In this study, a chemotaxonomic tool was created on the basis of ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS) for the identification of 13 common Finnish deciduous trees and shrubs from their leaf bud metabolites. The bud extracts were screened with UHPLC–ESI–QqQ–MS and UHPLC–ESI–Q–Orbitrap–MS to discover suitable markers for each species. Two approaches were tested in the marker selection: (1) unique species-specific markers to obtain selective fingerprints per species and (2) major markers to maximise the sensitivity of the fingerprints. The markers were used to create two selected ion-recording-based fingerprinting tools with UHPLC–ESI–QqQ–MS. The methods were evaluated for their selectivity, repeatability, and robustness in plant species identification by analysing leaf buds from several replicates of each species. The created chemotaxonomic tools were shown to provide unique chromatographic profiles for the studied species in less than 6 min. A variety of plant metabolites, such as flavonoids, triterpenoids, and hydroxycinnamic acid derivatives, were found to serve as good chemotaxonomic markers for the studied species. In 10 out of 13 cases, species-specific markers were superior in creating selective and repeatable fingerprints. The chemical diversity of plants and the special chemical features of different plant families, genera, and species provide exciting possibilities for their discrimination and chemotaxonomic classification. Such discrimination is useful for not only educational  and specBetula pubescens- and Betula pendula-type birch species, and HPTLC (high-performance thin-layer chromatography) was used by Melnyk et al. − ion at m/z 191 and a [caffeic acid–H]− ion at m/z 179, as well as its fragments at m/z 135 and 161. Monocaffeoylquinic acids have been reported in many species of the genus Sorbus − ion at m/z 315 was observed, indicating the loss of a caffeic acid moiety. Eyles et al. reported calceolariosides A and B in other Fraxinus species, and according to their data, calceolarioside A elutes before B in reverse-phase chromatography − ion at m/z 359, and it has been found in Acer saccharum Marsh. buds previously [− or [syringic acid–H]− ion at m/z 197, and an [M–H–44–30]− ion at m/z 123 due to carboxyl and methoxyl losses. Markers 6, 7, and 8 shared some fragment ions with the glucosyringic acid. Additionally, the MS/MS spectrum showed an [M–H–198]− ion at m/z 313 from the cleavage of syringic acid, an [M–H–152–14]− ion at m/z 345, and a [gallic acid–H]− ion at m/z 169. Therefore, the compounds were tentatively identified as monogalloyl syringyl glucoses. They eluted as three not fully separated peaks at 0.70, 0.79, and 0.88 min, suggesting three isomers. Marker 11 also showed evidence of galloyl and syringyl moieties. The MS/MS spectrum showed an [M–H–152]− ion at m/z 511 from the loss of gallic acid, an [M–H–198]− ion at m/z 465 from the loss of syringic acid, and an [M–H–152–198]− ion at m/z 313 . The compound was suggested to be digalloyl syringyl glucose. All four markers of eviously . The MS/15 at m/z 621 was the [M–2H]2– ion of heptagalloyl glucose previously identified in A. platanoides [− ion at m/z 1243. It eluted at 1.15 min and had UV maxima at 221 and 278 nm. The MS/MS spectrum showed an [M–H–152–152–170]− ion at m/z 769, an [M–2H–152–152]2– ion at m/z 469, an [M–H–152–152–152–170]− ion at m/z 617, an [M–2H–152–152–152]2– ion at m/z 393, and a [gallic acid–H]− ion at m/z 169. Marker tanoides . The MS A. platanoides samples. Glucosyringic acid was the most intensive peak at the earliest retention time. The second peak with a shoulder was monogalloyl syringyl glucose, the third peak was digalloyl syringyl glucose, and the fourth peak was heptagalloyl glucose.All four markers contributed to the LC–MS fingerprints of Sorbus samples were triterpenoids or triterpenoid derivatives. The UV and MS/MS spectra of markers 27 and 29 indicated that both of them included an additional hydroxycinnamic acid moiety. Marker 27 had a UV maximum at 309 nm and fragment ions characteristic of coumaric acid. The additional fragment ions of [M–H–18]− at m/z 615 resulting from the cleavage of water, [M–H–44] − at m/z 589 resulting from the loss of a carboxyl group, and [M–H–18–44]− at m/z 571 resulting from the loss of both could correspond for the triterpenoid part, which was tentatively identified as rotundic acid. De Tommasi et al. found similar triterpenoid derivatives in Eriobotrya japonica, including 3-O-trans-p-coumaroylrotundic acid − ion at m/z 605.4 and an [2M–H]− ion at m/z 1211.8. The MS/MS spectrum showed an [M–H–132]− ion at m/z 473 resulting from the cleavage of arabinose and a minor [M–H–132–18]− ion at m/z 455 resulting from the additional cleavage of water. Although curculigosaponin B has only been previously reported in Curculigo orchioides [Alnus species [32 and 34 did not contribute to the overall fingerprints of the A. glutinosa samples because of the higher intensity of the other markers. They were also identified as saponins, isomers, of alnustic acid arabinoside. The MS/MS spectrum showed aglycone at m/z 487. The loss of the carbon side chain yielding a fragment ion at m/z 389 and the following cleavage of carboxyl group or water resulting in the ions at m/z 345 and 371, respectively, were also detected in the MS/MS spectrum. Alnustic acid arabinoside was initially characterised in Alnus serrulatoides [Alnus species as well [In addition to the chioides , other t species . Markerslatoides , and lat as well . A. glutinosa, the MS/MS spectrum of markers 25 and 26 had similar characteristics. The lack of a UV spectrum and similar retention times suggested that markers 25 and 26 could be structurally similar to markers 31, 32, 33, and 34, meaning that they could also be triterpenoid saponins, although with a different sugar part because the neutral loss of glucose or arabinose could not be detected from the MS/MS spectrum. Markers 25 and 26 produced the first main peak in the fingerprints of the A. glutinosa samples.Compared with the other markers of 2, 28, 37, and 40–42 could not be reliably assigned to any of the compound classes discussed above. The molecular ion of marker 2 had an even m/z value, which suggests that it contains and odd number of nitrogen atoms. Marker 2 was a minor peak in the fingerprints of F. excelsior. Marker 28 was detected in all of the studied species. However, the combination of 28 and 37 detected as two peaks was not obtained for any other species than the Tilia species. Markers 40–42, found in A. incana, shared similar MS/MS characteristics, which could indicate that they belong to the same molecular family. The smallest fragments could have been derived from a coumaroyl moiety.Markers LC–MS-grade acetonitrile was purchased from Merck , and LC–MS-grade formic acid was purchased from VWR International . Water was purified with the Millipore Synergy water purification system . Ethanol  was purchased from Altia .v/v) solution and shaking for 10 min. The extract was filtered with a 0.20 µm PTFE filter.Leaf bud samples were collected from the Turku area, Southwest Finland, in spring of 2019, 2020, and 2021. After collection, the samples were frozen, lyophilised, and stored in a freezer. The extraction protocol was modified from Lahtinen et al. [v/v) (B). The elution profile was as follows: 0–0.3 min, 10% A in B; 0.3–3.1 min, 10–75% A in B (linear gradient); 3.1–3.5 min, 75% A in B; 3.5–3.6 min, 75–95% A in B; and 3.6–5.0 min column wash and stabilisation. The flow rate was 0.65 mL/min, and the injection volume was 5 μL. Mass analyses were performed using an ESI source and negative ionisation. The ESI conditions were as follows: capillary voltage, 1.8 kV; source temperature, 150 °C; desolvation temperature, 650 °C; desolvation and cone gas (N2), 1000 and 100 l/h, respectively; and collision gas, argon. The mass range for the full scan screening was set to m/z 150–2000. The selected ion recording (SIR) parameters are presented in The screening for markers and final fingerprinting analysis was performed using an Acquity UPLC system  coupled with a Xevo TQ triple−quadrupole mass spectrometer (Waters Corp.). The UPLC system consisted of a sample manager, a binary solvent manager, a column , and a diode array detector. The mobile phase consisted of acetonitrile (A) and water/formic acid  flow rate, 60 (arbitrary units); aux gas (N2) flow rate, 20 (arbitrary units); sweep gas flow rate, 0 (arbitrary units); and capillary temperature, +380 °C. The in-source collision-induced dissociation energy was 30 eV. A resolution of 35,000 and an automatic gain control of 3×106 were used for full scan MS data. The mass range was set to m/z 150−2250. MS/MS data, namely, dd-MS2 (Top N) data, were acquired using a resolution of 17,500; an automatic gain control of 1×105; a TopN of 7; and stepped normalised collision energies (NCEs) of 30, 50, and 80. The calibration was performed with a Pierce ESI Negative Ion Calibration Solution  and was the most accurate at m/z > 250. The data were processed with Thermo Xcalibur Qual Browser software .A similar Acquity UPLC system was configured with a hybrid quadrupole–Orbitrap mass spectrometer . The column and gradient were similar to the ones used in the QqQ analysis. The injection volume was 5 μL, and the flow rate was 0.65 mL/min. The heated ESI source  was operated in the negative ion mode. The parameters were set at as follows: spray voltage, −3.0 kV; sheath gas (N5 (for MS scans) and 1×104 (for MS/MS scans). MS chromatograms were built using the ADAP chromatogram builder [4, a minimum highest intensity of 3×105, and an m/z tolerance of 0.01. The chromatographic deconvolution was achieved by the local minimum search algorithm with the following settings: m/z range for MS/MS scan pairing of 0.01, RT range for MS/MS scan pairing of 0.1 min, chromatographic threshold of 75%, search minimum in RT range of 0.05 min, minimum relative height of 1%, minimum absolute height of 1×105, minimum ratio of peak top/edge of 1.5, and peak duration range of 0.2–1.0 min. Chromatograms were deisotoped using the isotopic peaks grouper algorithm with an m/z tolerance of 0.01 and an RT tolerance of 0.01 min. The chromatograms were then aligned together into a feature list with the join aligner module at an m/z tolerance of 0.01 . The aligned feature list was gap-filled using the multithreaded peak finder module  and filtered using the peak filter module by area (5 × 105–1 × 1010) and height (1×105–1×1010). Finally, the feature list was filtered using the feature list rows filter to remove peaks with fewer than two peaks in an isotope pattern.The MS/MS data files were converted from the Thermo Scientific .raw data format to .mzXML with the MS Convert Software included in the ProteoWizard package . The .mz builder , with a In this study, two alternative approaches were applied in the selection of marker candidates for the leaf buds of 13 common Finnish tree species. The approach using MZmine was found to be suitable for discovering species-specific markers, and it demonstrated differences in the chemical diversity of the studied species. The main ions chosen by the second approach were less specific to the species, but they produced repeatable fingerprints. The final UHPLC–MS fingerprinting tool utilised a combination of markers obtained with the different approaches. The fingerprinting tool enabled species identification from a single leaf bud in less than 6 min. The high-resolution mass data revealed that the markers belonged to many different plant metabolite subclasses such as flavonoids, hydroxycinnamic acid derivatives, and triterpenoids. The structural diversity of the markers qualitatively demonstrated the diversity in the leaf bud chemistry of different species.


