This study aimed to develop predictive models for δ13C values in soil using near infrared spectroscopy (NIRS) to increase overall sample size. In addition, this study aimed to assess the δ13C values between five vegetation classes.Stable carbon isotopes are important tracers used to understand ecological food web processes and vegetation shifts over time. However, gaps exist in understanding soil and plant processes that influence δThe Land Degradation Surveillance Framework (LDSF) was used to collect a stratified random set of soil samples and to classify vegetation. A total of 154 topsoil and 186 subsoil samples were collected and analyzed using NIRS, organic carbon (OC) and stable carbon isotopes.13C values, −26.1‰; followed by woodland, −21.9‰; cropland, −19.0‰; shrubland, −16.5‰; and grassland, −13.9‰. Prediction models were developed for δ13C using partial least squares (PLS) regression and random forest (RF) models. Model performance was acceptable and similar with both models. The root mean square error of prediction (RMSEP) values for the three independent validation runs for δ13C using PLS ranged from 1.91 to 2.03 compared to 1.52 to 1.98 using RF.Forested plots had the most negative average δ13C in soil, which will allow for landscape-scale assessments to better understand carbon dynamics.This model performance indicates that NIR can be used to predict δ Aboveground vegetation influences belowground carbon dynamics. Optimizing soil organic carbon (SOC) content is recognized as an essential component of ecosystem functioning Lal . The Uni13C values between –22 and –30‰, while about 15% of plants use the C4 photosynthetic pathway and have less negative δ13C values, generally ranging from –10 to – 14‰  use the C3 photosynthetic pathway and have δFarquhar ; Loomis Farquhar . The lat13C values in soil, including (i) microbial decomposition, (ii) the Suess effect  is now a well established methodology for the prediction of soil properties such as SOC, pH, base cations and texture Brown . In addi–1), can be analyzed to characterize the chemical, physical and mineralogical composition of the soil , with headquarters in Nairobi, Kenya has established a soil IR spectral database that currently has soil spectra from soil samples from across a wide range of landscapes that represent agricultural soils, forested landscapes, wetlands, and savannas Brown . These l13C values in soil using NIR spectra from soils collected from a well documented experimental station in Mexico (n = 100 soil samples). Using modified partial least squares (MPLS) regression, they developed calibration models with an R2 of 0.81  and without plant residues (n = 50 range – 19.1 to –22.1‰), which limits the application of these models beyond the specific case study they were developed for. Our study builds on this work to explore the potential for NIR to predict δ13C values across a varied set of soils that represent a wide range of chemical and physical characteristics, including different vegetation classes. Kleinebecker et al.  regression they developed calibration models obtaining positive results with R2 values of 0.89 and 0.99 for δ13C and δ15N, respectively  Assess the potential for NIRS to predict δ13C using a diverse set of soil samples; 2) Compare different predictive models  and 3) Better understand stable carbon isotope variation across various vegetation classes and smallholder farming systems in SSA.Recent advances in big data analytics and ensemble learning methods allow for the development of predictive models that are stable across a range of soil functional operating ranges. In this paper we present a case study where we use NIR spectroscopy of soils to develop predictive models for SOC and δ2 sites using the Land Degradation Surveillance Framework (LDSF). Top (n = 156) and sub (n = 184) soil samples from nine LDSF sites across Ethiopia (Mega), Kenya , Democratic Republic of Congo (DRC) , Uganda (Hoima), Madagascar (Didy) and Tanzania (Mbola) were included in the study , with 4 subplots (100 m2) within each sampling plot. Measurements and observations were made at the subplot and plot levels, respectively. Land use was classified at each plot using a simplified version of the FAO Land Cover Classification System (LCCS)  with a resolution of 4 cm–1 using a Bruker Multipurpose Analyzer (MPA) at the World Agroforestry Centre (ICRAF) Plant and Soil Spectroscopy Laboratory in Nairobi, Kenya .Soil samples were air-dried and sieved to 2 mm. Airdried soil samples were scanned in duplicate in nearinfrared spectral range . Soil samples from DRC were analyzed at Isotope Bioscience Laboratory  of Ghent University, Belgium using EA-IRMS , coupled to a 2020 IRMS ). Stable carbon isotopes were expressed as δ13C in parts per mile (‰ relative to the V-PDB (Pee Dee Belemnite) standard  using dry combustion on acidified samples and for stable carbon isotopes with an elemental analyzer isotope ratio mass spectrometer (EA-IRMS) at IsoAnalytics Laboratory .All calculations and statistical analysis were performed using R statistics  compared with Mega, which had the lowest (13 trees ha–1) (Basic site characteristics for the nine sites included in the study are shown in es ha–1) .13C in soil. Given the diversity of the spectra, the potential for applying these models across diverse landscapes is high.There was substantial variation in soil NIR spectra both between and within the sites . For exa–1. Mean topsoil OC across the sites was 18.9 g kg–1 and mean subsoil OC was 12.5 g kg–1. –1), followed by Mbola (4.1 g kg–1), OlLentille (8.3 g kg–1), Mpala (11.0 g kg–1), Mega (17.9 g kg–1), Burhale (25.0 g kg–1), Hoima and Didy (29.0 g kg–1), and then Luhihi (33.5 g kg–1) (Soil organic carbon varied across the nine sites from 0.9 to 98.3 g kg g kg–1) . A compa13C in topsoil was –18.8‰, and average δ13C in subsoil was –19.4‰, which indicates that these are mixed C3-C4 ecosystems, with the exception of Didy, which is dominated by C3 vegetation (forest). Didy topsoil had the most negative δ13C values with a median of –27.04‰, followed by Mbola (–21.77‰), Kipsing (–19.00‰), Luhihi (–18.68‰), Hoima (18.27‰), Mpala (–16.50‰), Burhale (–16.42‰), OlLentille (–15.70‰), and Mega (–13.60‰) , which may have implications for whether or not the organic matter is at steady state. However, the lack of a strong shift in isotopic signature with depth at most sites, could be due to the sampling intervals (0–20 and 20–50 cm), compared to sampling strategies that use smaller increments, or it could indicate that their have not been recent vegetative shifts.Average δ–13.60‰) . Further13C values , indicating a dominance of C3 vegetation, –26.1‰; followed by woodland, –21.9‰; cropland, –19.0‰; shrubland, –16.5‰; and finally grassland, –13.9‰  and grassland (p < 0.001) plots showed significant difference. These data have important implications for the use of stable carbon isotopes in East Africa, e.g., since both semi-natural and cropland systems have mixed C3-C4 signatures large sample sizes will be needed to develop robust models to assess vegetation shifts and soil organic matter turnover rates.Fifty-six plots were classified as forest, 41 plots as woodland, 135 plots as shrubland, 30 plots as grassland and 78 plots as cropland. Forested plots had the most negative average δ, –13.9‰ . In gene13C was similar for the PLS and RF models when tested on three different validation datasets. Root Mean Square Error of Prediction (RMSEP), which is a useful measure of accuracy reflecting the overall difference between measured and predicted values, was low for both PLS and RF models (1.95 for PLS and 1.77 for RF). The results further show average R2 for the validation runs using PLS of 0.80 compared to a slightly higher average R2 using RF of 0.84. Overall, R values were slightly higher for calibration runs compared to validation runs, which is expected, with average R2 for calibration using PLS of 0.91 compared to 0.97 for RF. It is important to test prediction models such as the ones presented here on datasets that are independent of that used for developing the model in order to generate some measure of model stability. In our case, validation samples were drawn randomly in each iteration and the calibration model was fitted to this dataset with low RMSEP in both cases indicating good performance for both models . We found the highest SOC concentrations in Didy (eastern Madagascar), Burhale and Luhihi (eastern DRC) and Hoima (western Uganda), which all represent sub-humid and humid environments. The lowest SOC values were found in semi-arid environments  and in Miombo woodlands (Mbola).There is a need to better understand the global distribution of C3 and C4 plants for a number of different reasons, including for improving global circulation models for COThere are many factors influencing SOC concentrations, including inherent soil properties such as sand content, land degradation status such as erosion prevalence  to C4- dominated vegetation  along a conversion gradient  systems. In addition to conversion studies, Billings and Richter  and PLS (average RMSEP = 1.95) models. Average R2 values for the three model validation runs were 0.84 and 0.80 for the RF and PLS models, respectively. Our results show that both PLS, which is a data-reduction technique, and RF, which is generally good for feature selection, can be successfully applied for the prediction of δ13C in soils. Further, the high performance of the RF model applied in our study indicates that this technique is suitable for detecting spectral features that are important for determining the relative abundance of 12C and 13C in soils. Viscarra Rossel and Behrens  and not merely predicting SOC. This further highlights the promise of spectroscopy for predicting stable isotopes in soil and shows the potential of new technological advances such as soil spectroscopic techniques for prediction of δ13C, which can lower the costs of analytical procedures and hence enable larger sample sizes and landscape-scale assessments of vegetation dynamics and SOC.Based on the validation predictions, model performances for predicting δ Behrens  also eva Behrens . Explora Behrens . Our res Behrens , and givBuilding on these results, the potential use of mid-infrared spectroscopy (MIRS) should also be explored and is recommended by the authors for future studies. These results have important implications for the use of stable carbon isotopes in East Africa since both seminatural and cropland systems have mixed C3-C4 signatures. Hence, large sample sizes will be needed to develop robust models to assess vegetation shifts and soil organic matter turnover rates. In conclusion, application of spectroscopic techniques would allow for more costeffective analysis and increased sample sizes, which are needed for landscape-scale ecological studies.


