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.

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.

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.

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.

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.

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.

Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs.

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3-interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via 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 domain architectures homologous to those of known RIM homologs, but with 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 placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. 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 homolog. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like 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.

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.

Septic arthritis of the hip and knee treated surgically in pediatric patients: Analysis of the Kids' Inpatient Database.

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.

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.

Noncontact sports participation in adolescent idiopathic scoliosis: effects on parent-reported and patient-reported outcomes.

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<0.05). Regression models revealed major Cobb angle (ß coefficient: -0.312) and sports participation (ß coefficient: 0.422) as significant predictors of SRS-30 Function score (R=0.434, P<0.05). Our data show that for AIS patients with statistically similar bracing status and coronal and sagittal deformities, patients who participated in sports were more likely to have improved functionality, self-image, expectations, and parental perception of deformity. Further investigation is warranted to acquire a comprehensive understanding of the relationship between AIS and patient participation in sports. Maintaining moderate levels of physical activity and participating in safe sports may benefit treatment outcomes. Level of Evidence III - Retrospective Comparative Study.

CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course.

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.

Intrasubstance Anterior Cruciate Ligament Injuries in the Pediatric Population.

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.

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.

Retinoid X receptor α downregulation is required for tail and caudal spinal cord regeneration in the adult newt.

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, RARß, 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 RARß), 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 RARß. Whereas RARß 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 RARß and inhibits tail and caudal spinal cord regeneration. Moreover, treatment with a selective antagonist for RARß, 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.

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.