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Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman–Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6–60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

In this article we describe the conceptual interactions between climate change adaptation and equity by analysing flooding adaptation strategies commonly used in the United States. We articulate the importance of an equity lens to support a more nuanced understanding of adaptation and inform adaptation strategy design that prioritizes equity as an outcome, rather than an externality. We include an illustration of what adaptation ‘looks’ like when applying an equity lens. We underscore the importance of such a lens for creating effective and just climate adaptation.

This study examined sleep and its cognitive and affective correlates in adults with and without autism spectrum disorder (ASD), utilizing UK Biobank data. There were no group differences in subjective sleep duration [n = 220 ASD; n = 2200 general population (GP)]. Accelerometer measures of sleep duration or nighttime activity did not differ by group, but sleep efficiency was marginally lower in ASD (n = 83 ASD; n = 824 GP). Sleep efficiency was associated with wellbeing and mental health, and pathways between accelerometer sleep measures and wellbeing and mental health were significantly stronger for adults with ASD (who also reported substantially poorer wellbeing and > 5 × likelihood of experiencing mental distress). These findings highlight the need to monitor sleep to maintain good mental health in adult ASD.

Biodiversity loss is one of the greatest global challenges and requires substantial investment in building the capacity of conservation professionals to design and implement robust conservation plans. In this study, we surveyed 155 past participants of training in facilitating species conservation planning processes given by the Conservation Planning Specialist Group of the IUCN Species Survival Commission. Based on a recently developed theory of change for the training, we examined how and to what extent the training contributed to the desired outcome of increasing trainees’ capacity for leading the design and facilitation of species conservation planning processes. Our results indicate that recall of training content, self-efficacy (an individual's belief they can complete a specific task or behaviour successfully) and peer network participation had significant impacts on the outcome of applying training content in the workplace. Furthermore, our results suggest that self-efficacy played a highly influential role in trainees' participation in species conservation planning post-training. The implications of this research point to designing conservation training that considers not only the skills and knowledge to be gained by learners but also the strategies that enhance trainees' self-efficacy in applying new skills and knowledge and in establishing peer networks to support trainees in turning training objectives into realities.

Dysregulated protein synthesis is seen in many aggressive cancers, including metastatic breast cancer. However, the specific contributions of certain translation initiation factors to in vivo disease remain undefined. This is particularly true of eIF4B, an RNA-binding protein and cofactor of the RNA helicase eIF4A and associated eIF4F cap-binding complex. While eIF4A, eIF4G, and eIF4E are well-known to contribute to the progression of many cancer types including metastatic breast cancers, the role played by eIF4B in breast cancer remains relatively unclear. We therefore explored how naturally divergent and experimentally modulated eIF4B levels impact tumor growth and progression in well-characterized murine triple negative breast cancer (TNBC) models. Surprisingly, we found that higher eIF4B levels in mouse and human breast cancers were associated with less aggressive phenotypes. shRNA-mediated eIF4B knockdown in TNBC lines failed to markedly alter proliferation and global translation in the cells in vitro and only modestly hindered their growth as primary mammary tumors growth in mice. However, eIF4B knockdown significantly enhanced invasive growth in vitro and exacerbated both tumor burden and mortality relative to nontargeting shRNA controls in a model of metastatic disease. Analysis of eIF4B levels and breast cancer patient survival reinforced a link to better outcomes. Interestingly, low eIF4B expression was also associated with more formidable immune evasion in vitro and in vivo, implicating a novel immunomodulatory role for this factor in the malignant setting that suggests a mode of action beyond its historical role as a co-activator of eIF4A/F.Significance StatementMetastasis is the leading cause of cancer-related mortality. Despite many advances in our understanding of this complex process and the molecular and cellular events involved, mechanisms that allow secondary tumors to arise and persist remain incompletely understood. Uncharacterized metastatic determinants active at the level of translational control may be exploitable as novel therapy targets or biomarkers predicting a tumor’s potential for spread and recurrence. Here we describe previously unrecognized consequences of dysregulated eIF4B levels in murine breast cancer that shed light on how this translation initiation factor contributes to disease outcomes. Our findings suggest that eIF4B levels direct metastatic risk and immune evasion, and further study should establish its value in personalized treatment decisions and development of future therapies.