Y. pestis infection of its flea and mammalian hosts. The functions of many of these genes remain predicted or unknown, necessitating their characterization, as this may provide a better understanding of Y. pestis specialized biological adaptations to the discrete environments of its two hosts. This study provides functional context to adjacently clustered horizontally acquired genes predominantly expressed in the flea host by deciphering their fundamental processes with regard to (i) transcriptional organization, (ii) transcription activation signals, and (iii) biochemical function. Our data support a role for these genes in osmoadaptation and aromatic amino acid metabolism, highlighting these as preferential processes by which Y. pestis gene expression is modulated during flea infection.Distinct gene repertoires are expressed during  Yersinia pestis, the flea-transmitted bacterium that causes plague, regulates its gene expression appropriately to adapt to these two physiologically disparate host environments. In fleas competent to transmit Y. pestis, low-GC-content genes y3555, y3551, and y3550 are highly transcribed, suggesting that these genes have a highly prioritized role in flea infection. Here, we demonstrate that y3555, y3551, and y3550 are transcribed as part of a single polycistronic mRNA comprising the y3555, y3554, y3553, y355x, y3551, and y3550 genes. Additionally, y355x-y3551-y3550 compose another operon, while y3550 can be also transcribed as a monocistronic mRNA. The expression of these genes is induced by hyperosmotic salinity stress, which serves as an explicit environmental stimulus that initiates transcriptional activity from the predicted y3550 promoter. Y3555 has homology to pyridoxal 5′-phosphate (PLP)-dependent aromatic aminotransferases, while Y3550 and Y3551 are homologous to the Rid protein superfamily (YjgF/YER057c/UK114) members that forestall damage caused by reactive intermediates formed during PLP-dependent enzymatic activity. We demonstrate that y3551 specifically encodes an archetypal RidA protein with 2-aminoacrylate deaminase activity but Y3550 lacks Rid deaminase function. Heterologous expression of y3555 generates a critical aspartate requirement in a Salmonella entericaaspC mutant, while its in vitro expression, and specifically its heterologous coexpression with y3550, enhances the growth rate of an Escherichia coli ΔaspC ΔtyrB mutant in a defined minimal amino acid-supplemented medium. Our data suggest that the y3555, y3551, and y3550 genes operate cooperatively to optimize aromatic amino acid metabolism and are induced under conditions of hyperosmotic salinity stress.While alternating between insects and mammals during its life cycle, IMPORTANCE Distinct gene repertoires are expressed during Y. pestis infection of its flea and mammalian hosts. The functions of many of these genes remain predicted or unknown, necessitating their characterization, as this may provide a better understanding of Y. pestis specialized biological adaptations to the discrete environments of its two hosts. This study provides functional context to adjacently clustered horizontally acquired genes predominantly expressed in the flea host by deciphering their fundamental processes with regard to (i) transcriptional organization, (ii) transcription activation signals, and (iii) biochemical function. Our data support a role for these genes in osmoadaptation and aromatic amino acid metabolism, highlighting these as preferential processes by which Y. pestis gene expression is modulated during flea infection. Yersinia pestis, is a severe zoonotic disease infamous for having caused the death of millions of people in three major pandemics. This disease primarily affects rodents which become infected mainly through blood feeding by rodent-associated infected fleas. Transmission by fleas is a recent adaptation that has likely evolved in the last 1,500 to 20,000 years during the transformation of Y. pestis from its clonal ancestor, Yersinia pseudotuberculosis, which is transmitted by the fecal-oral route and causes a mild enteric self-resolving illness , hyperosmolarity, nutrient limitation, and the immune response, are slowly being uncovered, along with how Y. pestis overcomes such stresses  which encodes proteins that generate the osmoprotectant glutamate  with lower GC content (39.8%) than that typically noted for Y. pestis (48%) genes and that ranked in the top 150 most highly expressed genes in the flea  having a similarly low GC content. The y3554, y3553, and y335x genes showed robust expression in the flea gut , y3554 , y3553 , y355x , y3551 , and y3550  suggest their acquisition by horizontal gene transfer. In silico analysis using FGENESB predicts that the continuous region of this genomic island comprises three transcriptional units, with y3555-y3554-y3553 and y355x-y3551 constituting two separate operons and y3550 representing a monocistronic transcriptional unit. Additionally, BPROM identifies distinct promoter sequences upstream of the genes y3555, y355x, and y3550 -dependent transaminases, including aromatic and aspartate aminotransferases encoded in some bacteria. These PLP-dependent aminotransferases typically catalyze the interconversion of an aromatic amino acid and α-ketoglutarate to oxaloacetate and glutamate. The Y. pestis KIM10+ genome encodes two PLP-dependent aromatic aminotransferases, encoded by y2760 (aspC) and y0579 (tyrB).The y3554 gene encodes a putative Na+/H+ antiporter (NhaC type), and there are two other antiporters (NhaA and NhaB) in the reported genome sequence of Y. pestis. A primary role for Na+/H+ cation exchange antiporters  domain protein superfamily, that are known to bind hydrophobic ligands and mediate signal transduction  sensory domain. PAS domains are ubiquitous in bacterial proteins involved in signal transduction and enable sensing of physical and chemical stimuli, like oxygen and redox potential  protein superfamily, originally called the YjgF/YER057c/UK114 family of proteins. Rid enzymes eliminate reactive intermediates that are produced by PLP-dependent enzymatic activity, which can alter the function of cellular targets and prevent the inhibition of transaminases  and y355x (pCS::P5x) showed ∼102- to 103-fold higher levels of luminescence expression than those of the strains carrying the empty vector (pCS26-Pac) or transcriptional fusions with potential regulatory regions of y3554 (pCS::P54), y3553 (pCS::P53), and y3550 (pCS::P50). This suggests that only the upstream regulatory regions of y3555 and y355x, and not y3550, are able to promote gene transcription using flea-matched temperature testing conditions. To confirm this observation, we generated two other transcriptional fusion constructs that allowed testing of transcription directed from the predicted upstream regulatory regions of y3555 or y355x. The transcriptional fusion construct pCS::P55-54 contains the region covered in pCS::P54 extended up to the regulatory region of y3555, and the construct pCS::P5x-51-50 contains the region of fusion pCS::P50 extended up to the regulatory region of y355x . A previously reported y3555-y3554-y3553 mutant was also used  glmS-pstS::Kanr, which was previously shown to exhibit equal fitness during competition with an unmarked parental strain  and at 7 days (T7) postinfection  and determined the steady-state mRNA expression levels of these genes. The expression levels for the genes y3555, y3554, and y3553 showed between 1.5- and 2.0-fold increases, while the expression of the genes determined to compose the second operon, i.e., y355x-y3551-y3550, showed between 4- and 6-fold increases in gene expression -inducible y3555 gene, was determined . Surprisingly, in the absence of aspartate, the addition of 100 μM IPTG, presumed to induce the expression of y3555, inhibited the growth of the S. entericaaspC789::Km mutant  mutant strain was transformed with the plasmid constructs pET28b::ridA, pET28a::y3551, and pET28a::y3550. Minimal defined medium supplemented with serine was used for growth analysis. In the S. entericaridA1::Tn10 (Tc) mutant, 2AA is generated from serine by the serine/threonine dehydratase IlvA (EC 4.3.1.19). In the absence of RidA, 2AA accumulates and damages some PLP-dependent enzymes, which can result in growth defects (S. entericaridA1::Tn10 (Tc) mutant. The S. entericaridA1::Tn10 (Tc) mutant strain carrying the empty vector control failed to grow in this medium, and as expected, it was complemented by the pET28b::ridA plasmid. Significantly, the pET28a::y3551 plasmid