Prunus armeniaca L.) in most of the countries is spring frost. Thus, the breeding efforts have concentrated on the use of late‐blooming genotypes as a means of frost avoidance. The aim of the present study was to identify late‐blooming genotypes with high fruit quality among seedling‐originated trees. Firstly, pre‐selections were done according to blooming time within 278 apricot seedling‐originated trees. Secondly, the late‐blooming selections were further evaluated according to their vegetative and fruit characteristics to determine superior types. Significant differences were observed among the late‐blooming genotypes in terms of the traits recorded. Fruit ground color was strongly variable, including white, yellow, yellow–green, light orange, orange, and dark orange. Fruit weight ranged from 27.37 to 33.99 g, fruit flesh thickness varied from 11.16 to 13.47 mm, and total soluble solids varied between 17.00% and 23.87%. Hierarchical cluster analysis (HCA) performed with Euclidean distance and Ward's method divided the genotypes into two main clusters based on morphological traits and in some cases, the genotypes belonging to an area were clustered into different clusters. All the 48 late‐blooming date genotypes selected could be useful as a parent to improve flowering season of cultivars. Also, among them, 10 genotypes were superior in terms of fruit quality‐related characters, such as fruit weight, fruit color, fruit taste, and TSS, and thus they can be singled out for cultivation.One of the major limiting factors in the intensive spread of apricot ( Prunus armeniaca L.) genotypes could be useful as a parent to improve flowering season of cultivars. Also, among them, 10 genotypes were superior in terms of fruit quality‐related characters, such as fruit weight, fruit color, and fruit taste, and thus they can be singled out for cultivation.All the 48 late‐blooming date apricot ( The generic term apricot includes four different species, viz, P. armeniaca L., the cultivated apricot; P. sibirica L., the Siberian apricot; P. mandshurica (Maxim.) Koehne, the Manchurian apricot; P. mume (Siebold) Siebold & Zucc., the Japanese apricot, and one naturally occurring interspecific hybrid, Prunus ×dasycarpa Ehrh., the black or purple apricot , healthy, and had a full crop. General orchard management, including irrigation, nutrition, pest, and disease control, was consistent with commercial practices.2.2The selected late genotypes were evaluated using 47 morphological and pomological traits to select superior selections. Length, width, and thickness for leaf, fruit, stone, and kernel were measured using a digital caliper. Weight for fruit, stone, and kernel was measured using an electronic balance with 0.01 g precision. Total soluble solids (TSS) content was determined using a refractometer , in ◦Brix. The remaining characters were qualitatively determined based on rating and coding according to the apricot descriptor  was performed to evaluate the variation among the genotypes based on the traits measured using SAS software , early (62 genotypes), moderate (148), and late (48). Significant variabilities in blooming time of genotypes under the same geographical conditions might be a result of the total exposure temperature required. Full blooming immediately follows the end of the dormancy period  to 90.18% (ripening date). The CV was more than 20.00% in 28 out of 47 characters recorded (Table Tree growth habit was standard (33 genotypes) and open 15). Tree growth vigor, tree height, branch density, and leaf density were predominantly moderate Table . The ran5. Tree gRipening date ranged from late May (40 genotypes) to early June (8). Extending the ripening season is interesting in breeding programs of apricot. The influence of growing degree‐day thresholds on ripening time is very important for apricot‐producing regions , yellow (15), yellow–green (7), light orange (6), orange (6), and dark orange (3). Also, there was significant diversity among the genotypes in terms of fruit flesh color, ranging from white to orange Table . The fruThe range of fruit‐related characters was as follows: fruit length: 34.99–38.20 mm, fruit width: 31.32–36.88 mm, fruit weight: 27.37–33.99 g, and fruit flesh thickness: 11.16–13.47 mm. The TSS varied between 17.00% and 23.87% Table . Thus, fStone length ranged from 28.13 to 30.92 mm, stone width varied from 20.65 to 24.37 mm, stone thickness varied from 10.40 to 11.78 mm, and stone weight varied from 1.48 to 2.71 g. The range of kernel‐related characters was as follows: kernel length: 18.27–20.19 mm, kernel width: 13.62–17.51 mm, kernel thickness: 6.65–7.41 mm, and kernel weight: 0.70–0.81 g. Kernel taste was bitter (17 genotypes), moderate (24), and sweet (7). Recently, sweet kernels of apricots have been used for direct consumption as a snack food like almond. Also, bitter kernels are used in the pharmaceutical and cosmetics industries , branch leaf width: (r = .53), spur leaf length (r = .58), spur leaf width (r = .51), and fruit length (r = .75), and fruit width (r = .73) and corresponded with the previous findings . Fruit weight was positively and significantly correlated with branch leaf length . The PC1 accounted for 6.24% of the total variance and was significantly correlated with tree growth habit, fruit ground color, and fruit flesh color. Three characters, including kernel color, kernel shriveling, and kernel taste, were placed into PC2 and accounted for 6.12% of the total variance. Fruit apex shape and fruit shape were correlated with PC3, which accounted for 5.69% of the total variance. Previously, PCA revealed that fruit‐related characters were important to distinguish the genotypes of apricot . Ali Khadivi: Formal analysis (lead); Methodology (lead); Project administration (lead); Software (lead); Supervision (lead); Validation (lead); Writing – review & editing (lead).None.None.


Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and gram-positive Staphylococcus aureus methicillin-resistant and susceptible strains. The most effective formula of AgNPs tested had single-digit (μg/mL) minimum inhibitory concentrations against gram-negative multidrug-resistant clinical bacterial isolates with novel and emerging mechanisms of resistance. The mode of killing was assessed in E. coli and was found to be bactericidal, which is consistent with previous studies using other AgNP formulations. We evaluated cytotoxicity by measuring physiological readouts using the Caenorhabditis elegans model and found that motility was affected, but not the lifespan. Furthermore, we found that at their antibacterial concentrations, AgNPs were non-cytotoxic to any of the mammalian cell lines tested, including macrophages, stem cells, and epithelial cells. More interestingly, our experiments revealed synergy with clinically relevant antibiotics. We found that a non-toxic and non-effective concentration of AgNPs reduced the minimum inhibitory concentrations of aminoglycoside by approximately 22-fold. Because both aminoglycosides and Ag are known to target the bacterial ribosome, we tested whether Ag could also target eukaryotic ribosomes. We measured the rate of mistranslation at bactericidal concentration and found no effect, indicating that AgNPs are not proteotoxic to the host at the tested concentrations. Collectively, our results suggest that AgNPs could have a promising clinical application as a potential stand-alone therapy or antibiotic adjuvants.As the threat of antimicrobial-resistant bacteria compromises the safety and efficacy of modern healthcare practices, the search for effective treatments is more urgent than ever. For centuries, silver (Ag) has been known to have antibacterial properties and, over the past two decades, Ag-based nanoparticles have gained traction as potential antimicrobials. The antibacterial efficacy of Ag varies with structure, size, and concentration. In the present study, we examined Ag nanoparticles (AgNPs) for their antimicrobial activity and safety. We compared different commercially-available AgNPs against gram-negative  All thefections , P. aerun wounds , both mefections , and A. dle East . E. colid deaths . Most ofd deaths . The avad deaths , drug red deaths , and sild deaths .Streptococcus pyogenes, ampicillin-resistant E. coli, MDR P. aeruginosa, Listeria monocytogenes, Klebsiella pneumoniae, and K. aerogenes  of amikacin, suggest a promising utility of AgNPs as antibiotic adjuvants. Furthermore, the lack of cytotoxicity to cells, no effect on 2.2.1.2.1.1.E. coli, P. aeruginosa, S. aureus, A. baumannii, Salmonella spp., K. pneumoniae, and Citrobacter freundii. More detailed information about each strain can be found in S. aureus and MRSA were cultured in Tryptic soy broth (TSB). All other strains were cultured in lysogeny broth (LB) Lennox.Bacterial strains used in this study include 2.1.2.2 in a humidified environment.Cell lines used in this study are found in 2.1.3.All experiments were performed at 25°C using the N2 wild-type strain . Nematod2.1.4.Chemicals and reagents are listed in 2.2.Ag concentrations were measured by atomic absorption spectroscopy (AAS). One milliliter of the sample was digested in 1 mL of 70% nitric acid using a microwave and then diluted to 10 mL using DI water. Triplicate measurements were performed for each sample.Transmission electron microscopy (TEM) samples were prepared using Formvar-C coated 300 mesh Cu grids. A 1.0 μl drop was deposited onto each grid and allowed to dry in a desiccator. Bright-field transmission electron microscopy (BF-TEM) imaging was performed using a Phillips CM100 TEM operated at 100 kV to analyze the particle size distribution. Two grids were imaged, and a minimum of three images per grid were used to obtain average particle size data. The particle outlines were identified using ImageJ software. Images were visually inspected for artifacts to ensure the maximum inclusion of actual particles. The particle diameters were calculated from the outlined areas assuming a circular shape.S probe correction operated at 200 kV. This allowed direct visualization of the atomic structures of the AgNPs. Immediately prior to HAADF-STEM imaging, each grid was plasma cleaned for a few seconds using a Fischione Instruments Model 1,020 plasma cleaner. This was required for successful atomic-resolution HAADF-STEM imaging to occur; otherwise, the build-up of carbonaceous contamination during imaging prohibited this.Additionally, atomic-resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging was performed using an FEI Themis scanning TEM (S/TEM) with C2.3.600) was adjusted to 1.0, then diluted 1:500 in LB media, followed by aliquoting 50 μl to each well, resulting in a final dilution of 1:1000. All concentrations of AgNPs were ultimately diluted by half as a result of the addition of the bacteria to each well, leaving the highest concentration as half of the stock. The plate was incubated at 37°C in the Tecan Infinite 200 Pro plate reader for 20 h. Every 30 min, the plate was shaken orbitally for 5 s, and the OD600 readings were taken. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that inhibited the growth of bacteria, as determined by a < 0.1 increase of OD600 over 20 h. The bactericidal or bacteriostatic activity of AgNPs was determined according to established protocols , where the FICI = FICAgNP + FICAmikacin; FICAgNP = MICAgNP combination/MICAgNP alone; FICAmikacin = MICAmikacin combination/MICAmikacin alone. The FICI values indicate synergy (FICI ≤ 0.5), partial synergy (0.5<FICI ≤ 0.75), additive (0.75<FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), or antagonistic (FICI>4.0) effect.Serial dilutions of amikacin were performed in water in a 96-well plate starting with 100 μl of 64 μg/mL of amikacin in row A and diluted into 50 μl of water until row G. Only water was added to row H. In a separate plate, 200 μl of 12.4 μg/mL AgNPs was added to column 12 and then diluted in 100 μl of water from columns 11–2. Only water was added to column 1. From the AgNP plate, 50 μl was transferred and added to the amikacin dilution plate. A culture of 2.4.4 cells per well and differentiated into macrophages for 72 h in the presence of 40 ng/mL PMA. A 150 μl aliquot of the AgNP stock mixed with 150 μl of RPMI 1640 medium was added to the top row of a new 96-well plate, and 150 μL of culture medium was added to rows B-H, followed by 1:2 serial dilutions from row A through G, leaving H as the untreated control. The medium was aspirated from the wells containing cells, washed twice with phosphate-buffered saline (PBS), followed by a transfer of 100 μl of the diluted AgNPs or plain medium, and incubated for additional 24 h. Following the incubation, the medium was aspirated, cells were washed twice with PBS, and fresh medium and CellTiter-Blue® viability reagent were added at 100 and 20 μl volumes, respectively. The plate was incubated for an additional 24 h. The absorbance was measured via optical density at OD570 and OD600 in the Tecan Infinite 200 Pro microplate reader. The cytotoxicity was determined by subtracting the OD570 values (absorption maximum for resorufin) from the OD600 (absorption maximum for resazurin reference) values, normalizing the values to the untreated control, and calculating the percent of cells alive. The viability was also tested using HeLa cells, RAW 264.7 macrophages, and mESCs. Each cell line was treated with AgNPs for times specified for each of the experiments. All cell lines were tested with three independent runs. RAW 264.7 and THP1 macrophages were tested with two replicates per run, while HeLa and mESCs were tested with three replicates per run.Cell viability was measured using the CellTiter-Blue® cell viability assay. The assay was performed according to the manufacturer’s instructions (Promega). Briefly, THP-1 monocyte-derived macrophages were seeded into tissue culture-treated 96-well plates at a density of 5 × 102.5.4 cells/mL. The cells were allowed to attach overnight. AgNPs were serially diluted from stock concentrations as described above. Cells were washed twice with PBS, and AgNPs dilutions were transferred to the 96-well plate containing cells and incubated for 4 h. An overnight culture of E. coli DH5α was adjusted to an OD600 of 1.0 and diluted in DMEM. AgNP-containing medium was aspirated, and cells were washed twice with PBS. Bacteria were added to the cells at a multiplicity of infection (MOI) of 10 and the plates were centrifuged at 2200 RPMs for 30 min at 37°C, followed by a 30-min incubation at 37°C and 5% CO2. After the incubation, the well content was replaced with a fresh medium containing 100 μg/mL gentamicin to eliminate extracellular bacteria. The plates were incubated for 1 h. The medium was then aspirated, cells were washed twice with PBS, and lysed with 100 μl of 0.05% Triton X-100 for 18 min. The lysate was serially diluted at 1:10 and 10 μl from each well was plated on LB agar plates. LB plates were incubated overnight at 37°C, followed by CFU enumeration. The procedure was repeated with THP-1 cells with a 24-h pre-treatment and PMA differentiation, as described above. Experiments were performed with three independent runs of three replicates per run.RAW 264.7 macrophages were seeded and incubated overnight as described above in tissue culture-treated 96-well plates at a density of 5 × 102.6.To measure the stop codon readthrough, a stop codon was inserted between Rluc and Fluc . The con2.7.E. coli OP50 was performed as described previously  killing of eviously . Briefly2.8.E. coli OP50. The worms were washed with M9 minimal medium to remove the bacteria from the surface, then incubated with an alkaline hypochlorite solution until the adult tissues began dissolving. The reaction was stopped with M9, and the alkaline hypochlorite solution was washed off with M9 three times. Worms were incubated at room temperature on a nutator in M9 overnight. Synchronized worms were plated onto plain and AgNP-supplemented (0.2 and 1.0 μg/mL) NGM plates seeded with PFA-killed E. coli OP50, followed by a 72-h incubation. Following the incubation, a single worm was placed into a drop of M9 on a microscope slide and allowed to acclimate for 1 min. The rate of motility was recorded as the number of body bends in 30 s. A body bend was defined as an approximately 90° bend along the midline with a decrease in nose-to-tail distance. The experiment was repeated twice with a total of 40 worms per treatment.Motility was assessed by adapting the thrashing assay described elsewhere . Worms w2.9.E. coli OP50 and cultured until the young adult stage (~42 h at 25°C). Twenty worms were picked onto NGM plates containing 100 μg/mL of 5′-fluorodeoxyuridine (FUDR), either 0, 0.2, or 1.0 μg/mL AgNPs, and a PFA-killed E. coli OP50 lawn. The experiment was performed using five plates (n = 100) per each condition. Prior to lifespan experiments, we assessed the effect of AgNPs and FUDR on E. coli OP50 using a method described in 2.3.L1 stage age-synchronized worms were plated onto fresh NGM seeded with live 2.10.E. coli samples were grown overnight, as previously described. Bacterial cells were incubated with water (control group), 0.2, 3.1, or 7.2 μg/mL AgNP for 6 h. Immediately after incubation, formvar–carbon square mesh grids were cleaned via glow discharge in a PELCO easiGlow Discharge Cleaning System. Ten microliters of each bacterial sample were spotted onto its respective grid and incubated at room temperature for 45–60 s. After incubation, each grid was tapped against filter paper to remove excess liquids from the grid surface. One drop of 0.5% uranyl acetate from a 0.22 μm syringe filter was placed on each grid, covered to shelter from light, and incubated for 15–30 s at room temperature. Grids were tapped against filter paper to remove excess liquids after incubation. Prepared EM grids were placed in a secured, ventilated, dark container overnight to allow the substrate to dry. All grids were imaged using the Tecnai FEI G2 Spirit 120 kV TEM.Cells treated with AgNPs were compared to control cells using TEM. 2.11.600 of an overnight culture of E. coli DH5α was adjusted to 1.0 and diluted at 1:500 in LB media containing 0.4 μg/mL of AgNPs. Aliquots of 50 μL were added to each well, further diluting AgNPs and bacteria to final concentrations of 0.2 μg/mL and 1:1,000, respectively. Bacterial growth was measured via OD600 using the Tecan Infinite Pro 200 for 20 h at 37°C every 30 min with a 5-s orbital shake before each read. Experiments were performed with three independent runs of three replicates per run. According to the European Committee on Antimicrobial Susceptibility Testing, the fractional inhibitory concentration (FIC) index was calculated and described as antagonistic (FIC ≥2), indifferent (1 < FIC <2), additive (0.5 < FIC ≤1), or synergistic . Error bars represent the standard error of the mean (SEM). All statistical analyzes were performed using GraphPad Prism version 9.4.0 for Windows (GraphPad Software).Student’s t-test, one-way analysis of variance (ANOVA), and Kaplan–Meier survival analyzes were used to determine statistical significance. The data were considered statistically significant when 3.3.1.While various formulations of AgNPs are available for research and public use, most, if not all of them, exhibit some level of antibacterial activity. We used commercially available AgNPs to investigate their antimicrobial properties and potential utility for future applications that could limit the need for traditional antibiotics. The average Ag concentrations of Formula 1 and Formula 2 measured by AAS were 12.3 ± 0.2 μg/mL and 26.8 ± 0.5 μg/mL, respectively . To dete3.2.E. coli DH5α. We found comparable MICs of ~3 μg/mL using all samples  and the MIC is >4, then the mode of inhibition is bacteriostatic  genes that confer resistance to colistin by modifying the bacterial cell membrane . Hyperlibacteria ; Table 1iostatic . We used killing .3.3.3.3.1.in vitro and in vivo models to assess cytotoxicity. We measured the effect of AgNPs on the viability of mouse RAW 264.7 and human THP-1 macrophages, human epithelial HeLa cells, and mouse embryonic stem cells, mESCs. While the epithelial cells were not affected by any of the tested concentrations, the viability of macrophages and mESCs was affected at higher AgNP concentrations; however, no cytotoxicity was detected at lower bactericidal concentrations (<1.6 μg/mL), which already exhibit moderate antibacterial effects at sub-MIC levels  concentration of AgNPs. Although this concentration slightly affected the viability of mESCs, we did not detect any significant effect on translation fidelity . Similarly, 25 μg/ml of 25 nm AgNPs shortened C. elegans lifespan, while at the same concentration, 75 nm particles had no effect  of AgNPs did not affect r plates . Smallerarticles . Howevero effect . The difmotility , a resulmotility . Since tin vivo efficacy.Our TEM results suggest that AgNPs bind to bacterial fimbriae. If this is the case, then one would expect detectable effects on bacterial attachment and biofilm formation. Given that fimbriae support bacterial virulent mechanisms , targetiThe original contributions presented in the study are included in the article/DC: conceptualization and supervision. DC, NQ, NR, and KF: methodology. AD and DC: software, manuscript draft preparation, and visualization. AD, DC, NR, and KF: validation. AD, DD, WD, NQ, CN, LA, GE, JB, NR, KF, and DC: investigation. AD, DD, WD, NQ, GE, JB, NR, KF, and DC: manuscript review and editing. All authors contributed to the article and approved the submitted version.Funding for this study was provided by Natural Immunogenics Corporation and the University of Florida Industry Partnerships Grant Program awarded to DC. KF was supported by the Japanese Science and Technology Agency grant JPMJPR2049.NQ, CN, and LA were employed by Natural Immunogenics Corporation.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors declare that this study received funding from Natural Immunogenics Corporation. The funder had the following involvement in the study: product characterization using AAS and TEM.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Adiposity evaluated by body mass index (BMI) is associated with glycometabolism. The aim of the investigation was to explore the correlation of visceral fat area (VFA), body fat percentage (BFP), BMI and waist circumference (WC) with type 2 diabetes mellitus (T2DM) and pre-diabetes.A total of 18,458 participates underwent physical examination in Nanjing Drum Tower Hospital from January 2018 to April 2022 was included in this study. Data were collected retrospectively. Regression analysis was used to evaluate the relationship of VFA, BFP, WC and BMI with diabetes status, fasting blood glucose (FBG) and glycohemoglobin (HbA1c).p < 0.001). HbA1c was also positively correlated with the four indexes with βs of 2.645, 0.328, 0.685 and 0.255(p < 0.001). Subgroup analysis shown that FBG and HbA1c were positively correlated with VFA, BFP, BMI and WC in normal and pre-diabetes group (p < 0.001). FBG was negatively correlated with BMI in T2DM group (p = 0.023). In T2DM, there were non-linear relationships of HbA1c with VFA, BFP, WC and BMI with the inflection points for about 7%. Before the inflection point, HbA1c was positively correlated with obesity-related indicators, and it was reversed after the inflection point. In the individuals with excessive VFA and normal BMI, the risk for glycometabolism disorder exceed compared with normal VFA and normal BMI. Every per-standard deviation increasing in VFA, BFP, WC and BMI, the corresponding risk increasing of glycometabolism disorder was 16.4, 14.6, 22.6 and 22.2%.After fully adjusted for multiple covariates, VFA, BFP, WC and BMI in T2DM and pre-diabetes group exceeded compared with normal group. FBG was positively correlated with VFA, BFP, WC and BMI with βs of 2.221,0.306,0.606 and 0.175, there might be a trend of fat loss. VFA could negatively affect glycometabolism independently from BMI. The optimum to evaluate the risk of glycometabolism disorder was WC. As two major public health events in the twenty-first century, obesity and T2DM go hand in hand . ObesityTo this day, the most frequently used index to evaluate obesity remains to be BMI . RecentlComputed tomography (CT), Magnetic Resonance Imaging (MRI) and Dual-emission X-ray Absorptiometry (DXA) are commonly used to evaluate VFA and BFP, but they are difficult to be widely used because of their high requirements of facilities, high expense and complex operation. In recent years, since its simple operation and low cost, bioelectrical impedance analysis (BIA) method is becoming more and more popular . BioelecParticipates included in the study who underwent physical examination in the health management center of Nanjing Drum Tower Hospital from January 2018 to April 2022 were screened according to inclusion and excluding criteria. Inclusion criteria. 