers' directives, particularly regarding a straightforwardly immoral demand (unjustified stealing), as wrong and very unfair, based primarily on moral justifications or coordinated justifications involving recognition of different competing moral (or moral and nonmoral) concerns. With age, children increasingly viewed directives to retaliate for intended harm as more fair and those regarding ambiguous harm as more unfair; justifications recognizing different concerns also increased with age, although more for retaliation for accidental and intended harm than for other situations. Children largely endorsed disobedience and attributed negative emotions to actors who were described as complying. Thus, children prioritized moral concerns over obedience to authority when mothers asserted authority unfairly, although their responses showed variability with age and the situational context.

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner.

HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity.

Does volunteering decrease burnout? Healthcare professional and student perspectives on burnout and volunteering.

Background: Burnout among healthcare providers is a significant crisis in our healthcare system, especially in the context of the COVID-19 pandemic. The aim of this study was to understand what motivates healthcare workers and students to volunteer in their community as well as examine how volunteering relates to burnout. These findings can help health organizations better meet the needs of healthcare workers, as well as provide insights for non-profits that rely on volunteer professionals. Methods: Healthcare providers (N = 8), graduate healthcare students (N = 10), and undergraduate students (N = 14) who volunteered at community health fairs completed the OLBI burnout assessment and an individual semi-structured interview to characterize their attitudes toward volunteering and its relationship with burnout. Interviews were recorded, transcribed, and analyzed using a phenomenological approach, comparing themes across levels of burnout among providers and students. Results: Participants described that feeling burnt out decreased one's likelihood to volunteer, but also that volunteering prevented burnout. The OLBI scores showed that 79.2 and 20.8% of students were low and moderately burnt out respectively, and 87.5 and 12.5% of health professionals were low and moderately burnt out, respectively. Students volunteered for professional development while healthcare professionals cited a desire for a change in their day-to-day work as a reason to volunteer. Both students and health professionals often volunteered because they wanted to make a difference, it made them feel good, and/or they felt a responsibility to volunteer. COVID-19 had a wide range of effects on burnout and motivations to volunteer. Conclusion: Volunteering may be useful for preventing burnout among healthcare workers and students, but may not be helpful for those already experiencing burnout. Interview responses and the fact that none of the volunteers had high burnout levels according to their OLBI scores suggest those who choose to volunteer may be less burnt out. Healthcare organizations and schools can encourage volunteering by emphasizing the difference healthcare students and professionals can make through volunteering in the community. Increasing convenience and emphasizing professional development can help recruit and retain healthcare student volunteers. Highlighting the chance to diversify their scope of practice may help recruit and retain healthcare professional volunteers.

Separate structural and functional domains of Tn4430 transposase contribute to target immunity.

Like other transposons of the Tn3 family, Tn4430 exhibits target immunity, a process that prevents multiple insertions of the transposon into the same DNA molecule. Immunity is conferred by the terminal inverted repeats of the transposon and is specific to each element of the family, indicating that the transposase TnpA is directly involved in the process.However, the molecular mechanism whereby this protein promotes efficient transposition into permissive targets while preventing transposition into immune targets remains unknown. Here, we demonstrate that both functions of TnpA can be uncoupled from each other by isolating and characterizing mutants that are proficient in transposition (T+) but impaired in immunity (I-). The identified T+/I- mutations are clustered into separate structural and functional domains of TnpA, indicating that different activities of the protein contribute to immunity.Combination of separate mutations had synergistic effects on target immunity but contrasting effects on transposition. One class of mutations was found to stimulate transposition, whereas other mutations appeared to reduce TnpA activity. The data are discussed with respect to alternative models in which TnpA acts as a specific determinant to both establish and respond to immunity.

LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

Lipid droplets (LDs) store lipids that can be utilized during times of scarcity via autophagic and lysosomal pathways, but how LDs and autophagosomes interact remained unclear. Here, we discovered that the E2 autophagic enzyme, ATG3, localizes to the surface of certain ultra-large LDs in differentiated murine 3T3-L1 adipocytes or Huh7 human liver cells undergoing prolonged starvation. Subsequently, ATG3 lipidates microtubule-associated protein 1 light-chain 3B (LC3B) to these LDs. In vitro, ATG3 could bind alone to purified and artificial LDs to mediate this lipidation reaction. We observed that LC3B-lipidated LDs were consistently in close proximity to collections of LC3B-membranes and were lacking Plin1. This phenotype is distinct from macrolipophagy, but it required autophagy because it disappeared following ATG5 or Beclin1 knockout. Our data suggest that extended starvation triggers a noncanonical autophagy mechanism, similar to LC3B-associated phagocytosis, in which the surface of large LDs serves as an LC3B lipidation platform for autophagic processes.