restored significant growth to the strain, indicating that Y3551 had 2AA deaminase activity (y3550 plasmid failed to restore growth to the S. entericaridA1::Tn10 (Tc) mutant strain  converts cysteine to 2AA, which can be deaminated to pyruvate by solvent water or a RidA enzyme. Thus, RidA activity is detected as an accelerated rate of pyruvate formation in a CdsH assay. The assay was performed using purified Y3551 and  greater . These dl-PSP family endonucleases by BLAST analyses  grew significantly faster than did the wild-type strain grown under the same conditions (E. coli aspartate (aspC) and tyrosine (tyrB) aromatic aminotransferase double mutant that requires either aspartate or tyrosine for growth  but not y3550 (μ = 0.0110 ± 0.0012) and y3551 (μ = 0.0105 ± 0.0002) relative to the empty vector-containing strain (μ = 0.0120 ± 0.0009) (y3555 with y3550 (μ = 0.0256 ± 0.0005) or y3551 (μ = 0.0154 ± 0.0007) increased the growth rate and biomass yield similar to those of the y3555-expressing strain, indicating that the increased growth rate and yield were y3555 dependent  . The coeependent . Howevery3555-y3554-y3553-y355x-y3551-y3550, that show exceptionally robust expression during flea infection, is accounted for by their cotranscription as a single polycistronic mRNA transcript. Adjacently clustered genes are often grouped transcriptionally as an operon and frequently encode functionally related proteins. This feature allows protein synthesis to be controlled coordinately in response to the needs of the bacterium only when and where the proteins are required. While the genomic island is expressed only at flea-matched temperatures . This suggested that absence of the genomic island is not critical for adaptation to osmotic stress in vitro. Nonetheless, y3555, a metabolic gene showing putative roles in amino acid metabolism, confers a striking ability to enhance the growth fitness of E. coli, a trait that is also made apparent during the overexpression of y3555 in Y. pestis strains. These two findings allude to a role of the genomic island in optimizing amino acid metabolism in the context of hyperosmotic stress. This is fitting since (i) Y. pestis primarily metabolizes amino acids during flea infection . Instead, Y3555 aligns with over 85% identity to uncharacterized PLP-dependent aminotransferases from foodborne isolates of S. enterica sequenced by the Centers for Disease Control and Prevention . The crystallization of Y3555 may help determine the substrates or products of the protein, and their characterization is ongoing.To begin to understand the function of Y3555, it was expressed in e growth , 52, butd growth . As suchY. pestis, the y3551 gene encodes an archetypal RidA protein that can deaminate the reactive enamine intermediate 2AA. Additionally, the Y3550 gene product likely belongs to the Rid5-6 subclass of the Rid family proteins whose functions have not yet been characterized. Further adding to its distinctive status is that while the promoters predicted immediately upstream of y3555 and y355x can drive the transcription of y3550, a dedicated promoter predicted immediately upstream of the Y3550 coding sequence is exclusively activated by hyperosmotic stress to inclusively drive y3550 transcription.During active metabolism, cellular function can be compromised by an anomalous accumulation of endogenously produced reactive intermediates of metabolism, e.g., 2AA. The elimination of such metabolic intermediates is therefore needed to optimize cellular function, growth, and fitness. The highly conserved Rid family proteins prevalent in all domains of life , 49 are y3555 and y3550 suggests a synergistic activity of these two proteins. Such a case can be explained by the specific Rid activity meted out by Y3550 being able to potentially eliminate a metabolic by-product that is toxic and/or produced by Y3555. One complementary observation within a context of Y3555 serving in aromatic amino acid metabolic processes is that BPROM predicts a binding box of the transcriptional factor TyrR (–y3550 (the predicted promoter region spans nucleotide positions −120 to −87). In many enteric bacteria, TyrR regulates genes involved in the uptake and biosynthesis of aromatic amino acids in response to the presence or absence of each of the three aromatic amino acids  which function as cofactors for TyrR  is otherwise apparent. It has been demonstrated that the infectious blood source influences Y. pestis infection in fleas  and y0579 (tyrB), which convert aspartate and tyrosine to glutamate, respectively, are induced at robust levels during a Y. pestis flea infection . Two ridA homologs, y2240 and y1830, and a putative Rid1 to -7 superfamily-encoding gene, y0163, may be able to compensate for the functions of their homologs in the genomic island  to minimize RNA degradation and then harvested at 4°C and stored at −80°C until RNA isolation.The bacterial strains used in this work are listed in Table S1 in the supplemental material. Bacterial cultures were grown at 28°C with shaking in brain heart infusion (BHI) medium. When necessary, media were supplemented with carbenicillin (100 μg/ml) or kanamycin (50 μg/ml). For the osmolarity assays, overnight cultures of parental KIM6Salmonella enterica serovar Typhimurium LT2 was the parental strain for all S. enterica mutants. Overnight cultures of S. enterica were grown in 8 g/liter Difco nutrient broth (NB) with NaCl (5 g/liter) and the appropriate antibiotic (150 μg/ml ampicillin or 50 μg/ml kanamycin). Minimal no-carbon E (NCE) was supplemented with MgSO4 (1 mM), trace minerals, glucose (11 mM), and, where indicated, serine (5 mM), isoleucine (0.3 mM), aspartate (1 mM), and/or isopropyl-β-d-1-thiogalactopyranoside  ( 100 μM) , 61. AllThe plasmids and primers used in this study are listed in Tables S1 and S2, respectively.luxCDABE operon . Lux transcriptional fusion plasmids were transformed into the Y. pestis KIM6+ parental strain.The Lux transcriptional fusions were generated by cloning a PCR product containing the corresponding regulatory region into the low-copy-number vector pCS26-Pac that carries the promoterless E operon . PCR proy3550, y3551, and y3555 genes from the p270/p271, p272/p273, and p274/p275 primer pairs, respectively. The PCR products were digested with BamHI and XhoI and ligated into pTrc99A or pET28a previously digested with BamHI and SalI restriction enzymes. The plasmid constructs generated from cloning y3550, y3551, and y3555 into pTRc99A are referred to as pTRc55, pTRc50, and pTRc51, and for pET28a cloning, they are referred to as pET28a::y3550, pET28a::y3551, and pET28a::y3555, respectively. To clone y3555 together with y3551 or y3550 into pTrc99A, y3555 was amplified with primers p508 and p509, digested with HindIII, and ligated into pTRc50 or pTRc51 such that y3555 was inserted downstream of y3550 or y3551. These plasmids were referred to as pTRc50_55 and pTRc51_55, respectively. Plasmids were transformed into transformed into E. coli DG44 and selected for on LB–100 μg/ml carbenicillin.The pTRc99A- and pET28a-inducible expression plasmids were generated by cloning PCR products obtained for the 7T previously digested with the same restriction enzymes, transformed into DH5α lambda pir chemical competent E. coli, and selected for on LB–50 μg/ml kanamycin. The plasmid, referred to as pUC18R6K-mini-Tn7T::kanr, was isolated and verified for sequence. Electrocompetent Y. pestis KIM6+ was transformed with pUC18R6K-mini-Tn7T::kanr and pTNS2 and selected for on heart infusion broth (HIB)–50 μg/ml kanamycin plates. Colonies were screened with the pstSup2 and pTn7R primers for insertion at the glmS-pstS site. This Y. pestis KIM6+ strain with the kanamycin cassette integrated at the glmS-pstS site was referred to as Y. pestis KIM6+glm-pstS::Kanr.The kanamycin resistance cassette from the plasmid pKD4 was amplified with primers p395 and p396 (Table S2). The PCR product was digested with BamHI and EcoRI, ligated into pUC18R6K-mini-TnY. pestis KIM6+ carrying plasmids containing the lux fusions was grown overnight in LB supplemented with appropriate antibiotics. An aliquot was used to inoculate fresh LB medium the next day. The cultures were grown on a shaker at 28°C to mid-logarithmic phase and then treated or not with 0.5 M NaCl for 100 min. Aliquots of 1 ml were taken out to determine the optical density of the culture by measuring the optical density at 600 nm (OD600). One-tenth of a milliliter of each culture was dispensed into a 96-well plate (Costar), and the luminescence