Myopia Control Efficacy and Long-Term Safety of a Novel Orthokeratology Lens (MESOK Study)-A Randomized Controlled Clinical Trial Combining Clinical and Tear Proteomics Data.

Myopia control efficacy and long-term safety of the Breath-O-Correct orthokeratology (OK) lens was evaluated in a 2-year randomized, single vision (SV) spectacle lens-controlled, single-blind clinical trial combining clinical and tear proteomics data. A total of 71 children (43 OK, 9.8 ± 1.3 years; 28 SV, 9.5 ± 1.4 years) completed the 2-year study. Axial length (AL), cycloplegic refraction, clinical safety parameters (best-corrected visual acuity, central cornea thickness, corneal endothelial health, ocular surface disease index), and quantitative tear proteomics were evaluated by masked examiners. Mean 2-year-normalized AL elongations in the OK and SV groups differed significantly (p = 0.03) and were 0.37 ± 0.37 mm and 0.60 ± 0.41 mm, respectively. OK-mediated myopia control efficacy was 37.1%. No significant difference was found in clinical safety parameters of both groups (p > 0.10), except for a thinner central corneal thickness in the OK group (p = 0.01). Proteomics revealed modest OK lens-mediated effects on immune response proteins, including an increased abundance of haptoglobin at 6 and 12 months and a decreased abundance of two proteins (neutrophil defensin 3 and histone 4) at 6 months. The changes were further validated using a high-resolution multiple-reaction monitoring (MRMHR) mass spectrometry. In summary, the Breath-O-Correct OK lens significantly reduced AL elongation in schoolchildren without adverse clinical effects or subclinical inflammatory responses.

Target Landscape of Conserved Plant MicroRNAs and the Complexities of Their Ancient MicroRNA-Binding Sites.

In plants, microRNA (miRNA)-target interactions (MTIs) require high complementarity, a feature from which bioinformatic programs have predicted numerous and diverse targets for any given miRNA, promoting the idea of complex miRNA networks. Opposing this is a hypothesis of constrained miRNA specificity, in which functional MTIs are restricted to the few targets whose required expression output is compatible with the expression of the miRNA. To explore these opposing views, the bioinformatic pipeline Targets Ranked Using Experimental Evidence was applied to strongly conserved miRNAs to identity their high-evidence (HE) targets across species. For each miRNA family, HE targets predominantly consisted of homologs from one conserved target gene family (primary family). These primary families corresponded to the known canonical miRNA-target families, validating the approach. Very few additional HE target families were identified (secondary family), and if so, they were likely functionally related to the primary family. Many primary target families contained highly conserved nucleotide sequences flanking their miRNA-binding sites that were enriched in HE homologs across species. A number of these flanking sequences are predicted to form conserved RNA secondary structures that preferentially base pair with the miRNA-binding site, implying that these sites are highly structured. Our findings support a target landscape view that is dominated by the conserved primary target families, with a minority of either secondary target families or non-conserved targets. This is consistent with the constrained hypothesis of functional miRNA specificity, which potentially in part is being facilitated by features beyond complementarity.

TRUEE; a bioinformatic pipeline to define the functional microRNA targetome of Arabidopsis.

Central to plant microRNA (miRNA) biology is the identification of functional miRNA-target interactions (MTIs). However, the complementarity basis of bioinformatic target prediction results in mostly false positives, and the degree of complementarity does not equate with regulation. Here, we develop a bioinformatic workflow named TRUEE (Targets Ranked Using Experimental Evidence) that ranks MTIs on the extent to which they are subjected to miRNA-mediated cleavage. It sorts predicted targets into high (HE) and low evidence (LE) groupings based on the frequency and strength of miRNA-guided cleavage degradome signals across multiple degradome experiments. From this, each target is assigned a numerical value, termed a Category Score, ranking the extent to which it is subjected to miRNA-mediated cleavage. As a proof-of-concept, the 428 Arabidopsis miRNAs annotated in miRBase were processed through the TRUEE pipeline to determine the miRNA 'targetome'. The majority of high-ranking Category Score targets corresponded to highly conserved MTIs, validating the workflow. Very few Arabidopsis-specific, Brassicaceae-specific, or Conserved-passenger miRNAs had HE targets with high Category Scores. In total, only several hundred MTIs were found to have Category Scores characteristic of currently known physiologically significance MTIs. Although non-exhaustive, clearly the number of functional MTIs is much narrower than many studies claim. Therefore, using TRUEE to numerically rank targets directly on experimental evidence has given insights into the scope of the functional miRNA targetome of Arabidopsis.

