Alternative Sequences Research Articles

Out of the blue: Prynne’s gifts

ABSTRACTA couple of years ago a friend presented me out of the blue with a gift: a little brown, blank envelope, and inside it Prynne’s newest poem at the time, Dune Quail Eggs. Any book given as a gift may feel like it belongs to the moment of its giving, but this one seemed to represent a special case, not just because of its surprisingness, but because the object itself in its particularity so obviously influenced the rhythm of my discovery of it. The envelope, though blank, seemed to begin in advance the sequence of alternations by which the text, when I read it later, would withhold and release information. The poem’s meaning needed the envelope; and maybe by implication it also needed my friend’s surprise gesture. But how could it need these features of a contingency belonging only to my personal experience and now to my fading memory? I am curious, in this piece, about the alter ego of the etext: the poem whose manner of being is so intrinsically physical that we never get hold of it.

Low-level point heteroplasmy detection in human mitogenomes amplified with different polymerases and sequenced on MiSeq FGx platform.

Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied. The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes. Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level. All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine. The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes.

Comparison of Miniperc and Standard Percutaneous Nephrolithotomy in the Treatment of Renal Stone: An Open Level Parallel Arm Randomized Control Trial

Background: Treatment of renal stone is very crucial for the outcomes of the patients. Objective: The purpose of the present study was to compare the Miniperc and standard percutaneous nephrolithotomy in the Treatment of Renal Stone. Methodology: This randomized control trial was conducted in the Department of Urology at National Institute of Kidney Diseases and Urology, Dhaka and some private hospitals in Dhaka city of Bangladesh from July 2016 to November 2017 for a period of one and half year. Patients from 18 to 65 years of age with renal calculi ( 2cm) were selected on the basis of plain X-ray and ultra-sonogram of KUB region, from Urology outpatient Department (OPD) in National Institute of Kidney Diseases and Urology (NIKDU), Dhaka and some private hospitals in Dhaka city. Patients were selected in every alternate sequence (odd numbers for Miniperc in Intervention group and even numbers for Standard PCNL, control group). All patients underwent PCNL of both the procedures under general anesthesia and received intravenous broad spectrum antibiotics. Results: A total number of 60 patients were selected for study of which 30 patients were underwent miniperc and 30 patients were undergoing standard PCNL. The mean age of miniperc group and PCNL group were 34.43±11.09 and 36.70 ± 12.27 years respectively. An overall stone clearance rates was 93.33 (28) in intervention group and 6.67 % (2) was not cleared. Among the control group an overall stone clearance rates were 96.67% (29) and 3.33% (1) was not cleared. Stone clearance rates were not significant. The mean operative time (min) of intervention group was 97.47±15.03 and the mean operative time (min) of control was 86.37±17.73 Operative time was significant between the groups (p<0.05). Conclusion: In conclusion the rate of clearance of intervention group is not statistically significant. Journal of Current and Advance Medical Research, January 2023;10(1):19-24

Thresholds and Adaptation Pathways of Nature-based Solutions for Sea-level Rise

Nature-based solutions serve as flexible, multi-functional, and adaptable actions aimed at promoting human well-being and socioeconomic benefits from climate risks by restoring natural ecosystem structures and functions. Due to uncertainties such as the magnitude/rate of sea-level rise (SLR), social politics, economic investment, etc., those strategies might be unfeasible and reach the tipping points of socio-ecological performance. The design of dynamic adaptation pathways contains a broad suite of actions that should be adopted in different SLR scenarios based on each method's thresholds to better manage uncertainty. The research question is, how to know the thresholds of nature-based strategies and create pathways to preserve multiple options in an uncertain future? To facilitate it, the paper defines the metrics to assess nature-based solutions performance under SLR and unpack the thresholds of each strategy based on case study analysis of the US, to design adaptation pathways over time. Results indicated that the feasible combination of nature-based strategies/pathways could bring more socio-ecological benefits. The number of adaptation options/pathways would decline with the extreme SLR, which shows coastal adaptation needs to start earlier than expected. These findings explore alternative sequences of decisions and illuminate the paths of alternative strategies to better adapt to SLR.

A long-awaited taxogenomic investigation of the family Halomonadaceae.