of each well was measured in the Infinite M1000 reader (Tecan), with an integration time of 1 s and using the Tecan i-control software version 1.7.1.12. Plates were first shaken with a 2-mm linear amplitude 5-s pulse. Data were normalized by dividing relative light units (RLU) by the OD600 to obtain the final RLU corrected for bacterial growth.Y. pestis KIM6+ strain was grown in Miller’s LB broth at 26°C to mid-logarithmic phase; the culture was split in two, and one-half received 0.5 M NaCl and the other distilled water vehicle control treatment. After 20 min, culture RNA was stabilized immediately with RNAprotect bacterial reagent (Qiagen). Total RNA was purified using the Quick-RNA miniprep kit (Zymo). Purified RNA was subject to Turbo DNase I (Ambion) treatment, according to the manufacturer’s instructions. Then, 800 to 1,600 ng of RNA was used in the first-strand cDNA synthesis reaction using Maxima H Minus reverse transcriptase (Thermo Fisher), according to the manufacturer’s instructions. Negative controls were performed by omitting the reverse transcriptase. The cDNA was then treated with RNase H (NEB) at 37°C for 30 min. The PCR amplification step was performed using Phusion polymerase (Thermo Fisher), 100 ng of cDNA, and the Y3555Set2Fw/5051RT-R primer pair  (Table S2); this pair amplifies a 5,014-bp region upstream of y3555 to 226 bp within y3550. RT-PCR cycles were as follows: one cycle of 1 min at 98°C for initial denaturation, and 31 cycles of 10 s at 98°C, 20 s at 68°C, and 1 min 40 s at 72°C for PCR amplification with the Y3555Set2Fw/5051RT-R primer pair. A final elongation step of 5 min at 72°C was included. Controls for the expected size of the PCR products were performed by using chromosomal DNA from Y. pestis KIM6+ as the template. The PCR products were analyzed by 1% agarose gel electrophoresis gels. The GeneRuler 1-kb DNA ladder (Thermo Scientific) was run alongside the PCR products.The y3550 and y3555 genes and the entire continuous region spanning genes y3555, y3554, y3553, y355x, y3551, and y3550  and the tandem genes y3551 and y3550 were replaced with a selectable kanamycin (Km) resistance cassette in the Y. pestis KIM6+ strain (Table S1). The Δy3550 Δy3555 double mutant was constructed similarly, but for this, the Δy3550 mutant was used. Then, the FLP recombination target (FRT)-flanked Km cassette was excised from these strains after transformation with the helper plasmid pFLP3 (Table S1), expressing the FLP recombinase that acts on the directly repeated FRT (sacB and pFLP3 were cured by sucrose counterselection by growing the strains on LB without NaCl and supplemented with 10% sucrose. The strains generated were Δy3550 (VV150), Δy3555 (VV151), Δy3550 Δy3551 (VV278), Δy3550 Δy3555 (VV279), and “all” (VV280) mutants (Table S1) and retained an FRT scar. To construct plasmids expressing y3550 or y3555, a DNA fragment containing the y3550 gene alone or y3555 with its native promoter sequence was amplified by PCR using the 3550-F/3555-R or 3550-F/3555-R primer pair (Table S2) and cloned into vector pCR4-TOPO, generating plasmids pCR4::3550 and pCR4::3555, respectively. All mutant strains were verified by PCR and sequencing.Nonpolar gene deletion mutant strains were generated by the lambda (λ) red recombinase system , 64 and ated FRT . Both pKY. pestis KIM6+ was purified using the Quick-RNA miniprep kit (Zymo). Chromosomal DNA was removed by incubating 5 μg of RNA from each strain with Turbo DNase I (Ambion), according to the manufacturer’s instructions. cDNA was synthesized in a reaction mixture containing 2 μg of each DNase-treated RNA, 100 pmol random hexamers, and 0.5 mM dinucleoside triphosphates (dNTPs), using RiboLock RNase inhibitor (Thermo Fisher) and Maxima H minus reverse transcriptase (Thermo Fisher), according to the manufacturer’s protocol. The obtained cDNA was used as the template for quantitative PCR (qPCR) assays, with 0.5 μM primer pairs for y3550, y3551, y3553, y3554, y3555, or gyrB (Table S2) and the SsoAdvanced Universal SYBR green Supermix (Bio-Rad). Real-time PCRs were performed with the c1000 Touch thermal cycler and the CFX96 real-time system (Bio-Rad), and data were collected using the CFX Manager software 2.1 (Bio-Rad). The reaction conditions were 95°C for 3 min, 95°C for 10 s, and 58°C for 30 s during 40 cycles. The level of gyrB mRNA was used as an internal control to normalize the results. The quantification technique used to analyze data from cultures was the 2–ΔΔCT method  and 7 days postinfection. All experiments involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington State University, Pullman, WA, and were conducted in strict accordance with institutional guidelines based on the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.For coinfections, tn7 site , called escribed . The infE. coli strains harboring IPTG-inducible constructs were grown overnight in LB containing 100 μg/ml carbenicillin with shaking at 37°C. For the E. coli DH5α strains, the overnight inoculum was diluted 1:200 into fresh M9 medium supplemented with 0.4% glucose and 0.1% Casamino Acids medium (Difco). For the E. coli D44 strains, the overnight inoculum was diluted 1:200 into fresh M9 medium supplemented with 0.4% glucose and 0.1% Casamino Acids medium (Difco), succinate (2.5 mg/ml), malate (2.5 mg/ml), α-ketoglutarate (1 mg/ml), and 100 μg/ml each asparagine, glutamine, and glutamic acid, as previously described . The cell suspension was used to inoculate growth medium (2% inoculum), and growth was monitored. The growth data were plotted in a log10 format using GraphPad Prism 7.0c, and the error bars represent the standard error of the mean for three biological replicates.The growth of EcRidA and Y3551 were purified from E. coli strain BL21-AI harboring either pET20-ridA (DM12740) or pET28b-y3551 (DM16279). Overnight cultures grown in superbroth with ampicillin were used to inoculate 3 liters of superbroth with ampicillin. Cultures were grown for ∼3 h at 37°C with shaking until an OD650 of 0.7 was reached. Fresh arabinose was added to a final concentration of 0.2%, and cultures were incubated with shaking at 37°C overnight. A clarified supernatant was injected onto a nickel-nitrilotriacetic acid (Ni-NTA) Superflow resin, and proteins were purified according to the manufacturer’s protocol (Qiagen). Protein aliquots were frozen in liquid nitrogen and stored at −80°C. Purified CdsH was a gift from Dustin Ernst and was purified similarly from E. coli strain BL21-AI containing pET14b-cdsH, as previously described . The reactions were monitored continuously in a 96-well quartz plate by measuring the absorbance at 340 nm for 2 min using a SpectraMax M2 microplate reader. The initial rate of pyruvate formation was calculated from the rate of NADH oxidation in the first 30 s, along with the molar extinction coefficient .2AA deaminase activity was determined using a coupled assay with purified cysteine desulfhydrase (CsdH), RidA, and Y3551, as previously described , 69. AssIn silico predictions of genomic island genetic organization and prediction of promoters were achieved using FGENESB and BPROM  freeware. Alignment of amino acid sequences of Rid proteins was accomplished using the EMBL-MUSCLE tool (https://www.ebi.ac.uk/Tools/msa/muscle/). The protein secondary structure prediction and structural alignments of Y3555 were made using Phyre2 (g Phyre2 .S. enterica strains,” above, or the legends to Statistical analysis was accomplished using GraphPad Prism 7.0, the details for which are provided in “Growth quantification of


