Novel phonotactic learning by children and infants: Generalizing syllable-position but not co-occurrence regularities.

Restrictions in the sequencing of sounds (phonotactic constraints) can be represented at the level of sound co-occurrences (e.g., in baF.Pev, F and P co-occur) and at the level of the syllable (e.g., F is syllable-coda/end, P is syllable-onset/start). Can children (5-year-olds) and infants (11-month-olds) represent constraints as sound co-occurrences and/or relative to syllable positions? Participants listened to artificial languages displaying both word-medial consonant restrictions in co-occurrence pairs (e.g., FP or DZ but not FZ) and in the position of consonants within syllables (e.g., P/Z onsets and D/F codas) in words like baF.Pev and tiD.Zek. Children responded similarly to novel words with the same (e.g., FP) versus different (e.g., FZ) co-occurrence pairs, but they were more misled (i.e., responded "heard it before") by novel words with consonants in the same (e.g., onset-P) versus different (e.g., coda-P) syllable positions (Experiment 1). With the same training stimuli, infants had similar orientation times for novel words with the same versus different co-occurrence pairs, but they had longer orientation times for novel words with consonants in the same versus different syllable positions (Experiment 2). Thus, across different methods and ages, syllable-position information was more readily available for generalization than consonant co-occurrence information. The results suggest that when multiple regularities are present simultaneously, some phonotactic constraints (e.g., consonants in particular syllable positions) may be spontaneously represented and generalized by children and infants, whereas others (e.g., consonant co-occurrences) might not be available. The results contribute toward understanding how children and infants represent sound sequences.

A Shift in the Direction of the Production Effect in Children Aged 2-6 Years.

Research has found mixed evidence for the production effect in childhood. Some studies have found a positive effect of production on word recognition and recall, while others have found the reverse. This paper takes a developmental approach to investigate the production effect. Children aged 2-6 years (n = 150) from a predominantly white population in Ottawa, Canada were trained on familiar words which were either seen, heard or produced, followed by a recall task. Results showed a developmental shift: younger participants showed a reverse production effect, recalling more words that were heard during training, while older children showed the typical production effect, recalling more produced words. The effect of production on recall is not unidirectional and varies by age.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

Arabidopsis CER1-LIKE1 Functions in a Cuticular Very-Long-Chain Alkane-Forming Complex.

Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A's to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Lipids in membrane dynamics during autophagy in plants.

Autophagy is a critical pathway for plant adaptation to stress. Macroautophagy relies on the biogenesis of a specialized membrane named the phagophore that maturates into a double membrane vesicle. Proteins and lipids act synergistically to promote membrane structure and functions, yet research on autophagy has mostly focused on autophagy-related proteins while knowledge of supporting lipids in the formation of autophagic membranes remains scarce. This review expands on studies in plants with examples from other organisms to present and discuss our current understanding of lipids in membrane dynamics associated with the autophagy pathway in plants.

Atg23 and Atg27 act at the early stages of Atg9 trafficking in S. cerevisiae.

Atg9 is a conserved multipass transmembrane protein with an essential role in autophagy. In Saccharomyces cerevisiae, it travels through the secretory pathway to a unique compartment, the Atg9 peripheral structures. These structures are then targeted to the phagophore assembly site (PAS), where they are proposed to help deliver membrane to the forming autophagosome. We used 'in vivo reconstitution' of this process in a multiple-knockout strain to define four proteins, Atg11, Atg19, Atg23 and Atg27, as the core minimal machinery necessary and sufficient for the trafficking of Atg9 to the PAS. Atg23 and Atg27 function in the formation of the Atg9 peripheral structures. Overexpression of Atg9 can bypass the need for Atg23, suggesting that the amount of Atg9 in each peripheral structure is a critical factor in their targeting to the PAS. In contrast, overexpression of Atg23 or Atg27 interferes with Atg9 trafficking, suggesting that these proteins must be present in the appropriate stoichiometry in order to function properly. These data allow us to resolve existing controversies regarding the role of Atg23 and Atg27, and propose a model that ties together previous observations regarding the role of Atg9 in autophagosome formation.

Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex.

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process.

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual T-DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

Limits to sustained energy intake. XVI. Body temperature and physical activity of female mice during pregnancy.