1) Age>30 years and ≤ 90 years; 2) Have complete personal holographic files, including general information , diastolic pressure (DBP), medical history), biochemical parameters , low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), serum uric acid (UA), etc.), and data of body composition parameters collected through bioelectric impedance analysis. Excluding criteria: 1) refuse to answer medical history; 2) diagnosed with type 1 diabetes; 3) pregnant; 4) taking steroids such as glucocorticoids; 5) Unreasonable data (e.g.WC of 0 cm) after data quality control.General information of participates, such as gender, age, medical history (including hypertension (HBP), diabetes, medication history, etc.) was retrospective collected. Body parameters, BIA results and serum biochemical indexes were retrospective collected. All tests were performed on empty stomach individuals in the morning. The sequence of different tests were allocated according to the call system of the health management center.Body parameters were measured by trained researchers. Blood pressure measurement: let the subject calm down in a quiet environment for 10–20 minutes, and then use an electronic sphygmomanometer to measure blood pressure. All subjects on an empty stomach relieve oneself, remove metal ornaments, and wear light clothes to measure their height, weight, waist circumference, and body composition in the morning. Use height and electronic scale to measure height and weight. WC was measured by a soft ruler.Use h-key350 human body composition analyzer  to measure the VFA of human body. During the measurement, the individuals stood on the instrument barefoot to ensure that both feet were in full contact with the foot electrode position of human body composition analyzer. Hold the handles on both sides with both hands and ensure that the five fingers are in full contact with the test electrode. Straighten arms to both sides and maintain an included angle of about 15 degrees with torso. Entered personal information such as age, gender, and height for correction. Clicked to start the detection, waited about 2 min, and then the BFP and VFA results were automatically transmitted to the connected computer for storage and archiving. The test requirements are as follows: (1) vigorous exercise or other physical activities are not recommended within 2 hours before the test; (2) Bathing within 2 hours before the test is not recommended; (3) The indoor temperature should be kept between 21 and 25 °C; (4) It is recommended to use the toilet before the test, because human excreta will temporarily change the body composition.Serum biochemical indexes: take 3 ml of peripheral venous blood under the fasting state of the subject, and use the automatic biochemical analyzer to detect FBG, TC, HDL-c, LDL-c, TG, etc.The research is based on the project “ construction of life cycle intelligent monitoring and management service system based on DRGs” approved by the Ethics Committee of Nanjing Drum Tower Hospital(2022–046-01).2 was defined as BMI obesity. VFA ≥ 100 cm2 was VFA obesity. BFP>25% for males or>35% for females was defined as BFP obesity [http://www.empowerstats.com). P < 0.05 indicated the difference of statistically significant.Continuous variables such as age, SBP and DBP were expressed in the form of mean ± standard deviation (SD), and classified variables such as sex, DM and HBP were expressed in the form of percentage(%). The diagnosis of diabetes came from previous history and/or FBG ≥ 7.0mmo/l and/or HbA1c ≥ 6.5% . The dia obesity , 13. WC  obesity . Regress2 (p < 0.001), respectively. BFP were 27.591 ± 6.449, 29.019 ± 6.687, 28.996 ± 6.898 (%) (p < 0.001), respectively. WC were 86.362 ± 9.478, 88.813 ± 9.805, 91.327 ± 10.116 cm (p < 0.001), respectively. BMI were 23.997 ± 3.145, 24.902 ± 3.231, 25.810 ± 3.360 kg/m2 (p < 0.001), respectively. See Table A total of 18,458 participants were included in the study. According to the different diabetes status, the population was divided into three groups . VFA in normal group, pre-diabetes group and T2DM group were 84.546 ± 30.795, 93.614 ± 33.489, 99.311 ± 36.381 cmp < 0.001), respectively. The BFP was 0.446 and 0.998% higher than normal (p < 0.001). The WC was 1.253 and 2.340 cm higher (p < 0.001), and the BMI was 0.410 and 0.853 kg/m2 higher than normal group (p < 0.001). Subgroup analysis showed that the difference between groups still existed. See Table The group with normal glycometabolism was taken as the reference. After fully adjusting for multiple covariates , the VFA of pre-diabetes group and T2DM group were 3.466 and 8.259 cm higher than normal group  < 0.001, 0.306  < 0.001, 0.606  < 0.001 and 0.175  < 0.001, respectively). After subgroup analysis, this correlation still existed in the normal group and the pre-diabetes group. But in T2DM group, VFA, BFP and WC had no correlation with FBG. BMI was negatively correlated with FBG. After fully adjustment for multiple covariate, VFA, BFP, WC, BMI were positively correlated with HbA1c(β (95% CI) p-value = 2.645  < 0.001, 0.328  < 0.001, 0.685  < 0.001 and 0.255  < 0.001, respectively). After subgroup analysis, this correlation still existed in the normal group and the pre-diabetes group. But in the T2DM group, VFA, BFP, WC and BMI had no correlation with HbA1c. See Tables After fully adjusting multiple covariates, VFA, BFP, WC, BMI were positively correlated with FBG(β (95% CI) Smooth fitting curve and threshold effect analysis explored the nonlinear relationship between HbA1c and VFA, BFP, WC and BMI. It was found that VFA, BFP, WC and BMI showed an inverted U-shaped curve with the change of HbA1c. VFA, BFP, BMI and WC had threshold effect with the change of HbA1c, with the inflection points of 7, 6.7, 7 and 7% respectively. See Table According to different obesity status for the indexes, the participants were divided into 4 groups. Group 1: normal VFA/BFP/WC and normal BMI. Group 2: VFA/BFP/WC obesity and normal BMI. Group 3: normal VFA/BFP/WC and BMI obesity. Group 4: VFA/BFP/WC obesity and BMI obesity. Taking group 1 as the reference group, explore the risk of glycometabolism disorder (pre-diabetes and T2DM) in groups 2 to 4. The study found that in group 2, the risk of glycometabolism disorder in the VFA obesity and BMI normal group was higher than that in group 1. However, there was no significant difference in the risk of glycometabolism disorder for the groups with BFP or WC obesity and normal BMI compared with group 1. See Table 2 increase in VFA corresponded for 0.005 increasing in the risk of glycometabolism disorder (β(95% CI) p-value = 1.005  < 0.001). For every 1% increase in BFP, the risk of glycometabolism disorder increased by 0.022(β(95% CI) p-value =1.022  < 0.001). For every 1 cm increase in WC, the risk of glycometabolism disorder increased by 0.023(β(95% CI) p-value =1.023  < 0.001). For every 1 kg/m2 increase in BMI, the risk of glycometabolism disorder increased by 0.068(β (95% CI) p-value =1.068  < 0.001). For per standard deviation increasing in VFA, BFP, WC and BMI, the risk of glycometabolism disorder increased by 16.4, 14.6, 22.6 and 22.2%. See details in Table After fully adjusting multiple covariates, every 1 cmAdiposity is closely related to glycometabolism. In addition to BMI, our study indicated that VFA, BFP, WC were associated with glycometabolism disorder. Individuals with glycometabolism disorder had higher BFP, VFA, WC and BMI compared with those with normal glycometabolism. FBG and HbA1c were positively correlated with VFA, BFP, WC and BMI, respectively. In patients with pre-diabetes or normal individuals, there is a positively correlation between fat distribution and deterioration of glycemic control. However in individuals with T2DM, the correlations disappeared or reversed. In T2DM, the association of HbA1c and VFA, BFP, WC and BMI showed a nonlinear inverted U shape with an inflection about 7%. Before 7%, there is a positive correlation, and after 7%, it was opposite, suggesting that patients with poorly controlled T2DM may be with a trend of fat loss. VFA independent from BMI could negatively affect glycometabolism. WC might be optimum to evaluate the risk of glycometabolism disorder.As we all know, adiposity is an independent risk factor for glycometabolism disorder since the accumulation of fat can affect the effects of insulin through a variety of mechanisms , 16. Pan2 and 8.3cm2 higher than those with normal glycometabolism, BFP was 0.4 and 1.0% higher, WC was 1.3 cm and 2.3 cm higher, and BMI was 0.4 kg/m2 and 0.9 kg/m2 higher, respectively. HbA1c and FBG were positively correlated with VFA, BFP, WC and BMI. This suggests that obesity-related indexes measured by BIA are consistent with BMI in judging the risk of glycometabolism disorders. It was found that the incidence of T2DM was positively correlated with VFA [After fully adjusting for multiple variables, VFA of individuals with pre-diabetes and T2DM were 3.5cmwith VFA . Among iwith VFA . The asswith VFA . The negwith VFA . Therefowith VFA , 24. A swith VFA . The viswith VFA . This mawith VFA . This suThe study demonstrated the correlation between adiposity related indicators measured by BIA and glycometabolism disorder. Our research has many advantages: 1) our study is a rare large sample population study for evaluating glycometabolism and body composition using BIA in Asia. 2) Studies have confirmed that the fat distribution in the pre-diabetes population is related to the deterioration of blood glucose control, which reminded the pre-diabetes patients to pay more attention to the fat distribution. 3) This study confirms that VFA can affect glycometabolism independently of BMI, and reminded clinicians that they should pay attention to using BIA to evaluate implicit obesity and improve glycemic control. However there were some limitation in this study: 1) Firstly, the cross-sectional research cannot confirm the causal relationship between visceral fat accumulation and glycometabolism disorder. 2) Secondly, the study explored the relationship between adiposity and HbA1c in T2DM, but lacked of data about the duration of diabetes and lean muscle mass.3) Lifestyle such as diet and exercise, was not involved in the study, which also limit the application of our conclusion.In conclusion, VFA, BFP, WC and BMI were positively correlated with the risk of glycometabolism disorder. Individuals of T2DM with poor glycaemic control seem to have a trend of fat loss. Even if BMI does not exceed, attention should be paid to VFA. BMI and WC cannot be completely replaced by VFA and BFP for evaluating the risk of diabetes and pre-diabetes.