APOE) ε4 allele are both associated with increased risk of the Alzheimer’s disease (AD). However, the extent of the joint contribution of APOE ε4 allele and age on the brain white matter integrity, cognition and their relationship are unclear. We assessed the age-related variation differences of major cognitions in 846 non-demented elderly, and brain major white matter tracts in an MRI sub-cohort of 111 individuals between ε4 carriers and noncarriers. We found that: (i) carriers showed a steeper age-related decline after age 50 in general mental status, attention, language, and executive function and performed worse than noncarriers at almost all ages; (ii) main effect of age on anterior fibers, but main effect of APOE ε4 on posterior fibers, and the interactive effect of them existed on anterior and posterior fibers; (iii) carriers showed an accelerated age-related integrity reduction of these fibers compared to noncarriers who had a slight decrease but not significant; and (iv) significant associations of the higher white matter integrity with better multi-cognitive performance in old ε4 carriers. Overall, combining APOE status with age may be useful in assessing possible mechanisms of disease development in AD.Advanced age and apolipoprotein E ( APOE) ε4 allele is a well-established one. Comparing with ε4 allele noncarriers, individuals carrying one copy of ε4 allele had 2- to 3-fold, individuals carrying two copies of ε4 allele had about 12-fold AD risk . For each DTI scan, images covering the whole brain were acquired by an echo-planar imaging sequence with the following scan parameters: TR=9500 ms, TE=92 ms, 30 diffusion-weighted directions with a b-value of 1000 s/mm2, and a single image with a b-value of 0 s/mm2, slice thickness=2 mm, no inter-slice gap, 70 axial slices, acquisition matrix =128×128, FOV=256×256 mm2, averages=3.Among the participants, 111 subjects (53 https://www.fmrib.ox.ac.uk/fsl). First, the DICOM files of all subjects were converted into NIfTI using the dcm2nii tool embedded in MRIcron. Second, the brain mask was estimated, and the resulting brain mask was required for the subsequent processing steps. Third, the non-brain spaces in the raw images were cut off leading to a reduced image size, reducing the memory cost and speeding up the processing in subsequent steps. Fourth, each image was coregistered to the b0 image using an affine transformation to correct the eddy-current induced distortions and simple head-motion artifacts. The diffusion gradient directions were adjusted accordingly. Fifth, a voxel-wise calculation of the tensor matrix and the diffusion tensor metrics were yielded for each subject .DTI data were processed with the FDT toolbox in FSL (http://cmrm.med.jhmi.edu/), a probabilistic atlas generated by mapping the DTI data of 78 subjects to a template image. The atlas contains 20 main WM bundles and has three individual sets of sub-templates with different probability levels in the probability tractography map: 0%, 25% and 50%. In this study, we chose to use the 25% threshold subtemplate, which contains 20 major tracts. The JHU-WM atlas was overlaid on the WM skeleton of each subject in the CBM-DTI-81 space, such that each skeleton voxel could be categorized into one of the major tracts. Then, the mean FA at the skeleton voxels within each tract was calculated.To identify major white matter tracts, we used the digital WM atlas JHUICBM-DTI-81 (APOE ε4 carriers vs. noncarriers) in age and education, two-sample t-test were performed. For gender and medical history, Chi-square test was applied. For each cognitive domain, we first tested the APOE ε4 genotype and age interaction by using a general linear model (GLM), and then used curvilinear regression models to assess associations between each cognition and age in the APOE ε4 carrier and noncarrier separately. For the multiple nodal efficiencies, we applied the FDR procedure to correct the multiple comparisons at a q-value of 0.05.To evaluate the group difference (APOE ε4 genotype × age interaction on each cognitive domain (raw MMSE score or composite cognitive domain scores). Specifically, a GLM with “age”, “APOE ε4” and “APOE ε4 × age” as predictor variables were applied, in which gender and education were included as covariates. Curvilinear regression models were used to assess associations between cognitive functions and age in the APOE ε4 carriers and noncarriers respectively. We use linear, quadratic, or sigmoidal regression curve to fit different cognitive functions and judge from the goodness of fit to data (R2).We assessed the APOE ε4 × age interaction on fiber diffusion metrics  at each tract was tested using GLM. Specifically, for each fiber, we applied a GLM with “age”, “APOE ε4” and “APOE ε4 × age” as predictor variables. Also, gender and education were included as covariates.The influence of the APOE ε4 carriers and noncarriers separately. Since there is no easy way to plot a two-way interaction with two continuous variables , we first equally divided all carriers or noncarriers into four sub-groups in terms of the rank of age: low-age carriers  and high-age carriers , low-age noncarriers  and high-age noncarriers . And then calculate the partial correlation of white matter integrity and cognitive scores, accordingly. Gender and education were included as covariates.We further investigated how the age influences correlation between the above-identified white matter tracts and the cognitive performance in Supplementary Tables


Brassica rapa genomes recently sequenced and assembled using long-read technology and optical mapping, we investigated structural variants and repetitive content between the two accessions and genome size variation among a core collection. We explored the structural consequences of the presence of large repeated sequences in B. rapa ‘Z1’ genome vs. the B. rapa ‘Chiifu’ genome, using comparative genomics and cytogenetic approaches. First, we showed that large genomic variants on chromosomes A05, A06, A09, and A10 are due to large insertions and inversions when comparing B. rapa ‘Z1’ and B. rapa ‘Chiifu’ at the origin of important length differences in some chromosomes. For instance, lengths of ‘Z1’ and ‘Chiifu’ A06 chromosomes were estimated in silico to be 55 and 29 Mb, respectively. To validate these observations, we compared using fluorescent in situ hybridization (FISH) the two A06 chromosomes present in an F1 hybrid produced by crossing these two varieties. We confirmed a length difference of 17.6% between the A06 chromosomes of ‘Z1’ compared to ‘Chiifu.’ Alternatively, using a copy number variation approach, we were able to quantify the presence of a higher number of rDNA and gypsy elements in ‘Z1’ genome compared to ‘Chiifu’ on different chromosomes including A06. Using flow cytometry, the total genome size of 12 Brassica accessions corresponding to a B. rapa available core collection was estimated and revealed a genome size variation of up to 16% between these accessions as well as some shared inversions. This study revealed the contribution of long-read sequencing of new accessions belonging to different cultigroups of B. rapa and highlighted the potential impact of differential insertion of repeat elements and inversions of large genomic regions in genome size intraspecific variability.Traditionally, reference genomes in crop species rely on the assembly of one accession, thus occulting most of intraspecific diversity. However, rearrangements, gene duplications, and transposable element content may have a large impact on the genomic structure, which could generate new phenotypic traits. Comparing two  Brassica napus varieties correlating with genes and phenotypic traits such as silique length and seed weight (Flowering Locus C (or FLC) genes  genes . HoweverBrassica pangenomes has started to reveal the extensive number of structural variants within one of the two diploid progenitors Brassica oleracea  is one of the diploid progenitors of the important allotetraploid oilseed crops, B. juncea , and B. napus   and ssp. trilocularis (yellow sarson with ‘Z1’). Chinese cabbage has originated in central China by natural hybridization between Pak-choi (ssp. chinensis) and turnip rape (ssp. oleifera) 1,200–2,100 years ago  . Therefoears ago . Diploid process . The maj process , and ove species . B. rapa2.18 Mya , but its in Asia . This re in Asia  and phenears ago . Yellow Brassica genomes, we compared structural rearrangements between two varieties of B. rapa that have contrasted morphotypes, evolutionary history and reproductive mode. Specifically, we identified differential insertion of ribosomal DNA and LTR retrotransposons and reconstructed the evolution of these repeats during B. rapa domestication. Finally, we unraveled wide variation in DNA content among different accessions belonging to B. rapa core collection.Using established protocols (flow cytometry and cytogenetics), along with state-of-the-art whole genome sequencing and optical mapping for assembly of complex B. rapa genotypes, B. rapa var. pekinensis cabbage ‘Chiifu’ (pure line), and B. rapa var. trilocularis ‘Z1’ (doubled haploid line), as well as their F1 hybrid (‘Z1’ × ‘Chiifu’), were grown in semi-controlled conditions in a greenhouse. Seven to 10 individuals of each B. rapa accession representing biological replicates were harvested for leaves and roots. Leaf tissue was used for molecular work and flow cytometry, whereas root meristem samples were used to perform fluorescent in situ hybridization (FISH) experiments. In addition, 10 accessions representing the different groups of B. rapa core collection . The resulting GFF3 files were hand curated using custom python scripts (Python version 2.7.12).The latest genome assemblies available for apa ‘Z1’  and ‘Chiapa ‘Z1’  were retPET V2.5 . BrieflyB. rapa ‘Z1’ and ‘Chiifu,’ as well as the A subgenome of B. napus ‘Darmor Bzh’ V4.1, were retrieved from the Brassica database2 and compared. The package MUMmer V3.23  isolated from Triticum aestivum. pTa-71 was labeled with Alexa-488 dUTP by random priming. Biotinylated probes were immunodetected by Texas red avidin DCS (Vector Laboratories), and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories) containing 2.