Reprocessable Networks from Vegetable Oils, Salts, and Food Acids: A Green Polymer Outreach Demonstration for Middle School Students.

Massive amounts of mismanaged plastic waste have led to growing concerns about their adverse impacts on the environment, ecosystem, and human health. Enabling efficient plastic recycling is a key component for developing a sustainable future, which requires cohesive efforts in technology innovations, public awareness, and workforce development. Particularly, outreach activities to inform the broader community about current efforts to fabricate sustainable polymeric materials can play a central role in inspiring future generations while also improving their knowledge, viewpoints, and behaviors to address plastic waste challenges. Herein, this account demonstrates an effort to educate middle school students about a key emerging concept in polymer science for sustainable material development: reprocessable polymer networks. Background information is provided to the students about the need to transition from petroleum-based chemical feedstocks to their bioderived counterparts. We note that the materials used in this demonstration lesson are all produced from common household foods, with which students routinely interact in various applications, making them not only safe but also compelling for the middle school classroom.

Genetic architecture of childhood speech disorder: a review.

Severe speech disorders lead to poor literacy, reduced academic attainment and negative psychosocial outcomes. As early as the 1950s, the familial nature of speech disorders was recognized, implying a genetic basis; but the molecular genetic basis remained unknown. In 2001, investigation of a large three generational family with severe speech disorder, known as childhood apraxia of speech (CAS), revealed the first causative gene; FOXP2. A long hiatus then followed for CAS candidate genes, but in the past three years, genetic analysis of cohorts ascertained for CAS have revealed over 30 causative genes. A total of 36 pathogenic variants have been identified from 122 cases across 3 cohorts in this nascent field. All genes identified have been in coding regions to date, with no apparent benefit at this stage for WGS over WES in identifying monogenic conditions associated with CAS. Hence current findings suggest a remarkable one in three children have a genetic variant that explains their CAS, with significant genetic heterogeneity emerging. Around half of the candidate genes identified are currently supported by medium (6 genes) to strong (9 genes) evidence supporting the association between the gene and CAS. Despite genetic heterogeneity; many implicated proteins functionally converge on pathways involved in chromatin modification or transcriptional regulation, opening the door to precision diagnosis and therapies. Most of the new candidate genes for CAS are associated with previously described neurodevelopmental conditions that include intellectual disability, autism and epilepsy; broadening the phenotypic spectrum to a distinctly milder presentation defined by primary speech disorder in the setting of normal intellect. Insights into the genetic bases of CAS, a severe, rare speech disorder, are yet to translate to understanding the heritability of more common, typically milder forms of speech or language impairment such as stuttering or phonological disorder. These disorders likely follow complex inheritance with polygenic contributions in many cases, rather than the monogenic patterns that underly one-third of patients with CAS. Clinical genetic testing for should now be implemented for individuals with CAS, given its high diagnostic rate, which parallels many other neurodevelopmental disorders where this testing is already standard of care. The shared mechanisms implicated by gene discovery for CAS highlight potential new targets for future precision therapies.

Large-scale sequencing of plant small RNAs.

Deep sequencing technologies have become very powerful tools in the identification and quantification of small RNAs involved in gene regulation. Small interfering RNA (siRNA) and miRNA are two classes of DCL-dependent small RNAs known to affect phenotype, developmental regulation, and various traits in plants. These small RNAs function by selectively repressing gene expression mainly by guiding cleavage, resulting in degradation of target transcripts. In this chapter, we describe a method for preparation of 5(')-phosphate-dependent small RNA libraries, a hallmark of RNase III-like DCL products, for high-throughput sequencing, and recommendations for small RNA analysis. This method is useful for determining small RNA involvement in critical pathways in plants, identifying and quantifying novel small RNAs, and examining small RNA global expression patterns.

miRNAs in the biogenesis of trans-acting siRNAs in higher plants.

Multicellular eukaryotes utilize many complex small RNA mechanisms to regulate gene expression from DNA modifications to RNA stability. RNA interference also regulates exogenous gene expression by degrading invading pathogen RNAs or preventing expression of foreign DNA incorporated into the host genome. Here we review the mechanisms for trans-acting (ta)-siRNA biogenesis and function, including pathways that utilize components of the miRNA and transitive RNAi defense. There are several distinguishing features of ta-siRNA pathways including the requirement for a miRNA-guided cleavage event that sets a processing register, RDR6 dependent dsRNA production, and DCL4 dependent processing to create unique, phased 21 nucleotide small RNAs. These phased small RNAs function to suppress target genes that only show similarity at the ta-siRNA recognition site, and act in trans to repress expression non-cell autonomously of specific target genes. Since the advent of high throughput sequencing technologies, phased siRNAs have been identified in a number of organisms [Heisel SE, Zhang Y, Allen E, Guo L, Reynolds TL, Yang X, et al. Characterization of unique small RNA populations from rice grain. PLoS One 2008;3:e2871. Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang XJ, et al. A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev 2007;21:1190-203. Johnson C, et al. Clusters and superclusters of phased small RNAs in the developing inflorescence of rice. Genome Res 2009;19:1429-40. Zhu QH, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F, et al. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 2008;18:1456-65. Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, et al. Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and ta-siRNA-directed targeting. Plant Cell 2007;19:926-42.]. These include transcripts generated either from non-protein-coding or protein-coding transcripts, long imperfect dsRNA or through an unknown mechanism; therefore some of these may not necessarily be classified as canonical ta-siRNAs.

