This study describes the internal structure of materials used to produce medical stents. A two-level elastoviscoplastic mathematical model, which sets the parameters and describes the processes at the grain level, was developed and numerically implemented. A separate study was conducted to identify the most dangerous deformation modes in the balloon-expandable stent placement using the finite-element method in COMSOL Multiphysics. As a result, the challenging strain state type required for setting the kinematic loading on a representative macrovolume in the two-level model was obtained. A yield surface for different deformation paths in the principal deformation space for stainless steel AISI 316L was obtained and the effect of grain size on the deformation behavior of this material was explored using the developed model. Coronary stents, made of metal alloys and biopolymers, are used to expand blocked blood vessels and maintain sufficient blood flow in the human body, . The useBalloon-expandable stents, unlike the self-expanding stents, experience large plastic deformations resulting in their self-hardening ,15. SuchGrain boundaries play a significant role in the plastic-deformation and fracture processes of polycrystalline materials. The grain (or intergrain) boundary is the interface between two crystals with different crystallographic orientations. The intergrain boundary layer has a pronounced defect structure, with a characteristic thickness of approximately two or three interatomic distances in pure metals . High stThe fact that the grain boundaries are linked to GBS, and accommodation mechanisms defines the parameters of our model that characterizes the influence of the boundaries on the material’s deformation character. The impact of intergrain boundaries is apparent in the Hall–Petch law , which rIn models of inelastic deformation of polycrystals, hardening laws are usually formulated as evolution equations for critical shear stresses of the dislocation motion ,39,40,41A literature review on the grain structure used for the modeling of metals demonstrated that the grain sizes in a polycrystal follow the lognormal distribution law, which is consistent with experimental data ,44,45,46The features of the grain structure in coronary stents were studied with images of a 316L stainless steel stent strut obtained with scanning electron microscopy ,50 Figu. These iTexture formation is another problem for grain-structure modeling of such components. As noted in ,50, a teFractures are the most common problem related to the use of stents, as well as the arterial damage from direct contact between the arteries and stent. Various sources report that stent rupture occurs in 1–18% of cases, which is quite a serious challenge for their wide applications ,51,52. IThe properties and behavior of a material at the sample level (macrolevel) significantly depend on the evolution of its meso- and microstructure and should be properly considered in a model of the inelastic deformation processes of polycrystals . So, theIn this paper, a two-level (macro-meso) elastoviscoplastic model of inelastic deformation of a polycrystalline aggregate was developed. A macrolevel element is the representative volume of a polycrystal consisting of many mesolevel elements, i.e., grains . Apparently, a representative volume is the minimum volume of a material with enough elements responsible for the considered process mechanisms for the statistical description of its state .It is worth noting that the additivity hypothesis of the elastic and inelastic components of the strain-rate measure at the meso- and macrolevel is accepted in most elastoviscoplastic models ,53:(1)ζ=Hooke’s law in the rate form serves as the constitutive relation at the mesolevel:b in the slip direction and the slip plane’s normal n. Assuming a uniform dislocation distribution in grains, the inelastic component of the strain rate measure can be calculated [K is the total number of the slip systems and k-th slip system.The main mechanism of inelastic deformation in the material is the intragrain dislocation slip along the most closely packed planes and directions. The combination of the plane and direction forms the slip system (SS), which is characterized by the Burgers vector lculated ,53:(5)ζik-th slip system, m is the constant of the rate sensitivity of the material. k-th slip system, defined asTo establish the shear rates in the slip systems, the following power law can be applied:The transition between the scale levels is performed by employing an averaging procedure for the mesolevel element’s characteristics. The constitutive relation at the macrolevel is Hooke’s law in the rate form:N is the grain number):Thus, the systems of equations at the meso- and macrolevel have the form {k-th SS, respectively, and P is the number of regions approximating the grain boundaries. The misorientation measure The rate of increase in the critical shear stresses due to the grain-boundary hardening mechanism is defined as follows ,41:(11)fThe behavior of the balloon-expandable stent  was anax-axis. Outside the stent, the boundary conditions were free. Thus, the stent expanded up to a radius of 2 mm evenly along its entire length. The stent length changed from 10 to 6.7 mm when expanding along the x-axis.Often, balloon-expandable stents unevenly deform during stent placement. As a result, these deformations can lead to stent damage up to full rupture. The radius of the stent before expansion is 0.75 mm . The simThe elastoplastic flow model was used to describe stent deployment; the values of the model parameters for stainless steel are given in In the modeling of the stress-strain state of a stent, it was hard to specify the deformation conditions, for which individual parts of the structure could accumulate plastic deformations and collapse at relatively low stresses. Based on the analysis of the stent model, built in the COMSOL Multiphysics software package, the relationship between the values of the principal deformations were determined and the principal deformation directions of the structure parts vulnerable to fracture were found. Considering the coaxiality of the principal stresses and deformation vectors in the elastic region, the value characterizing the strain state type was obtained:ki of the deformation tensor in pressure. This was necessary to determine the deformed state of the structure and the deformation directions in the principal stress space to use the developed model (1)–(12).To describe the behavior of the structural material in the plastic deformation process, an elastoviscoplastic two-level model was employed with explicit consideration of the main physical mechanisms and processes at the macro- and mesolevel. The macrolevel polycrystalline aggregate consisted of 350 grains, with uniformly distributed orientations. The grain-size distribution followed the lognormal law; its parameters are discussed in more detail in ,45,46. Nki are the orthogonal unit vectors along the corresponding xi-axes.To identify hardening parameters, a numerical experiment of the uniaxial tensile test of the polycrystals was carried out using the hardening laws (10) and (11). A representative volume experienced uniaxial tensile deformation along the x1-axis at the following rate:The dependence of the Cauchy stress tensor intensity, ulations  demonstrIn order to assess the influence of grain size on the deformation behavior of the material, the dependence of the yield strength uggested . The relThe range of the law parameters was chosen in such a way so that the grain size in the material changed from 3 to 150 μm. The grain-size distributions in a polycrystal with 350 grains for some parameter values are shown in Using the Lode parameter (13) calculated in the simulation in COMSOL Multiphysics, a relationship between the quantities, obtained  for the X-axis of principal deformation E1 on a given plane;Angle φ between the deformation direction and the projection of the The radius vector drawn from the origin to the considered point, which is equal to the yield strength of the material.Each point of the diagram has two polar coordinates:The minimum yield strength, with a value of about 258 MPa, was obtained for the unit vectors, {0.998, 0, −0.062}, {−0.586, 0.537, 0.607}, {−0.998, 0, 0.062}, and {0.586, −0.537, −0.607}, setting the deformation direction in the principal deformation space . These vectors defined the relationship between the principal deformations and formulated the kinematic loading conditions, at which plastic deformation occurred at the lowest stress in the material. Thus, the most dangerous deformation directions for structure parts with the highest stresses  in the pThe influence of the 316L stainless steel stent microstructure on the mechanical strength and durability in the process of design deployment was studied in this paper. Modeling the process of stent expansion under linearly distributed loading using the COMSOL Multiphysics software package found non-uniform stress-strain fields with the most dangerous zones of design in terms of highest plastic deformation and stresses. To describe the mechanisms and processes at the grain level in the plastic deformation process in these zones, a two-level elastoviscoplastic model was built, allowing to directly control the internal structure of the material, such as grain orientation and grain size in a polycrystal. The numerical simulation based on the developed model showed a good correlation with experimental data. It was found that an increase in the parameters μ and σ of the lognormal distribution law led to the grain-size growth in the polycrystal and, consequently, caused a significant drop in the yield strength. The most dangerous deformation direction for the structure part with the highest stresses and plastic deformation in the process of stent expansion were obtained along the unit vectors {0.998, 0, −0.062}, {−0.586, 0.537, 0.607}, {−0.998, 0, 0.062}, and {0.586, −0.537, −0.607} in the principal deformation space. The yield strength for these deformation directions had a minimum value of about 258 MPa.