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

The inclusion of stakeholders and knowledge systems is increasingly valued in research to address complex socio-ecological challenges around the world. Often these projects take place in cross-cultural setting where external researchers risk perpetuating historically extractive research models that not only harm local communities but damage the validity of research projects. Responsible community engagement is increasingly recognized as a practice that can improve researcher-community relationships and research quality by incorporating principles of ethics, reciprocity, and power sharing. In partnership with local community leaders, researchers from a U.S. university coded 76 research articles for indicators of responsible community engagement conducted in the Samburu, Kenya community since 2000. Findings from this study suggest that most of the research in Samburu has followed extractive models. Of the 76 articles reviewed many failed to acknowledge ethical protocols, did not address relevance of the study to the local community, and did not report any indication of outreach or community change as a result of the research. While a portion of articles showed evidence of community involvement in the studies, the involvement was primarily limited to secondary roles with little to no shared decision-making power over the research process. We discuss methodological considerations for future research and steps that must be taken in order to shift the norm and practice of academic research in sites such as Samburu, Kenya.

Frameworks for understanding well-being play an important role in designing and evaluating climate change adaptation intervention and policy. To be effective, frameworks must capture the complexiti...

During development and regeneration, growth cones at the tips of extending axons navigate through a complex environment to establish accurate connections with appropriate targets. Growth cones can respond rapidly to classical and non-classical guidance cues in their environment, often requiring local protein synthesis. In vertebrate growth cones, local protein synthesis in response to classical cues can require regulation by microRNAs (miRNAs), a class of small, conserved, non-coding RNAs that post-transcriptionally regulate gene expression. However, less is known of how miRNAs mediate growth cone responses to non-classical cues (such as retinoic acid (RA)), specifically in invertebrates. Here, we utilized adult regenerating invertebrate motorneurons to study miRNA regulation of growth cone attraction to RA, shown to require local protein synthesis. In situ hybridization revealed the presence of miR-124 in growth cones of regenerating ciliary motorneurons of the mollusc Lymnaea stagnalis. Changes in the spatiotemporal distribution of miR-124 occurred following application of RA, and dysregulation of miR-124 (with mimic injection), disrupted RA-induced growth cone turning in a time-dependent manner. This behavioural regulation by miR-124 was altered when the neurite was transected, and the growth cone completely separated from the soma. miR-124 did not, however, appear to be involved in growth cone attraction to serotonin, a response independent of local protein synthesis. Finally, we provide evidence that a downstream effector of RhoGTPases, ROCK, is a potential target of miR-124 during RA-induced growth cone responses. These data advance our current understanding of how microRNAs might mediate cue- and context-dependent behaviours during axon guidance.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo lineage tracing allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3’UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.Summary StatementAfter spinal cord injury, miR-200 fine-tunes expression levels brachyury and β-catenin to direct spinal cord stem into cells of the mesodermal or ectodermal lineage.

Comparing risks against benefits of adolescent idiopathic scoliosis (AIS) patients participating in sports represents a controversial topic in the literature. Previous studies have reported sports participation as a possible risk factor for AIS development, while others describe its functional benefits for AIS athletes. The objective of this study was to determine if sports participation had an impact on pain, function, mental status, and self-perception of deformity in patients and their parents. Patients had full spine radiographs and completed baseline surveys of demographics, socioeconomics, and patient-reported outcomes (PRO): Scoliosis Research Society (SRS)-30, Body Image Disturbance Questionnaire, and Spinal Appearance Questionnaire (SAQ: Children and Parent). Patients were grouped by their participation (sports) or nonparticipation (no-sports) in noncontact sports. Demographics, radiographic parameters, and PRO were compared using parametric/nonparametric tests with means/medians reported. Linear regression models identified significant predictors of PRO. Forty-nine patients were included (sports: n=29, no-sports: n=20). Both groups had comparable age, sex, BMI, bracing status, and history of physical therapy (all P>0.05). Sports and no-sports also had similar coronal deformity (major Cobb: 31.1° vs. 31.5°). Sagittal alignment profiles (pelvic incidence, pelvic incidence minus lumbar lordosis, thoracic kyphosis, and sagittal vertical axis) were similar between groups (all P>0.05). Sports had better SRS-30 (Function, Self-image, and Total) scores, SAQ-Child Expectations, and SAQ-Parent Total Scores (P