Pattern formation via small RNA mobility.

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

AGO1-miR173 complex initiates phased siRNA formation in plants.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack.

Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation.

Trans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5' adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5' nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps.

Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting.

Posttranscriptional RNA silencing of many endogenous transcripts, viruses, and transgenes involves the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 (RDR6/DCL4)-dependent short interfering RNA (siRNA) biogenesis pathway. Arabidopsis thaliana contains several families of trans-acting siRNAs (tasiRNAs) that form in 21-nucleotide phased arrays through the RDR6/DCL4-dependent pathway and that negatively regulate target transcripts. Using deep sequencing technology and computational approaches, the phasing patterns of known tasiRNAs and tasiRNA-like loci from across the Arabidopsis genome were analyzed in wild-type plants and silencing-defective mutants. Several gene transcripts were found to be routed through the RDR6/DCL4-dependent pathway after initial targeting by one or multiple miRNAs or tasiRNAs, the most conspicuous example of which was an expanding clade of genes encoding pentatricopeptide repeat (PPR) proteins. Interestingly, phylogenetic analysis using Populus trichocarpa revealed evidence for small RNA-mediated regulatory mechanisms within a similarly expanded group of PPR genes. We suggest that posttranscriptional silencing mechanisms operate on an evolutionary scale to buffer the effects of rapidly expanding gene families.

High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes.

In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.

Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis.

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms . Three ta-siRNA gene families (TAS1, TAS2, and TAS3) are known in Arabidopsis thaliana. Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 ) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER-LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant (ARF3mut) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning.

A tolerance of DNA heterology in the mammalian targeted gene repair reaction.

Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.

Double displacement loops (double d-loops) are templates for oligonucleotide-directed mutagenesis and gene repair.

Appreciable levels of gene repair result from the hybridization of two oligonucleotides at a specific site in a mutated gene and subsequent correction by a form of oligonucleotide-directed mutagenesis known as gene repair. The incorporation of the two oligonucleotides into superhelical plasmid DNA leads to the formation of double d-loops, structures shown to be templates for the repair of both frameshift and point mutations. Structural limitations placed on the template indicate that correction is influenced significantly by the positioning of the second oligonucleotide, known as the annealing oligonucleotide. Complexes constructed with two oligonucleotides directly opposite each other exhibit the highest levels of gene repair activity. Blocking the 3'-end of either oligonucleotide with an amino C7 group does not diminish the performance of the double d-loop as a template for correction of the point mutation, suggesting that primer extension does not play a pivotal role in the mechanism of gene repair.

DNA pairing is an important step in the process of targeted nucleotide exchange.

Modified single-stranded DNA oligonucleotides can direct the repair of genetic mutations in yeast, plant and mammalian cells. The mechanism by which these molecules exert their effect is being elucidated, but the first phase is likely to involve the homologous alignment of the single strand with its complementary sequence in the target gene. In this study, we establish the importance of such DNA pairing in facilitating the gene repair event. Oligonucleotide-directed repair occurs at a low frequency in an Escherichia coli strain (DH10B) lacking the RECA DNA pairing function. Repair activity can be rescued by using purified RecA protein to catalyze the assimilation of oligonucleotide vectors into a plasmid containing a mutant kanamycin resistance gene in vitro. Electroporation of the preformed complex into DH10B cells results in high levels of gene repair activity, evidenced by the appearance of kanamycin-resistant colonies. Gene repair is dependent on the formation of a double-displacement loop (double-D-loop), a recombination intermediate containing two single-stranded oligonucleotides hybridized to opposite strands of the plasmid at the site of the point mutation. The heightened level of stability of the double-D-loop enables it to serve as an active template for the DNA repair events. The data establish DNA pairing and the formation of the double-D-loop as important first steps in the process of gene repair.

Superior colliculus and active navigation: role of visual and non-visual cues in controlling cellular representations of space.

To begin investigation of the contribution of the superior colliculus to unrestrained navigation, the nature of behavioral representation by individual neurons was identified as rats performed a spatial memory task. Similar to what has been observed for hippocampus, many superior collicular cells showed elevated firing as animals traversed particular locations on the maze, and also during directional movement. However, when compared to hippocampal place fields, superior collicular location fields were found to be more broad and did not exhibit mnemonic properties. Organism-centered spatial coding was illustrated by other neurons that discharged preferentially during right or left turns made by the animal on the maze, or after lateralized sensory presentation of somatosensory, visual, or auditory stimuli. Nonspatial movement-related neurons increased or decreased firing when animals engaged in specific behaviors on the maze regardless of location or direction of movement. Manipulations of the visual environment showed that many, but not all, spatial cells were dependent on visual information. The majority of movement-related cells, however, did not require visual information to establish or maintain the correlates. Several superior collicular cells fired in response to multiple maze behaviors; in some of these cases a dissociation of visual sensitivity to one component of the behavioral correlate, but not the other, could be achieved for a single cell. This suggests that multiple modalities influence the activity of single neurons in superior colliculus of behaving rats. Similarly, several sensory-related cells showed dramatic increases in firing rate during the presentation of multisensory stimuli compared to the unimodal stimuli. These data reveal for the first time how previous findings of sensory/motor representation by the superior colliculus of restrained/anesthetized animals might be manifested in freely behaving rats performing a navigational task. Furthermore, the findings of both visually dependent and visually independent spatial coding suggest that superior colliculus may be involved in sending visual information for establishing spatial representations in efferent structures and for directing spatially-guided movements.