Lactation is the most energy-demanding phase of mammalian reproduction, and lactation performance may be affected by events during pregnancy. For example, food intake may be limited in late pregnancy by competition for space in the abdomen between the alimentary tract and fetuses. Hence, females may need to compensate their energy budgets during pregnancy by reducing activity and lowering body temperature. We explored the relationships between energy intake, body mass, body temperature and physical activity throughout pregnancy in the MF1 mouse. Food intake and body mass of 26 females were recorded daily throughout pregnancy. Body temperature and physical activity were monitored every minute for 23 h a day by implanted transmitters. Body temperature and physical activity declined as pregnancy advanced, while energy intake and body mass increased. Compared with a pre-mating baseline period, mice increased energy intake by 56% in late pregnancy. Although body temperature declined as pregnancy progressed, this served mostly to reverse an increase between baseline and early pregnancy. Reduced physical activity may compensate the energy budget of pregnant mice but body temperature changes do not. Over the last 3 days of pregnancy, food intake declined. Individual variation in energy intake in the last phase of pregnancy was positively related to litter size at birth. As there was no association between the increase in body mass and the decline in intake, we suggest the decline was not caused by competition for abdominal space. These data suggest overall reproductive performance is probably not constrained by events during pregnancy.

Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach.

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana , which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues. Graphical abstract.

Phosphatidylinositol-4-phosphate joins the dance of plant autophagosome formation.

In plants, macroautophagy/autophagy is a key mechanism that contributes to their ability to cope with a wide range of environmental constraints such as drought, nutrient starvation or pathogen resistance. Nevertheless, the molecular mechanisms of plant autophagy, and notably that of autophagosome formation, remain poorly understood. As the starting point of our recent paper, we considered the potential functional contribution of lipids in the numerous membrane-remodeling steps involved in this process. By combining biochemistry, genetics, cell biology and high-resolution 3D imaging, we unraveled the function of the lipid phosphatidylinositol-4-phosphate (PtdIns4P) in autophagy in Arabidopsis thaliana, thus providing novel insights into the assembly of autophagosomes in plant cells.

Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses.

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.

Phosphatidylinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana.

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion.

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.

How Lipids Contribute to Autophagosome Biogenesis, a Critical Process in Plant Responses to Stresses.

Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer.

Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.

The impact of water deficiency on leaf cuticle lipids of Arabidopsis.

Arabidopsis (Arabidopsis thaliana) plants subjected to water deficit, sodium chloride (NaCl), or abscisic acid treatments were shown to exhibit a significant increase in the amount of leaf cuticular lipids. These stress treatments led to increases in cuticular wax amount per unit area of 32% to 80%, due primarily to 29% to 98% increases in wax alkanes. Of these treatments, only water deficit increased the total cutin monomer amount (by 65%), whereas both water deficit and NaCl altered the proportional amounts of cutin monomers. Abscisic acid had little effect on cutin composition. Water deficit, but not NaCl, increased leaf cuticle thickness (by 49%). Electron micrographs revealed that both water-deprived and NaCl-treated plants had elevated osmium accumulation in their cuticles. The abundance of cuticle-associated gene transcripts in leaves was altered by all treatments, including those performed in both pot-grown and in vitro conditions. Notably, the abundance of the ECERIFERUM1 gene transcript, predicted to function in alkane synthesis, was highly induced by all treatments, results consistent with the elevated alkane amounts observed in all treatments. Further, this induction of cuticle lipids was associated with reduced cuticle permeability and may be important for plant acclimation to subsequent water-limited conditions. Taken together, these results show that Arabidopsis provides an excellent model system to study the role of the cuticle in plant response to drought and related stresses, and its associated genetic and cellular regulation.

The Effects of Confinement on Neuropsychiatric Symptoms in Alzheimer's Disease During the COVID-19 Crisis.

BACKGROUND: Neuropsychiatric symptoms, such as depression, anxiety, apathy, agitation, and hallucinations, are frequent in Alzheimer's disease (AD) and their prevalence tends to increase with external stressors. OBJECTIVE: We offer the first investigation of the effects of confinement during the COVID-19 crisis on neuropsychiatric symptoms in patients with AD. METHODS: We contacted caregivers of 38 patients with AD who were confined to their homes for nearly two months and asked them to report whether patients experienced any change in neuropsychiatric symptoms during, compared to before, the confinement and rate its severity and impact on themselves using the Neuropsychiatric Inventory-Questionnaire. RESULTS: Among the 38 patients, only 10 demonstrated neuropsychiatric changes during the confinement. Cognitive function of these 10 patients, assessed with the Mini-Mental State Examination, was worse than that of patients who did not demonstrate neuropsychiatric changes. Interestingly, among the 10 patients with neuropsychiatric changes, the duration of confinement significantly correlated with the severity of symptoms as well as with their caregivers' distress. DISCUSSION: The confinement seems to impact neuropsychiatric symptomatology in AD patients with low baseline cognitive function.