Scaphoid fractures in the pediatric population represent ∼3% of all hand and carpal fractures. Cast immobilization has been shown to yield excellent results in the acute phase, however some patients develop nonunions. Currently, there is no consensus regarding the best surgical treatment after development of a pediatric/adolescent scaphoid nonunion.A comprehensive literature review was performed utilizing Medline, Ovid, and Embase databases to compare surgical techniques for adolescent scaphoid nonunions on the basis of union rates, functional outcomes, and operative complications. Our initial search returned 2110 publications. Inclusion criteria consisted of a scaphoid fracture with3 months of no clinical or radiographic improvement after cast immobilization and age less than 18 years. Ultimately, 11 studies met our criteria and were included in the final analysis.A total of 176 surgically treated pediatric/adolescent scaphoid nonunions were identified from the 11 studies, including 157 nonvascularized bone graft procedures and 19 nongrafted rigid fixation procedures. Patients treated with a nongrafted method achieved union with a total random effects model revealing a union rate of 94.6%, whereas the grafted cohort had a union rate of 94.8%. Functional outcomes including range of motion and grip strength were significantly improved in both cohorts. Patients managed operatively with bone graft had 4 complications, in contrast those without bone grafting did not report complications (P=0.9).Surgical treatment of pediatric/adolescent scaphoid fracture nonunions produce excellent union rates and functional outcomes after surgical intervention, using both grafted and nongrafted techniques. Future prospective studies are needed to assess if the outcomes of a specific technique are more favorable, as well as to determine if differences exist based on fracture location.Level III. This study is a meta-analysis of studies containing level of evidence of III or greater.

The spinal cord is one of the most important structures for all vertebrate animals as it connects almost all parts of the body to the brain. Injury to the mammalian spinal cord has devastating consequences, resulting in paralysis with little to no hope of recovery. In contrast, other vertebrate animals have been known for centuries to be capable of functionally regenerating large lesions in the spinal cord. Here, we will review the current knowledge of spinal cord regeneration and recent work in different proregenerative animals that has begun to shed light on the cellular and molecular mechanisms these animals use to direct cells to rebuild a complex, functional spinal cord.

Biodiversity is under threat at a global level, and many of the most biodiverse hotspots are in developing regions of the world. In many of these communities, livelihoods are often dependent on the same natural landscapes that support biodiversity. As a result, achieving global conservation and development goals is a priority in these regions, and therefore they attract the interest of both local and international researchers. However, research by outside, Western-based researchers can present ethical and practical challenges in these areas. Fortunately, community-based participatory research (CBPR), if managed well, can contribute to responsible conservation research in these regions. In this article, we investigate strategies to address ethical issues associated with cross-cultural conservation and development research. Our analysis draws on the experiences of a women’s village in northern Kenya and six Western researchers. Using qualitative methodologies, we identify common themes in ethical conservation and develop research including critical consciousness, relationship-building, reciprocity, and adaptive research processes. We discuss the implications for ethical CBPR and, specifically, the need for both researchers and funders to only conduct such research if they can devote the resources required to do so ethically.

Evidence suggests that new vocabulary undergoes a period of strengthening and integration offline, particularly during sleep. Practical questions remain, however, including whether learning closer to bedtime can optimize consolidation, and whether such an effect varies with vocabulary ability. To examine this, children aged 8–12-years-old ( n 59) were trained on written novel forms (e.g. BANARA) in either the morning (long delay) or the evening (short delay). Immediately after training and the next day, lexical competition (a marker of integration) was assessed via speeded semantic decisions to neighbouring existing words (e.g. BANANA); explicit memory was measured via recognition and recall tasks. There were no main effects indicating performance changes across sleep for any task, counter to studies of spoken word learning. However, a significant interaction was found, such that children with poorer vocabulary showed stronger lexical competition on the day after learning if there was a short delay between learning and sleep. Furthermore, while poorer vocabulary was associated with slower novel word recognition speed before and after sleep for the long delay group, this association was only present before sleep for the short delay group. Thus, weak vocabulary knowledge compromises novel word acquisition, and when there is a longer period of post-learning wake, this disadvantage remains after a consolidation opportunity. However, when sleep occurs soon after learning, consolidation processes can compensate for weaker encoding and permit lexical integration. These data provide preliminary suggestion that children with poorer vocabulary may benefit from learning new words closer to bedtime.