Emotional labor: The role of organizational dehumanization.

In a permanent quest for profit, employees can be reduced to a mere function or instrument, dissociated from their quality as individuals for the organization's ends. Experiencing such a feeling as an employee has been called organizational dehumanization. Scholars have recently suggested that organizational dehumanization may play a key role in the development of emotional labor. However, how organizational dehumanization and two main emotional labor strategies (i.e., surface and deep acting) are causally related remains unclear in this literature. In the present research, we argue that employees who experience organizational dehumanization and whose self is thus threatened then engage in surface acting to "conserve" their self or in deep acting to "give up" their self in service of the role. Overall, the combined results of three studies offer strong evidence that organizational dehumanization leads employees to perform more surface acting, but not more deep acting. Unexpectedly, our findings also indicate that deep acting reduces the perception of being dehumanized by the organization. In showing this, the present research sheds light on the potential dark side of deep acting, by suggesting that this strategy can change employees' perspectives in a way that may encourage them to stay in an organization that treats them as a means to an end. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Examining the role of fundamental psychological needs in the development of metadehumanization: A multi-population approach.

In the present paper, we investigate dehumanization processes from a victim perspective. We propose that dehumanization experiences, that is metadehumanization, arise from people's feelings that their fundamental human needs are thwarted and that such experiences influence their emotions, self-esteem, and coping strategies. Our model is put at test in three contexts involving different types of dehumanization victims: Women (Study 1a, N = 349), patients with severe alcohol use disorder (Study 1b, N = 120), and employees in organizations (Study 1c, N = 347). Our integrated model of metadehumanization, which considers both its antecedents and consequences, proved stable across contexts and populations and therefore helps building bridges between different psychological disciplines in which dehumanization occurs.

Aeromedical Implications of Long-Term COVID-19 Sequelae.

BACKGROUND: While many COVID-19 studies focus on acute effects of the infection, few examine the intermediate and long-term sequelae of the illness. Studies have shown that a good portion of patients have chronic effects in several body systems for several months or longer. Such effects can potentially adversely impact pilot performance in flight. We sought to determine the long-term effects of COVID-19 infection, how such effects can affect pilot performance, and how to best evaluate pilots for aeromedical flight clearance.METHODS: We used the PubMed literature search engine to review peer-reviewed articles that focused on the intermediate and long-term effects of COVID-19 infection. Chronic signs and symptoms were subdivided based on the particular body organ system affected. Merging information obtained from case reviews, article reviews, and aeromedical standards, we created a risk stratification guide to assist with the aeromedical disposition of affected pilots.RESULTS: Long-term effects of COVID-19 infection can last for several months or longer. The most common effects are fatigue, weakness, pulmonary diffusion defects, depression, and anxiety.DISCUSSION: This review article focuses on the most common intermediate- and long-term COVID-19 conditions of aeromedical significance and the corresponding course of actions recommended for the aeromedical examiner. Aeromedical evaluation should take into consideration factors related to the pilot, aircraft type, and specific aviation environment. Such evaluation may include diagnostic testing, medical specialist consultation, preflight simulation in an altitude chamber, human centrifuge testing, and/or a flight simulator checkride.Ko SY, Nguyen NK, Lee CL, Lee LA, Nguyen KUT, Lee EC. Aeromedical implications of long-term COVID-19 sequelae. Aerosp Med Hum Perform. 2021; 92(11):898-907.

Evaluation and accurate diagnoses of pediatric diseases using artificial intelligence.