The family Halomonadaceae is the largest family composed of halophilic bacteria, with more than 160 species with validly published names as of July 2023. Several classifications to circumscribe this family are available in major resources, such as those provided by the List of Prokaryotic names with Standing in Nomenclature (LPSN), NCBI Taxonomy, Genome Taxonomy Database (GTDB), and Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB), with some degree of disagreement between them. Moreover, regardless of the classification adopted, the genus Halomonas is not phylogenetically consistent, likely because it has been used as a catch-all for newly described species within the family Halomonadaceae that could not be clearly accommodated in other Halomonadaceae genera. In the past decade, some taxonomic rearrangements have been conducted on the Halomonadaceae based on ribosomal and alternative single-copy housekeeping gene sequence analysis. High-throughput technologies have enabled access to the genome sequences of many type strains belonging to the family Halomonadaceae; however, genome-based studies specifically addressing its taxonomic status have not been performed to date. In this study, we accomplished the genome sequencing of 17 missing type strains of Halomonadaceae species that, together with other publicly available genome sequences, allowed us to re-evaluate the genetic relationship, phylogeny, and taxonomy of the species and genera within this family. The approach followed included the estimate of the Overall Genome Relatedness Indexes (OGRIs) such as the average amino acid identity (AAI), phylogenomic reconstructions using amino acid substitution matrices customized for the family Halomonadaceae, and the analysis of clade-specific signature genes. Based on our results, we conclude that the genus Halovibrio is obviously out of place within the family Halomonadaceae, and, on the other hand, we propose a division of the genus Halomonas into seven separate genera and the transfer of seven species from Halomonas to the genus Modicisalibacter, together with the emendation of the latter. Additionally, data from this study demonstrate the existence of various synonym species names in this family.

DDX5 maintains cardiac function by regulating CamkII delta alternative splicing

Abstract Background Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease. Dead-box protein 5 (DDX5) is a master regulator of many RNA processes, though its functions in heart physiology and disease remain unclear. Purpose To elucidate the function of DDX5 in HF and the mechanisms underlying its role in regulating alternative splicing. Methods and Results We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. Ddx5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. By integrating alternative splicing analysis and RNA immunoprecipitation sequencing data, we identified that DDX5 regulated the aberrant splicing of CamkIIδ, thus preventing the production of CaMKIIδA and maintaining Ca2+ homeostasis. In line with this, we found an increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5 depleted cardiomyocytes. Conclusions Our findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes.

Annotating otoliths with a deep generative model

Abstract Otoliths are a central information source for fish ecology and stock management, conveying important data about age and other life history for individual fish. Traditionally, interpretation of otoliths has required skilled expert readers, but recently deep learning classification and regression models have been trained to extract fish age from images of otoliths from a variety of species. Despite high accuracy in many cases, the adoption of such models in fisheries management has been slow. One reason may be that the underlying mechanisms the model uses to derive its results from the data are opaque, and this lack of legibility makes it challenging to build sufficient trust in the results. Here, we implement a deep learning model that instead of age predicts the location of annotation marks for each of the annuli. This allows an expert to evaluate the model’s performance in detail. The quality of the annotations was judged by a panel of four expert otolith readers in a double-blinded randomized survey. Using a scale from 1 to 5, the generated marks received an average quality score of 4.22, whereas expert annotations received an average score of 4.33. By counting the marks to determine fish age, we obtained an agreement between expert and model annotations of 64% on our test set, which running the model stochastically increased to 69%. Stochastic sampling yields further benefits, including an explicit measure of the model’s uncertainty, the post hoc likelihood of the different age classes for each otolith, and a set of alternative annotation sequences that highlight the structure of the annuli.

Detection of regularities in auditory sequences before and at term-age in human neonates

During the last trimester of gestation, fetuses and preterm neonates begin to respond to sensory stimulation and to discover the structure of their environment. Yet, neuronal migration is still ongoing. This late migration notably concerns the supra-granular layers neurons, which are believed to play a critical role in encoding predictions and detecting regularities. In order to gain a deeper understanding of how the brain processes and perceives regularities during this stage of development, we conducted a study in which we recorded event-related potentials (ERP) in 31-wGA preterm and full-term neonates exposed to alternating auditory sequences (e.g. “ba ga ba ga ba”), when the regularity of these sequences was violated by a repetition (e.g., ``ba ga ba ga ga''). We compared the ERPs in this case to those obtained when violating a simple repetition pattern (“ga ga ga ga ga” vs. “ga ga ga ga ba”). Our results indicated that both preterm and full-term neonates were able to detect violations of regularity in both types of sequences, indicating that as early as 31 weeks gestational age, human neonates are sensitive to the conditional statistics between successive auditory elements. Full-term neonates showed an early and similar mismatch response (MMR) in the repetition and alternating sequences. In contrast, 31-wGA neonates exhibited a two-component MMR. The first component which was only observed for simple sequences with repetition, corresponded to sensory adaptation. It was followed much later by a deviance-detection component that was observed for both alternation and repetition sequences. This pattern confirms that MMRs detected at the scalp may correspond to a dual cortical process and shows that deviance detection computed by higher-level regions accelerates dramatically with brain maturation during the last weeks of gestation to become indistinguishable from bottom-up sensory adaptation at term.

