Regulation of stem cell identity by miR-200a during spinal cord regeneration.

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 cell tracking 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.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

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.

Context-Dependent Role of miR-124 in Retinoic Acid-Induced Growth Cone Attraction of Regenerating Motorneurons.

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.

Reconstitution and analyses of RNA interactions with eukaryotic translation initiation factors and ribosomal preinitiation complexes.

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.

Operative Management for Pediatric and Adolescent Scaphoid Nonunions: A Meta-analysis.

INTRODUCTION: 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. METHODS: 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 with >3 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. RESULTS: 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). CONCLUSION: 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 OF EVIDENCE: Level III. This study is a meta-analysis of studies containing level of evidence of III or greater.

The 5'-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway.

Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5' cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.

Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System.

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 Lymnaeastagnalis, 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.

Tetrazine-Responsive Self-immolative Linkers.

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.

Active yeast ribosome preparation using monolithic anion exchange chromatography.

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

Identification and characterization of functionally critical, conserved motifs in the internal repeats and N-terminal domain of yeast translation initiation factor 4B (yeIF4B).

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.

Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo.

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP-eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G-mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP-eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed-loop mRNP formation via PABP-eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

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.

Yeast eukaryotic initiation factor 4B (eIF4B) enhances complex assembly between eIF4A and eIF4G in vivo.

Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

Magnetic resonance imaging predictors of treatment response in late-life depression.

In older adults, depression not only results in more years lived with disability than any other disease but it also carries additional risks of suicide, medical comorbidities, and family caregiving burden. Because it can take many months to identify an effective treatment regimen, it is of utmost importance to shorten the window of time and identify early on what medications and dosages will work effectively for individuals having depression. Late-life depression (LLD) has been associated with greater burden of age-related changes (eg, atrophy, white matter ischemic changes, and functional connectivity). Depression in midlife has been shown to alter affective reactivity and regulation, and functional magnetic resonance imaging (fMRI) studies in LLD have replicated the same abnormalities. Effective treatment can normalize these alterations. This article provides a review of the current literature using structural and functional neuroimaging to identify MRI predictors of treatment response in LLD. The majority of the literature on structural MRI has focused on the vascular depression hypothesis, and studies support the view that loss of brain volume and white matter integrity was associated with poorer treatment outcomes. Studies using fMRI have reported that lower task-based activity in the prefrontal cortex and limbic regions was associated with poorer outcome. These imaging markers may be integrated into clinical decision making to attain better treatment outcomes in the future.

Palladium-catalyzed direct C-H functionalization of benzoquinone.

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.

Effect of 100% Orange Juice and a Volume-Matched Sugar-Sweetened Drink on Subjective Appetite, Food Intake, and Glycemic Response in Adults.

Dietary recommendations to reduce the consumption of free sugars often group 100% fruit juice with other sugar-containing beverages. The objective of this study was to determine the effect of consuming 100% orange juice compared to an orange drink on next-meal food intake (FI), glycemic response, average appetite, emotions, and sensory characteristics in normal-weight adults. Thirty-six normal-weight adults (age: 26.8 ± 0.9 years) consumed, in random order and at least 5 days apart, three 240 mL test beverages as follows: (a) 100% orange juice, (b) orange drink, or (c) water. Subjective sweetness and pleasantness were determined immediately after test beverage consumption. Glycemic response, average appetite, and subjective emotions were measured every 15 min for 60 min. Food intake was determined at a pizza lunch 60 min later. Rest-of-day glycemic response and energy intake (EI) were determined using a continuous glucose monitor and food record, respectively. Lunch FI (p = 0.054) and total EI (p = 0.01) were both lower after 100% orange juice compared with the orange drink. Caloric compensation was 84% after 100% orange juice and -25% after the orange drink (p = 0.047). Average appetite was not significantly different between the test beverages (p > 0.05). Blood glucose iAUC adjusted for available carbohydrate was lower after 100% orange juice compared with the orange drink (p < 0.001). Rest-of-day blood glucose concentrations were lower after 100% orange juice compared with the orange drink (p = 0.03) and water control (p < 0.001). In conclusion, consumption of 100% orange juice as a preload resulted in higher caloric compensation, lower total daily EI, and lower blood glucose concentrations compared to the orange drink.