Pain is a globally prevalent problem, and a comprehension of its pathophysiology is important with respect to patient's health. Musculoskeletal pain conditions (MPs) may be associated with physical, lifestyle, and nutrition status, while dietary acid load (DAL) may be inversely associated with musculoskeletal health in adults. This cross‐sectional study consisted of 175 adults experiencing pain. Anthropometric measurements, physical activity (PA), and pain intensity were assessed via specific questionnaires. Dietary data were collected using a 7‐day 24‐h recall. Foods and beverages were analyzed with Nutritionist IV software for extracting the total energy and nutrients. Net endogenous acid production (NEAP) and potential renal acid load (PRAL) were evaluated for assessing the DAL. Linear regression and Spearman correlation were used to investigate the association of exposure and input variables. Linear regression showed a positive relationship between PRAL and NEAP and pain intensity in the crude model. This significant positive relationship remained after adjusting for all confounders. A lower consumption of potassium, magnesium, vitamin B9 and C, and fiber was seen in the following quartiles of PRAL and NEAP. In addition, MPs intensity and PRAL and NEAP had a weak, positive correlation. This study suggests that a higher DAL may be associated with MPs. However, further research is needed. This study demonstrates a significant, positive, relationship between higher dietary acid load (DAL) and pain intensity among adults with musculoskeletal pain. The inclusion criteria were having MPs. Exclusion criteria were having a bone fracture in the last 3 months, pregnancy and/or lactation, and psychosomatic disorders. Information on age, gender, education, job, and marital status was collected. In addition, delivery type undergone by of women  was assessed via questionnaire. This study was approved by the National Committee for Ethics in Biomedical Research under code IR.IAU.SRB.REC.1399.084. All volunteers were informed about the study and provided written informed consent, prior to participation in the study.Where 2.22) were measured for all participants according to standard techniques.The weight was measured by digital scales, when participants were fasted for 8 h and were in light clothing, to the nearest 0.1 kg. The height was measured with Seca 216, to the nearest 0.1 cm, with participants in a standing position and unshod. The waist circumference (WC) (cm) and body mass index (BMI)  of the participants was evaluated using the short form of the International Physical Activity Questionnaire (IPAQ) , a software program modified for Iranian foods  and number (%), respectively. PRAL and NEAP were divided into quartiles based on the trends. To compare the differences between quantitative and qualitative variables, one‐way analysis of variance (ANOVA), analysis of covariance (ANCOVA), and chi‐square tests were used among quartiles, respectively. Linear regression was used, in crude and adjusted models, to understand the relationship between DAL and pain intensity. Model 1 was adjusted for age, PA, energy intake, BMI, WC, while model 2 was adjusted for model 1 + gender, education, job, marital status, and delivery type. In addition, Spearman correlation was conducted to complement linear regression analyses. Statistical significance was accepted, a priori, at p < .05.Data analysis was conducted using SPSS version 26 . The Kolmogorov–Smirnov test was used to determine the normality of the data, and quantitative and qualitative variables were reported as the mean ± standard deviation , 69.35 ± 15.19 (kg), 24.84 ± 4.32 (kg/m²), and 17.06 ± 17.37 across quartiles of DAL, respectively (Table SD of NEAP and PRAL were 45.13 ± 24.07 (mEq/day) and −0.43 ± 27.52 (mEq/day), respectively. A significant difference was found between job, marriage, sex, delivery type, age, PA, height, and pain intensity among PRAL quartiles (p < .05). Among NEAP quartiles, there was a significant difference in job, marriage, sex, delivery type, age, and height (p < .05).The mean ± 3.2SD of total energy intake was 2230 ± 651  and there was a significant difference in potassium, magnesium, vitamin C, B9, and E, and fiber intake among PRAL quartiles (p < .05). Energy, protein, carbohydrate, and fat consumption increased across PRAL groups. Furthermore, dietary calcium, potassium, magnesium, vitamin C and B9, carbohydrate, and fiber intake were significantly different among NEAP quartiles (p < .05). Protein and sodium intake increased among NEAP quartiles.Dietary intakes of participants are shown between quartiles, after adjusting energy intake, in Table 3.3β = 4.67, 95% CI = 2.15–7.19, p < .001; β = 4.03, 95% CI = 1.49–6.58, p = .002), respectively, after adjusting all confounders in model 2. In addition, we saw a negative, weak correlation between age, gender, and PRAL and NEAP. Moreover, MPs pain and PRAL and NEAP had a positive weak correlation  and high in processed red meat, may be responsible for reducing nociceptive sensitivity and increasing chronic pain in obese, inflamed, mice (Totsch et al., Some studies have shown a significant relationship between DAL and inflammation. For instance, higher DAL may cause metabolic acidosis, which can lead to the production of various inflammatory markers (Wu et al., Aligned with increasing PRAL, protein, carbohydrate, and fat intakes were concurrently elevated. In line with this study, the consumption of high amounts of carbohydrates can reportedly play an important role in oxidative stress, specifically via glucose oxidation (Kaushik et al., To the best of our knowledge, the current study represents the first to have investigated DAL and MPs intensity. Nevertheless, one of the limitations of the present study is the imbalanced sample, with a disproportionate number of women versus men, which could impact our findings. Women have been posited to report greater pain compared with men due to greater nerve density (Paller et al., 5This study demonstrates a significant, positive, relationship between DAL and pain intensity among adults with musculoskeletal pain.The author declares that there is no competing interest.The National Committee for Ethics in Biomedical Research approved this study under code IR.IAU.SRB.REC.1399.084. The specifics of the study were told to all qualified participants and written consent was obtained. The data are not publicly available because of containing information that could compromise the privacy of the research. Data are available from the authors upon reasonable request and with permission.Committee for Ethics in Biomedical Research approved this study under code IR.IAU.SRB.REC.1399.084.The author listed approved the final manuscript and consent for publication.