Cost-utility of 20-valent pneumococcal conjugate vaccine compared to no vaccination and recommended alternative vaccines among Belgian adults

ABSTRACTBackgroundThe Belgian Superior Health Council (SHC) preferentially recommended the 20-valent pneumococcal conjugate vaccine (PCV20) for adults aged ≥65 years, immunocompromised patients, and patients aged ≥50 years suffering from conditions that increase their risk for pneumococcal infections. The objective of this paper is to present the cost-utility of PCV20 compared to no vaccination and the alternative sequence of PCV15 followed by the 23-valent pneumococcal polysaccharide vaccine (PPV23) in this population.Research Design and MethodsThe analysis employed a static Markov model capturing lifetime risk of pneumococcal infections, associated disutility, mortality, and costs from different healthcare payer perspectives.ResultsResults indicated use of PCV20 among Belgian older and at-risk adults is highly cost-effective compared to no vaccination, with an incremental cost per quality-adjusted life-year (QALY) of €4,164. Compared to the sequential regimen (PCV15+PPV23), PCV20 vaccination is a cost-saving strategy. Subgroup analysis indicated PCV20 vaccination of at-risk adults aged 65–84 years would also be cost-saving from the national healthcare perspective.ConclusionBased on current knowledge, this analysis suggests that access to PCV20 should be proposed in all adults recommended for vaccination by the SHC as PCV20 prevents additional hospitalizations and deaths caused by pneumococcal infection at an affordable cost.

Behaviour of hybrid fibre reinforced polymer (HyFRP) laminates on high strength concrete (HSC) beams under flexural loading

In this paper, experimental studies of hybrid fibre reinforced polymer (HyFRP) laminates consisting of carbon ply fibre reinforced polymer (CFRP) and glass ply fibre reinforced polymer (GFRP) are reported on in conjunction with studies of concrete beams with high strength (HSC) under the category of with and without HyFRP laminates. The aim of this experimental work is to examine how alternative sequences of CFRP and GFRP ply of hybrid FRPs effect the enhancement of the high strength concrete beams. For the purpose of this study, there are a total of seventeen HSC beams cast. One of the beams was cast without HyFRP to act as a guiding beam, and the residual sixteen beams were cast with HyFRP using reinforced concrete and various combinations of carbon ply fibre reinforced polymer (CFRP) and glass ply fibre reinforced polymer (GFRP). The sequences in which hybrid FRP layers are attached serve as the primary variables in this test. Concrete with reinforcements after laminating beams with two, three, and even four layers of HyFRP, a load was applied in the form of static to the beams until they failed. The findings of the tests lead one to the conclusion that the repercussions of hybrid FRPs on the ductility of HSC beams are strengthened differently depending the order of the FRP layers.

ClO2-Mediated Oxidation of the TEMPO Radical: Fundamental Considerations of the Catalytic System for the Oxidation of Cellulose Fibers.

The reaction mechanism of ClO2-mediated TEMPO oxidation was investigated by EPR spectroscopy and UV-Vis spectroscopy in the context of an alternative TEMPO sequence for cellulose fiber oxidation. Without the presence of a cellulosic substrate, a reversibility between TEMPO and its oxidation product, TEMPO+, was displayed, with an effect of the pH and reagent molar ratios. The involvement of HOCl and Cl-, formed as byproducts in the oxidation mechanism, was also evidenced. Trapping HOCl partly inhibits the reaction, whereas adding methylglucoside, a cellulose model compound, inhibits the reversibility of the reaction to TEMPO.

On de novo Bridging Paired-end RNA-seq Data.