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which,Rab3 interactingmolecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channelsin vitrovia a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing homologous domain architectures as known RIM homologues, but some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoanTrichoplax adhaerens, and molluscs. RNA expression and localization studies inTrichoplaxand the mollusc snailLymnaea stagnalisindicate differential regional/tissue type expression, but overlapping expression in single isolated neurons fromLymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homologue. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOHlike PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

Pastoral women in the semi-arid rangelands of East Africa are significantly burdened by the vulnerability to and responsibility for responding to changing climates. Consequently, understanding how adaptation and coping strategies impact pastoral women's well-being is critical for supporting the climate resilience of communities and the landscapes on which they rely. We used a household survey, guided by a multi-dimensional framework of well-being, to investigate how the use of drought-related coping and adaptation strategies by Samburu households influenced livestock loss and women's well-being in northern Kenya. Coping and adaptation strategies predicted numerous social-cognitive components of well-being, although not livestock loss. We conjecture these results are a product of a gendered division of labor within households and the community. We argue that interventions aimed at supporting drought resilience must consider the gendered implications of climate response strategies, multiple indicators for evaluation, and the influence of community and place.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):592-601, 2018.

Some adult vertebrate species, such as newts, axolotls and zebrafish, have the ability to regenerate their central nervous system (CNS). However, the factors that establish a permissive CNS environment for correct morphological and functional regeneration in these species are not well understood. Recent evidence supports a role for retinoid signaling in the intrinsic ability of neurons, in these regeneration-competent species, to regrow after CNS injury. Previously, we demonstrated that a specific retinoic acid receptor (RAR) subtype, RARβ, mediates the effects of endogenous retinoic acid (RA) on neuronal growth and guidance in the adult newt CNS after injury. Here, we now examine the expression of the retinoid X receptor RXRα (a potential heterodimeric transcriptional regulator with RARβ), in newt tail and spinal cord regeneration. We show that at 21 days post-amputation (dpa), RXRα is expressed at temporally distinct periods and in non-overlapping spatial domains compared to RARβ. Whereas RARβ protein levels increase, RXRα proteins level decrease by 21 dpa. A selective agonist for RXR, SR11237, prevents both this downregulation of RXRα and upregulation of RARβ and inhibits tail and caudal spinal cord regeneration. Moreover, treatment with a selective antagonist for RARβ, LE135, inhibits regeneration with the same morphological consequences as treatment with SR11237. Interestingly, LE135 treatment also inhibits the normal downregulation of RXRα in tail and spinal cord tissues at 21 dpa. These results reveal a previously unidentified, indirect regulatory feedback loop between these two receptor subtypes in regulating the regeneration of tail and spinal cord tissues in this regeneration-competent newt.

Shared storybook reading is a key aid to vocabulary acquisition during childhood. However, word learning research has tended to use unnaturalistic (explicit) training regimes. Using a storybook paradigm, we examined whether children (particularly those with weaker vocabularies) are more likely to retain new words if they learn them closer to sleep. Parents read their children (5- to 7-year-olds; N = 237) an alien adventure story that contained 12 novel words with illustrations at one of two training times: at bedtime or 3–5 h before bedtime. Using online tasks, parents tested their children’s ability to recall the new words (production) and associate them with pictures (comprehension) immediately after hearing the story and again the following morning. As hypothesized, we replicated two findings. First, children showed overnight improvements in their ability to produce and comprehend new words when tested again the next day. Second, children with better existing vocabulary knowledge showed larger overnight gains in new word comprehension. Counter to expectations, overnight gains in comprehension were larger if the story was read 3–5 h before bedtime rather than at bedtime. These ecologically valid findings are consistent with theories that characterize word learning as a prolonged process supported by mechanisms such as consolidation and retrieval practice, with existing vocabulary knowledge acting as an important source of variability in retention. The findings provide preliminary evidence that encountering new words in stories later in the day (but not too close to sleep) may help to harness vocabulary growth and may be more beneficial than leaving shared storybook reading just for bedtime.

