Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Do Viruses From Managed Honey Bees (Hymenoptera: Apidae) Endanger Wild Bees in Native Prairies?

Populations of wild and managed pollinators are declining in North America, and causes include increases in disease pressure and decreases in flowering resources. Tallgrass prairies can provide floral resources for managed honey bees (Hymenoptera: Apidae, Apis mellifera Linnaeus) and wild bees. Honey bees kept near prairies may compete with wild bees for floral resources, and potentially transfer viral pathogens to wild bees. Measurements of these potential interactions are lacking, especially in the context of native habitat conservation. To address this, we assessed abundance and richness of wild bees in prairies with and without honey bee hives present, and the potential spillover of several honey bee viruses to bumble bees (Hymenoptera: Apidae, Bombus Latrielle). We found no indication that the presence of honey bee hives over 2 yr had a negative effect on population size of wild bee taxa, though a potential longer-term effect remains unknown. All levels of viruses quantified in bumble bees were lower than those observed in honey bees. Higher levels of deformed wing virus and Israeli acute paralysis virus were found in Bombus griseocollis DeGeer (Hymenoptera: Apidae) collected at sites with hives than those without hives. These data suggest that the presence of honey bees in tallgrass prairie could increase wild bee exposure to viruses. Additional studies on cross-species transmission of viruses are needed to inform decisions regarding the cohabitation of managed bees within habitat utilized by wild bees.

Systematic Review of Recent Pediatric Down Syndrome Neuropsychology Literature: Considerations for Regression Assessment and Monitoring.

OBJECTIVES: To conduct a systematic review of recent pediatric Down syndrome (DS) neuropsychology research that may be useful to clinicians and researchers examining regression in this population. METHODS: We reviewed original peer-reviewed articles published between 2013 and 2018 studying neuropsychological profiles in DS. RESULTS: Thirty-one articles (of 1231 included in the original search) passed all inclusion criteria, were evaluated for bias, and were included in the analysis. CONCLUSION: Findings argued against a single "DS profile" and revealed multiple within-group differences as well as expected and unexpected differences relative to typically developing children and children with other intellectual and developmental disabilities. Areas identified as most germane to regression monitoring included working memory, inhibition, letter and word identification, navigational route learning, motor skills (when strong at baseline), single word receptive/expressive vocabulary, and adaptive function.

Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation.

The extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by the E. coli BirA protein, has been fused to proteins-of-interest, making them substrates for in vivo biotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in the Drosophila female germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secreted Drosophila proteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirA in vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretion in vivo, providing access to powerful experimental approaches for secreted proteins-of-interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.

Developmental Regression, Depression, and Psychosocial Stress in an Adolescent with Down Syndrome.

CASE: Kristen is a 13-year-old girl with Down syndrome (DS) who was seen urgently with concerns of cognitive and developmental regression including loss of language, social, and toileting skills. The evaluation in the DS clinic focused on potential medical diagnoses including atlantoaxial joint instability, vitamin deficiency, obstructive sleep apnea (OSA), and seizures. A comprehensive medical evaluation yielded only a finding of moderate OSA. A reactive depression was considered in association with several psychosocial factors including moving homes, entering puberty/onset of menses, and classroom change from an integrated setting to a self- contained classroom comprising unfamiliar peers with behavior challenges.Urgent referrals for psychological and psychiatric evaluations were initiated. Neuropsychological testing did not suggest true regression in cognitive, language, and academic skills, although decreases in motivation and performance were noted with a reaction to stress and multiple environmental changes as a potential causative factor. Psychiatry consultation supported this finding in that psychosocial stress temporally correlated with Kristen's regression in skills.Working collaboratively, the team determined that Kristen's presentation was consistent with a reactive form of depression (DSM-IV-TR: depressive disorder, not otherwise specified). Kristen's presentation was exacerbated by salient environmental stress and sleep apnea, rather than a cognitive regression associated with a medical cause. Treatment consisted of an antidepressant medication, continuous positive airway pressure for OSA, and increased psychosocial supports. Her school initiated a change in classroom placement. With this multimodal approach to evaluation and intervention, Kristen steadily improved and she returned to her baseline function.

Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torso-like in receptor activation.

Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase Torso. Both Torso and Torso's presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torso-like, which is expressed by follicle cells adjacent to the ends of the developing oocyte. We find that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torso-like or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torso-like were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torso-like were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torso-like alone were ineffective but acted synergistically to stimulate Torso signaling. Our results suggest that Torso interacts with both Trunk and Torso-like, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo.

Autism associated with early institutionalization, high intelligence, and naturalistic behavior therapy in a 7-year-old boy.

CASE: Paul is a 7-year-old boy with a history of cerebral palsy and left-side weakness secondary to perinatal injury. He was adopted to the United States at 19 months from a baby home in Eastern Europe, where the caregiver to child ratio was 7:1. Paul spent most of his early developmental period in a crib. On adoption, he was nonverbal and nonambulatory, but these skills developed within 1 year. Paul was noted at 4 years of age to be struggling socially and also to exhibit restricted interests (e.g., memorizing maps and world leaders). He was referred for neuropsychological testing at age 5 and was found to have cognitive skills in the gifted range (verbal intelligence quotient, IQ =143; 99.8%) but exhibited markedly reduced social reciprocity with high levels of restricted interests and repetitive behaviors, leading to a diagnosis of autism spectrum disorder (ASD) in the context of early institutionalization. Given his cooperative and attentive presentation, high IQ, and ability to imitate, Floortime, a more naturalistic behavioral therapy for ASD, was recommended rather than traditional applied behavior analysis, which is more commonly available in the region. In addition, Paul was provided with group speech and language therapy with a social/pragmatic focus. After 1 year, Paul's socialization improved but he struggled to initiate interactions and maintain friendships. He focused instead on his restricted interests and played alone. After 2 years of intervention, Paul presents as highly sociable with well-sustained eye gaze, interactive play, and successful friendships. Still, without direction and structure, Paul will happily draw maps for hours at a time. He is hyperlexic and working far above grade level across subjects. His mother now questions--is this still truly institutional autism or is he simply too intelligent to relate to same-age peers?

"Complex" attention-deficit hyperactivity disorder, more norm than exception? Diagnoses and comorbidities in a developmental clinic.

OBJECTIVE: Current recommendations for evaluation and diagnosis of attention-deficit hyperactivity disorder (ADHD) are meant for primary care settings and may not adequately address the needs of children seen in subspecialty developmental-behavioral pediatric settings who may have higher rates of comorbid developmental, learning, and psychiatric disorders. The authors sought to characterize the diagnostic complexity of school-aged children diagnosed with ADHD after comprehensive multidisciplinary evaluation in a subspecialty developmental-behavioral pediatric clinic. METHODS: The authors conducted a retrospective medical record review of 144 patients aged 7 to 11 years who were consecutively evaluated by an interdisciplinary team (developmental-behavioral pediatrician, psychologist, educator) in a school-age clinic within a developmental-behavioral pediatrics tertiary care center from January 1, 2009 to December 31, 2009. RESULTS: After comprehensive evaluation, rates of ADHD diagnosis increased from 32.6% (n = 47) preevaluation to 54.2% (n = 78) postevaluation (p < .0001). Rates of learning disorders among children receiving a final diagnosis of ADHD increased from 2.6% (n = 2) preevaluation to 50% (n = 39) postevaluation. (p < .0001). Among children receiving a final diagnosis of ADHD, 73.1% (n = 57) were diagnosed with at least 1 comorbid psychiatric, developmental, or learning disorder. CONCLUSIONS: Among school-aged children diagnosed with ADHD in a developmental-behavioral pediatric subspecialty setting, a comprehensive evaluation including developmental, neuropsychological, and educational assessments yielded high rates of comorbid psychiatric, developmental, and learning disorders. This supports the need to provide comprehensive interdisciplinary assessment for such children to ensure the identification and treatment of not only the core symptoms of ADHD but also the comorbidities that may otherwise go unrecognized and therefore not optimally treated.