The high-throughput short-reads RNA-seq protocols often produce paired-end reads, with the middle portion of the fragments being unsequenced. We explore if the full-length fragments can be computationally reconstructed from the sequenced two ends in the absence of the reference genome-a problem here we refer to as de novo bridging. Solving this problem provides longer, more informative RNA-seq reads, and benefits downstream RNA-seq analysis such as transcript assembly, expression quantification, and splicing differential analysis. However, de novo bridging is a challenging and complicated task owing to alternative splicing, transcript noises, and sequencing errors. It remains unclear if the data provides sufficient information for accurate bridging, let alone efficient algorithms that determine the true bridges. Methods have been proposed to bridge paired-end reads in the presence of reference genome (called reference-based bridging), but the algorithms are far away from scaling for de novo bridging as the underlying compacted de Bruijn graph (cdBG) used in the latter task often contains millions of vertices and edges. We designed a new truncated Dijkstra's algorithm for this problem, and proposed a novel algorithm that reuses the shortest path tree to avoid running the truncated Dijkstra's algorithm from scratch for all vertices for further speeding up. These innovative techniques result in scalable algorithms that can bridge all paired-end reads in a cdBG with millions of vertices. Our experiments showed that paired-end RNA-seq reads can be accurately bridged to a large extent. The resulting tool is freely available at https://github.com/Shao-Group/rnabridge-denovo.

Prediction of Mycotoxin Response of DNA-Wrapped Nanotube Sensor with Machine Learning

DNA-wrapped single-walled carbon nanotubes (SWCNTs) have demonstrated great versatility in their use as optical sensors. SWCNTs emit a near-infrared fluorescence that is responsive to even the slightest changes in the nanotube environment, enabling sensors that can respond to single-molecule fluctuations within the vicinity of their surfaces. The fluorescence response and surface interactions of these sensors are determined by the DNA wrapping sequence. However, the lack of information on the relationship between the DNA sequence and its effect on the SWCNT fluorescence remains a bottleneck for designing sequences that are specific to analytes of interest. We have recently demonstrated the use of directed evolution to control the fluorescence response of SWCNTs through DNA design. Iterative cycles of DNA mutation, screening, and selection allowed us to evolve sequences that yield DNA-wrapped SWCNT sensors with a desired fluorescence response to mycotoxins.In this work, we apply the screening results of the DNA libraries used in this approach to train machine learning (ML) algorithms. Artificial neural network (ANN) and support vector machine (SVM) methods were used to predict the response of ssDNA-SWCNT sensors to a specific mycotoxin. The reliability of these models was further assessed through cross-validation. The ANN and SVM models with cross-validation were able to accurately classify the various DNA sequences as yielding either a high or low fluorescence response with an accuracy of 73 and 81%, respectively. The models were further used to predict the performance of alternative DNA sequences outside the initial training dataset, using the Hierarchy and k-means ++ clustering methods to evaluate the similarity and dissimilarity of each DNA sequence. Compared to the SVM model, the ANN model showed an improved ability to predict high responses for dissimilar DNA sequences. We further applied a combinatorial approach based on SVM and ANN models to design new DNA sequences for improving sensor performance. The success of this approach was validated experimentally, demonstrating the rational design of improved sensors with 95% prediction accuracy.The application of ML algorithms to directed evolution libraries of DNA thus allows one to accurately map the performances of these sensors within a particular sequence space. The computational success of this mapping provides a framework for replacing current empirical approaches with the rational design of DNA sequences for SWCNT sensing. Keywords: Nano Biosensor, DNA-SWCNT, Mycotoxin, Machine learning, Directed evolution method

Emergent layer stacking arrangements in c-axis confined MoTe2

The layer stacking order in 2D materials strongly affects functional properties and holds promise for next-generation electronic devices. In bulk, octahedral MoTe2 possesses two stacking arrangements, the ferroelectric Weyl semimetal Td phase and the higher-order topological insulator 1T′ phase. However, in thin flakes of MoTe2, it is unclear if the layer stacking follows the Td, 1T′, or an alternative stacking sequence. Here, we use atomic-resolution scanning transmission electron microscopy to directly visualize the MoTe2 layer stacking. In thin flakes, we observe highly disordered stacking, with nanoscale 1T′ and Td domains, as well as alternative stacking arrangements not found in the bulk. We attribute these findings to intrinsic confinement effects on the MoTe2 stacking-dependent free energy. Our results are important for the understanding of exotic physics displayed in MoTe2 flakes. More broadly, this work suggests c-axis confinement as a method to influence layer stacking in other 2D materials.

