MULTIMODAL IMAGING OF CORNEAL NEOVESSELS: OCT ANGIOGRAPHY IN COMPARISON TO FLUORESCEIN ANGIOGRAPHY ON 10 EYES AND REVIEW OF THE LITERATURE

Introduction:The use of OCT angiography for the analysis of neovessels of the ocular surface is currently at the experimental stage. Through this work we evaluate the benefits of OCTA in the detection of corneal neovessels, their depth and surface, the signs suggestive of activity / quiescence in comparison to fluorescein angiography. Materials And Methods:10 patients (10 eyes) with corneal neovessels (CNV) were prospectively recruited to Ophthalmology B at the Rabat Specialty Hospital between June and September 2019. All patients received OCT A fluorescein angiography (AF) at two-week intervals. Results:The results of the OCT A matched the AF data by showing immature neovessel (NV) characteristics for the early-diffusing NVs, allowing the study of the structure : trunk, numbers of segments, and fractal divisions, the existence of apical loop. As well as the detection of the flow level, exudative phenomena and associated lesions in high resolution OCT B. Discussion:Few studies have evaluated the OCTA in the evaluation of NVC, the published articles analyze the feasibility and reproducibility of this technique. Our initial analyzes suggest that scans provide better penetration and resolution of NVCs, but this requires direct comparative studies between OCTA systems used to scan the same eyes. The learning curve seemed fast for this technique. Conclusion :The evaluation of antiangiogenic treatments and the risk of graft rejection, it should be noted when interpreting OCTA scans of the anterior segment that artifacts may appear in dense scars areas in addition to motion artifacts.Future software enhancements and optimization for anterior segment acquisition may reduce these artifacts even more and improve the resolution of the image.

HIV-1 integrase binding to genomic RNA 5′-UTR induces local structural changes in vitro and in virio

Background During HIV-1 maturation, Gag and Gag-Pol polyproteins are proteolytically cleaved and the capsid protein polymerizes to form the honeycomb capsid lattice. HIV-1 integrase (IN) binds the viral genomic RNA (gRNA) and impairment of IN-gRNA binding leads to mis-localization of the nucleocapsid protein (NC)-condensed viral ribonucleoprotein complex outside the capsid core. IN and NC were previously demonstrated to bind to the gRNA in an orthogonal manner in virio; however, the effect of IN binding alone or simultaneous binding of both proteins on gRNA structure is not yet well understood. Results Using crosslinking-coupled selective 2′-hydroxyl acylation analyzed by primer extension (XL-SHAPE), we characterized the interaction of IN and NC with the HIV-1 gRNA 5′-untranslated region (5′-UTR). NC preferentially bound to the packaging signal (Psi) and a UG-rich region in U5, irrespective of the presence of IN. IN alone also bound to Psi but pre-incubation with NC largely abolished this interaction. In contrast, IN specifically bound to and affected the nucleotide (nt) dynamics of the apical loop of the transactivation response element (TAR) and the polyA hairpin even in the presence of NC. SHAPE probing of the 5′-UTR RNA in virions produced from allosteric IN inhibitor (ALLINI)-treated cells revealed that while the global secondary structure of the 5′-UTR remained unaltered, the inhibitor treatment induced local reactivity differences, including changes in the apical loop of TAR that are consistent with the in vitro results. Conclusions Overall, the binding interactions of NC and IN with the 5′-UTR are largely orthogonal in vitro. This study, together with previous probing experiments, suggests that IN and NC binding in vitro and in virio lead to only local structural changes in the regions of the 5′-UTR probed here. Accordingly, disruption of IN-gRNA binding by ALLINI treatment results in local rather than global secondary structure changes of the 5′-UTR in eccentric virus particles. Graphical Abstract