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using an ORCA-Flash4  on an Axioplan 2 microscope  and analyzed using Zen 2 PRO software . Finally, the lengths of both A06 chromosomes in the hybrid ‘Z1’ × ‘Chiifu’ were estimated on 14 cells at the mitotic metaphase stage and measured using the visualization software Zen 2 PRO .To validate the presence of repeated sequences on the chromosome A06 of  B. rapa  were lab B. rapa , which cB. rapa var. pekinensis ‘Chiifu’ (from2) and B. rapa var. trilocularis ‘Z1’ (from3), applying a method based on a calibration using the CNV (based on read depth approach) of single copy genes (scripts available at github/juboutte). First, 17,954 putative single copy genes in ‘Chiifu’ and ‘Z1’ were identified using custom-made python scripts based on PCK genes from B. rapa  and 96 gypsy sequences downloaded from the gypsy database 4, were retrieved as reference sequences. Then, rDNA and gypsy sequences of ‘Chiifu’ and ‘Z1’ were identified by performing BLASTn and tBLASTn algorithms  and gypsy LTR Retrotransposons were obtained using Shotgun data for  B. rapa . Second,B. rapa ‘Chiifu’ and ‘Z1,’ phylogenetic analyses were performed. First, 45S rDNA and gypsy sequences (first translated to protein sequences and merged to the 96 gypsy database sequences) of ‘Chiifu’ and ‘Z1’ were aligned separately using Mafft alignment v.7.245; option –auto  were harvested and transferred to a Petri dish. This material was chopped using a sharp razor blade in 500 μL of staining buffer (from Cystain PI OxProtect) and incubated at room temperature for 30–90 s. The solution was then filtered through a 50 μm nylon mesh, and 1.5 mL of solution (0.0166 mg of RNase A and 10 μL of propidium iodide) was added per sample. Incubation at room temperature was made for 30–60 min, protected from light. Estimation of genome size for each accession was obtained using a CyFlow space cytometer (Partec Inc.). This instrument was equipped with a 488 nm blue laser 50 mW and a band-pass filter LP590 used as an emission filter. Also, prior to running the samples, gain and linearity of the instrument were adjusted by using DNA control PI from Sysmex. Finally, G1 peaks in B. rapa accessions and P. sativum were collected for each sample to calculate nuclear DNA content (1C in pg) and haploid genome size (in Mbp).In order to explore the intraspecific variability of genome size among the different varieties of B. rapa ‘Z1’ and B. rapa ‘Chiifu’ and proportions of sequences within each LTR gypsy clade were investigated using χ2-test. Genome size variations were investigated using analysis of variance (ANOVA) and Tukey pairwise comparisons [honestly significant difference (HSD)] tests. Normality of data was first tested by Shapiro test. Statistical tests were performed using R software v.3.5.1 , A06, A08, and A09  were composed of a mean number of TEs, rDNA, and genes of 4,244.5 , 25.5 (median = 19), and 160.5 (median = 135.5), respectively  and ‘Chiifu’  reciprocally . InversiB. rapa ‘Z1’ and B. rapa ‘Chiifu.’ TEs were annotated along the 10 chromosomes of both genomes with higher proportions in the centromeric regions. Considering a genome size of 529 Mb for B. rapa ‘Z1’ and ‘Chiifu’ after The proportions of repeat elements  were estimated in B. rapa ‘Z1,’ several chromosomes  had a higher proportion of TEs compared to ‘Chiifu’ assembled chromosomes. As expected, regions rich in TEs had a lower proportion of genes. Ribosomal sequences were observed in the 10 chromosomes of both genomes with a higher proportion on the A01, A05, A06, and A09 compared to other chromosomes of ‘Z1’ . Indeed, chromosomes A05 and A06 had a higher percentage of LTR TEs in comparison to the other chromosomes (In  of ‘Z1’ . As the  of ‘Z1’ . These r of ‘Z1’ . Proportomosomes . CNV anaB. rapa samples (clade 3) and two containing only ‘Z1’ sequences (clades 1 and 2). These two clades contained 67 and 96 ‘Z1’ rDNA sequences, respectively, coming from chromosomes A01, A05, A06, and A09 .As the large specific sequences of ‘Z1’ were made predominantly of rDNA and LTR gypsy, phylogenies of both repeated elements were performed to explore their evolutionary dynamics. Ribosomal DNA phylogeny of the ‘Chiifu’ and ‘Z1’ genomes allowed us to identify three clades, one common to the two  and A09 . LTR gyp and A09 . Proportin situ hybridization was performed on ‘Z1,’ ‘Chiifu,’ and F1 ‘Z1’ × ‘Chiifu’ individuals to compare their A06 chromosomes, presenting high length variation. The 45S probe (green fluorescence) on B. rapa marked five different chromosome pairs . The chromosome A06 from ‘Chiifu’ was on average 17.6% (±6.96%) smaller compared to the chromosome A06 of ‘Z1’ , the significative impact of accessions was tested , followed by Tukey pairwise comparisons and group assignment based on P < 0.05. Of particular interest, the two focal varieties ‘Chiifu’ and ‘Z1’ have significantly different 1C DNA content with 0.557 (median = 0.565) and 0.590 (median = 0.588), respectively . All accessions can be divided in seven subspecies and six groups structured within the core collection  of the genome presenting such variations  of genome-specific sequences based on all possible pairwise comparisons  represent dispensable genes  exhibited wider standard error than any other Brassicaceae species. By comparison in A. thaliana, the analyses of DNA content in 10 ecotypes showed little variation with less than 1% among the different accessions . In cessions , which svariance .Usually, genome size variation is often associated with TE content and ribosomal repeats as these elements could be highly dynamic. The large specific insertions identified in ‘Z1’ allowed improving our understanding of centromeric and pericentromeric regions that are usually difficult to sequence and assemble in complex genomes such as plant genomes, because of their high density in repeats. Interestingly, apart from some dispensable genes, specific ‘Z1’ insertions exhibited mostly both rDNA copies and LTR elements.Brassica species, the number of 45S rDNA loci is variable. For example, B. oleracea, B. nigra, and B. rapa have 4, 6, and 10 rDNA loci, respectively  ‘Schneeball’ (six loci). Here, we investigated the 45S rDNA in two other B. rapa varieties, ‘Z1’ and ‘Chiifu.’ The number of 45S rDNA loci identified in these two varieties  is highly similar to the number of rDNA copies previously estimated in this variety  act as modifiers of recombination that create different patterns of recombination rate across the genome (B. rapa composing the core collection. Thus, exploring this diversity will deeply improve our understanding of the species adaptive processes and could be used in the creation of new interspecific and intraspecific Brassica hybrid varieties.Our results unveil the necessity for long-read sequencing strategies and developing various whole-genome references for agronomically important species . This ree genome , leadinge genome . Similare genome . First hhttp://www.genoscope.cns.fr/plants and http://brassicadb.org.Publicly available datasets were analyzed in this study. This data can be found here: JF and JB conceived and designed the experiments with inputs from LM. TC, SL, and LM implemented the REPET pipeline and performed the work on TE annotation. JB, LM, CF, and JM performed the bioinformatics analyses. J-MA and CB analyzed the generated optical maps and verified regions of interests. AB, VH, and OC performed the cytological analyses. GT performed the flow-cytometry experiments. GD and ML performed the DNA extractions and PCR to verify inversions. A-MC and MR-G helped supervise the project and participated in critical thinking of the results with LM, FB, CF, OC, JB, and JF. JF and JB wrote the manuscript. All authors approved the final manuscript.The 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.