Secondly, we performed 16S rRNA targeted metagenomic approach (n = 72) to compare the vaginal microbiota between these groups. We found a wide variety of bacteria, with Proteobacteria, Firmicutes, and Bacteroidota being the most abundant phyla in both groups, as well as Escherichia-Shigella, Streptococcus, and Bacteroides at the genus level. Most agents identified in the sequencing method also grew in the culture-dependent method, showing the viability of these bacteria. Alpha diversity did not differ between HE and VD sows, regarding sample richness and diversity, but a beta-diversity index showed a different microbiota composition between these groups in two tested herds. ANCOM analysis revealed that Bacteroides pyogenes were more abundant in VD females and can be a marker for this group. Other agents also require attention, such as the Streptococcus dysgalactiae and Staphylococcus hyicus found in remarkably greater relative abundance in VD sows. Network analysis revealed important positive correlations between some potentially pathogenic genera, such as between Escherichia-Shigella, Trueperella, Streptococcus, Corynebacterium, and Prevotella, which did not occur in healthy sows. We conclude that the alteration of the vaginal microbiota between healthy and purulent vulvar discharge sows, although not extreme, could be due to the increase in the relative abundance of specific agents and to associations between potentially pathogenic bacteria.Purulent vulvar discharges, primarily caused by genito-urinary tract infections, are an important source of economic loss for swine producers due to sow culling and mortality. However, the agents that compose the vaginal microbiota of sows and their changes during infections are not well understood. The first goal of this study was to characterize and compare the vaginal bacterial content of healthy (HE,  Metritis and endometritis are the most common cause for these purulent discharges1 and are an important source of sow culling and mortality, which is increasing worldwide2. Infectious agents causing these conditions have been identified by traditional methods that rely on bacterial culture and isolation6, but few studies have characterized the microbiota in the vaginal canal of sows by high-throughput sequencing methods10, and less so compared between healthy sows and those with vulvar discharge12.Purulent vulvar discharges caused by bacterial infections are a compelling cause of the decreased reproductive performance of sows15. Thus, a better understanding of the bacterial vaginal composition in healthy and diseased sows has the potential to mitigate urogenital infections with more specific treatments and better prevention strategies, improving sow welfare and reducing economic losses.Previous studies show that the vaginal microbiota can play a significant role as a biological barrier against disease, due to its constitution and secretion of antimicrobial components11 characterized the vaginal microbiota of sows with and without endometritis and found higher abundances of Escherichia-Shigella, Bacteroides, Fusobacterium, and Clostridium_sensu_stricto_1 genera in diseased than in healthy sows, suggesting that the microbiota of these groups could be different. Zhang et al.12 investigated the effect of intestinal microbiota on vaginal bacterial composition in sows and indicated that some genera such as Escherichia-Shigella and Bacteroides may be linked to the onset of endometritis. However, Wang et al.11 did not perform a differential abundance analysis test and both studies evaluated a small number of females (n = 8) and should be of limited value. Our study hypothesizes that the vaginal microbiota of sows with and without vulvar discharge may present differences in bacterial composition. Therefore, we evaluated 310 vaginal samples by culture and MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) mass spectrometry identification, and 72 of these samples were further characterized by targeted metagenomic sequencing to compare the vaginal microbiota between healthy and purulent vulvar discharge sows.Wang et al.Bacterial culture and identification by MALDI-TOF mass spectrometry were able to identify a wide variety of species in sows presenting vulvar discharge. Of the 270 females with this clinical sign, 98.5% (266/270) were positive for bacterial isolation. Eighty-nine percent of the samples generated mixed cultures, with up to eight distinct species isolated from the same animal. For the 40 healthy females, only one did not present bacterial growth and 82% (32/39) of the samples generated mixed cultures.Enterococcus faecalis , Streptococcus hyovaginalis  and Acinetobacter Iwolffii . Although HE sows had a higher frequency of Escherichia coli, we observed no statistically significant difference between groups (p = 0.06). VD sows also showed high frequencies for some species, but also without significant difference, such as Streptococcus dysgalactiae  and Staphylococcus hyicus . Trueperella pyogenes (5.3% vs. 0%), Bacteroides pyogenes (3.4% vs. 0%) and Corynebacterium diphtheriae (7.9% vs. 0%) were only isolated in VD sows.As there was no statistical difference between the frequencies of the main agents for the different herds in the culture method (Supplementary Table S1), we analyzed the herd data together. The culture method showed that HE sows had a higher frequency of some bacterial species than VD sows . This was followed by E. coli and Streptococcus suis (12.8%), Escherichia coli and E. faecalis (9.4%), E. coli, and S. hyicus (7.1%). Meanwhile in HE sows, E. coli and E. faecalis (23%), E. coli and S. dysgalactiae (10.2%), E. coli and Trueperella abortisuis (10.2%) were the most frequent associations. Furthermore, the Venn diagram generated for the culture and MALDI-TOF MS identification results .p < 0.001, Chao1 index, see Supplementary Fig. S1 online). However, only herd 3 had higher diversity compared to all other herds . There was also a difference in the beta-diversity analysis comparing herds. In the UniFrac PCoA analysis , only herd 1 and 2 did not show a significant difference from each other, while the other pairwise PERMANOVA results  indicated significant differences between bacterial community (p < 0.05 and q < 0.05).A primary analysis to compare bacterial diversity among herds showed a difference for alpha diversity indices. Herds 1 and 3 had higher richness compared to herds 2 and 4 . For herd 4 there was a tendency for difference between clusters   Fig. B. For thProteobacteria had the highest relative abundance in healthy and vulvar discharge sows, which accounted for 62.1% and 48.2% of relative abundance, respectively, followed by Firmicutes, which accounted for 29% and 34% of relative abundance, respectively, and Bacteroidota, which accounted for 8.4% and 16.6% of relative abundance, respectively  and herd-2  with a lower relative abundance of Bacteroidota . In contrast, herd-3 had a lower relative abundance of Proteobacteria  but a higher relative abundance of Bacteroidota . Herd-3 still had a higher relative abundance of Firmicutes  compared to other herds. Herd-4 had a high relative abundance of Proteobacteria  and Bacteroidota , but a lower relative abundance of Firmicutes compared to other herds . Except for herd-3, the relative abundance of Proteobacteria was higher in HE females than in VD females in all herds. Conversely, the phylum Bacteroidota was higher in VD females in all herds, especially in herd-3 and herd-4.When herds were analyzed separately, some differences in relative abundance were observed Fig. B. A highEscherichia-Shigella was the most abundant in both groups but was higher in healthy than in vulvar discharge sows, accounting for 52.2% and 30.5% of relative abundance when data from all herds were analyzed together. Acinetobacter, Aerococcus, Prevotella, and Pseudomonas also showed a slightly higher relative abundance in healthy sows. Conversely, Streptococcus, Bacteroides, Pasteurella, Porphyromonas, and Staphylococcus were more abundant in sows with purulent vulvar discharge. For Pasteurella, however, all sequences came from a single sample in the VD group.At the genus level Fig. A, EscherEscherichia-Shigella, Streptococcus, and Bacteroides remain the main genera, present in all herds. But remarkable differences can be seen for Porphyromonas in herd-3, Bacteroides in herd-3 and herd-4, and Staphylococcus in herd-1 and herd-2.The graph in Fig. B. pyogenes and S. dysgalactiae were found in remarkable greater relative abundance in females with purulent vulvar discharge than in healthy females . When the test was applied to each farm separately, B. pyogenes were significantly more abundant in VD females only in herd-3 . S. dysgalactiae was also found in greater abundance in VD females  and should be avoidedEscherichia-Shigella and Trueperella, and Escherichia-Shigella and Streptococcus27. Significantly, the association between these species also occurred in the culture-dependent method. A positive correlation between Corynebacterium and Prevotella may also be important since these agents have already been described in reproductive disorders of sows20 and women40. Such correlations did not occur for HE females, in which Escherichia-Shigella and Enterococcus are dominant nodes, with negative correlations, showing dominance for these agents. This is also corroborated by the MALDI-TOF MS and relative abundance graphs since Escherichia and Enterococcus present higher frequency and relative abundance in HE females in all herds. The Enterococcus genus also has a strong positive correlation with the Veillonella genus commonly found in considerable abundance in the vaginal microbiota of sows, between 2 and 6%, but without a relationship with disease41.Network analysis correlations between the genera identified by metagenomic sequencing show that VD sows had more positive correlations between agents recognized as pathogenic for the reproductive tract, such as , especially B. pyogenes, which can be proposed as a marker for VD sows. Higher frequencies or relative abundances of specific agents in VD sows, and the association between them, also require attention. Evidence suggests that dysbiosis in the vaginal canal of female swine that presents purulent vulvar discharge occurs by the association and increase of some potentially pathogenic bacteria such as B. pyogenes, S. dysgalactiae, S. hyicus, C. diphtheriae, and T. pyogenes.This study provides new evidence on the vaginal bacterial composition of sows with and without purulent vulvar discharge. Most bacteria obtained in culture were also present in the metagenomic sequencing, showing the viability of the detected agents. The vaginal microbiota of these groups shares most of the features identified by 16S rRNA sequencing, but punctual changes in the abundance of certain agents differentiated VD and HE femalesThree hundred ten vaginal samples were collected from commercial crossbred sows housed in four commercial farms in different Brazilian states: São Paulo, Minas Gerais, Paraná, and Mato Grosso. Two hundred seventy samples were collected from sows presenting purulent vulvar discharge (VD) and 40 were collected from healthy sows (HE).Animals were inspected for purulent vulvar discharge with the use of a sterile and disposable vaginal speculum during pregnancy or in the lactation period, at least three days after the farrowing process, combined with the observation of the presence of purulent material on the floor and in the bars below the female. The