For centuries, communities around the globe have worked together to manage resources—a process often referred to as community-based conservation. More recently in the US, diverse stakeholders have partnered to address complex environmental issues that span land ownership or administrative boundaries, calling these efforts collaborative conservation. Reviews of community-based and collaborative conservation have historically focused on developing countries. To synthesize published information on U.S.-focused groups, we conducted a literature review with the following objectives: 1) characterize geographic distribution, types of participants, and sources of funding for these groups; 2) assess issues motivating group formation, goals, activities to achieve those goals, and outcomes; 3) identify whether these goals, activities, and outcomes were biophysical, social, and/or economic. To accomplish these objectives, we searched for relevant peer-reviewed journal articles, book chapters, and reports in online databases from 1800's-2017. Our review resulted in 245 documents describing 296 collaborative conservation groups across all 50 states (primarily the Western U.S.) and the District of Columbia. The top three issues motivating collaborative group formation included impacts of land and resource degradation on livelihoods, water quality and management, and managing land and resources across ownership boundaries. We found that there was little published information on the outcomes of group activities (16% of groups). For the limited number of groups for which the literature reported outcomes, there was no relationship between positive outcomes and group characteristics. Our findings suggest that renewed efforts to evaluate the impact of collaboration on conservation are warranted and could support learning and improved action.

Purpose Establishing stable and flexible phonological representations is a key component of language development and one which is thought to vary across children with neurodevelopmental disorders affecting language acquisition. Sleep is understood to support the learning and generalization of new phonological mappings in adults, but this remains to be examined in children. This study therefore explored the time course of phonological learning in childhood and how it varies by structural language and autism symptomatology. Method Seventy-seven 7- to 13-year-old children, 30 with high autism symptomatology, were included in the study; structural language ability varied across the sample. Children learned new phonological mappings based on synthesized speech tokens in the morning; performance was then charted via repetition (without feedback) over 24 hr and followed up 4 weeks later. On the night following learning, children's sleep was monitored with polysomnography. Results A period of sleep but not wake was associated with improvement on the phonological learning task in childhood. Sleep was associated with improved performance for both trained items and novel items. Structural language ability predicted overall task performance, though language ability did not predict degree of change from one session to the next. By contrast, autism symptomatology did not explain task performance. With respect to sleep architecture, rapid eye movement features were associated with greater phonological generalization. Conclusions Children's sleep was associated with improvement in performance on both trained and novel items. Phonological generalization was associated with brain activity during rapid eye movement sleep. This study furthers our understanding of individual differences in the acquisition of new phonological mappings and the role of sleep in this process over childhood. Supplemental Material https://doi.org/10.23641/asha.11126732

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA binding. Recent evidence suggests mRNAs have variable dependence on eIF4B, suggesting this factor could promote changes in mRNA selection for translation, in order to adapt to stressors. However, the importance of eIF4B and its constituent domains for mRNA selection under diverse cellular and environmental conditions remain undefined. Here we compared the effects of disrupting eIF4B RNA- and ribosome-binding motifs under ~1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to various stressors. In particular, the NTD conferred a strong growth advantage in the presence of urea. Ribosome profiling revealed that the NTD promoted translation of mRNAs with long and highly structured 5-prime untranslated regions, both with and without urea exposure. Our results suggest eIF4B controls mRNA loading and scanning as a part of the PIC, rather than by activating mRNPs prior to ribosome binding. Furthermore, our data indicate the yeast response to urea includes a translational component, driven by production of proteins associated with the cellular periphery. Together our analyses suggest general eIFs can promote diverse cellular responses.

Sleep is known to support the neocortical consolidation of declarative memory, including the acquisition of new language. Autism spectrum disorder (ASD) is often characterized by both sleep and language learning difficulties, but few studies have explored a potential connection between the two. Here, 54 children with and without ASD (matched on age, nonverbal ability and vocabulary) were taught nine rare animal names (e.g., pipa). Memory was assessed via definitions, naming and speeded semantic decision tasks immediately after learning (pre‐sleep), the next day (post‐sleep, with a night of polysomnography between pre‐ and post‐sleep tests) and roughly 1 month later (follow‐up). Both groups showed comparable performance at pre‐test and similar levels of overnight change on all tasks; but at follow‐up children with ASD showed significantly greater forgetting of the unique features of the new animals (e.g., pipa is a flat frog). Children with ASD had significantly lower central non‐rapid eye movement (NREM) sigma power. Associations between spindle properties and overnight changes in speeded semantic decisions differed by group. For the TD group, spindle duration predicted overnight changes in responses to novel animals but not familiar animals, reinforcing a role for sleep in the stabilization of new semantic knowledge. For the ASD group, sigma power and spindle duration were associated with improvements in responses to novel and particularly familiar animals, perhaps reflecting more general sleep‐associated improvements in task performance. Plausibly, microstructural sleep atypicalities in children with ASD and differences in how information is prioritized for consolidation may lead to cumulative consolidation difficulties, compromising the quality of newly formed semantic representations in long‐term memory., This polysomnography study examined the sleep‐associated consolidation of words for real but rare animals, in children with and without autism. Children with autism showed increased forgetting of novel features (e.g., an asp is a hairy caterpillar), relative to typical peers, one month after exposure. They also showed reduced sigma power (within the sleep spindle frequency range), and showed more general associations between overnight changes in task performance (for both novel and particularly familiar words) and sleep spindle parameters, in stark contrast to typical peers who showed selective associations between spindles and overnight changes in novel memory traces.

Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5' UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of the effects of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence- and structure-changes. In addition to providing streamlined and detailed protocols, we describe a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G.

Pair living and pair bonding are rare in mammals, and the mechanisms of their maintenance remain a puzzle. Titi monkeys, a ‘textbook example’ for ‘monogamous’ primates, have strong pair bonds and extensive male care. To investigate mechanisms of pair-bond maintenance, we studied seven wild groups of red titis ( Plecturocebus cupreus ) in Peruvian Amazonia over a period of 14 months. We analysed pair bonds by measuring proximity, grooming and approaches/leaves within pairs, and collected data on intergroup encounters. Females contributed to grooming more than males, especially during infant dependency, when most of the grooming within pairs was done by females. Females were also more active in controlling proximity between pair mates, making most of the approaches and leaves. Males, on the other hand, invested more in territorial defences. They participated in more intergroup encounters than females and were more active during these encounters. Our data is most consistent with the ‘male-services’ hypothesis for pair-bond maintenance, where a female contributes more to the proximity and affiliation maintenance while a male provides beneficial services.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):592-601, 2018.

Pediatric intrasubstance anterior cruciate ligament (ACL) tears have a significant epidemiologic impact as their numbers continue to grow globally. This review focuses on true pediatric intrasubstance ACL tears, which occur >400,000 times annually. Modifiable and non-modifiable risk factors include intercondylar notch width, ACL size, gender, landing mechanisms, and hormonal variations. The proposed mechanisms of injury include anterior tibial shear and dynamic valgus collapse. ACL tears can be associated with soft tissue and chondral defects. History and physical examination are the most important parts of evaluation, including the Lachman test, which is considered the most accurate physical examination maneuver. Imaging studies should begin with AP and lateral radiographs, but magnetic resonance imaging is very useful in confirming the diagnosis and preoperative planning. ACL injury prevention programs targeting high risk populations have been proven to reduce the risk of injury, but lack uniformity across programs. Pediatric ACL injuries were conventionally treated nonoperatively, but recent data suggest that early operative intervention produces best long term outcomes pertaining to knee stability, meniscal tear risk, and return to previous level of play. Current techniques in ACL reconstruction, including more vertically oriented tunnels and physeal sparing techniques, have been described to reduce the risk of physeal arrest and limb angulation or deformity. Data consistently show that autograft is superior to allograft regarding failure rate. Mean durations of postoperative therapy and return to sport were 7 ± 3 and 10 ± 3 months, respectively. These patients have good functional outcomes compared to the general population yet are at increased risk of additional ACL injury. Attempts at primary ACL repair using biological scaffolds are under investigation.

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By miRNA sequencing analysis, we identify 482 miRNAs in the regenerating central nervous system (CNS) of the mollusc Lymnaea stagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up- or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate, for the first time, that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (pedal A), whereas expression in the growth cones of another class of respiratory motorneurons (right parietal A) was absent in vitro. These findings support our hypothesis that miRNAs are important regulators of retinoic acid-induced neuronal outgrowth and regeneration in regeneration-competent species.

Retinoic acid (RA) is the biologically active metabolite of vitamin A,and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By R-Seq analysis, we identify 482 miRNAs in the regenerating CNS of the mollusc Lymnaea stagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate for the first time that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (Pedal A), whereas expression from the growth cones of another class of respiratory motorneurons (RPA) was absent in vitro. These findings support our hypothesis miRNAs are important regulators of retinoic acid induced neuronal outgrowth and regeneration in regeneration-competent species.