Lymphatic Tissue and Organ Engineering for In Vitro Modeling and In Vivo Regeneration.

The lymphatic system has an important role in maintaining fluid homeostasis and transporting immune cells and biomolecules, such as dietary fat, metabolic products, and antigens in different organs and tissues. Therefore, impaired lymphatic vessel function and/or lymphatic vessel deficiency can lead to numerous human diseases. The discovery of lymphatic endothelial markers and prolymphangiogenic growth factors, along with a growing number of in vitro and in vivo models and technologies has expedited research in lymphatic tissue and organ engineering, advancing therapeutic strategies. In this article, we describe lymphatic tissue and organ engineering in two- and three-dimensional culture systems and recently developed microfluidics and organ-on-a-chip systems in vitro. Next, we discuss advances in lymphatic tissue and organ engineering in vivo, focusing on biomaterial and scaffold engineering and their applications for lymphatic vessels and lymphoid organ regeneration. Last, we provide expert perspective and prospects in the field of lymphatic tissue engineering.

Integrating Clinical Data and Tear Proteomics to Assess Efficacy, Ocular Surface Status, and Biomarker Response After Orthokeratology Lens Wear.

Purpose: This study evaluated the efficacy and ocular surface status of Breath-O Correct, novel orthokeratology (OK) lenses, worn overnight for 3 months. Lens-induced changes in the tear proteome were evaluated. Methods: Thirty-one subjects, aged 19 to 26 years with refractive error from -1.00 to -5.00 D, were randomly assigned 1:1 to the treatment or control group. Refraction, visual acuity, corneal integrity, biomechanics and endothelial health, ocular surface changes, and subjective symptoms were assessed at the baseline, one-month, and three-month visits. The tear proteome was characterized over time using sequential window acquisition of all theoretical ion spectra mass spectrometry. Results: Lenses improved uncorrected visual acuity and reduced spherical powers with similar efficacy to other OK lenses. Significant reductions (P < 0.05) in corneal hysteresis (11.12 ± 1.12 to 10.38 ± 1.36 mm Hg) and corneal resistance factor (11.06 ± 1.32 to 9.90 ± 1.45 mm Hg) were observed in the treatment group after one month of lens wear, whereas other assessed factors remained unchanged. Thirteen and eight differentially expressed proteins were found after one month and three months of lens wear, respectively. Two proteins (proline-rich protein 27 and immunoglobulin V regions) were differentially expressed at both visits. Conclusions: Over a three-month period, Breath-O Correct lenses were overall safe, well tolerated, efficacious in refractive power reduction, and comparable with other OK lenses. Furthermore, their use caused only minor noninflammatory protein expression changes in the tear proteome. Translational Relevance: This study investigated the safety of orthokeratology contact lenses on the ocular surface in molecular aspects and standard clinical parameters.

Biology and Function of miR159 in Plants.

MicroR159 (miR159) is ancient, being present in the majority of land plants where it targets a class of regulatory genes called GAMYB or GAMYB-like via highly conserved miR159-binding sites. These GAMYB genes encode R2R3 MYB domain transcription factors that transduce the gibberellin (GA) signal in the seed aleurone and the anther tapetum. Here, GAMYB plays a conserved role in promoting the programmed cell death of these tissues, where miR159 function appears weak. By contrast, GAMYB is not involved in GA-signaling in vegetative tissues, but rather its expression is deleterious, leading to the inhibition of growth and development. Here, the major function of miR159 is to mediate strong silencing of GAMYB to enable normal growth. Highlighting this requirement of strong silencing are conserved RNA secondary structures associated with the miR159-binding site in GAMYB mRNA that promotes miR159-mediated repression. Although the miR159-GAMYB pathway in vegetative tissues has been implicated in a number of different functions, presently no conserved role for this pathway has emerged. We will review the current knowledge of the different proposed functions of miR159, and how this ancient pathway has been used as a model to help form our understanding of miRNA biology in plants.

The Use of MicroRNA Decoy Technologies to Inhibit miRNA Function in Arabidopsis.

The study of gene function is best achieved through the generation of loss-of-function mutants. However, for many plant microRNAs (miRNAs), this has proven challenging, as they often belong to sequence-related families, which are encoded by multiple genes that are functionally redundant. To overcome this issue, transgenic methods have been developed that express miRNA decoys, which can sequester and inhibit families of sequence-related miRNAs. This includes miRNA MIMICs, SHORT TANDEM TARGET MIMICs, and miRNA SPONGEs. Here, we describe the methods to generate transgenic Arabidopsis that express these miRNA decoys in order to determine miRNA function.