introduction of the speculum was always performed by two veterinarians, one dealing with the vulva and exposing the vaginal canal and the other introducing the speculum to avoid external contamination Samples were only collected if purulent secretion was present in the deep region of the vaginal canal, after the passage of the speculum, which also excludes the possibility that this purulent material originates from vulvar lesions or vaginitis. For the collection, two sterile swabs (one for culture and one for metagenomic approaches) were collected, placed in transport media, and immediately refrigerated until arrival at the laboratory for further processing. Secretions considered to be normal-appearing and lochia were not collected. Only secretions very suggestive of infectious processes, intensely purulent and with lumps were collected and were considered in further analyses. Sows that had just urinated were not collected. If antimicrobials were used in any of the animals in the previous 30 days before collection, these females were not included in further analysis. Healthy females were collected if they did not present vulvar discharge, hyperthermia, low body condition score, had been medicated, or had any other disease condition.https://arriveguidelines.org).This study was evaluated and approved by the Animal Use Ethics Committee (CEUA) of the Faculty of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil (CEUA Process Number 1875170317). This Committee follow all international guidelines and regulations to evaluate studies and experiments on live vertebrates. The authors declare that the methods are in accordance with ARRIVE guidelines  and incubated at 37ºC for 24 to 48 h. After growth, the isolated bacterial colonies were transferred to 3.0 ml of BHI broth (Brain Heart Infusion – Difco), and from this culture, aliquots were separated for storage at -80º C and for protein extraction for identification by Matrix Associated Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS).Swabs were plated on MacConkey agar, Chromagar Orientation®, and Columbia agar with 5% sheep blood , and the plates were incubated at 37ºC for 24 to 48 h under aerobic conditions. For anaerobic growth, swabs were seeded on 42. The Microflex™ mass spectrophotometer  from the São Paulo State Environmental Company (CETESB) was used. To capture the protein spectra, 1.0 µl of protein suspension was transferred to a 96-well stainless-steel plate and, after drying at room temperature, 1.0 µl of the polymer matrix (α-cyano-4-hydroxycinnamic acid) was added. Each strain was distributed in three wells and for each plate, two readings were performed with the FlexControl™ software  using the MTB_autoX method. Finally, the BioTyper™ 3.0 software  compared the captured spectra for each strain with the manufacturer's library for bacterial identification. By comparing the presence/absence of specific peaks, a log (score) value was obtained. The criteria for interpreting the standards used in this study were those of the manufacturer Bruker Daltonik: scores ≥ 2.0 were accepted for species attribution, and scores ≥ 1.7 and < 2.0 were used only for genus identification.For bacterial identification by MALDI-TOF MS, the colonies were subjected to ribosomal protein extraction using the protocol described by Hijazin et al.For the metagenomic approach, 72 swabs (40 VD and 32 HE) had their bacterial DNA extracted using the DNeasy® Blood & Tissue Kit (QIAGEN), following the manufacturer's recommendations. DNA concentration and integrity were verified in the NanoDrop® 2000 equipment and by agarose gel electrophoresis before being sent to the sequencing facility. Briefly, 16S rRNA amplicons were produced using specific primers for the 16S-V4 region (515F-806R) along with barcodes. All PCR reactions were conducted with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The mixed PCR products were purified with Qiagen Gel Extraction Kit  and the libraries generated with NEBNext® UltraTM DNA Library Prep Kit for Illumina and quantified via Qubit and qPCR. Amplicons were sequenced on the Illumina MiSeq platform to generate 250 bp paired-end reads.43. Paired-end reads were assigned to samples based on their unique barcodes and truncated by cutting off the barcode and primer sequences with the cutadapt tool44 and the union of reads was performed with the Vsearch plugin45. A quality filter was applied with the quality-filter plugin by the q-score-joined method46 and a denoising step was performed using the deblur plugin with the denoise-16S method47. Sequences with ≥ 97% or more similarity were assigned to the same OTU (Operating Taxonomic Unit) with the Vsearch plugin45 and were taxonomically classified using a pre-trained classifier trained on the SILVA database49.Downstream analyses were performed using the QIIME 2 platformp-value  was considered. Differential abundance analysis was performed using the ANCOM37 test on the QIIME 2 platform43. Figures and graphics were performed with the qiime2R package50 and ggplot2 package51 in the R-Studio software52.Fisher's exact test was used to compare the frequencies of the main agents isolated in the culture method and identified by MALDI-TOF MS, between herds and between HE and VD sows, under the significance level of 5%. For statistical analysis of alpha diversity comparing the herds and groups of healthy females and those with purulent vulvar discharge, the Kruskal–Wallis test was performed, with a significance level of 5%. For the comparison of beta diversity, the permutational multivariate analysis of variance (PERMANOVA) was performed with 999 permutations. A significance level of 5% for false discovery rate adjusted 53 to build a correlation matrix based on Pearson correlation. Plots were generated using the qgraph package54.For network analysis, the absolute abundance data were submitted to non-paranormal transformation according to Liu et al.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of  Antisense oligonucleotides (ASOs) are being developed as therapeutics for disorders of the central nervous system (CNS) . Due to in vivo activity of ASOs preclinically is by direct bolus injection into the cerebral lateral ventricles of mice [A common method for evaluating  of mice ,5. We haASOs are short, chemically modified oligonucleotides, designed to bind by Watson-Crick base pairing to perfectly complementary regions in RNA targets of interest, thereby modulating their function . BesidesCellular assays of hepatotoxicity and cytotoxicity have been developed to evaluate this hepatotoxic potential of ASOs before dosing in mice . The molin silico as well as in vitro, before dosing systemically in mice. In comparison, potential toxic effects of ASOs after intracerebroventricular (ICV) dosing in mice have only been sporadically reported to date. In the scientific literature, to our knowledge, only unintended activation of astroglial or microglial responses or inflammation have been reported for some ASOs [Taken together, the hepatotoxic potential of ASOs can be controlled ome ASOs ,19.However, neurofunctional assessment of ASOs in rodents to evaluate potential acute effects has been recommended by the Oligonucleotide Safety Working Group , and an In this study, we report on the systematic assessment and scoring of acute tolerability behavior of mice for 1 h after ICV bolus injection of 148 different LNA-modified ASOs with full phosphorothioate (PS) backbone. We find that about 40% of the ASOs evaluated have moderate to severe acute tolerability observations that preclude further development.Certain sequence features, most prominently the number and position of guanine (G) nucleotides relative to the 3′-end of the ASO, which increase the toxic potential of the ASO, and the number of adenine nucleotides, which decrease it, can be summarized as a sequence score and used to predict the acute neurotoxic potential with >80% accuracy. These sequence features were identified in a much larger set of 1,645 LNA-modified PS ASOs evaluated for changes in spontaneous calcium oscillations when incubated with neuronal cells, since this cellular assay was found to accurately predict the acute neurotoxic potential in mice. Elimination of acutely toxic ASOs early in a program allows for more robust preclinical efficacy studies, improves animal welfare practice, and reduces safety concerns should these side effects translate to higher species.Single-stranded DNA oligonucleotides with complete PS backbones and LNA-modified flanks were synthesized on a MerMade 192 × synthesizer  following standard phosphoramidite protocols. The final 50-dimethoxytrityl (DMT) group was left on the oligonucleotide for later use as lipophilic handle and chromatographic retention probe. The oligonucleotides were cleaved from the controlled pore glass solid support by means of aqueous ammonia and subsequently deprotected at 65°C for 5 h. The oligonucleotides were purified by solid phase extraction in TOP cartridges  using the DMT group, after which the DMT group was removed. As the last purification step, the oligonucleotides were eluted from the cartridge and evaporated to dryness.The oligonucleotides were dissolved in sterile 0.9% saline solution, and the concentration of oligonucleotide in solution confirmed by calculating the Beer-Lambert extinction coefficient and measuring ultraviolet absorbance. Oligonucleotide identity and purity were validated by reversed-phase ultra-performance liquid chromatography coupled to mass spectrometry. Nucleobase sequences and chemical modification patterns for all synthesized oligonucleotides are available in Adult C57BL/6J female mice weighing between 20 and 30 g  were housed with 3 to 4 mice per cage. Animals were held in colony rooms maintained at constant temperature (21 ± 2°C) and humidity (50 ± 10%) and illuminated for 12 h per day (lights on at 06:00 h). Behavioral studies were conducted between 07:00 and 15:00 h. Animals were maintained in accordance with the guidelines of the Animal Care and Use Committee of the Bristol Myers Squibb Company, and the “Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health. Research protocols were approved by the Bristol Myers Squibb Company Animal Care and Use Committee.ICV, freehand injections of ASO were performed using a Hamilton microsyringe fitted with a 27- or 30-gauge needle . The neen = 4–6 mice per treatment group). The needle was left in place for 10 s before removal. This procedure required no surgery or incision. Animals were warmed on heating pads until they recovered from the procedure.Once the needle was positioned, 100 μg ASO was given in a volume of 5 μL in 0.9% saline vehicle and injected into the right (or left) lateral ventricle over 20–30 s  hyperactivity, (2) decreased activity and arousal, (3) motor dysfunction/ataxia, (4) abnormal posture and breathing, and (5) tremor/convulsions. Each category was scored on a scale of 0 to 4. Scores for each category were summed to a final acute tolerability score going from 0 (no side effects) to 20 . The acute tolerability scores for each mouse were averaged to produce a representative score for each treatment group. The average acute tolerability scores for all ASOs dosed in mice are available in 2 and fed with 25 μL of additional media on days 4 and 8. On the day of the experiment, media was removed from the plate and the cells were washed once with 50 μL/well of 37°C assay buffer -1-piperazineethanesulfonic acid) pH 7.4). To evaluate the role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, neuronal