Objective This study evaluated incidence over time, any association between race and demographics, and hospital-related parameters in pediatric patients with septic hip or knee arthritis. Methods The Kids’ Inpatient Database was used to identify all children with a diagnosis of septic hip or knee arthritis who underwent incision and drainage (1997–2012). Results Between 1997 and 2012, overall incidence of septic arthritis of the knee (0.20–0.33 per 100,000) and hip (0.12–0.18 per 100,000) increased. Conclusion Incidence of pediatric septic joint arthritis, an emergent orthopaedic condition, has increased over time. Patient demographics may vary with respect to both age and race.

Molecules that undergo activation or modulation following the addition of benign external small-molecule chemical stimuli have numerous applications. Here, we report the highly efficient "decaging" of a variety of moieties by activation of a "self-immolative" linker, by application of water-soluble and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.

A direct Pd-catalyzed C=H functionalization of benzoquinone (BQ) can be controlled to give either mono- or disubstituted BQ, including the installation of two different groups in a one-pot procedure. BQ can now be directly functionalized with aryl, heteroaryl, cycloalkyl, and cycloalkene groups and, moreover, the reaction is conducted in environmentally benign water or acetone as solvents.

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

Objectives: Female youth ice hockey players are an overlooked population. No national study has established incidence rates for injuries in female youth ice hockey. The objective of this study was to establish incidence rates by injury location, diagnosis, and mechanism of injury using USA Hockey sanctioned age divisions. Methods: The National Electronic Injury Surveillance System (NEISS) was queried for all ice hockey injuries (product code 1279) from January 1, 2007 to December 31, 2016. Cases involving players over the age of 19 and males were excluded. Each injury’s narrative text field was reviewed to determine mechanism of injury. Data was analyzed using (IBM®, v24). Comparisons of incidence by age were made using student’s two sample t-test with 95% confidence interval. Trend analyses were performed using a linear regression. USA Hockey membership statistics were used to establish population at risk and calculate incidence rates. All incidence rates were reported per 10,000 person-years. Results: A total of 384 patients, representing an estimated 10,398 ice hockey-related injuries, presented to NEISS-participating United States emergency departments. During the study period, female youth ice hockey players increased significantly from 44,678 in 2007 to 57,792 in 2016 (p=3.9x10-5, R-squared=0.89, ß =0.94). The overall number of injuries, however, only slightly increased from 992 in 2007 to 1,042 in 2016 (p=ns). Thus, the incidence rate (IR) of injuries fell from 222.1 to 180.2 during the study period. The most commonly injured body parts were the head (n=3048, IR=554.5), trunk (n=1399, IR=256.4), knee (n=1127, IR=169), shoulder (n=704, IR=153.3) and ankle (n=591, IR=120.2). The most common diagnoses were strain/sprain (n=2002, IR=417.7), contusion (n=1877, IR= 348), internal organ injury (n=1863, IR=320), concussion (n=1112, IR=218) and fracture (n=1255, IR=202). The top mechanisms of injury were player-to-player contact (n= 3016, IR=535), falls (n=2249, IR=380.9), and contact with boards (n=942, IR=165.8). The incidence rate of injuries increased with age; The 0-8, 9-10, 11-12, 13-14, 15-16 and 17-19 age divisions had IR’s of 24, 84, 226, 381, 360, and 750, respectively. The player-to-player mechanism of injury also increased with age. The largest IR gap between ages fell between the 11-12 and 13-14 age groups, similar to what has been observed in male ice hockey studies. Player-to-player contact is the leading mechanism of injury in all age groups except the 0-8 age group. Head injuries increased with age division: 0-8 (n=15, IR=1), 9-10 (n=153, IR=17.4), 11-12 (n=598, IR=67.2), 13-14 (n=885, IR=115.1), 15-16 (n=650, IR=121.6) and 17-19 (n=746, IR=232.2). The two most common diagnoses of head injuries were concussion (36%) and internal organ injury (61%), both of these diagnoses increasing with age. In fact, concussion diagnosis increased significantly between each age group (pConclusion: We established the first collection of injury incidence rates for female ice hockey gleaned from a national database. Though body checking is illegal at all levels of women’s ice hockey, player-to-player contact prevailed as the leading mechanism of injury in all but the 0-8 age division. It was also the leading mechanism for the most common injury sites, including the head. This study laid bare an unspoken but long understood fact of the girl’s game that body checking is common and major contributor to the game’s injury burden.