Meloidogyne javanica second-stage juveniles (J2), causing J2 mortality up to 89%. Through gas chromatography–mass spectrometry, eight compounds were identified in the cassava wastewater volatiloma, with alcohols being the major class. The compounds ethyl butanoate and butyric acid identified in cassava wastewater volatiloma were selected for in vitro nematicidal activities and substrate fumigation tests. The lethal concentration predicted LC50–48 hr values (effective doses to kill 50% of M. javanica J2 population after 48 h of exposure) were 172.6 μg ∙ mL-1 and 301.2 μg ∙ mL-1 for butyric acid and ethyl butanoate, respectively. In a pot assay, the application of butyric acid and ethyl butanoate as a soil fumigant, at a dose of 0.5 mL ∙ kg-1 substrate, significantly (P < 0.05) decreased M. javanica infectivity and reproduction compared to the negative control (water). However, ethyl butanoate proved to be a more efficient soil fumigant (P < 0.05) than butyric acid, as its application reduced the number of galls and eggs to the level of the commercial fumigant Dazomet. This study is the first to demonstrate the potential of ethyl butanoate as a soil fumigant against M. javanica.As a result of more restrictive legislation regarding the use of pesticides, over the last two decades, the demand for new soil fumigants has increased. These compounds can come from a variety of sources, including organic residues. In this study, we identified in the cassava wastewater volatiloma a molecule with potential to be developed as a new fumigant nematicide. Cassava wastewater (20 mL) releases volatile compounds toxic to  Meloidogyne spp.) are major threat to food production  volatilome, erucin was identified; this is a volatile compound with LC50–24 hr (effective doses to kill 50% of M. javanica J2 population) against M. incognita of 3.2 mg ∙ mL-1, which value is close to the predicted value for metam sodium, which is the a.i of several commercial nematicides (Fumigant nematicides were the first synthesized products to be used to control plant-parasitic nematodes (PPNs) and they continue to be highly effective in reducing nematode populations in soil . Howeveraticides . An examaticides .Manihot esculenta) roots into flour or starch results in different waste products, which includes a yellowish liquid called cassava wastewater, referred to in Brazil as manipueira  followed by gas chromatography–mass spectrometry (GC–MS) analysis. Finally, °C, until it was used in the tests.The cassava wastewater used in this study was collected directly from a cassava flour factory located at Alpinópolis, Minas Gerais State, Brazil. The cassava used for the production of the flour was of the wild type, cultivar IAC 13. The collected waste yellowish liquid was kept in closed polyethylene terephthalate (PET) bottles, in a cold chamber at a temperature of 10M. javanica was originally collected from lettuce (Lactuca sativa) roots at Ijaci town, Minas Gerais State, Brazil . The nematodes were multiplied in pots (2 L) containing commercial substrate  and tomato seedling (Solanum lycopersicum L. cv. Santa Clara) for 2 mon in a greenhouse with temperature ranging from 20°C to 28°C. Eggs were extracted as described by A population of ® vials, 35 g of autoclaved sand was deposited. On the sand surface, 20 mL of cassava wastewater was added. As a negative control, 20 mL of sterile water was used. Then, an empty microtube with a volume of 1.5 mL was partially buried in the sand. To concentrate the volatiles released by the organic residue, the vials were sealed and incubated at 28°C (± 2°C) in the dark for 3 d. After this period, an aqueous suspension of 1 mL, containing approximately 150 J2 of M. javanica, was injected, using a syringe, inside the microtube. Then the Supelco® vials were sealed and incubated at 28°C (± 2°C) for 24 hr. At the end of this period, the vials were opened and the J2 transferred to wells of a 96-well polypropylene plate. Then, the technique developed by -1 sodium hydroxide solution (NaOH) was added to the J2 suspension contents of each well. Juveniles that remained immobile for, approximately, 3 min were classified as dead.The technique used to perform this test was developed by -1. The temperature setting of the GC oven was 40°C to 130°C at 3°C ∙ min-1 and then up to 240°C at 10°C ∙ min–1. The mass spectrum of each chromatogram peak was extracted through the Automated Mass Spectral Deconvolution and Identification System (AMDIS) v. 2.63. The VOC identification was performed by comparing the mass spectra of the sample peaks with NIST library spectra by the Mass Spectral Search Program, and by comparing experimentally obtained retention indices (RI Exp.) with the retention indices in the literature (RI Lit.)  fiber . The temRI Lit.) . The exp50). Butyric acid was chosen taking into account the largest peak area in the chromatogram. On the other hand, ethyl butanoate was selected due to the lack of studies of this compound against M. javanica. For butyric acid the concentrations used to determine the LC50 were: 0 μg mL-1, 150 μg mL-1, 165 μg mL-1, 180 μg mL-1, 210 μg mL-1, 280 μg mL-1, 330 μg mL-1, and 400 μg mL–1; for ethyl butanoate the concentrations used were: 0 μg mL-1, 100 μg mL-1, 150 μg ∙ mL-1, 200 μg ∙ mL-1, 250 μg mL-1, 350 μg ∙ mL-1, 450 μg mL-1, and 550 μg mL-1. These values were defined considering previous tests where the compounds were tested at concentrations of 100 μg mL-1 and 500 μg mL-1, data not shown. The different concentrations of the molecules were prepared using an aqueous solution of Tween 80 at 0.01 g mL-1 as a solubilizing agent. The assays were performed using 1,000-μL microtubes, in which 300 μL of aqueous suspension containing, approximately, 300 J2 were mixed to 300 μL of the stock solution of each molecule to achieve the desired final concentrations. Then, the microtubes were sealed and incubated at 28°C for 48 h. After this time period, the microtubes were homogenized, opened, and 100 μL of the suspension were transferred to a 96-well polypropylene plate for J2 observation. To find out if the immobile nematodes were dead, 10 μL of a 1.0-mol L-1 sodium hydroxide (NaOH) solution was added to the contents of each well. Juveniles that remained immobile for approximately 3 min were classified as dead. In this way, the mortality percentage was determined.Based on the analysis by GC–MS, butyric acid and ethyl butanoate  were selected for the tests to determine the lethal concentration . The treatments consisted of the application of butyric acid or ethyl butanoate at a dose of 0.5 mL per liter of substrate, following the methodology described by -1 and 2.0 mL ∙ kg-1 of soil  was used, at a dose of 0.25 g ∙ L-1 of substrate. As a negative control, water was used. The bottles containing the mixtures were sealed with a lid and plastic film, homogenized, and kept at 28°C for 3 d. After this period, the bottles were opened and the substrate transferred to plastic trays, where it remained for 7 d to release the remaining volatiles. After this period, the substrate was then poured into 300-mL plastic cups. A 20-d-old Santa Clara tomato seedling, susceptible to M. javanica, was transplanted into each cup. After 60 d, the following parameters were evaluated: fresh root mass and number of galls and eggs in the root systems.For this experiment, PET bottles of 2-L volume were used, in which 1 L of multiplant of soil . Each boP > 0.05), a combined analysis was performed. In the soil fumigant assays, the data of galls and eggs per gram of root were subjected to transformation via Log (x + 1) before statistical analysis to normalize the data. Once the assumptions were met, the F-test was applied through ANOVA. When the significance level in the F-test (P < 0.05) was reached, means of each treatment were compared with the Tukey’s test at 5% probability. In the cassava wastewater volatile experiment, the Student’s t-test (P < 0.05) was used to compare the means between the two groups. To determine the lethal concentration of ethyl butanoate and butyric acid required to kill 50% of M. javanica J2, the generalized linear regression logistic model was used through the drc package in the R program . All the data sets were tested for normality (Shapiro–Wilk’s test) and homogeneity (Levene test). The experiment repetitions (experiments 1 and 2) were submitted to analyses of variance (ANOVA) and if there was no difference between them ( program . RStudioP < 0.001) toxic to M. javanica J2, causing mortality above 89% The butyric acid and ethyl butanoate LCrameters . Butyricutanoate . None ofutanoate . ConcentM. javanica, caused a significant (P< 0.05) reduction in the number of galls and eggs per gram of tomato roots, compared to the negative control (water) (P< 0.05) more effective than butyric acid, the former being statistically similar to the nematicide Dazomet, used at a dose of 0.25 g ∙ L-1. The application of butyric acid or ethyl butanoate did not affect the tomato root fresh mass (RFM), being similar to the negative control (water)  . However (water) . The plaM. javanica J2. Since cassava wastewater contains, among other things, HCN, nematicidal activity from this plant residue was expected  and harbors several microorganisms corroborates the production of this class of chemical compounds (–1) between 40 a.m.u. and 350 a.m.u. were monitored in order to obtain the complete mass spectra for identification purposes. Therefore, any compound with a molecular mass lower than 40 a.m.u., such as HCN, water (H2O), or carbon dioxide (CO2), will not be detected even though it is present in the sample.To confirm our suspicion, a chromatographic analysis was carried out, which identified eight compounds, most of them belonging to the alcohol class. The fact that cassava wastewater is a liquid with high organic content  is effective in reducing root-knot nematodes in soil at rates of 0.5 mL ∙ kg–1 and 2.0 mL ∙ kg–1 of soil  with the best performance in the LCM. javanica J2, with alcohols being the main active volatile components of this residue. Among the two volatiles studied against M. javanica, ethyl butanoate stood out and showed good potential for use in the field. These studies will be done in the near future.In conclusion, our results demonstrated that the cassava wastewater volatiloma is toxic to


In the original article, there was an error. The AUC of IVA + tumor size predicting VA improvement was incorrectly stated. A correction has been made to Results, Paragraph 6:p < 0.001), and the AUC of IVA + tumor size predicting VA improvement was 0.831 . For medium to large tumors, the AUC of tumor volume predicting VA outcome was 0.748 . The cutoff point of IVA level was 4.5 . The cutoff point of tumor volume was 43.50 cm3  (Table 4).In the ROC analysis, the AUC of IVA predicting VA improvement was 0.787  (13). Tumors were divided into small, medium, and large size groups by tertiles. Tumor volume ≤13.80 cm3 was categorized as a small-size tumor, 13.80 cm3 < tumor volume ≤34.60 cm3 was categorized as a medium-size tumor, and the tumor volume > 34.60 cm3 was categorized as a large-size tumor.According to the range of vision loss reported by the International Council of Ophthalmology (12), we converted the categories of vision impairment to a seven-level scale: level 7 to level 1 represent normal vision (≥0.8), mild visual impairment (0.32–0.63), moderate visual impairment (0.125–0.25), severe visual impairment (0.05–0.1), profound visual impairment (0.02–0.04), near-blindness (<0.02), and light perception or blindness, respectively. The VA outcome of patients was categorized into “VA improvement” and “no VA improvement.” If the LVA level was higher than the IVA level, the outcome was defined as “VA improvement.” If not, the outcome was defined as “no VA improvement.” “Maintained” was used when the LVA level was equal to the IVA level, and “deteriorated” was used when the LVA level was lower than the IVA level. Tumor volume was calculated using the ellipsoid volume formula “A × B × C × π/6” (The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