spontaneous calcium oscillations were measured under two conditions, in the presence and absence of 1 mM MgCl2, as described previously [Primary cortical neurons were prepared from Sprague-Dawley rat embryos on embryonic day 19. Cells were plated with 25,000 cells/well onto 384-well poly-D-lysine-coated fluorescent imaging plate reader (FLIPR) plates (Greiner Bio-One) in 25 μL/well neurobasal media containing B27 supplement and 2 mM glutamine. Cells were grown for 11 days at 37°C in 5% CO2. After 1 h, 20 μL of room temperature assay buffer was added and the cells were allowed to equilibrate to room temperature for 10 additional minutes and placed in the FLIPR.Cells were loaded with a cell permeant fluorescent calcium indicator dye, fluo-4 AM (Life Technologies). Fluo-4 AM was prepared at 2.5 mm in dimethyl sulfoxide containing 20% plutonic F-127 and then diluted 1:1,000 in assay buffer. Cells were incubated for 1 h with 20 μL of 2.5 mM fluo-4 AM at 37°C in 5% CO2+ containing buffer (NMDA-independent), the total number of peaks in a 3-min period were calculated for vehicle and cyanquixaline (CNQX)-treated cells. Data were expressed as percent of vehicle-treated control.For studies confirming the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor dependency of calcium oscillations in MgFor studies used to identify ASOs for advancement to evaluation in mice, a different system to evaluate calcium oscillations was used. This system was designed to capture both increases and decreases in oscillation frequency following ASO treatment. First, baseline signal of intracellular calcium levels was read for 100 s (1 reading/s) before the addition of ASO. ASOs were added with a 384-well head in the FLIPR in 20 μL of assay buffer at 75 μM for a final concentration of 25 μM. FLIPR signal was read for an additional 200 s (1 reading/s) after the addition of ASO. After 5 min, a second read of FLIPR signal was conducted for 300 s (1 reading/s) to allow for additional data capture.Spike amplitude and frequency of calcium oscillations were calculated from FLIPR signal reads. An average control value was established by measuring the average FLIPR spike amplitude signal over a 300 s read for nontreated wells. A scoring system was developed where a score of 1 was given for each 1 s read where signal increase was >50% of the average control amplitude value. A score of 0 was given for each 1 s read, which increased <50% of average control amplitude value. For each ASO, the total summed score was calculated and converted to percent of control. Calcium oscillation scores for all ASOs are available in t-distribution. The calculated features included ASO length, number of LNA-modified sugars, the number of each type of nucleobase, and the length of the stretch of nucleotides from each end of the ASO, which did not include guanine nucleobases. Sequence features analyzed in this study are available in The association between sequence features of each ASO and calcium oscillation scores was analyzed by nonparametric Spearman's rank order correlation. Test for significance of this correlation was done using an asymptotic approximation to the Student's t-statistic. The trained model is implemented as an R function with source code available in The relation between informative sequence features and calcium oscillation scores was modeled as a weighted linear combination (Eqn. 1). Parameters of the linear model were estimated by the method of least squares and solved numerically by QR decomposition using Householder reflections. This numerical method is implemented in the lm function in the R language for statistical programming . SignifiMAPT) for behavioral side effects after ICV injection in mice. Five neurobehavioral categories were observed up to 1 h after dosing, and each scored from 0 to 4. Scores in each category were summed to a final acute tolerability score between 0 (no signs observed) to 20 (severe side effects requiring euthanasia).We assessed 148 LNA-modified ASOs targeting the premessenger RNA (mRNA) of human microtubule-associated protein Tau , and with and without 1 mM Mgllations . However2+ is known to inhibit NMDA receptors [2+ were primarily AMPA dependent. To test this, cells were incubated with AMPA receptor antagonist CNQX at five different concentrations, with or without 1 mM Mg2+ in the culture media. In the absence of Mg2+, CNQX had minimal inhibitory effects on calcium oscillations .2+) and found a significant correlation between acute tolerability scores and the calcium oscillation scores evaluated in cells .In particular, for the marked and severe tolerability classes with tolerability scores above 7, the calcium oscillations scores were almost always found to be reduced below pretreatment levels. Taken together, the data presented in MAPT pre-mRNA. These ASOs had not been selected for dosing in mice because of weaker on-target activity when evaluated in cells (data not shown). We observed a broad range of AMPA-dependent calcium oscillation scores in this set of ASOs, indicating a highly variable association between the ASO sequence and its effect on spontaneous calcium oscillations  (P < 4 × 10–10 for each parameter based on the t-statistic).where ing set) . All terThe trained linear model reproduces the observation that similar sequences in general have similar scores . ExpansiWe used the model to calculate scores for the 148 ASOs tested in mice that were set aside and not used to train the model . We observed a significant correlation with the calcium oscillation scores measured for this set of ASOs .Interestingly, the calculated scores correlated even more significantly with the acute tolerability scores observed in mice . SimilarSupporting this, a receiver operating characteristic curve analysis of classification performance of the calculated score showed an area under the curve (AUC) at 87% . ApplyinThe specificity, or negative predictive value, of the classification is at 91% in the test set. That is, if only ASOs with calculated scores above 70 had been assessed in mice, 91% of these ASOs would have acceptable acute neurotoxic potentials in mice with tolerability scores at or below 4.The test set of 148 ASOs targets 44 different and nonoverlapping regions on the MAPT pre-mRNA and in total cover 882 nts . AlthougHowever, when excluding the ASOs from the training set that overlaps with the regions covered by ASOs from the test set, and re-training the model on this reduced training set, the classification performance remains exactly the same. To further establish this, we next validated the model by calculating scores for ASOs designed to target a different human pre-mRNA. Specifically, we calculated scores for 19 ASOs designed to target the human stanniocalcin 1 pre-mRNA (STC1), which we had also assessed in mice for behavioral side effects. The 19 ASOs target 7 unique and nonoverlapping regions on the STC1 pre-mRNA . In thisFor this set of ASOs, the calculated scores correlated significantly with the acute behavioral scores, and at a cutoff at 70, achieved an overall classification accuracy of 89% . We alsoThe analysis of sequence features  as well To further test the generality of this observation, we designed 13 ASOs with no G nucleotide . For theAmong the random base sequences fulfilling the abovementioned criteria, preference was given to those where the proportion of A nucleotides roughly equaled the sum of T and C nucleotides. Finally, designs with slightly different lengths and LNA modification patterns were chosen. As expected, the calculated scores for these 13 ASOs were all >70, and, when evaluated in mice by ICV dosing, were all found to be well tolerated with acute tolerability scores below 1 .We have, in this study, reported on the serious neurobehavioral side effects we observed for two out of every five LNA-modified PS-ASOs injected into cerebral lateral ventricles in mouse brain. Across hundreds of ASOs evaluated in this manner, for those ASOs where the adverse effects manifested, they typically occurred within minutes after dosing. We have found that ASOs with high potential for this type of acute neurotoxicity often cause reductions in spontaneous calcium oscillations in primary neuronal cells, and that their nucleobase composition differs significantly from ASOs with lower acute neurotoxic potential.in silico and in vitro before ICV dosing in mice, resulting in high impact on drug discovery campaigns: (1) the identification of safe tool compounds needed to test disease hypotheses in vivo, (2) a significant reduction in animals needed for ASO testing and improvement in animal welfare, and (3) reduced risk of testing potentially toxic ASOs in higher species.We expect these results to apply to ASOs irrespective of their intended function, be that directing enzymes to act on the RNA target, blocking motifs in the RNA sequence, or affecting the RNA structure. These insights allow the acute neurotoxic potential of LNA-modified PS ASOs to be managed both We speculate that additional factors beside the impact on spontaneous calcium oscillations may also contribute to the acute neurotoxic potential observed in mice. In particular, for calcium oscillation scores ∼100 , in miceA total of 1,825 ASOs were evaluated in cells and mice in this study, all with LNA-modified flanks and a central DNA region supporting RNase H binding. We did not see any difference between sugar-modified (LNA) nucleotides and DNA nucleotides with respect to their contribution to the acute neurotoxic potential, suggesting that they impact this potential to the same extent. However, future work, for example with fully sugar-modified oligonucleotides as well as with oligonucleotides with no sugar modifications, should explore this further.Future work should also explore a wider range of sugar modifications to establish whether some sugar modifications impact the frequency and severity of these side effects to a different extent than others. Acute toxicity of ASOs with deoxy-, cET-, and 2′-O-methoxyethyl modifications to the sugar ring has been reported in patent publications in both mice and rats regardless of route of administration  . ASOs inWe only evaluated ASOs with full PS backbones in these studies. Future work should consider the extent to which this backbone modification chemistry contributes to the acute neurotoxic potential compared to other types of backbone chemistries. Similarly, ASOs where ligands have been conjugated at one or both ends might present a different profile compared to the unconjugated ASOs evaluated in this study.Comparing the informative features identified in this work for predicting acute neurotoxic potential with earlier work identifying features for predicting hepatotoxic potentials , the modWhen evaluating decision tree ensemble models similar to what was reported for hepatotoxic potentials , but traThe underlying mechanism explaining why G nucleotides increase the acute neurotoxic potential the most, and A nucleotides the least, is unclear to us. We speculate that it could be connected to basic chemical properties of the four different nucleotides. For example, guanine has the lowest redox potential of the four nucleobases, and is therefore the most easily oxidized ,31. It cIn support of this, it has previously been described that oligonucleotides can inhibit oxidation reactions primarily by a mechanism of metal ion chelation . The negin vivo tolerability. While the mechanism of this remains to be determined, one possibility is that ASOs, when taken up into neurons in mouse brain, transiently affect the membrane trafficking of AMPA receptors. This could be either by direct interactions, or indirectly, perhaps mediated by some of the chemical properties discussed above. This disruption of AMPA receptor trafficking and normal calcium transients could dynamically disrupt neuronal network activity, leading to an imbalance between excitatory and inhibitory neuronal firing in the mouse brain. Future work could explore if the findings reported in this study directly translate into changes in neuronal activity such as depression of the CNS.Our data also suggest a link between AMPA receptors, ASO sequence, and acute These or other chemical properties potentially impacting the acute neurotoxic potential are likely affected by whether nucleobases are paired as double-stranded oligonucleotides or not. We consequently speculate that small interfering RNAs ,35 or ASFurthermore, it may be possible to “dampen” the chemical properties affecting the acute neurotoxic potential, either by formulating ASOs in complex solutions instead of the simple saline solution used in these studies or by chemically modifying the guanine without impacting its binding properties negatively . This la