Inequality is rife throughout South Africa. The first wave of COVID-19 may have affected people in lower socioeconomic groups worse than the affluent. The SARS-CoV-2 seroprevalence and the specificity of anti-SARS-CoV-2 antibody tests in South Africa is not known.We tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott SARS-CoV-2 IgG assay. We assessed the association between antibody positivity and COVID-19 symptom status, medical history, and sociodemographic variables. We tested 137 serum samples from healthy controls collected in Cape Town prior to the COVID-19 pandemic, to confirm the specificity of the assay in the local population.Of the 405 volunteers tested one month after the first peak of the epidemic in Cape Town, 96(23.7%) were SARS-CoV-2 IgG positive. Of those who tested positive, 46(47.9%) reported no symptoms of COVID-19 in the previous 6 months. Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The specificity of the assay was 98.54%(95%CI 94.82%-99.82%) in the pre-COVID controls.There is a high background seroprevalence in Cape Town, particularly in people of lower socioeconomic status. Almost half of cases are asymptomatic, and therefore undiagnosed by local testing strategies. These results cannot be explained by low assay specificity. Despite the early implementation of a stringent nationwide lockdown protocol, within 6 months South Africa (with a population of ¬60 million people) had registered over 620 000 cases of COVID-19, recording over 12 000 new cases per day at the peak of the first wave Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is an interesting article and informs policy formulation in combating Covid-19. The paper looks at two aspects, firstly the SARS-CoV-2 seroprevalence in workers with lower socioeconomic status in Cape Town and then checking the specificity of the Abbott SARS-CoV-2 IgG Assay. These are very important issues as the whole world suffers from the Covid-19 pandemic.In the first part of the study, the authors tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott 29 SARS-CoV-2 IgG assay. They assessed the association between antibody positivity and COVID- 19 symptom status, medical history, and sociodemographic variables.The study found that Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The main strength of this paper is that it addresses an interesting and timely question of whether there is an association between antibody positivity and COVID- 19 symptom status, medical history, and sociodemographic variables. It is a pertinent issue in Africa as most families have low-income jobs and live in high densely populated towns. Therefore, it was a noble idea to find out if there was a link between lower socioeconomic and SARS-CoV-2 seroprevalence.In the second part of the study, the specificity of the Abbott SARS-CoV-2 IgG Assay. The study also noted a high specificity of the assay with regard the South African population. The sample size for the verification exercise validates the results of the assay specificity. However, it would have been good to compare the serum sample results with SARS-CoV-2 RT-PCR as it is currently the gold standard instead of assuming the absence of SARS-CoV-2 from the local population at the time of sample collection. It was also ideal to check Sensitivity of the test kit to complete the process of kit verification. This would enhance the outcome of the study as the accuracy of the test kit in the given population would have been determined.Overall, the paper is good and concise and brings valuable information.Reviewer #2: In general, the paper is logically presented and is easy to follow. A few spelling and grammar mistakes are present as outlined in my comments below.The title and abstract cover the main aspects of the work. The results uncover the high seroprevalence that are not detected by the current strategies, this is novel. This provides useful insights for strategies review. The methods are clear and replicable.All the results presented match the methods described and statistical analysis were appropriate to the research question and study design. Data presentation was clear and easy to follow. Control selection was appropriate for the study design.There is correlation of results and conclusion. I am satisfied with the validity of the manuscript.A. Major Comment1. No attempt has been made to address potential bias through analytic methods, eg., sensitivity analysis.B. Minor Comments.1. Line 99: It is important to write how serum is obtained since you can not directly collect serum from a patient.2. Line 148: It is not clear if these 17 participants are out of the 180 or the whole sample of 405.3. Line 151: It is not clear if these 67 are out of the total sample or 180, make it clear.4. L-211-212-213-214. From your study there has been no significant association between designated essential workers and sero-positivity. Is it possible that in the discussion of this finding you add and highlight some studies of sero-prevalence done among essential workers such as Healthcare workers as they have been found out to be associated with risk of infection from SARS-CoV 2?5. This paper highlights the issues of socio-economic and demographic status in relation to SARS CoV 2 sero-positivity and as such did you by any means collect data on the races of the participants as it would be interesting to know and link to prior studies done of who is marginalised and of low socio-economic status in Western Cape and their perceived associated risk to SARS-CoV 2 infection.6. The discussion section gave comparative analysis to UK and USA and Nigeria; please check if there are any other sero-prevalence studies in Africa, or Sub Sahara Africa that corroborates to your findingsC. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Authors may consider the below small changes as applicable.1. Line 23: South Africa has a “higher” degree not South Africa has a “high” degree since this is comparison.2. Line 40: Should instead be “Almost half of cases are asymptomatic” not “Half of cases are asymptomatic” because it is not exactly 50% according to your findings.3. Line 82: Put comma  between “2020” and “we”.4. Line 88: should read “During the stringent lockdown” add “the”.5. Line 88: Add comma between “lockdown” and “most”.6. Line 88-89: Add comma between “work” and “either.7. Line 98: should read “directly into” not “directly “on to”.8. Line 101: Add these commas “Abbott Laboratories, South Africa, Pty Ltd”.9. Line 103: Should be in past tense and read “An index of >1.4 was considered” not “is considered”.10. Line 105: Should read “of between 87.5% and 100%”,11. Line 105: Should read “of between 99.63% and 100%12. Line 105: should read “Using a 99% specificity” not “Using this 99% specificity”.13. Line 107: consider the word “ascertain” not “assert”.14. Line 121-122: Should read: Out of 410 volunteers screened, 5  were excluded from the study15. Line 134: rather “pre-existing condition” not “pre-existing diagnosis”.16. Line 143: remove “any” from “having had any”.17. Line 188: put a comma between the closing bracket and a18. Line 204: Remove comma between “epidemic” and “demonstrates”.19. Line 218: Put a comma between “City” and “the”20. Line 222: Put comma between “pandemic” and “the counties”.21. Line 223: Put comma between “pandemic” and “these”.22. Line 223: Put “a” between “had” and “higher”.23. Line 224: Put “a” between “UK,” and “larger”.24. Line 227: Should be “smaller increases” not “small increases” since this is comparison.25. Line 231: Remove comma between “wave” and “is higher”.26. Line 232: add “a” between “with” and “high”.27. Line 232” use “higher” and “lower” since this is a comparison.28. Line 242: Add comma between “study” and “almost”.29. Line 250: Add comma between “study” and “41”.30. Line 251: Rather make it read “of which 24” instead of “of whom 24”..31. Line 265: Should be “markers” instead of “marker” since statins is plural too.32. Line 267: Put comma between “group” and “the”.33. Line 269: Remove comma between “method” and “which”34. Line 284: Put comma between “study” and “we”**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  12 Feb 2021We have attached a 'response to reviewers' letter which responds to all comments from the reviewers and editor. We also attach revised manuscripts with and without tracked changes. The figures were uploaded to, and reviewed by PACE before the first submission, and so this process has not been repeated. Thanks for the opportunity.AttachmentResponse to Reviewers_PONE_11.02.2021.pdfSubmitted filename: Click here for additional data file. 15 Feb 2021Higher SARS-CoV-2 seroprevalence in workers with lower socioeconomic status in Cape Town, South Africa.PONE-D-21-00967R1Dear Dr. Shaw,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Exploring the chemical composition and biological activity of different fruit varieties is essential for the valorization of their health claims. The current study focuses on a detailed comparative analysis of three early- and two mid-ripening peach varieties: “Filina” (peach), “July Lady” (peach), “Laskava” (peach), “Gergana” (nectarine), and “Ufo 4” (flat peach). They were characterized in terms of essential nutrients such as carbohydrates (sugars and dietary fibers), amino acid content, and lipids as well as mineral content, fat-soluble vitamins, carotenoids, and chlorophyll. Polyphenolic compounds and the related antioxidant activity were also assessed. The methanolic extract of the peel seems to be richer in the studied biologically active substances compared to the fleshy part of the fruit. Anthocyanins were most abundant in “Gergana” and “July Lady” extracts . The total phenol content of the samples varied from 34.11 ± 0.54 to 157.97 ± 0.67 mg gallic acid equivalents (GAE)/100 g fw. “Filina” and “July Lady” varieties possessed the highest antioxidant activity. Overall, the results of this study confirm that the studied peach varieties have satisfactory nutritional value and are potential sources of biologically active substances. Each variety represents an individual palette of nutrients that should be considered separately from the other. Nutrition can be seen as balanced when eating the appropriate amounts of a large variety of foods . Fruits Stone fruits are highly prized for their unique aesthetic and organoleptic characteristics. Traditionally, fruit quality indicators include appearance, sugar, and acid content. However, fruits also contain innumerable phytochemicals, which, although at relatively low concentrations, play a key role in overall quality ,6. Some Prunus persica L. Batsch (peach) is the second most important fruit in Europe after the apple [Prunus