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection

Short, structured fragments of non-coding mRNA may act as molecular switches upon binding specific ligands, regulating the translation of proteins encoded downstream this mRNA sequence. One switch, called riboswitch N1, is regulated by aminoglycosides such as neomycin. Nucleobase mutations in the apical loop, although distant from the binding pocket, significantly affect neomycin affinity and riboswitch regulatory efficiency. To explain this influence, we conducted molecular dynamics simulations using generalized replica exchange with solute tempering (gREST). Translation assay of a reporter protein in a yeast system shows that mutating A17 to G in the riboswitch apical loop reduces 6-fold the translation regulation efficiency of the mutant. Indeed, simulations of the unbound riboswitch show that G17 frequently stacks with base 7, while base 8 is stabilized towards the binding site in a way that it may interfere with the conformational selection mechanism and decrease riboswitch regulatory activity. In the riboswitch complexes, this single-point A to G mutation disrupts a strong hydrogen bond between nucleotides 5 and 17 and, instead, a new hydrogen bond between residue 17 and neomycin is created. This change forces neomycin to occupy a slightly shifted position in the binding pocket, which increases neomycin flexibility. Our simulations of the U14C mutation suggest that the riboswitch complex with neomycin is more stable if cytosine 14 is protonated. A hydrogen bond between the RNA phosphate and protonated cytosine appears as the stabilizing factor. Also, based on the cell-free translation assay and isothermal titration calorimetry experiments, mutations of nucleotides 14 and 15 affect only slightly the riboswitch ability to bind the ligand and its activity. Indeed, the simulation of the unbound U15A mutant suggests conformations preformed for ligand binding, which may explain slightly higher regulatory activity of this mutant. Overall, our results corroborate the in vivo and in vitro experiments on the N1 riboswitch-neomycin system, detail the relationship between nucleobase mutations and RNA dynamics, and reveal the conformations playing the major role in the conformational selection mechanism.

Systolic thickening fraction of inerventricular septum as a predictor of superresponse to cardiac resyncronisation therapy - concept of a helical ventriсular band

Aim: to assess morpho-functional properties of left ventricle (LV) in patients with superresponse (SR) to CRT using the helical ventriсular band concept (HVB).Materials and methods: 56 patients were examined (mean age 54.0±9.9 years) at baseline and during follow-up visit: 48.8±25.6 months. Patients were divided into groups: I group (n=34) with decrease of LV end-systolic volume (ESV) ≥30% (superresponders) and II group (n=22) - decrease of LV ESV ˂30% (nonsuperresponders).Results: apical loop descendens segment (DS) and ascendens segment (AS) of HVB were evaluated according to the concept of F. Torrent-Guasp et al. According to the logistic regression mechanical interventricular delay (MID) (OR 1.072, 95% CI 1.017-1.131; p=0.01) and systolic thickening fraction (STF) of interventricular septum (IVS) DS (OR 0.944, 95% CI 0.895 - 0.995; p = 0.033) had an independent relationship with CRT SR. According to the ROC analysis the sensitivity and specificity of this model were 72.7% and 66.7% (AUC=0.769; р=0.001). AS STF of IVS was higher in SR (22.5 [0.0;40.0]% и 0.0 [0.0;25.0]%; р=0.005). The survival rate in group I was 85.1%, in group II was 63.6% (Log-Rank test p=0.019).Conclusion: SR is associated with a higher AS STF of IVS, higher MID, also with a higher survival rate.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity

ABSTRACT The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

Effect of apical portion of T-, sloped L-, and reversed L-closing loops on their force systems

ABSTRACT Objective: To investigate the effect of the position of the apical portion of closing loops on the force system at both loop ends. Materials and Methods: T-loops were compared with backward-sloped L-loops (SL) and reversed L-loops (RL). SL-loops were directed toward the anterior side; RL-loops were directed toward the posterior side. Loop response to loop pulling was determined with finite element analysis at six positions of the apical loop portion for 12-mm interbracket distance and 8-mm loop length and height. Three-dimensional models of the closing loops were created using beam elements with the properties of stainless steel. Loop responses (horizontal load/deflection, vertical force, and moment-to-force ratio) at both loop ends were calculated as well as at 100 g and 200 g activation forces. Results: T-, SL-, and RL-loops with the same position of the apical portion showed approximately the same force system at both loop ends. This behavior was found across the investigated range through which the loops were moved (interbracket center to posterior bracket). Conclusions: The center of the apical portion determined the force system of the closing loops regardless of the position of the loop legs. The centers of the apical portion of the T-, SL-, and RL-loops acted like V-bend positions.