Sharp-wave-ripple-associated activity in the medial prefrontal cortex supports spatial rule switching

Previous studies have highlighted an important role for hippocampal sharp-wave ripples in spatial alternation learning, as well as in modulating activity in the medial prefrontal cortex (mPFC). However, the direct influence of hippocampal sharp-wave ripples on mPFC activity during spatial alternation learning has not been investigated. Here, we train Long Evans rats on a three-arm radial maze to perform a sequence of alternations. Three alternation sequences needed to be learned, and while learning a sequence, the activity in the mPFC was inhibited either directly following sharp-wave ripples in the hippocampus (on-time condition) or with a randomized delay (delayed condition). In the on-time condition, the behavioral performance is significantly worse compared to the same animals in the delayed inhibition condition, as measured by a lower correct alternation performance and more perseverative behavior. This indicates that the activity in the mPFC directly following hippocampal sharp-wave ripples is necessary for spatial rule switching.

Towards autonomous adaptive disassembly of permanent-magnet synchronous motors with industrial robots

On the way to a circular economy, it is necessary to develop new end-of-life (EOL) solutions for critical components and materials. Due to the electrification of personal transportation a rise in production numbers of electric motors is to be expected, necessitating the development of EOL solutions for such motors. Such solutions, like the reuse or remanufacturing of components, necessitate in term the disassembly of the motors. To economically facilitate such disassembly operations, new automation solutions need to be developed. One possible approach is the disassembly with industrial robots coupled with process-specific end effectors. This makes it possible to flexibly execute a variety of disassembly processes without the need for cost-intensive specialized machines. However, due to the inherent uncertainties of products returning from the field, an ideal disassembly procedure cannot be assumed. To address this issue, in this paper we present a new concept for the autonomous adaptive disassembly of permanent-magnet synchronous motors (PSM) with industrial robots. The system will be able to plan possible disassembly sequences while taking different process alternatives for individual steps into account. During the actual disassembly the system will check the outcome of the individual steps, as well as the resulting state of the motor to be disassembled. In case of an unsuccessful operation the system will then be able to autonomously adapt to the actual situation and generate an alternate disassembly sequence utilizing alternative processes. This paper presents the necessary infrastructure, both in hard- and software, to achieve this autonomous adaptability for the example of PSM, thereby making a contribution on the way to intelligent automatic disassembly systems and the change to an economic and ecological circular economy.

Alternative Sequence Classification of Neural Networks for Bridge Deck Condition Rating

Alternative Sequence Classification of Neural Networks for Bridge Deck Condition Rating

SARS-CoV-2 Recombination and Coinfection Events Identified in Clinical Samples in Russia.

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line.

Decision Making in Determining Improve Margin at the PT XYZ Using the Analytic Hierarchy Process

PT XYZ is one of 6 largest refineries in Indonesia whose business is engaged in oil and gas which processes crude oil into products with high selling value. By changing the business pattern of PT XYZ from cost oriented to profit oriented so that it encourages every business operation to be able to increase the company's margins by implementing optimization, efficiency and strategies that encourage the avoidance of events that hinder operations so that it has an impact on changes in processing planning. The profitability of refineries is greatly affected by the price spread of crude oil and the products produced which are very unstable in nature, therefore efforts are needed to improve operational reliability, efficiency and optimization which can maintain the stability of the potential increase in the company's margins. This final project is used to determine the best alternative that will be chosen by PT XYZ for improvements that can be carried out by PT XYZ alone without any involvement from the Head Office or third parties so that these improvements can be controlled directly by PT XYZ. This improvement will provide benefits by increasing the company's margins. The decision making used is by combining Value Focused Thinking with the Analytical Hierarchy Process. Value Focused Thinking is used to determine criteria and sub-criteria with the interview pattern with the Subject Matter Expert and followed by determining alternatives with the Focused Group Discussion pattern, followed by AHP to determine the best alternative. Based on the analysis that has been carried out with the help of expert choice software, the best alternative sequence is Reformat Maximizing, Replacement of the Steam Turbine into a Motor, Increasing LPG Production and Utilization of Factory own use.

Quick Fix: ArcGIS and a Sense of Place in the Humanities Classroom

ArcGIS is an online tool which allows users to create and curate digital StoryMaps which contain a variety of geographic points, text, images, and web links. Instructors can turn to this software for a variety of uses in the humanities classroom, including incorporating a sense of place and movement to the study of various genres of texts. Through ArcGIS, students can create digital artifacts which bridge the gap between text and the world around them in alternative assignment sequences which include substantial writing and research components and offer more potential for creativity and collaboration than the standard argumentative essay.