Escherichia coli reduction in wheat flour using atmospheric cold plasma (ACP) with three types of gas. The jet-type atmospheric cold plasma wand system was used with a 30 W power supply, and three gases  were applied as the treatment gas. The operating parameters for process optimization considered were wheat flour mass (g), treatment time (min), and gas flow rate (L/min). The wheat flour samples were artificially contaminated with E. coli at a concentration of 9.25 ± 0.74 log CFU/g. ACP treatments with argon, air, and nitrogen resulted in 2.66, 4.21, and 5.55 log CFU/g reduction of E. coli, respectively, in wheat flour under optimized conditions. The optimized conditions to reduce E. coli were 0.5 g of the flour mass, 15 min of treatment time, and 0.20 L/min of nitrogen gas flow rate, and the predicted highest reduction level from modeling was 5.63 log CFU/g.In this study we aimed to derive the response surface models for  Escherichia coli (STEC) O121 and O26, caused by consumption of uncooked baking flour [1et al. [E. coli contamination in wheat flour has been reported in other countries as well, including Turkey and Australia [E. coli outbreaks linked to wheat flour, the Canadian Food Inspection Agency (CFIA) recommended that the product should not be consumed raw [Wheat flour had not drawn attention in terms of food safety as the cooking process inactivates most of the pathogenic bacteria in wheat flour. However, concerns were raised after outbreaks between 2015-2017 in Canada and the USA of the shiga toxin-producing g flour 1. Accordiustralia . Followiet al. [et al. [et al. [Various treatment methods  have been reported to reduce microbial contamination in wheat flour -8. Li etet al.  measured [et al.  studied ACP has attracted attention as one of the more promising non-thermal technologies for the reduction of viable bacteria in difficult-to-treat foods such as dry flours. Plasma refers to a partially or entirely ionized gas composed of free electrons, photons, and ions . Additio14In particular, several studies proved that ACP application on wheat flour altered rheological parameters, but showed limited microbial reduction ability due to insufficient period of application , 18. We Escherichia coli ATCC 14763 was obtained from the American Type Culture Collection, grown according to ATCC instructions, and stored in 20% (w/v) glycerol at -80°C prior to experimental use. Thawed E. coli inoculant was transferred into full-strength tryptic soy broth  and incubated at 37°C for 24 h with constant shaking at 200 rpm. The cultured cells were centrifuged at 3,220 ×g for 15 min at 4°C, and the pellets were washed twice with sterile saline water . A 5 ml volume of 10% sucrose solution was added to the washed pellets and stored overnight at -80°C prior to freeze-drying the inoculum using a Free Zone 6-L Stoppering Tray Dry System  at -50°C for 50 h. The obtained freeze-dried powder of the inoculant (~30 mg) was stored at -20°C. For the preparation of contaminated wheat flour by E. coli, 30 g of flour and freeze-dried inoculant were placed in a sterile stomacher bag . Mixing of wheat flour and inoculants was completed by a vortex shaker  for 5 min or more until a uniform mixture was observed.All-purpose wheat flour  was purchased at a local market. The pre-inoculated flour sample was stored in a sealed container at -20°C for subsequent experiments. The ACP equipment used in this study is shown in E. coli was determined from the difference between viable cell counts (log CFU/g) of non-treatment (control) and after-treatment flour. The wheat flour samples of control and after-treatment were weighed and mixed with 9 times the volume of saline water . Then, the samples were serially diluted to an appropriate level, spread-plated on tryptic soy agar , and incubated at 37°C for 24 h.The reduction level of E. coli reduction in wheat flour with ACP treatment using the Design-Expert 13.0.2 software package . Three experimental designs were employed and consisted of different carrier gases that included argon (≥ 99.9%), air (O2 concentration 20-22%), and nitrogen (≥ 99.9%) to compare the reduction effectiveness among gas types. x1), treatment time (x2), and gas flow rate (x3). The flour samples  were treated for three durations  through various gas flow rates  using the three gases . The reduction of E. coli  was selected as a response parameter (dependent variable) for modeling with the three gases. The Box-Behnken design (BBD) is an independent, rotatable quadratic design that consists of a central point with the middle points of the edges of the cube circumscribed on a sphere [Three factors were selected for investigation of their interactions to optimize a sphere . This api and xj are the independent variables and k is the number of variables. Y is the predicted response. b0, bi, bii, and bij are constant, linear, quadratic, and interaction coefficients, respectively. Lastly, e is the random error [where xom error .p < 0.05).The validation of the regression models was performed for the optimized conditions determined by the Design-Expert program. The validation experiment was conducted for each gas. The actual combination used in the validation was determined to be the closest integer to the derived values. The predicted responses and actual responses were compared through analysis of variance (ANOVA) at the confidence level of 95% (p < 0.05). The significant p-value for ANOVA analysis and insignificant lack-of-fit value validated the significance of the model. The model equations were derived and examined after the statistical analysis.The average and standard deviations of the results were calculated based on triplicate experiments. The statistical analysis was conducted using ANOVA and Tukey’s multiple comparisons with the PRISM 9.3.1  at the confidence level of 95% , time (min), and gas flow rate (L/min), respectively. The sum of squares, mean square, F-value, and p-value, as well as insignificant lack-of-fit values, accounted for the significance of the models , but the interaction with mass and time was statistically significant. As in the current study, Mošovská et al. [E. coli inoculated on 5 g of black pepper by more than 7.65 log CFU/g. Thus, ACP treatment with air has shown effectiveness for reducing E. coli in contaminated wheat flour; however, the flow rate of air used in the ACP treatment did not influence E. coli reduction.12 L/min  were use12 L/min . The floá et al.  found thNitrogen. All of the coefficients in the nitrogen gas model were statistically significant and the most effective at reducing the number of viable E. coli among the three gases. The maximum and minimum log CFU/g reductions were 4.94 and 1.01 under conditions of 0.5 g, 15 min, and 0.12 L/min, and 1.5 g, 10 min, and 0.04 L/min, respectively. All of the coefficients such as mass, time, flow rate, and their interactions were shown to be statistically significant in the model unlike the other two types of gases. The flow rate of nitrogen gas had an effect on microbial reduction, which is shown in E. coli in wheat flour.et al. [2 : 80% N2 produced a higher bactericidal effect than using the ambient air. In addition, Patil et al. [2 : 90% N2 in carrier gases showed a higher bactericidal effect on Bacillus astrophaeus spores in comparison to atmospheric air. Finally, Han et al. [Consistent with the current study, Jeon et al.  found thl et al.  found thn et al.  reported2 value and the lack-of-fit test were determined to check the significance of the selected models. Responses of variables were plotted in a 3D response surface to interpret the correlation among different combinations. Statistical values from developed models are shown in 2 values of models for argon, air, and nitrogen were 0.9060, 0.9217, and 0.9685, respectively, and corresponding to adjusted R2 values were 0.7852, 0.8210, and 0.9279. These results indicate a high level of correlation between the actual and predicted values in the model. Values of adequate precision for the three gases were higher than 4, confirming that the models were fitted. Zhuang et al. [The Rg et al.  reportedE. coli reduction in wheat flour using atmospheric cold plasma (ACP) with three types of gas. Simultaneously, the interactions between ACP treatment factors  were evaluated. As a result, the most important factors of ACP treatment were shown to be the treatment factors mass and time. Interestingly, the interactions between factors differed by the type of gas used. The interactions of mass × time, and mass × flow rate using nitrogen gas were significantly effective on E. coli reduction but time × flow rate was not. Nitrogen gas was the most effective treatment gas on E. coli reduction in wheat flour using ACP treatment. In the validation test, ACP with nitrogen gas achieved the maximum E. coli reduction at the level of 5.55 log CFU/g. This study proposed ACP treatment models for reducing E. coli in wheat flour, and indicated that nitrogen was the most effective treatment gas after evaluation of three carrier gases. Lastly, the results of the present study have proposed the basic data for the industrial sterilization of wheat flour using ACP.In this study we aimed to derive the response surface models for