When the energy was elevated between 0.08 MeV and 1.408 MeV, the highest values were attained for concrete A, with values ranging between 0.451 cm−1 and 0.179 cm−1. The lowest half-value layer (Δ0.5) values were achieved for concrete C, where the Δ0.5 values varied between 1.53 cm and 3.86 cm between 0.08 MeV and 1.408 MeV. The highest Δ0.5 values were achieved for concrete A, where the Δ0.5 varied between 1.77 cm and 4.67 cm between 0.08 MeV and 1.408 MeV. According to this investigation, concrete A has the highest promise in radiation shielding purposes because it has the most desirable properties of the concretes studied.We aimed, in this investigation, to prepare novel concretes which can be used in gamma-ray shielding applications. The experimental approach was performed using a NaI (Tl) detector to measure the concrete’s shielding features for different energies, ranging from 0.081 MeV to 1.408 MeV. The density of the fabricated concretes decreased with increasing W/C ratio, where the density decreased by 2.680 g/cm Gamma-ray is electromagnetic radiation that is frequently utilized in diverse fields, including nuclear energy, nuclear medicine, agriculture, industry, dental centers, and many other fields. It is known that gamma photons are massless and chargeless, travel at the speed of light, and have high penetration ability; therefore, the shielding of the gamma ray is very difficult. Thus, the development of novel materials, with sufficient attenuation ability, is an important challenge facing workers in the field of radiation shielding materials. When nuclear engineers plan to fabricate shielding materials, many factors must be taken into account, in order to obtain materials with interesting attenuation performance. These factors include the density, the thickness of the medium type, and energy of the radiation ,3,4,5,6.3 and it is a good choice as a protective material in some applications. It is also used for the storage of high-level nuclear waste. The low cost and simple preparation method are the main reasons that encourage civil and nuclear engineers to focus on developing certain kinds of concrete with different aggregates as shielding materials against low and medium γ-ray energy (Eγ). Moreover, some types of concrete are composed of a mixture of light and heavy nuclei, so can be applied not only to shield the γ-photons, but also to shield the neutrons [Heavyweight concrete is concrete that possesses a density higher than 2.6 g/cmneutrons ,8.Several previous works have shown that when the density of the medium is increased, the attenuation performance of this medium is improved. One common way to increase the density is by adding heavy materials. Accordingly, a few years ago, researchers were interested in enhancing the attenuation performance of concrete by adding heavy aggregates, such as hematite , magnetiTamayo et al. prepared a high-density concrete, utilizing siderurgical aggregates, and experimentally studied the radiation attenuation factors for the prepared concretes. They also investigated the impact of the W/C ratio on the attenuation parameters . Abo-El-Enein et al. prepared ordinary Portland cement pastes with hematite nanoparticles and ZnO nanoparticles and studied the influence of these nanoparticles on the thermal resistance and mechanical properties features. According to their results, the addition of 0.05% ZnO nanoparticles had a negative impact on the radiation protection efficiency of the prepared samples .Roslan et al. fabricated high-density concretes with ferro-boron aggregates and studied their radiation attenuation ability. They found that the density was enhanced with the addition of ferro-boron, and that the concrete mixed with ferro-boron has superior shielding properties to the control specimens. They concluded that the addition of ferro-boron causes an enhancement in photon attenuation capabilities .Liu et al. fabricated barite concretes using waste materials and studied the lead leaching, drying shrinkage, and mechanical properties. They used MCNP simulations to examine the gamma-ray shielding features. They demonstrated a reduction in the photon shielding features with the addition of funnel glass .In this investigation, we aim to fabricate new concretes that may be utilized in the radiation shielding field, using a NaI (Tl) detector to predict the concretes’ attenuation properties between 0.081 and 1.408 MeV. 3 (pV) to the bulk volume (bV). The pV was obtained by comparing the difference in mass between the saturated and dry specimen. The porosity was determined according to Equation (1), with resulting values listed in The concretes were prepared as a mixture of Portland cement, granite (coarse aggregate), and red sand (fine aggregate) with ratios of 1:3:2 by volume. Firstly, the ratio of each component used in the preparation was determined using an electric balance with an accuracy of ±0.01 g. After that, the weighted ratios of granite, cement, and sand were manually mixed well for 15 min. Thereafter, the chosen water ratio was added to the mixture and the effect of water/cement (W/C) on the porosity, density, and radiation shielding parameters was studied. The W/C ratios selected in the study were 0.4, 0.6, and 0.8. The mixture was then molded in the steel cubic molds with dimensions of 7 cm (length) × 7 cm (width) × various thicknesses and the prepared concretes, with different thicknesses, were left to dry for 15 days. Finally, the density was determined using the MH-300A densimeter, with an accuracy of 0.001 g/cm3 . The matµ) describes the capacity of the shielding material to tolerate crossing photons, and it is the most important protecting feature  detector. The detector recorded incident and transmission intensities, both with and without the constructed concrete (I and oI). The produced concrete samples were all the same thickness. We plotted the relation between (I/oI) and the sample thickness, and from the slope of the obtained curve determined the µ value for each concrete sample [The linear attenuation coefficient  to transmitted (I)) and sample thickness . It is the most important operator for predicting a shield’s propensity to attenuate γ-radiation. The calculation for the linear attenuation coefficient (µ) is shown in Equation (2) [The experimental tion (2) .(2)μ(cm0.5) can be calculated from µ values, namely [The half-value layer Δ0.5TF, %) is a parameter that can be used to calculate the rate of γ- ray passing through a known thickness of concrete and is shown in Equation (4) [The transmission factor (tion (4) . MoreovepV, bV, Φ, and the density of the fabricated concretes. Raising the W/C ratio from 0.4 to 0.8 increased the pV values from 6.68 to 8.83% and was accompanied by a slight increase in bV values from 53.75 to 54.90%. The increase in pore volume combined with the increase in the bulk volume caused an increase in the porosity of the concrete from 12.4 to 16.1%, as shown in 3), where the ρ values decreased slightly, from 2.68 to 2.56 g/cm3.The W/C ratio has a great impact on the structural properties γ) from the applied radiation sources affected the µ values. µ diminished with the elevation of the incoming energy from the γ-photons. At low energies, the photoelectric effect (PE) is the reason for high µ values, and one must remember that the PE cross-section is inversely proportional with the Eγ (σPE α Eγ−3.5) [γ to above 0.1 MeV, the Compton scattering effects (CS) began to change the behavior of the µ values, which fell progressively in line with the rising Eγ values. These changes in the µ behavior were attributed to the proportionality of Cs cross-section with the incoming Eγ (σcom α E−1). As clarified in µ values among the fabricated concretes, followed by concretes B and C. The µ values for all concretes decreased when the energy was increased from 0.081 to 1.408 MeV. The highest µ values were achieved for concrete A, where the µ varied between 0.451 cm−1 and 0.179 cm−1 when the energy was raised between 0.08 and 1.408 MeV. The lowest µ values were achieved for concrete C, where the µ values diminished to between 0.390 cm−1 and 0.148 cm−1 when the applied Eγ was raised between 0.08 and 1.408 MeV, respectively. There were some small anomalies observed in the µ values at some discrete gamma-ray energies; thus, the measurements were repeated four times and the uncertainty in measurements was calculated. Radiation shielding factors were measured experimentally, to determine the concretes’ ability to resist the γ-photons.  Eγ−3.5) ,24,25. Wµ values. µ values, in the Eγ, range from 0.081 to 1.408 MeV, as a result of raising the concretes’ ρ values. µ values were enhanced, from 0.215 to 0.270 cm−1, raising the concretes ρ values from 2.56 to 2.68 g/cm3, respectively, at Eγ of 0.344 MeV. The same trend was observed for other Eγ values, where the µ value was raised from 0.157 to 0.186 cm−1 at Eγ of 1.332 MeV. The present study showed that increasing the W/C ratio in fabricated concrete caused a notable reduction in the concretes’ ρ, which consequently caused a negative effect on the µ values and other shielding properties. The ρ values were reduced, ranging between 2.68, and 2.564 g/cm3 for concretes A, B, and C, as the W/C ratio was increased to values of 0.4, 0.6, and 0.8. This decrease in the ρ values, with increases in the W/C ratio, may be attributed to the hypothesis that “raising the W/C ratio leads to the formation of air bubbles inside the concrete layers”. These air bubbles were destroyed and formed pore holes after drying the fabricated concretes. Raising the W/C ratio was usually associated with an increase in the number of pore holes created inside the fabricated container, which had a negative effect on gamma-ray shielding.The fabricated concretes’ Φ and ρ were strongly affected by the concretes’ measured µ values for the fabricated concretes. At Eγ of 0.081 MeV, the measured µ changed from 0.45 cm−1 to 0.39 cm−1, due to the increase in the Φ values, from 12.71 to 16.02%, respectively. Similarly, at Eγ of 0.662 MeV, the µ values decreased from 0.237 to 0.197 cm−1 with the rise in Φ values. This decrease in the µ ay be attributed to the increase in the pore volume inside the concrete.In order to confirm this hypothesis, the Φ values were studied see b. Figureµ values of the fabricated concrete and those for the other previously reported concretes was performed . For all fabricated samples, the TF values were highly increased when raising the incoming Eγ. This increase was attributed to the predominant CS interaction inside the concrete samples, where CS interaction allowed the photons to accumulate inside the sample, throughout the experiment, consuming only a small part of the incoming Eγ during the interaction. The TF values for concrete A (for example), increased from 40% to 70 % when Eγ varied between 0.081 and 1.408 MeV  and from 55 to 25% (for concrete C), when Eγ varied between 0.081 MeV and 1.408 MeV.The calculated RPE values for the fabricated concrete are also shown in 0.5 can describe the fabricated concretes’ ability to reduce the number of transmitted γ- photons. As shown in 0.5 values fluctuated with the rise in incoming Eγ, allowing the required thickness of the concrete composition for radiation shielding to be identified. Conversely, the Δ0.5 of the fabricated concretes was increased by raising the Eγ and the W/C ratio. The lowest Δ0.5 values were achieved for concrete A, where the Δ0.5 values varied between 1.53 cm and 3.86 cm when the Eγ was raised from 0.08 to 1.408 MeV. The increase in the Δ0.5 for the fabricated concrete samples can be attributed to the same reasons discussed in the µ section.The Δ3, when the W/C ratio was raised from 0.4 to 0.8. Moreover, the porosity calculation showed that the Φ values inside the fabricated concrete increased when the W/C ratio was raised. Furthermore, an experimental approach was performed in order to assess the radiation protection features of the fabricated concretes with the Eγ range extended from 0.081 to 1.408 MeV. The highest µ values observed for concrete A decreased from 0.451 cm−1 to 0.180 cm−1 at values of 0.081 and 1.408 MeV, respectively. The examinations were confirmed using the computed other shielding parameters, such as TF, RPE, and Δ0.5. The TF of the fabricated concretes was observed to be significantly diminished with the raising of the incoming Eγ. Conversely, the RPE values were reduced due to the increase in the incoming Eγ. As can be seen, the fabricated concrete A had the highest values of TF  and RPE (60% at 0.081 MeV). Based on the measurements of RPE and Δ0.5, it was established that fabricated concretes are preferred in many radiation shielding applications.The present work studied the W/C ratio effect on the radiation shielding properties of concretes. Therefore, three samples of concrete were fabricated, using a mixture of Portland cement, red sand, and granite aggregates, with ratios of 1:2:3. The W/C ratios were adjusted to be 0.4, 0.6, and 0.8. The measurements for the fabricated concretes’ ρ values showed that the ρ values slightly decreased from 2.68 to 2.56 g/cm


