The human innate host defense molecules, SP-A1 and SP-A2 variants, differentially affect survival after infection in mice and in lung transplant patients. SP-A interacts with the sentinel innate immune cell in the alveolus, the alveolar macrophage (AM), and modulates its function and regulation. SP-A also plays a role in pulmonary surfactant-related aspects, including surfactant structure and reorganization. For most (if not all) pulmonary diseases there is a dysregulation of host defense and inflammatory processes and/or surfactant dysfunction or deficiency. Because SP-A plays a role in both of these general processes where one or both may become aberrant in pulmonary disease, SP-A stands to be an important molecule in health and disease. In humans (unlike in rodents) SP-A is encoded by two genes (SFTPA1 and SFTPA2) and each has been identified with extensive genetic and epigenetic complexity. In this review, we focus on functional, structural, and regulatory differences between the two SP-A gene-specific products, SP-A1 and SP-A2, and among their corresponding variants. We discuss the differential impact of these variants on the surfactant structure, the alveolar microenvironment, the regulation of epithelial type II miRNome, the regulation and function of the AM, the overall survival of the organism after infection, and others. Although there have been a number of reviews on SP-A, this is the first review that provides such a comprehensive account of the differences between human SP-A1 and SP-A2.

Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.

Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy.

BackgroundSurfactant protein A (SP-A) contributes to lung immunity by regulating inflammation and responses to microorganisms invading the lung. The huge genetic variability of SP-A in humans implies that this protein is highly important in tightly regulating the lung immune response. Proteomic studies have demonstrated that there are differential responses of the macrophages to SP-A1 and SP-A2 and that there are sex differences implicated in these responses.MethodsPurified SP-A variants were used for administration to alveolar macrophages from SP-A knockout (KO) mice for in vitro studies, and alveolar macrophages from humanized SP-A transgenic mice were isolated for ex vivo studies. The actin cytoskeleton was examined by fluorescence and confocal microscopy, and the macrophages were categorized according to the distribution of polymerized actin.ResultsIn accordance with previous data, we report that there are sex differences in the response of alveolar macrophages to SP-A1 and SP-A2. The cell size and F-actin content of the alveolar macrophages are sex- and age-dependent. Importantly, there are different subpopulations of cells with differential distribution of polymerized actin. In vitro, SP-A2 destabilizes actin in female, but not male, mice, and the same tendency is observed by SP-A1 in cells from male mice. Similarly, there are differences in the distribution of AM subpopulations isolated from SP-A transgenic mice depending on sex and age.ConclusionsThere are marked sex- and age-related differences in the alveolar macrophage phenotype as illustrated by F-actin staining between SP-A1 and SP-A2. Importantly, the phenotypic switch caused by the different SP-A variants is subtle, and pertains to the frequency of the observed subpopulations, demonstrating the need for single-cell analysis approaches. The differential responses of alveolar macrophages to SP-A1 and SP-A2 highlight the importance of genotype in immune regulation and the susceptibility to lung disease and the need for development of individualized treatment options.

Psychology researchers have long attempted to identify educational practices that improve student learning. However, experimental research on these practices is often conducted in laboratory contexts or in a single course, which threatens the external validity of the results. In this article, we establish an experimental paradigm for evaluating the benefits of recommended practices across a variety of authentic educational contexts—a model we call ManyClasses. The core feature is that researchers examine the same research question and measure the same experimental effect across many classes spanning a range of topics, institutions, teacher implementations, and student populations. We report the first ManyClasses study, in which we examined how the timing of feedback on class assignments, either immediate or delayed by a few days, affected subsequent performance on class assessments. Across 38 classes, the overall estimate for the effect of feedback timing was 0.002 (95% highest density interval = [−0.05, 0.05]), which indicates that there was no effect of immediate feedback compared with delayed feedback on student learning that generalizes across classes. Furthermore, there were no credibly nonzero effects for 40 preregistered moderators related to class-level and student-level characteristics. Yet our results provide hints that in certain kinds of classes, which were undersampled in the current study, there may be modest advantages for delayed feedback. More broadly, these findings provide insights regarding the feasibility of conducting within-class randomized experiments across a range of naturally occurring learning environments.

Podocalyxin represents a Wilms' tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced "silencing" of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.

Endocytosis, and the subsequent trafficking of endosomes, requires dynamic physical alterations in membrane shape that are mediated in part by endophilin proteins. The endophilin B family of proteins contains an N-terminal Bin/amphiphysin/Rvs (N-BAR) domain that induces membrane curvature to regulate intracellular membrane dynamics. Whereas endophilin B1 (SH3GLB1/Bif-1) is known to be involved in a number of cellular processes, including apoptosis, autophagy, and endocytosis, the cellular function of endophilin B2 (SH3GLB2) is not well understood. In this study, we used genetic approaches that revealed that endophilin B2 is not required for embryonic development but that endophilin B2 deficiency impairs endosomal trafficking, as evidenced by suppressed endosome acidification, EGFR degradation, autophagic flux, and influenza A viral RNA nuclear entry and replication. Mechanistically, although the loss of endophilin B2 did not affect endocytic internalization and lysosomal function, endophilin B2 appeared to regulate the trafficking of endocytic vesicles and autophagosomes to late endosomes or lysosomes. Moreover, we also found that despite having an intracellular localization and tissue distribution similar to endophilin B1, endophilin B2 is dispensable for mitochondrial apoptosis. Taken together, our findings suggest that endophilin B2 positively regulates the endocytic pathway in response to growth factor signaling, autophagy induction, and viral entry.

The human SFTPA1 and SFTPA2 genes encode the surfactant protein A1 (SP-A1) and SP-A2, respectively, and they have been identified with significant genetic and epigenetic variability including sequence, deletion/insertions, and splice variants. The surfactant proteins, SP-A1 and SP-A2, and their corresponding variants play important roles in several processes of innate immunity as well in surfactant-related functions as reviewed elsewhere [1]. The levels of SP-A have been shown to differ among individuals both under baseline conditions and in response to various agents or disease states. Moreover, a number of agents have been shown to differentially regulate SFTPA1 and SFTPA2 transcripts. The focus in this review is on the differential regulation of SFTPA1 and SFTPA2 with primary focus on the role of 5′ and 3′ untranslated regions (UTRs) and flanking sequences on this differential regulation as well molecules that may mediate the differential regulation.