This paper addresses a puzzle involving two modes of superlative modification in definite relatives in Jordanian Arabic Absolute/Relative superlatives; Definite relative clauses; Late merger. This degree argument composes in two ways: first, it can be directly saturated by degree-denoting measure phrases of typed which combines with the gradable adjectives via function application . Alterna5The superlative operator in (3) compares the set of degrees originated in the matrix clause with the sets of degrees denoted by the contextually-determined comparison class C and it asserts that the relevant individual has the scalar relation to a degree d that no other element in the comparison class has . For exaThe superlative construction in (4) has another reading; the so-called relative reading . On the the longest article and it creates a degree abstract over the degree d in its original position. The LF that delivers the truth conditions of the absolute reading has the following representation:Under the head-external view .On the relative reading, the [-est C] moves out of the DP 1C2C3'with the template sequence ‘ʔC1C2 aC3’  may follow or precede the noun it modifies as in (7). The two forms of superlative structure vary with respect to the morphosyntax of definiteness and Case: when the superlative adjective post-modifies the noun, both the noun and the post-modifying superlative adjective have the overt definite article l as in (7.a). By contrast, the pre-modifying superlative adjective never incorporates definite morphology as shown in (7.b).Superlative adjectives , the post-modifying superlative adjective inherits the Case feature of the NP head.On the other hand, the head NP that is pre-modified by the superlative adjective bears a default genitive Case mark as in 9)  and (10)’  and (9.b) have different Case assignment domains: while the nominative and accusative Case is valued at the sentential level via an Agree operation with the c-commanding tensed TFor the ease of exposition, we will refer to the first form of superlative structure in (9.a) as A(ttributive) superlative and the second form in (9.b) as G(enitive) superlative.1.2yalli relative clause that dominates a propositional attitudinal verb, the so-called high/low ambiguity of the relative clause arises.yalli relative clause as the result of the A- and G- superlative modification .yalli relative. More specifically, it proposes a unified analysis that explains the semantic puzzle represented by the facts in (12) and (13).The facts in (12) and (13) present a puzzle: why is the relative low reading of G-superlatives blocked and such a reading is available in A-superlatives?! This paper aims at analyzing the two forms of superlative modification under the low reading of the (ya)illi relative clause modified by the superlative is a matching structure with an amount interpretation that serves as the complement comparison class of the QRing superlative operator by later merger. Section The paper is structured as follows. Section 22.1yalli relative in (14) may be analyzed relative to two main lines of analysis: the head external and head NP raising structures.Recent grammatical investigations into relative clauses have examined their properties with respect to two cases of structural ambiguity. The first case is a syntactic ambiguity in which relative clauses such as the On the head external analysis, the head NP is base-generated outside the relative clause and an internal relative clause operator undergoes A-bar movement as in (15). The operator's movement creates a predicate of individuals that composes with the NP via predicate modification. In this way, the external NP head has no representation within the relative clause .Image 3A variant of head external analysis is the matching structure in (16). On the matching analysis, the head NP is base generated in the left periphery of relative clause and an internal representation of the NP gets interpreted within the relative clause through an operation of ellipsis-based movement deletion under identity. Therefore, the head NP and its internal representation are not connected via movement chain so that the external NP doesn't exhibit reconstruction effects. It is still represented within the relative clause by a copy that is left by movement deletion. Such a copy is identical or partially similar to the external NP head . Given A-bar movement, the head NP reconstructs and gets interpreted in its base-generation position within the relative clause  modifies relative clauses that dominate a propositional attitudinal verb as exemplified in (18) .(18)the As argued in 12In (19), the –est operator compares the predicate of degrees [λd. Tolstoy wrote the d-long book x] with the predicates of degrees denoted by a contextually-determined comparison class C and it asserts that the relevant book has a length to a degree d that no other element in the comparison class has as represented in (20).13The matching analysis of (20) is represented by the following tree (21).This tree has the following output:As shown in , the preWar & Peace was the longest book written by Tolstoy, we want the predicate ‘[longest book] λx [John said that –est λd [Tolstoy had written [ the d-long book x ]]]’ to denote the singleton set consisting of War & Peace. Since this set has a unique element in its denotation, it can compose felicitously with the definite description operator.the Cossacks was the longest book written by Tolstoy and yesterday he said that War and Peace was the longest). Since the predicate of individuals, ‘[λx [that John said that –est λd [Tolstoy wrote [the d-long book x]]]’, which can be taken to refer to the set of books that John claimed to be the longest, may compose directly with the NP ‘longest book’ to yield a uniquely singleton set that can saturate the definite description, it can be judged felicitous in this situation, meaning that the entire DP doesn't actually has the denotation that the x such as John said that the longest book written by Tolstoy was x, which expresses the low reading.The composition, however, may wrongly produce unattested readings. Consider the situation where John made several claims about which book of Tolstoy was the longest from time to time  may not derive the low reading.x long (d) (w’) book(w’)’ induces a presupposition failure: it presupposes the existence of a unique pair  such that the book x is actually long to the degree d. But since the structure cannot be false for any  if d is longer than x's actual length, the definite description results into a presupposition failure and hence it renders the second argument of –est operator  (w) and book(w) …….. thex long (d) (w’) book(w’)]) undefined for the sake of comparison.14This is the case because the definite description that corresponds to the trace of movement deletion ‘thelongest is adjoined in the left periphery of the embedded clause ‘[Tolstoy wrote thex book(w’)]’. Third, the world lambda operator λw’ of the intensional verb ‘believe’ applies via intensional function application .0 on the basis of definiteness agreement.17Since the modified relative clause triggers the high/low ambiguity, it should be the case that the (ya)illi relative clause has in addition to the head NP raising analysis (26) a head external structure . Abu Helal (in press) argued that the yalli relative clause is ungrammatical in contexts where the NP head raising is required.18As argued in Abu Helal (in press), the One piece of evidence comes from the interpretation of idiomatic expressions such as (27).yalli relative clause is taken to be a raising structure: a head NP raising structure would wrongly rule in (27.c) given that the locality condition of constituency in the idiomatic expressions would be satisfied under reconstruction.The ungrammaticality of (27.c) is unexpected if the yalli relative clause in PA.Further, the following binding-theoretic facts present even stronger evidence that argues against head NP raising in the yalli relative involves a raising syntax based on A-bar movement with reconstruction effects. A head NP raising structure would wrongly rule in the ungrammatical sentence in (28.b) by inducing a condition A bleeding effect. Similarly, a head NP raising structure would wrongly rule out the grammatical sentence in (29.b) by inducing a condition C violating effect.Again, the ungrammaticality of (28.b) is unexpected if we assume that the yalli relative may have a head external matching structure in addition to the analysis in (26).Based on the facts (27–28), we can safely conclude that the 3.2yalli relative clause with the low reading in A- and G-superlatives. First, the yalli relative has an amount interpretation . Second, the superlative morpheme that modifies the yalli relative clause is not internally represented within the yalli relative clause. These two facts suggest that the yalli relative clause with the low reading involves a relation of degree operator-variable dependency that is derived via raising of a null degree operator.19This subsection argues for two facts about the yalli relative involves predication of degrees based on a degree operator movement.First, we observe that the emergence of low reading in A-and G superlatives is sensitive to constraints on the degree operator-variable dependency formed by movement. The fact that the low reading is blocked under locality and islandhood conditions of degree movement strongly suggests that the One constraint is what kul wahed ‘every one’ blocks the low reading in (31) and (32) is. That is, the dependency between the degree operator and its associate scalar term which is expressed by the lower copy of the movement deletion  is interrupted by an intervening QP. By contrast, the degree operator-variable dependency that gives rise to the high reading has no such intervening effect and hence the high reading obtains in (31) and (32).This generalization restricts the scopal behavior of degree operator in such a way that it cannot scope over other quantificational operators which c-command the degree variable that is quantified over by the degree operator. Consider the A-and G-superlatives in (31) and (32). In the two structures, the low reading is absent: the two structures are unambiguous and the only reading they have is the high reading. Clearly, the presence of the quantificational NP As such, we predict that syntactic islands such as adjuncts and wh-constituents block the low reading as well. This is indeed the case. In (33), for example, the wh- island blocks the low reading.Similarly, the adjunct island blocks the low reading as in (34).(ya)illi relative clause with the low reading has an amount reading based on quantification of degrees and the low reading of the (ya)illi relative involves a dependency that is subject to constraints on degree movement.These facts show that the low reading in the A-and G-superlative is sensitive to the locality conditions that constrain the formation of degree operator-variable dependencies. We conclude that the (ya)illi relative clause in A- and G-superlatives doesn't involve an internal representation of the external superlative operator. This degree operator is base-generated in the external position of the relativized head NP and it is not interpreted as part of the internal representation of the external NP derived by movement deletion within the (ya)illi relative clause.Second, the degree operator-variable dependency detected in the ʔktar ‘most’ is interpreted in the scope of the intensional operator bidu ‘want’ as represented by the infelicitous structure (36).In the context that Robert met with Ali, the superlative in (35) is false. It can only be true if the superlative operator the student the most.21Therefore, the superlative structure in (36) only has the logical form in (37) where the superlative operator has scope at or above the surface position of the relativized NP head yalli relative clause in A-superlatives. The overall conclusion of this section is that the yalli relative clause with the low reading has amount reading derived by the movement of a null degree operator that creates degree operator-variable dependency.These facts suggest that the superlative operator is base-generated in its surface position in the external head NP and it is never represented within the 3.3Johni is interpreted within the c-command domain of the R-expression's referential pronoun he, resulting into condition C violation.Under the copy theory of movement , 1995, thisi is interpreted in the scope of its quantificational binder