Spinal cord regeneration - the origins of progenitor cells for functional rebuilding.

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.

Disseminated Intravascular Coagulation in Pediatric Scoliosis Surgery: A Systematic Review.

BACKGROUND: Disseminated intravascular coagulation (DIC) is a rare but serious complication of pediatric scoliosis surgery; sparse current evidence warrants more information on causality and prevention. This systematic review sought to identify incidence of DIC in pediatric patients during or shortly after corrective scoliosis surgery and identify any predictive factors for DIC. METHODS: Medline/PubMed, EMBASE, and Ovid databases were systematically reviewed through July 2017 to identify pediatric patients with DIC in the setting of scoliosis surgery. Patient demographics, medical history, surgery performed, clinical course, suspected causes of DIC, and outcomes were collected. RESULTS: Eleven studies met inclusion criteria. Thirteen cases from 1974 to 2012 (mean age: 15.3 ± 4.3 years, 72% women) were identified, with neuromuscular (n = 7; 54%) scoliosis as the most common indication. There were no prior bleeding disorder histories; all preoperative labs were within normal limits. Procedures included 8 posterior segmental fusions (54%), 3 Harrington rods (31%), 1 Cotrel-Dubousset, and 1 unit rod. Eight patients experienced DIC intraoperatively and 5 patients experienced DIC postoperatively. Probable DIC causes included coagulopathy following intraoperatively retrieved blood reinfusion, infection from transfusion, rhabdomyolysis, hemostatic matrix application, heparin use, and hypovolemic shock. Most common complications included increased intraoperative blood loss (n = 8) and hypotension (n = 7). The mortality rate was 7.69%; one fatality occurred in the acute postoperative period. CONCLUSIONS: Prior bleeding disorder status notwithstanding, this review identified preliminary associations between variables during corrective scoliosis surgery and DIC incidence among pediatric patients, suggesting multiple etiologies for DIC in the setting of scoliosis surgery. Further investigation is warranted to quantify associated risk. CLINICAL RELEVANCE: This study brings awareness to a previously rarely discussed complication of pediatric scoliosis surgery. Further cognizance of DIC by scoliosis surgeons may help identify and prevent causes thereof.

Role of hybrid tRNA-binding states in ribosomal translocation.

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steady-state kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k(trans)), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K(1/2)). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K(1/2) conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k(trans) without increasing K(1/2). These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.

Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea.

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA recruitment. Recent evidence indicates yeast mRNAs have variable dependence on eIF4B under optimal growth conditions. Given the ability of eIF4B to promote translation as a function of nutrient conditions in mammalian cells, we wondered if eIF4B activities in translation could alter phenotypes in yeast through differential mRNA selection for translation. Here we compared the effects of disrupting yeast eIF4B RNA- and 40S-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 stressors requiring robust cellular integrity. In particular, the NTD conferred a strong growth advantage in the presence of urea, which may be important for pathogenesis of related fungal species. Ribosome profiling indicated that similar to complete eIF4B deletion, deletion of the NTD dramatically reduced translation, particularly of those mRNAs with long and highly structured 5-prime untranslated regions. This behavior was observed both with and without urea exposure, but the specific mRNA pool associated with ribosomes in response to urea differed. Deletion of the NTD led to relative increases in ribosome association of shorter transcripts with higher dependence on eIF4G, as was noted previously for eIF4B deletion. Gene ontology analysis indicated that proteins encoded by eIF4B NTD-dependent transcripts were associated with the cellular membrane system and the cell wall, while NTD-independent transcripts encoded proteins associated with cytoplasmic proteins and protein synthesis. This analysis highlighted the difference in structure content of mRNAs encoding membrane versus cytoplasmic housekeeping proteins and the variable reliance of specific gene ontology classes on various initiation factors promoting otherwise similar functions. Together our analyses suggest that deletion of the eIF4B NTD prevents cellular stress responses by affecting the capacity to translate a diverse mRNA pool.