P < 0.01). Mean VLAS, M‐VLAS, and the RLAD values in our study were 1.79 ± 0.3 , 2.23 ± 0.44 , and 1.2 ± 0.34 , respectively. These were significantly less than previously published reference interval values (P < 0.001). The VHS, M‐VLAS, and the RLAD were not affected by sex, body weight, or BCS; whereas the VLAS was moderately affected by body weight. Findings from this study can be used as background for future thoracic radiographic assessments in CKCS.Cavalier King Charles Spaniels (CKCS) are predisposed to developing myxomatous mitral valve disease (MMVD), with radiographs frequently used to screen for evidence of left‐sided cardiomegaly secondary to MMVD. Vertebral heart size (VHS), vertebral left atrial size (VLAS), modified VLAS (M‐VLAS), and radiographic left atrial dimension (RLAD) are reported as objective measurements of global heart size and left atrial size. Normal VHS in CKCS (10.6 ± 0.5) is reportedly higher than the non‐breed‐specific value (9.7±0.5). Breed‐specific VLAS, M‐VLAS, and RLAD cut‐offs have not been reported in CKCS. The aim of this prospective reference interval study was to describe the VHS, VLAS, M‐VLAS, and RLAD values for 30 clinically healthy adult CKCS. Inclusion criteria were unremarkable physical examination, normal echocardiography, and thoracic radiographs without malposition/abnormalities. There were 22 female and eight male dogs. Ages ranged from 1 to 6 years. The VHS mean value in our sample was 10.08 ± 0.56 . This was significantly greater than a previously published general canine reference value of 9.7 ± 0.5 and significantly less than a previously published CKCS breed‐specific value of 10.6 ± 0.5 ( ACVIMAmerican college of veterinary internal medicineBCSbody condition scoreCKCSCavalier King Charles SpanielsDVdorso‐ventral viewE/AE and A waves ratioLAleft atriumLA/Aoleft atrium to aorta ratioLA/Ao_Sxshort‐axis left atrium indexed to the short‐axis aortic rootLAD/AoD_Lxlong‐axis left atrial dimension indexed to the long‐axis aortic valve annulus diameterLLleft lateral viewLVleft ventricleLVIDDleft ventricular internal diameter at end‐diastoleLVIDDNnormalized left ventricular internal diameter in diastoleLVIDSleft ventricular internal diameter at end‐systoleMMVDmyxomatous mitral valve diseaseM‐VLASmodified vertebral left atrial sizeMVPmitral valve prolapseRLright lateral viewRLADradiographic left atrial dimensionT4fourth thoracic vertebraTD/TWthoracic depth – thoracic width ratiovvertebral unitVHSvertebral heart scoreVLASvertebral left atrial size.1Myxomatous mitral valve disease (MMVD) is a common cardiovascular disease affecting dogs, progressing to mitral regurgitation, and eventually heart failure.The VLAS, M‐VLAS, and RLAD have recently been proposed as new radiographic methods for quantifying left atrial size in dogsThe first aim of this study was to describe breed‐specific reference interval values for VHS, VLAS, M‐VLAS, and RLAD in a sample population of healthy adult CKCS. The second aim was to test the hypothesis that these radiographic measures of cardiac size in CKCS would differ from previously published reference values. The third aim was to test the hypotheses that radiographic measures of cardiac size in CKCS would not be significantly affected by sex, body condition score (BCS), or chest conformation; but would be significantly affected by recumbency.2This study was a prospective, single‐center, reference interval study design. Thirty, healthy CKCS referred for preoperative evaluation or cardiologic screening were recruited from June 2019 to March 2021 at the Veterinary Medicine Department of the University of Milan. The sample size was based on convenience sampling.2.1This study being a part of a larger project, informed consent was obtained from all the clients before starting any examination procedures for included dogs. The radiographic examinations were carried out as per the hospital's standard procedures.2.2All included CKCS underwent a complete physical examination, thoracic radiography, and echocardiography. Dogs were considered healthy based on the absence of prior clinical conditions/abnormalities documented by the owners and unremarkable physical examination, cardiovascular assessment, and transthoracic echocardiogram.2.3All echocardiographic examinations were performed in unsedated dogs by a third‐year PhD veterinary cardiology student (M.B.) under the supervision of a veterinarian with more than 15 years of clinical experience in veterinary cardiology (C.L.), using the same ultrasonographic unit [MyLab50 Gold cardiovascular ultrasound machine ] equipped with multi‐frequency phased array probes (3.5–5 and 7.5–10 MHz), chosen according to the weight of the subject. Each dog underwent a complete echocardiographic examination, which included transthoracic two‐dimensional, M‐mode, and Doppler imaging.2.4Radiographic examinations and measures were performed by a third‐year veterinary radiology PhD student (M.M.) supervised by a veterinarian with more than fifteen years of experience in veterinary radiology and clinical practice (D.Z.). They were blinded to echocardiographic findings. The assessment of the interobserver variability of VHS, VLAS, M‐VLAS, and RLAD by multiple observers was not the intent of the study and was previously evaluated.A dedicated image analysis workstation  was used for all radiographic measurements. The radiographic evaluations of VHS, VLAS, M‐VLAS, and RLAD were performed using digital calipers for both RL and LL radiographic views.Specifically for VHS, the long‐axis dimension (L) was measured from the ventral border of the largest of the main stem bronchi seen in cross section to the most ventral point of the cardiac apex. The short‐axis dimension (S) was drawn perpendicular to the long‐axis dimension from the caudal border of the cardiac silhouette at the dorsal aspect of the caudal vena cava to the cranial border of the cardiac silhouette. The two lengths (L and S) were then repositioned over the thoracic vertebrae, parallel to the vertebral canal, beginning to the fourth thoracic vertebrae T4). The VHS was the sum of the two lines in vertebral body units Figure . For VLA. The VHS[Correction added on 29 March 2022, after first online publication: The citations for Figure 2.5TM 27.0, IBM, SPSS, USA). Descriptive statistics were generated. The distribution of data for continuous variables was assessed for normality by means of the Kolmogorov‐Smirnov test. Variables were normally distributed, and results were reported as mean ± standard deviation (SD) unless otherwise specified. A paired Student's t‐test was used to compare mean VHS, VLAS, M‐VLAS, and RLAD values between right and left lateral recumbency. An unpaired Student's t‐test was used to compare mean VHS, VLAS, M‐VLAS, and RLAD values between males and females, between dogs with different TD/TW ratios, particularly TD/TW higher or lower than 0.9, and between dogs with different BCS and body weights. The TD/TW 0.9 cut off was arbitrarily chosen due to the CKCS’ intermediate thoracic conformation. A Pearson correlation coefficient (r) was applied to study the correlation between all radiographic measurements, clinical  and echocardiographic data . The correlation was considered weak, moderate, strong, or perfect respectively when the value of the correlation coefficient was 0.1‐0.3, 0.4‐0.6, 0.7‐0.9, or 1.t‐test was then used to test whether the VHS, VLAS, M‐VLAS, and RLAD in our population of CKCS differed from the mean reference values proposed by literature.P‐value < 0.05 was considered significant for all analyses.Statistical analyses were performed by a veterinarian with coursework training in statistics (C.L.) using commercially available statistics software (SPSS3There were 22 of 30 (73.3%) females (n. 3 neutered) and eight of 30 (26.7%) males n. 1 neutered); with a mean age of 2.66 ± 1.42 years (range: 1‐6 years) and a mean body weight of 7.92 ± 1.68 kg (range: 5.1‐12 kg). Twelve subjects (40%) weighed more than the proposed breed standard (5‐8 Kg). The mean BCS was 5/9 ± 1/9 (range: 4/9‐7/9). Nineteen dogs (63.33%) had Blenheim coat color type, n.1 (3.33%) ruby, n. 3 (10%) black and tan, and n. 7 (23.34%) tricolor. The small number of subjects with coats other than Blenheim did not permit statistical analysis against this physical parameter. All the clinical and the echocardiographic data are reported in Table  neuteredP = 0.002) and higher than the reference value of 9.7 ± 0.5 proposed by Buchanan and Bücheler in 1995 (P < 0.001).P = 0.000), Lam et al , and Salguero et al .In our study, the CKCS had a significantly lower VHS  than the reference value of 10.6 ± 0.5 established by Lamb et al in 2001 for this breed . Both RL and LL VHS, VLAS, M‐VLAS, and RLAD did not significantly differ between males and females (P > 0.05). All radiographic measurements did not significantly differ between different BCS groups (P > 0.05). Mean TD to TW ratio was 0.91 ± 0.08 (95% range 0.88‐0.94). Fifteen dogs (50%) had a TD/TW ratio < 0.9 and n. 15 (50%) > 0.9. All CKCS of this study had an intermediate chest conformation (0.75 < TD/TW < 1.25).P = 0.26).Table P > 0.05), and body weight (P > 0.05). Only LL VLAS showed a moderate positive correlation with BCS . There was no significant correlation between the type of chest and VHS, VLAS, M‐VLAS, and RLAD in all included dogs and no significant differences were observed for dogs with different TD/TW ratio (higher and lower than 0.9). Conversely, CKCS with TD/TW ratio lower than 0.9 had greater LA/Ao_Sx ratio and lower sphericity index (both P = 0.001).The VHS, VLAS, M‐VLAS, and RLAD showed no correlation with BCS ,Buchanan and Bücheler did not detect any differences in the VHS between males and females.The VLAS found in our study population is lower than the values proposed by Malcolm et al and more similar to those reported by Puccinelli et al.We must also emphasize that all the published data about VLAS, M‐VLAS, and RLAD reported a cut off able to discriminate among subject with or without left atrial enlargement and not the normal radiographic size of the left atrium. It is therefore likely that values obtained in our study are lower for this reason. Further studies including a larger population of healthy and affected by MMVD at different stages CKCS are needed to better clarify the normal reference interval of the VLAS, M‐VLAS, and RLAD in this breed and a cut‐off value useful for discriminate left atrial enlargement. In addition, breed‐specific differences in the VLAS, M‐VLAS, and RLAD, like the VHS, should be considered.The main limitation for the current study was the small sample size. A larger population could have led to possible differences in the proposed reference range because the CKCS breed is very inhomogeneous in terms of size and morphotype. Body weight in CKCS may vary with different genetic lineages; however, our range of body weight was wide (5‐12 kg), and no correlation was found between the VHS and body weight. Thus, it is reasonable to believe that even increasing the sample population, comparable results would be found. In addition, most previous studies reporting breed‐specific reference values of the VHS included a maximum of 30 cases per breed, as the present study.In conclusion, results of this study support previous studies indicating that breed‐specific reference values for the VHS are needed. Furthermore (as underlined by 2019 ACVIM MMVD guidelines) VHS, VLAS, M‐VLAS, and RLAD breed‐specific reference values should be introduced in the evaluation of thoracic radiographs, also in healthy subjects. In CKCS, the VLAS, M‐VLAS, and RLAD values found in this study can be used as references to avoid misinterpretation of cardiomegaly in this breed. Further studies evaluating the VLAS, M‐VLAS, and RLAD in different canine breeds are warranted.(a)Conception and Design: Locatelli, Bagardi, Brambilla(b)Analysis of Data: Bagardi, Manfredi, Bassi, Spediacci, Ghilardi, Zani(c)Interpretation of Data: Locatelli, Bagardi(a)Drafting the Article: Bagardi, Locatelli, Manfredi, Bassi(b)Revising It Critically for Important Intellectual Content: Bagardi, Locatelli, Manfredi, Spediacci, Ghilardi, Zani, Brambilla(a)Final Approval of the Version to be Published: Bagardi, Locatelli, Manfredi, Bassi, Spediacci, Ghilardi, Zani, Brambilla(a)Agreement to be Accountable for All Aspects of the Work in Ensuring that Questions Related to the Accuracy or Integrity of Any Part of the Work are Appropriately Investigated and Resolved: Bagardi, Locatelli, Manfredi, Bassi, Spediacci, Ghilardi, Zani, BrambillaThe authors declare that there were no conflicts of interest