every professor in LF.Such a reconstruction effect can be found in A-movement as well. For example, the structure in (39) has a bound-variable reading in which the pronoun that is dominated by the lower copy of the A- moving DP that dominates the pronoun Based on the data in (42) and (43), it can be concluded that movement, be it A-or A-bar movement, may leave fully interpretable copies and hence it may trigger reconstruction effects.Nevertheless, it has been observed that A- and A-bar movement differ with respect to Condition C. Unlike the A-bar movement structure in (40) which has Condition C effect, the A-movement structure in (41) doesn't induce such an effect.The grammaticality of (41) is unexpected under the copy theory of movement. If the A-moving expression may leave an interpretable copy ), a condition C effect is predicted. Contrary to the fact, the sentences in (41) are grammatical and it appears to be the case A-movement bleeds Condition C.John from the c-command domain of the third person singular masculine pronoun. As for (40), independent properties of the grammar may prevent the application of the late merger of the sentential restrictor of the A-moving wh-phrase and hence a reconstruction effect arises (The standard solution to this asymmetry between A- and A-bar movement is to assume that the expression dominating the R-expression in (41) undergoes Late Merger. Such a limited countercyclic merger removes the DP containing the R-expression i made’ undergoes late merger and such an operation yields a semantically interpretable output. Assume that the relative clause ‘that Johni made’ denotes a predicate of individuals of type <et>. Assume further that the DP ‘which argument’ undergoes QRing. The relative clause, ‘that Johni made’, can compose with the denotation of argument via predicate modification.On the LF interpretability approach , late mei is a genius’ in (45.b), on the other hand, fails to produce a compositionally interpretable representation and hence late merger should not be an option for cases like (42.b). Let us see how the late merger of the complement ‘that Johni is a genius’ doesn't yield a compositionally interpretable output.The late merger of the complement ‘that JohnOn (ii) determiner replacement where the Det is replaced by a definite description operator that applies to the predicate that is introduced by predicate modification to yield (44), which is an expression of type e.Given this assumption, we can see how the complement in (42.b) may not undergo late merger. Assume that the lower copy of the wh-phrase ‘which argument’ in (42.b) is of type <t,et> .The LF interpretability approach  may exteTo illustrate, They analyzed the fact in (45) as follows. The DP ‘a picture’ undergoes rightward QRing across the adverbial yesterday. The movement forms a chain in which the DP is pronounced in the tail. Then the adjunct merges late to compose with the restrictor of the DP ‘argument’ as represented in (46).In this way, late merger removes the R-expression dominated by the relative clause from the c-command domain of its co-referential pronoun and Condition C can be circumvented by extraposition of adjuncts. Notice that the tree (46) is a semantically interpretable output. Interestingly, i] merges late to compose as the restrictor of the degree operator as schematized in (48).Again, the late merger of the comparative complement removes the R-expression dominated by the comparative complement from the c-command domain of its co-referential pronoun and Condition C can be circumvented by extraposition of comparative complements. The tree in (48) is semantically interpretable as well.i is a genius] as schematized in (50):Back to the asymmetry between A- and A-bar movement w.r.t. Condition C, As shown in (50), the restrictor dominating the R-expression may undergo wholesale late merger with the QRed determiner into a position that is higher than the c-command domain of the co-referential pronoun, but still lower that the Case assigning functional head T.i is a genius] is blocked in A-bar movement in (51), repeated from (49.b) and therefore a Condition C effect arises.In the contrary, i is a genius]’ is achieved structurally by a Goal-Probe Agree operation based on c-command relation with the Case assigning functional head v .They proposed that it is Case Filter that prohibits this operation. On the assumption that the accusative Case marking assignment of the wh-phrase ‘which’ along with its sentential complement ). Intuitively, this set of predicates of degrees include the set that PROx and other relevant articles are long to.27Since the sister of ⟦ -est C ⟧ in (63) denotes the predicate of degrees . Therefore, the members of ⟦ C ⟧ are the predicates of degrees such that Ali and other article-readers read a d-long article. On this way, the comparison class complement ⟦ -est C ⟧ denotes predicates of predicates of degrees of type <dt, t> which represents the comparison class complement of the first argument of the ⟦ -est ⟧ operator G.yalli relative clause undergoes late merger to compose with the comparison class complement C via predicate modification as represented. The type shifted yalli relative clause with the denotation. λD’<dt> . ∃d’ [Ahmad said that λw’ Einstein wrote [ιx. x is d’-long y ] in w’] & D’ = λd’’. d’’≤ d’] composes with ⟦ C ⟧ intersectively as represented in (71).We propose that the The LF representation in (72) expresses the truth conditions of the relative low reading of A-superlative.the longest paper along with its nominal modifier the yalli relative clause YALLI Ahmad said that Einstein wrote requires Case. Since the superlative head undergoes QRing into a Case-assigning position , the yalli relative can be introduced at this position to receive Case.We assume that the whole DP yalli relative clause to compose with the denotation of the comparison class complement ⟦ C ⟧ via predicate modification. The unavailability of this reading is well predicted under the current analysis: since the NP head along with its yalli relative clause reading requires Genitive Case which is assigned by the Case assigning functional head D0,yalli relative clause cannot be introduced outside the c-command domain of D0 . In this way, deriving the relative low reading of G-superlative is blocked by Case Filter as shown in (73).So what blocks the relative reading of G-superlatives with the low reading?! Recall that under the late merger analysis, the relative low reading of the G-superlative is derived through the DegP ⟦ -est C ⟧ QRing into Spec-VP followed by the late merger of the Our analysis makes one more correct prediction: it explains the asymmetry between A-superlative and G-superlative with respect to Condition C as exemplified in (74), repeated from (53).‘told-him’. Such a reading holds true if the teacher told Ali to read the article x to a degree d and there is no other article among the ones that Ali read which is as long or longer than d. A late merger analysis where the est-operator undergoes QRing as high as the VP ‘told-him’ followed by the late merger of the yalli relative clause can derive this new scopal reading and it also removes the R-expression Alii that is dominated by the yalli relative clause out of the c-command domain of its co-referential pronoun. Therefore, the A-superlative in (74) gives rise to a Condition C bleeding effect. Notice that this operation is not blocked by Case filter: the superlative operator QRs into a Case assigning position  has a reading in which the comparative operator –est takes scope over the VP yalli relative as high as the matrix VP is blocked by the need of the yalli relative clause to be Case-marked at the lower DP level, a condition C effect is inevitable.As for the ungrammatical G-superlative in (74.b), since the late merger of the 5yalli relatives in Jordanian Arabic: while the A-superlative is ambiguous between the absolute and relative readings under the low reading of the yalli relative ttributive and (G)enitive superlatives that modify relative , the G-syalli relatives. Our analysis is based on the following well-motivated working assumptions that were used to derive the low reading of the yalli relative clause: (i) the yalli relative clause undergoes late merger to compose with the comparison class complement of the QRed operator via predicate modification. (ii) Such composition may take place at the DP level to form the LF associated with the absolute low reading of the A- and G superlative and at the VP level to form the LF associated with the relative low reading in A- superlatives. (iii) Since late merger is controlled by Case filter  such a Case requirement explains the asymmetry between the A- and G-superlatives with respect to Condition C.To resolve the puzzle, we proposed a compositional analysis that explains the asymmetry between these two modes of superlative modification in e filter : 164, thAbdel-Rahman Abu Helal: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Al-Zaytoonah University of Jordan [grant number QF21/1803-3.0].This work was supported by the Deanship of Scientific Research at Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


P. alba  clones and their characterization to be used as non-conventional sources of potential functional ingredients. Seed flour and PEE obtained from Argentinian P. alba cultivars were chemically characterized. The antioxidant capacity was also determined. The results showed variability in macronutrient composition of seed flour obtained from different clones. Among them, seed flour obtained from P4, P5, P6, P10, P12, and P13 clones showed a higher protein and fiber content than the other clones. On the other hand, PEE obtained from P6, P7, and P10 clones showed the highest content of phenolic component (7.32–8.58 mg GAE/g flour). The extracts obtained from them also showed high antioxidant activity . C-glycosyl flavones, including vicenin II, isoschaftoside, schaftoside, vitexin, and isovitexin were the major components extracted in all clones. These compounds have remarkable properties for disease prevention linked to oxidative stress. Therefore, the P. alba seed could be considered as functional food waste with a great potential to be used as a novel renewable and sustainable material for the production of bioactive food formulations.The present study describes how flour and phenolic enriched extracts (PEE) are obtained from seed (food waste) of 10 different  Prosopis includes 45 species that belong to the Mimosoideae subfamily within the Fabaceae family, distributed in America, Asia, and Africa [Prosopis grows easily in tropical, subtropical, and semi-arid soils, enduring high temperatures, drought, alkalinity, and soil salinity [Prosopis spp. [Prosopis wood is harvested annually, to be used in the making of doors, floors, blinds, and windows [Prosopis pods have been used since pre-Hispanic times to feed both livestock and human. They are used to prepare drinks , and numerous foods such as syrups, flour, and sweets  [P. alba whole pod flour (Article 681 tris) and P. alba seed flour (Article 681) are included in the Argentine Food Code [Prosopis flours were obtained from whole pods, although in this process the seed were discarded as waste. The pods flours were evaluated functionally and chemically and its properties, i.e., antioxidant, hypoglycemic, antilipase, and anti-inflammatory activities were demonstrated, but the P. alba seed flours were not yet fully characterized [P. alba whole pod flour is growing, thus increasing