MicroRNA MIMIC binding sites: Minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis.

In plants, microRNA (miRNA) target MIMICs (MIMs) have been widely used to inhibit miRNA function. They are based on the Arabidopsis INSENSITIVE TO PHOSPATE STARVATION 1 (IPS1) gene that corresponds to a non-coding RNA containing a miR399 binding site that can be modified to sequester and inhibit any miRNA of interest. However, the efficacy of miRNA inhibition of these different MIMs can vary greatly. Using MIMs that have strong efficacy (MIM159) and poor efficacy (MIM165), we investigate the underlying cause of this variation. Firstly, sequence alignments of IPS1 homologs from the Brassicaceae identified a highly conserved sequence immediately downstream of the miRNA binding site. Mutating this sequence in the context of the MIM159 attenuates its strong efficacy. This conserved flanking region contains a predicted stem-loop structure that is also predicted to be present in most modified MIMs that appear to have a strong efficacy, but not in MIM165 that has a poor efficacy. Restoring this predicted stem-loop in MIM165 via mutation of only three or five nucleotides within the conserved flanking region resulted in MIM165 variants that have very strong efficacies of miRNA inhibition. However, specifically mutating this predicted stem-loop in the MIM159 context failed to significantly reduce efficacy, and additional mutations to restore this predicted stem-loop weakened efficacy further. Although this shows there is no simple correlation between this predicted stem-loop and efficacy, these results add to the growing evidence that the sequence context of miRNA binding sites is important, and that minor nucleotide substitutions to flanking sequences of miRNA binding sites can strongly enhance or attenuate the miRNA-target interaction.

Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy.

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159.

Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses.

BACKGROUND: The microR159 (miR159) - GAMYB pathway is conserved in higher plants, where GAMYB, expression promotes programmed cell death in seeds (aleurone) and anthers (tapetum). In cereals, restriction of GAMYB expression to seeds and anthers is mainly achieved transcriptionally, whereas in Arabidopsis this is achieved post-transcriptionally, as miR159 silences GAMYB (MYB33 and MYB65) in vegetative tissues, but not in seeds and anthers. However, we cannot rule out a role for miR159-MYB33/65 pathway in Arabidopsis vegetative tissues; a loss-of-function mir159 Arabidopsis mutant displays strong pleiotropic defects and numerous reports have documented changes in miR159 abundance during stress and hormone treatments. Hence, we have investigated the functional role of this pathway in vegetative tissues. RESULTS: It was found that the miR159-MYB33/65 pathway was ubiquitously present throughout rosette development. However, miR159 appears to continuously repress MYB33/MYB65 expression to levels that have no major impact on rosette development. Inducible inhibition of miR159 resulted in MYB33/65 de-repression and associated phenotypic defects, indicating that a potential role in vegetative development is only possible through MYB33 and MYB65 if miR159 levels decrease. However, miR159 silencing of MYB33/65 appeared extremely robust; no tested abiotic stress resulted in strong miR159 repression. Consistent with this, the stress responses of an Arabidopsis mutant lacking the miR159-MYB33/65 pathway were indistinguishable from wild-type. Moreover, expression of viral silencing suppressors, either via transgenesis or viral infection, was unable to prevent miR159 repression of MYB33/65, highlighting the robustness of miR159-mediated silencing. CONCLUSIONS: Despite being ubiquitously present, molecular, genetic and physiological analysis failed to find a major functional role for the miR159-MYB33/65 pathway in Arabidopsis rosette development or stress response. Although it is likely that this pathway is important for a stress not tested here or in different plant species, our findings argue against the miR159-MYB33/65 pathway playing a major conserved role in general stress response. Finally, in light of the robustness of miR159-mediated repression of MYB33/65, it appears unlikely that low fold-level changes of miR159 abundance in response to stress would have any major physiological impact in Arabidopsis.

Cost impact of using patients' own multidose medications in hospital.