IntroductionPediatric Intensive Care Unit (PICU) resources are overwhelmed in disaster as the need to accommodate influx of critically-ill children is increased. A full-scale chlorine overexposure exercise was conducted by the New York Institute for All Hazard Preparedness (NYIAHP) to assess the appropriateness of response of Kings County Hospital Center's (KCHC's) PICU surge plan to an influx of critically-ill children. The primary endpoint that was assessed was the ability of the institution to follow the PICU surge plan, while secondary endpoints include the ability to provide appropriate medical management.MethodsThirty-six actors/patients (medical students or emergency medicine residents) were educated on presentations and appropriate medical management of patients after a chlorine overexposure, as well as lectures on drill design and expected PICU surge response. Victims presented to the hospital after simulated accidental chlorine overexposure at a public pool. Twenty-two patients with 14 family members needed evaluation; nine of these patients would require PICU admission. Three of nine PICU patients were low-fidelity mannequins. In addition to the 36 actor/patient evaluators, each area had two to four expert evaluators (disaster preparedness experts) to assess appropriateness of global response. Patients were expected to receive standard of care. Appropriateness of medical decisions and treatment was assessed retrospectively with review of electronic medical record.ResultsThe initial PICU census was three of seven; two of these patients were transferred to the general ward. Of the nine patients that required Intensive Care Unit (ICU) admission, six actor/patients were admitted to the PICU, one was admitted to the Surgical Intensive Care Unit (SICU), one went to the Operating Room (OR), and one was admitted to a monitored-surge general pediatric bed. The remaining 13 actor/patients were treated and released. Medical, nursing, and respiratory staffing in the PICU and the general ward were increased by two main mechanisms (extension of work hours and in-house recruitment of additional staff). Emergency Department (ED) staffing was artificially increased prior to the drill. With the exception of ocular fluid pH testing in patients with ocular pruritus, all necessary treatments were given; however, an unneeded albuterol treatment was administered to one patient. Chart review showed adequate discharge instructions in four of 13 patients. Nine patients without respiratory complaints in the ED were not instructed to observe for dyspnea. All patients were in the PICU or alternate locations within 90 minutes.DiscussionThe staff was well versed in the major details of KCHC's PICU surge plan, which allowed smooth transition of patient care from the ED to the PICU. The plan provided for a roadmap to achieve adequate medical, nursing, and respiratory therapists. Medical therapy was appropriate in the PICU; however, in the ED, patients with ocular complaints did not receive optimal care. In addition, written discharge instruction and educational material regarding chlorine overexposure to all patients were not consistently provided. The PICU surge plan was immediately accessible through the KCHC intranet; however, not all participants were cognizant of this fact; this decreased the efficiency with which the roadmap was followed. An exaggerated ED staff facilitated evaluation and transfer of patients.ConclusionDuring disasters, the ability to surge is paramount and each hospital addresses it differently. Hospitals and departments have written surge plans, but there is no literature available which assesses the validity of said plans through a rigorous, structured, simulated disaster drill. This study is the first to assess validity and effectiveness of a hospital's PICU surge plan. Overall, the KCHC PICU surge plan was effective; however, several deficiencies (mainly in communication and patient education in the ED) were identified, and this will improve future response.ShahVS, PierceLC, RoblinP, WalkerS, SergioMN, ArquillaB. Waterworks, a full-scale chemical exposure exercise: interrogating pediatric critical care surge capacity in an inner-city tertiary care medical center. Prehosp Disaster Med. 2014;29(1):1-7.

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Retinoid signaling plays an important role in hippocampal-dependent vertebrate memories. However, we have previously demonstrated that retinoids are also involved in the formation of long-term implicit memory following operant conditioning of the invertebrate mollusc Lymnaea stagnalis. Furthermore, we have discovered an interaction between environmental light/dark conditions and retinoid signaling and the ability of both to convert intermediate-term memory into long-term memory. In this study, we extend these findings to show that retinoid receptor agonists and environmental darkness can both also extend the duration of long-term memory. Interestingly, exposure to constant environmental darkness significantly increased the expression of retinoid receptors in the adult central nervous system, as well as induced specific changes in a key neuron mediating the conditioned behaviour. These studies not only shed more light on how retinoids influence memory formation, but also further link environmental light conditions to the retinoid signaling pathway.