the generation of biomass that should be used. In this sense, an important aspect regarding economic benefit is to recover the high-added-valued constituents present in fruit processing wastes. The availability of this vegetable raw material, together with the problem represented by the accumulation of waste and its unexploited potential, has encouraged researchers to develop projects on the value-addition potential of fruit processing waste, such as enrichment in food formulation.d Africa . Prosopisalinity . In Argepis spp. . In our , patay) . In factood Code . Until nArticle 61 tris ancterized ,12,13,14P. alba flour is its high variability, for both biomass and pod production [Prosopis are pollinated by insects and are self-incompatible. Therefore the seeds of the same tree are highly likely to have multiple crosses. In this way, clonal propagation is an essential tool for the genetic improvement of P. alba. Fortunately, it can be propagated clonally by grafting, rooting the cuttings, and air layering [P. alba have been made for its economically valuable characteristics, such as rapid growth rate, high pod production, and improved flavor (sweeter pods without bitter characteristics) [One of the main difficulties faced by the industrialization of oduction . Regardilayering . Clonal ristics) ,16. Progristics) ,16,17.P. alba clones.The present study describes for the first time the chemical characterization and antioxidant capacity of flour and phenolic enriched extracts (PEE) obtained from seeds of previously selected Prosopis alba were collected in an experimental field of the Universidad Católica de Santiago del Estero in a town called Fernández, in the province of Santiago del Estero, Argentina. The mature pods of 10 clones of The pods with the same ripening stage were cleaned so as to remove any strange material and dried in a forced-air oven at 50 °C until reaching constant weight. The pods were named P1 to P13. The pods P2, P8, and P11 were discarded because they had abundant polluting plant material. The dried pods were milled and then sieved to separate both the mesocarp flour and whole seeds with endocarp (considered as waste). The seeds B,C were P. alba seed flour was carried out to obtain proteins and sugars [A differential extraction from d sugars . Crude pd sugars . Total cd sugars . Crude fd sugars . Resultsd sugars . The latd sugars .Fiber content was determined by using acid and alkaline digestion and heating . Crude fP. alba seed flour was mixed with 50 mL of 70° ethanol in an ultrasonic bath for 10 min at 25 °C. Then, the samples were placed in a shaker for 24 h at 25 °C and 120 rpm. Then, each sample was vacuum filtered to obtain the phenolic enriched extract (PEE). Then, the PEE was dried under reduced pressure at 40 °C and freeze-dried. The powder obtained was stored at −20 °C until use. Total phenolic compounds content was determined by using Folin-Ciocalteu reagent  [Five g of g for 5 min , and non-flavonoid phenolic compounds were quantified in the supernatant with the Folin–Ciocalteau reagent. Flavonoid phenolic compounds were calculated by using the difference between the total phenolic and non-flavonoid phenolic content. Flavone and flavonol content were determined by using the aluminum chloride colorimetric method [Total non-flavonoid phenolic compounds were determined by using the method described by Isla et al. (2014) . First, c method . Hydrolyc method .The chromatographic profiles of PEE were obtained by HPLC coupled to a diode array detector (HPLC-DAD)  in an analytical C18 column (XBridge) by using a linear gradient solvent system consisting of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol as follows: 90% to 43% A for 45 min, 43% to 0% A for 45 to 60 min, holding 100% B for 5 min. The injected sample volume was 20 µL with a flow rate of 0.5 mL/min. The identification of phenolic compounds was carried out by comparing the retention times and spectral data (220–600 nm) of each peak with those of standards  from Sigma-Aldrich  and Fluka Chemical Corp. .●+ scavenging activity was measured [●+ solution and 100 µL of sample to final concentrations between 2 and 10 µg GAE/mL of phenolic compounds. Absorbance was measured at 1 and 6 min at 734 nm. The sample concentration required to scavenge 50% ABTS●+ was determined as 50% ABTS●+ scavenging capacity (SC50). Results are expressed in µg gallic acid equivalent (GAE)/mL of PEE.ABTSmeasured  on a mic2O2) scavenging activity of PEE obtained from seed flours was carried out through the optimized enzymatic colorimetric assay [2O2, phenol, and 4-aminoantipyrine reaction system in the presence of horseradish peroxidase (HRP), at 37 °C. This reaction was measured at 504 nm. SC25 values denote the sample concentration required to scavenge 25% H2O2. Results are expressed in µg gallic acid equivalent (GAE)/mL.Hydrogen peroxide (Hic assay  adapted 3 and different extract concentrations (up to 20 µg GAE/mL) with and without EDTA. The reaction was started with H2O2 and ascorbic acid at 37 °C for 60 min. Then, 2-thiobarbituric acid was added and incubated for 20 min at 100 °C. Absorbance was recorded at 532 nm. Results are presented as SC50 values in µg GAE/mL required to inhibit the degradation of 50% 2-deoxy-D-ribose. Quercetin (5–50 µg/mL) was used as a reference compound.The hydroxyl radical scavenging activity of PEE was determined according to Cattaneo et al. (2016) . The reap ≤ 0.05 was considered significant.The comparison of mean values was made by ANOVA, followed by a Tukey post-test. A value of 50 values of free radicals by several methodologies for each flour sample were exported in a matrix of 14 columns and 30 rows, where each column represents a variable and each row represents a clone. Principal component analysis (PCA) and hierarchical clusters (HCA) using the Infostat software version 2015 [The chemical components concentration and the SCion 2015   [P. alba mesocarp flour [Clones of gentina) . The tregentina) ,16. The rp flour . The floProsopis species is rich in sugars [The carbohydrate content of seed flour exhibits the total carbohydrate content between 5.16 g/100 g and 23.15 g/100 g. P6, P7, P9, and P10 showed the highest sugar content. The total sugar content of seed flour was lower than that of mesocarp flour (73.95 g/100 g), data reported by Cardozo et al., 2010 [n sugars ,30 and tP. alba seed flour varied between 7.98 and 13.34 g/100 g. P4 and P5 clone seed flours showed even greater total protein content than P. alba mesocarp flour [P. alba seed flours showed this condition; only P3 and P9 clones exhibited less than 10% protein values. Fat values were lower in seed flour (1.70 to 3.98 g/100 g) than in mesocarp flour (11.3 g/100 g) [The protein content  of P. alrp flour . Accordirp flour , when a P. alba seed flours had a higher fiber content than that reported for mesocarp and cotyledon flour of P. alba [Crude dietary fiber content varied between 13.90 and 27.69 g/100 g. P1, P4, and P5 showed the highest crude fiber content. The deficiency of dietary fiber in the human diet has been associated with constipation, diverticulosis, cardiovascular diseases, and cancer, so its study is important . In addi P. alba . P4 flouThe low moisture content (less than 10%) would improve its storage stability by preventing mold growth and other biochemical reactions and extending the final product shelf life. All seed flours have this condition, except for P10 and P12 .P. alba cotyledons, seeds, and mesocarp flour [P. alba. The total phenolic compound content extracted from P. alba seed flour was between 5.05 and 8.58 mg GAE/g of flour. These values are higher than that obtained by Sciammaro et al. (2016) [P. alba seeds collected in Ingeniero Juárez, Formosa in the northeast of Argentina, but were lower than the total phenolic content of P. alba cotyledon flour (11 mg GAE/g of flour) reported by Cattaneo et al., (2016) [P. alba collected in Amaicha del Valle . Flours of P6, P7, and P10 clones showed the highest phenolic content, higher than P. alba mesocarp flour (4.6 mg GAE/g flour), [Ceratonia siliqua pulp flour [P. alba seed flour (2.68 to 4.01 mg GAE/g of flour). P10 flour showed the highest flavones and flavonol content. The flavonoid content was between 0.098 and 0.308 mg of quercetin equivalent (QE)/g flour (mg) and was lower than those found in P. alba cotyledons flour (3.9 mg EQ/g flour) and mesocarp flour (33–87.5 mg EQ/g flour) [The secondary metabolites play an important role in the defense systems of plants against environmental stress and pathogenic attacks. In addition, these compounds have various biological activities, so they are used as medicinal, food, and cosmetic ingredients . There arp flour ,12,13,14, (2016)  for P. alp flour . Non-fla2E)/100 g flour. The highest content was observed in P10, P12, and P13 clones. P7 presented a similar content of these compounds to that reported to P. alba cotyledon flours (175 mg PB2E/100 g flour) [Regarding the content of condensed tannins, the values varied between 44.75 and 223.73 mg of procyanidin B2 equivalent  [•+ scavenging activity by the PEE was lower than that of a commercial natural antioxidant such as quercetin (SC50 = 1.4 µg/mL) but similar to BHT, a synthetic antioxidant used in the food industry (SC50 = 3.52 µg/mL).This study analyzed the antioxidant activity of PEE obtained from seed flour . The sam GAE/mL) . The ABTP. alba seed flour also showed scavenging activity of H2O2 with SC25 values between 5.9 and 13.6 for the most active extracts and between 20.5 and 23.3 µg GAE/mL for the less active extracts. Scavenging activity of HO• was also found with SC50 values lower than those found for PEE from P. alba mesocarp flour [C-glycosyl flavonoids, compounds with demonstrated antioxidant capacity [The PEE of rp flour . Phenolicapacity ,38,39,40Considering the macronutrient components as variables, the principal component analysis (PCA) showed two components that explain 74.2% of the variability A. PrinciWhen attempting to determine the groups of secondary metabolites, two well-marked—besides three individual ones—appear A.The group with the highest number of individuals  is determined by the highest condensed tannin concentration. The second group, P9 and P3, is characterized by the lowest content of all phytochemicals. On the other hand, clones P1, P7, and P10 are each considered to correspond to a different group. P10 showed the highest content in flavones and flavonols, total phenolic, and non-flavonoids. P1 and P7 showed lower values of these compounds concerning P10 but higher than the other groups.Hierarchical cluster analysis by proximal composition B suggestP. alba fruit waste.The results obtained in this study established that principal component analysis and hierarchical cluster analysis were efficient tools for clone selection according to the chemical composition and functional properties of C-glycosyl flavones and high antioxidant activity.Seed flour obtained from P4, P5, P6, P10, P12, and P13 clones showed a higher protein and fiber content than the other clones. On the other hand, PEE obtained from P6, P7, and P10 showed the highest content of phenolic component, mainly P. alba fruit wastes have a great potential to be used as a novel renewable and sustainable material for the production of bioactive food formulations.Considering the potential use of seed flour for nutritional properties or phytochemical extraction, the selection of adequate clones is possible. Therefore, both the flour and the PEE obtained from