Maternal control of the Drosophila dorsal-ventral body axis.

UNLABELLED: The pathway that generates the dorsal-ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.

Teaching adults-best practices that leverage the emerging understanding of the neurobiology of learning.

It is important in teaching adults to recognize the essential characteristics of adult learners and how these characteristics define their learning priorities and activities. The seven key premises and practices for teaching adults provide a good guide for those interested in helping adults learn. The emerging science of the neurobiology of learning provides powerful new insights into how learning occurs in the complex integrated neural network that characterizes the adult. Differentiation of the two types of thinking: System 1 (fast, intuitive, and, often, emotional) and System 2 (slower, deliberate, and logical). System 1 thinking helps explain the basis for quick decisions and reliance of humans on heuristics (or rules of thumb) that leads to the type of convenient thinking associated with errors of thinking and judgment. We now know that the learning experience has an objective location-in the temporal and parietal lobes-as persistent dynamic networks of neurons and neuronal connections. Learning is initially stored in transient working memory (relatively limited capacity and time frame) and then moved under the right conditions to more long-lasting/stable memory (with larger capacity) that is stored for future access and development. It is clear that memories are not static and are not destined, once developed, to forever remain as stable constructs; rather, memories are dynamic, always available for modulation and alteration, and heavily invested with context, emotion, and other operant factors. The framework for such neural networks involves new neuronal connections, enhanced neuronal synaptic transmission, and neuron generation. Ten key teaching and learning concepts derived from recent neurobiology studies on learning and memory are presented. As the neurobiology of learning is better defined, the basis for how adults best learn, and even the preferences they display, can be employed as the physiological foundation for our best methods to effectively teach adults and facilitate their learning.

Developmental regression, depression, and psychosocial stress in an adolescent with Down syndrome.

CASE: Kristen is a 13-year-old girl with Down syndrome (DS) who was seen urgently with concerns of cognitive and developmental regression including loss of language, social, and toileting skills. The evaluation in the DS clinic focused on potential medical diagnoses including atlantoaxial joint instability, vitamin deficiency, obstructive sleep apnea (OSA), and seizures. A comprehensive medical evaluation yielded only a finding of moderate OSA. A reactive depression was considered in association with several psychosocial factors including moving homes, entering puberty/onset of menses, and classroom change from an integrated setting to a self-contained classroom comprising unfamiliar peers with behavior challenges.Urgent referrals for psychological and psychiatric evaluations were initiated. Neuropsychological testing did not suggest true regression in cognitive, language, and academic skills, although decreases in motivation and performance were noted with a reaction to stress and multiple environmental changes as a potential causative factor. Psychiatry consultation supported this finding in that psychosocial stress temporally correlated with Kristen's regression in skills.Working collaboratively, the team determined that Kristen's presentation was consistent with a reactive form of depression (DSM-IV-TR: depressive disorder, not otherwise specified). Kristen's presentation was exacerbated by salient environmental stress and sleep apnea, rather than a cognitive regression associated with a medical cause. Treatment consisted of an antidepressant medication, continuous positive airway pressure for OSA, and increased psychosocial supports. Her school initiated a change in classroom placement. With this multimodal approach to evaluation and intervention, Kristen steadily improved and she returned to her baseline function.

Developmental and behavioral disorders through the life span.

Developmental and behavioral disorders including intellectual disability, learning disabilities, and attention-deficit/hyperactivity disorder are highly prevalent, chronic health conditions. Despite being versed in caring for children with these conditions, pediatricians might be less prepared for challenging questions from families about the long-term course of these conditions and what can be done to improve outcomes. Through this state-of-the-art review, we provide clinicians with an understanding of the course of these conditions and adult outcomes in several areas including vocational, social, and health domains. We also provide a review of the most current research examining factors that predict or mediate adult outcomes for people with intellectual disability, learning disabilities, and attention-deficit/hyperactivity disorder. On the basis of the current literature, we offer practice recommendations aimed at optimizing adult outcomes for those with these disorders.

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila.

Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation.

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster.

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.