Artificial intelligence (AI)-based methods have emerged as powerful tools to transform medical care. Although machine learning classifiers (MLCs) have already demonstrated strong performance in image-based diagnoses, analysis of diverse and massive electronic health record (EHR) data remains challenging. Here, we show that MLCs can query EHRs in a manner similar to the hypothetico-deductive reasoning used by physicians and unearth associations that previous statistical methods have not found. Our model applies an automated natural language processing system using deep learning techniques to extract clinically relevant information from EHRs. In total, 101.6 million data points from 1,362,559 pediatric patient visits presenting to a major referral center were analyzed to train and validate the framework. Our model demonstrates high diagnostic accuracy across multiple organ systems and is comparable to experienced pediatricians in diagnosing common childhood diseases. Our study provides a proof of concept for implementing an AI-based system as a means to aid physicians in tackling large amounts of data, augmenting diagnostic evaluations, and to provide clinical decision support in cases of diagnostic uncertainty or complexity. Although this impact may be most evident in areas where healthcare providers are in relative shortage, the benefits of such an AI system are likely to be universal.

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

Sensing Membrane Curvature in Macroautophagy.

In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo formation of a new organelle called the autophagosome, which either surrounds and sequesters random portions of the cytoplasm or selectively targets individual intracellular challenges. Thus, the autophagosome is a flexible membrane platform with dimensions that ultimately depend upon the target cargo. The intermediate membrane, termed the phagophore or isolation membrane, is a cup-like structure with a clear concave face and a highly curved rim. The phagophore is largely devoid of integral membrane proteins; thus, its shape and size are governed by peripherally associated membrane proteins and possibly by the lipid composition of the membrane itself. Growth along the phagophore rim marks the progress of both organelle expansion and ultimately organelle closure around a particular cargo. These two properties, a reliance on peripheral membrane proteins and a structurally distinct membrane architecture, suggest that the ability to target or manipulate membrane curvature might be an essential activity of proteins functioning in this pathway. In this review, we discuss the extent to which membranes are naturally curved at each of the cellular sites believed to engage in autophagosome formation, review basic mechanisms used to sense this curvature, and then summarize the existing literature concerning which autophagy proteins are capable of curvature recognition.

The Tn3-family of Replicative Transposons.

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase.

The homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1.

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.

Ruptured intracranial mycotic aneurysm in infective endocarditis: a natural history.

Mycotic aneurysms are a rare cause of intracranial aneurysms that develop in the presence of infections such as infective endocarditis. They account for a small percentage of all intracranial aneurysms and carry a high-mortality rate when ruptured. The authors report a case of a 54-year-old man who presented with infective endocarditis of the mitral valve and acute stroke. He subsequently developed subarachnoid hemorrhage during antibiotic treatment, and a large intracranial aneurysm was discovered on CT Angiography. His lesion quickly progressed into an intraparenchymal hemorrhage, requiring emergent craniotomy and aneurysm clipping. Current recommendations on the management of intracranial Mycotic Aneurysms are based on few retrospective case studies. The natural history of the patient's ruptured aneurysm is presented, as well as a literature review on the management and available treatment modalities.

Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model.

CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.

Interaction of hepatocyte growth factor and non-steroidal anti-inflammatory drugs during gastric epithelial wound healing.

BACKGROUND/AIMS: Expression of the hepatocyte growth factor (HGF) and cyclooxygenase-2 (COX-2) is upregulated at the margins of healing gastric ulcers. We investigated in vitro the interference of HGF, the selective COX-2 inhibitor NS-398 and the nonselective COX inhibitor indomethacin with gastric epithelial wound healing and actin microfilament (actin-MF) formation. METHODS: Standardized gastric epithelial wounds, created in confluent RGM1 rat cell monolayers were treated with: HGF (10 ng/ml), NS-398 (1-100 microM) or indomethacin (0.01- 0.5 mM). The areas of re-epithelialization and cell proliferation were measured 24 h after wounding. Actin-MFs were labeled with fluorescein-conjugated phalloidin and their distribution was examined using a Nikon epifluorescence microscope. RESULTS: HGF caused a significant increase in gastric monolayer wound re-epithelialization and this was not affected by mitomycin C. Both indomethacin and NS-398 inhibited HGF-stimulated re-epithelialization, but the basal wound re-epithelialization rate and cell proliferation was only significantly inhibited by indomethacin. HGF triggered actin stress fiber formation which was inhibited by both indomethacin and NS-398, but only indomethacin interfered with actin-MF formation at the baseline condition. CONCLUSIONS: HGF significantly increased gastric wound re-epithelialization by activating cell migration which may be mediated by the COX-2 pathway.