BACKGROUND: The use of patients' own medications may allow minimization of drug wastage and costs. However, the cost impact of this practice, taking into account the time that pharmacy personnel spend on verification, is unknown. OBJECTIVES: To determine the cost impact of using patients' own multidose medications within a surgical population, relative to the cost of routine dispensing; to describe the prescribing of multidose medications with regard to type, prevalence, quantity, and formulary status; and to determine the percentage of medications suitable for use after verification. METHODS: In this prospective, consecutive, time-and-motion case series, admission orders for patients newly admitted to 6 surgical units were screened to identify patients' own multidose medications that required verification. The total time required for all verification-related activities was captured. Data were collected over 3 weeks in early 2011. RESULTS: Of the 250 patients admitted, 51 (20.4%) had a prescription for one of their own multidose medications. Verification was completed for 67 (79%) of 85 prescribed items, of which 61 (91%) were deemed suitable for use. Thirty-five different medication types were identified. Of the 85 prescribed medications, 57 (67%) were on formulary. The most common routes of administration for these 85 prescribed items were inhalation (56 [66%]), nasal (9 [11%]), and ophthalmic (8 [9%]). The average cost ± standard deviation was $24.54 ± $32.33 per multi-dose item. The average time required for verification was 10.5 ± 6.7 min per patient (4.8 ± 3.3 min per medication). The cost impact was calculated as the difference between the drug cost with routine hospital dispensing and the cost of verifying home medications, where a positive value indicated a lower cost with verification of home medications (i.e., a saving for the hospital). The average cost impact was $40.05 ± $42.60 per patient (p < 0.001 by 1-sample t test) ($18.85 ± $15.42 per medication). A total cost saving of $1601.85 was realized. CONCLUSIONS: Using patients' own multidose medications instead of routine dispensing resulted in a cost saving of 74%, including labour costs for verification by the pharmacist.

CONTEXTE: L'utilisation des propres médicaments des patients peut permettre de réduire le gaspillage et les coûts de médicaments. Par contre, la répercussion sur les coûts de cette pratique, en tenant compte du temps passé par le personnel de la pharmacie à la vérification, est inconnue. OBJECTIFS: Déterminer la répercussion sur les coûts de l'utilisation des propres médicaments multidoses des patients chez une population de personnes ayant subi une intervention chirurgicale, par rapport aux coûts de la distribution habituelle; décrire la prescription de médicaments multidoses relativement au type, à la prévalence, à la quantité et à l'inscription sur la liste des médicaments (formulaire thérapeutique); et déterminer le pourcentage de médicaments pouvant être utilisés, après vérification. MÉTHODES: Dans cette série prospective analysant les temps et mouvements de cas consécutifs, les ordonnances d'admission de patients nouvellement admis dans six différentes unités de soins chirurgicaux ont été examinées afin d'identifier quels étaient les propres médicaments multidoses des patients devant être vérifiés. Le temps total nécessaire pour effectuer l'ensemble des activités de vérification a été noté. Les données ont été recueillies sur une période de trois semaines au début de 2011. RÉSULTATS: Des 250 patients admis, 51 (20,4 %) avaient une prescription pour un de leurs propres médicaments multidoses. Une vérification a été effectuée pour 67 (79 %) des 85 médicaments prescrits dont 61 (91 %) ont été jugés aptes à l'utilisation. On a identifié 35 différents types de médicament. Des 85 médicaments prescrits, 57 (67 %) étaient inscrits sur la liste des médicaments. Les voies d'administration les plus courantes parmi ces 85 médicaments étaient l'inhalation (56 [66 %]), la voie nasale (9 [11 %]) et la voie oculaire (8 [9 %]). Le coût moyen ± l'écart-type était de 24,54 $ ± 32,33 $ par médicament multidose. Le temps moyen nécessaire pour la vérification était de 10,5 ± 6,7 minutes par patient (4,8 ± 3,3 minutes par médicament). La répercussion sur les coûts a été calculée comme étant la différence entre le coût des médicaments s'ils avaient été délivrés selon le circuit habituel de l'hôpital et le coût de la vérification des médicaments apportés par les patients. Une valeur positive indiquait que la vérification des médicaments apportés par les patients était moins coûteuse, donc une économie pour l'hôpital. La moyenne de la répercussion sur les coûts était de 40,05 $ ± 42,60 $ par patient (p < 0,001 au moyen du test T pour échantillon unique) (18,85 $ ± 15,42 $ par médicament). Une économie de coûts de 1601,85 $ a été réalisée. CONCLUSIONS: Utiliser les propres médicaments multidoses des patients plutôt que de faire appel à la délivrance habituelle de médicaments par l'hôpital s'est traduit par une économie de coûts de 74 %, ce qui tient compte des frais de personnel pour la vérification par le pharmacien. [Traduction par l'éditeur].