Gold inlays bonded with a resin cement: a clinical report.

Because the marginal gap of finished gold inlays luted with Panavia 21 luting agent and zinc phosphate are comparable and the low viscosity and minimal film thickness of Panavia 21 cement allows the clinician to easily seat restorations without compromising fit and marginal integrity, the favorable physical properties of low solubility, high retention, and improved marginal seal would appear to enhance the longevity of gold inlays seated with Panavia 21 cement.

Fetal transplants alter the development of function after spinal cord transection in newborn rats.

Pieces of fetal spinal tissue were transplanted into the site of complete midthoracic spinal transections in neonatal rat pups (transplant rats). The development of locomotion in these animals was compared with that of unoperated control rats and rats that received spinal transections alone (spinal rats). Reflex, treadmill and overground locomotion, staircase descent, and horizontal ladder crossing for a water reward were tested in control, spinal, and transplant rats from 3 weeks to adulthood. All tests were readily performed by control animals. Most spinal rats were unable to make many linked weight-supported steps on these tasks. Transplant rats were variable in their locomotor capabilities, but a subset of rats were able to demonstrate coordinated and adaptable locomotion on these tasks. Some transplant rats performed better on more challenging tasks, suggesting that motor strategies for these tasks used different information, perhaps from descending systems. Transplanted tissue survived, and in most cases there was immunocytochemical staining of serotonergic fibers passing into and caudal to the transplant, supporting the conclusion that descending systems grew through the transplanted tissue. Integration with the host tissue was often poor, suggesting that nonspecific or trophic effects of the transplant might also contribute to the development of locomotor function. Therefore several mechanisms may contribute to the repair of injured spinal cord provided by transplants that permit the development of useful locomotion.

Embryonic spinal cord transplants enhance locomotor performance in spinalized newborn rats.

The results of the present experiments demonstrate that fetal spinal cord transplants placed into the site of a complete transection in newborn rats permit the development of complex patterns of locomotion. These patterns differ in some respects from normal, but include weight support, appropriate postural adjustment, and coordination between forelimbs and hindlimbs. 5-HT agonists administered to transplanted rats can further modify these motor patterns in ways that may prove able to enhance locomotion. When placed into lesion cavities in adult spinal cord, cells genetically modified to express neurotrophins can survive, differentiate, and mimic at least one consequence of fetal transplants, rescue of axotomized neurons from retrograde cell death.

Reversible inactivation of the lateral dorsal thalamus disrupts hippocampal place representation and impairs spatial learning.

Place-specific discharge of hippocampal cells was monitored while rats performed daily 15 trials of a spatial memory task. During the intertrial interval between trials 5 and 6, the lateral dorsal nucleus of the thalamus (LDN) was reversibly inactivated. Choice accuracy on the maze became impaired, and many hippocampal place fields became disrupted. These data support the proposition that the LDN passes onto hippocampus important (spatial) information that is used for accurate maze navigation.

Analgesic effects of intraventricular and intrathecal injection of morphine and ketocyclazocine in the infant rat.

Little is known of the neural bases of analgesia in immature animals. This experiment examined the effects of intracerebroventricular (i.c.v.) and intrathecal (i.t.) administration of morphine or ketocyclazocine in tests of antinociception in rats aged 3 to 14 days of age. Analgesia tests were conducted using both thermal and mechanical (pressure) noxious stimuli applied to the forepaw, hindpaw or tail. In the 3-day-old morphine-injected i.c.v. produced analgesia in the forepaws when either the mechanical or thermal noxious stimulus was used. There was no effect when the hindpaw or tail was tested. At 10 days of age, when the mechanical stimulus was used, morphine was analgesic in tests on all three appendages but was only effective in the forepaw when the thermal stimulus was used. Morphine was fully effective in all tests with both stimuli at 14 days of age. Ketocyclazocine had no consistent effect when given i.c.v. When injected i.t., morphine produced analgesia in the forepaws in the thermal test at 4 days of age and in all appendages by 10 days. When the mechanical test was used, morphine was effective in all appendages at all ages tested. Ketocyclazocine was analgesic at all appendages for the mechanical stimulus at all ages but was only transiently effective in the thermal test. The results demonstrate differential development of analgesia mediated at different levels of the neural axis and are consistent with the development of descending inhibitory that may mediate analgesia induced by i.c.v. injections of morphine. Neural mechanisms that are involved in the analgesic effects of these drugs against the two types of stimuli are also developmentally distinct.