persica is suitable for direct consumption, possessing a pleasant and refreshing taste. Peaches are traditionally cultivated crops in Bulgaria. Due to the support of different EU funds, the amount of stone fruit gardens continues to grow. Stone fruits are Bulgaria’s largest fruit category, accounting for 40 percent of Bulgarian total fruit production. Since 2017, Bulgaria has ranked fifth in the EU in the area of peach orchards according to Eurostat data. There are plenty of Bulgarian varieties with excellent properties that could be presented to the EU market [he apple . Italy aU market .The nutritional profile of peaches depends on the presence of organic acids, minerals, carbohydrates, and dietary fiber that are among the major constituents of the fruit . Apart fInformation about the nutritional value of peach fruit from Bulgaria in different ripening stages and varieties is scarce. Moreover, three of the studied varieties have been relatively recently introduced. In this context, the present paper aims at characterizing and comparing three early- and two mid-ripening peach varieties: “Filina” (peach), “Ufo 4” (flat peach), “Gergana” (nectarine), “July Lady” (peach), and “Laskava” (peach) first, and then making propositions about the most beneficial variety in terms of nutrient quality. They were specified in terms of essential nutrients, such as carbohydrates , proteins (including amino acid content), and lipids as well as fat-soluble vitamins, carotenoids, and chlorophyll. Mineral content was also determined. In addition, polyphenolic compounds and the related antioxidant activity were studiedby Ferric Reducing Antioxidant Power (FRAP), Cupric Reducing Antioxidant Capacity (CUPRAC), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) methods.The following early-ripening peach and nectarine varieties were used: “Filina” (peach), “Ufo 4” , and “Gergana” (nectarine). “Filina” B is a BuPrunus persica (L.) Batsch and Prunus ferganensis (Kost. and Rjab.) from the parent combination “Hale” × (“Elberta” × “Fergana Yellow”). “July Lady” is an American peach variety spread worldwide (The mid-ripening “Laskava” (peach) and “July Lady” (peach) were investigated in the present study. “Laskava” D is a Buorldwide E.The trees were grafted on seedlings and were planted in the springs of 2012–2013 at two densities—50 and 100 trees per ha. The varieties were clingstone and semi-clingstone types. No bactericides were applied to plantings during testing.The undamaged peach, nectarine, and flat fruit were harvested at eating ripeness in the Fruit-growing Research Institute, Plovdiv, BG (lat. 42.10384828045957 and long. 24.72164848814686). Fruit were considered ripe, on the trees, when the growth of the fruit had stopped, the fruit began softening, exhibited a yellow or orange ground color , and was easily detached. Extraction procedures were performed and described for each analysis. The moisture content of the peach samples in fresh state was as follows: “Filina” 84.57%, “Gergana” 84.74%, “Ufo 4” 87.03%, “July Lady” 84.57%, and “Laskava” 85.26%.Peach fruit (peel and pulp) was analyzed in a fresh state for macro- and microelements and heavy metals by the microwave mineralization method. The analysis was performed in the accredited laboratory complex of the Agricultural University, Plovdiv. Trace elements have been determined by atomic absorption spectrometry after ash drying according to EN 14082:2003 . Sodium,The amino acid composition was determined by the method described by Tumbarski et al. . Fresh pTotal nitrogen content was determined using the Kjeldahl method according to ISO 1871 and protein content was calculated by multiplying the result by a conversion factor of 6.25.w/v)) in an ultrasonic bath  with a frequency of 45 kHz and 30 W power at 45 °C in triplicate. The samples were filtered. The contents of sugars and sorbitol were determined using a Shimadzu HPLC, coupled with an LC-20 AD pump, and a Shimadzu RID-10A refractive index detector (RID). The separation was done on a Shodex® Sugar SP0810 (300 mm × 8.0 mm i.d.) column with Pb2+ and a Shodex SP-G guard column   operating at 85 °C. The mobile phase was ultra-purified water  with a flow rate of 0.5 mL/min. The injection volume was 20 μL [The preparation of sample extracts was performed with distilled water  methods (2012) using Soxhlet apparatus. Each sample (around 2 to 3 g) was packed in a pre-weighed, oven-dried thimble. The thimbles were stapled and placed in a Soxhlet apparatus, and extracted for 6 h with n-hexane. The extracts were evaporated under vacuum and the residues were weighed. The results are expressed as g/100 g fw.The total dietary fibers were determined using a K-TDFR-100A , according to AOAC method 991.43  “Total, Three extraction procedures with respect to phenolic compounds were carried out as follows:v/v) at 50 °C by ultrasonication for 30 min (MEF). The residues and the extracts were separated by filtering through a filter paper; the obtained residues were re-extracted with a fresh portion of extractant in the same conditions.Homogenized fresh whole fruit (peel and pulp) from each variety (each 20 g) cut into small pieces was extracted with 80% aqueous methanol  at 50 °C by ultrasonication for 30 min (MEP). The residues and the extracts were separated by filtering through a filter paper; the obtained residues were re-extracted with a fresh portion of extraction solvent in the same conditions.The peel of fresh fruit cut into small pieces (each 15 g) was extracted with 80% aqueous methanol  from each variety (each 20 g) cut into small pieces was extracted with 100 mL water by ultrasonication at 50 °C for 15 min (WEF). The extract was then subjected to heat reflux extraction for 30 min and afterwards the residues and the extracts were separated by filtering through a filter paper.®-Werke GmbH & Co., Germany) at 50 °C. The obtained semi-liquid extracts were preserved at 4 °C, until used for further experiments.The extracts recovered from each of the extraction procedure were subjected to removing the excess of the solvent by distilling off in a vacuum rotary evaporator , coupled with diode-array detector  and EZChrom Elite™ software . Separation of the phenolic acids was performed by a Supelco Discovery HS C18 column , operated at 30 °C under gradient conditions with mobile phase consisting of 2% (v/v) acetic acid (solvent A) and acetonitrile (solvent B), as reported by Mihaylova et al. [The qualitative and quantitative determination of phenolic acids in the extracts was performed by using a Hitachi LaChrom Elitea et al. . The graw/v), and then a 5 mL tetrachlormethane:methanol mixture  with 0.5% butylated hydroxytoluene added. After the extraction in an ultrasonic bath  for 15 min, 1 mL of 10% NaCl was added. The samples were centrifuged, and the tetrachlormethane fraction was separated, filtered through anhydrous Na2SO4, and used for carotenoid analysis. Qualitative and quantitative determination of carotenoids was performed by using a LaChrom Elite  high-performance liquid chromatography (HPLC) system equipped with a diode array detector (DAD) and LaChrom Elite  software. The assay was performed according to the method described by Mihaylova et al. [v/v, solvent A) and tert-butyl methyl ether . The gradient elution program was performed as follows: 0–0.5 min 95% solvent A, at 3 min 80% solvent A, from 4.5 to 10 min 65% A/35% B, and at 20 min 95% A/5% B. The detection of β-carotene was carried out at 450 nm and the detection of lutein and lycopene at 470 nm. The results are expressed as μg/g fw, according to the established percentage of moisture content for each peach variety.Freeze-dried plant material (0.2 g) was extracted with 4 mL methanol 1:20, , w/v, anv/v) were added. The organic phase was separated, vacuum evaporated to dryness, and then dissolved in 1 mL HPLC grade methanol (Sigma) for further analyses.Freeze-dried plant material (1 g) was saponified with a 10 mL solution  in a water bath at 70 °C under reflux for 30 min. After the saponification process, 15 mL 1% NaCl and a 15 mL mixture of n-hexane and ethyl acetate  column  at 30 °C in isocratic mode with a mobile phase of methanol:water  with a flow rate of 2 mL/min [Separation of tocopherols was performed on a Symmetry2 mL/min . The toc2CO3. The mixture was vortexed and left for 5 min at 50 °C. After incubation, the absorbance was measured at 765 nm. The TPC is expressed as mg gallic acid equivalents (GAEs) per 100 g fw.The TPC was analyzed following the method of Kujala et al.  with som3)3, 0.1 mL of 1 M CH3COOK, and 3.8 mL of ethanol. After incubation at room temperature for 40 min, the absorbance was measured at 415 nm. Quercetin (QE) was used as a standard and the results are expressed as mg quercetin equivalents (QE)/100 g fw.The total flavonoid content was evaluated according to the method described by Kivrak et al. . An aliq3COONa  with an appropriate dilution factor. Absorbance (A) was measured using a UV–Vis spectrophotometer at 520 and 700 nm after a 15 min incubation at room temperature, and the results were calculated as follows:The total monomeric anthocyanin content was determined using the pH differential method . ProperlThe monomeric anthocyanin (MA) pigment concentration in the samples was calculated as:−4 M solution of DPPH was mixed with the samples in a ratio of 2:0.5 (v/v). The light absorption was measured at 517 nm after a 30 min incubation. The DPPH radical scavenging activity is presented as a function of the concentration of Trolox—Trolox equivalent antioxidant capacity (TEAC) and is defined as the concentration of Trolox with equivalent antioxidant activity expressed as μM TE/100 g fw.The ability of the extracts to donate an electron and scavenge 2,2-diphenil-1-picrylhydrazyl (DPPH) radicals was determined by the slightly modified method of Brand-Williams et al.  as descr•+) was estimated according to Re et al. [•+) was produced by reacting ABTS stock solution (7 mM) with 2.45 mM potassium persulfate  and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. Afterward, the ABTS•+ solution was diluted with ethanol to an absorbance of 0.7 ± 0.02 at 734 nm and equilibrated at 30 °C. After the addition of 1.0 mL of diluted ABTS•+ solution to 0.01 mL of samples, the absorbance reading was taken at 30 °C after 6 min. The results are expressed as the TEAC value (μM TE/100 g fw).The radical scavenging activity of the extracts against 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)  was mixed with 1 mL of neocuproine methanolic solution (7.5 × 10−3 M), 1 mL of CH3COONH4 buffer solution (pH 7.0), and 0.1 mL of herbal extract (sample) followed by the addition of 1 mL of water  and mixed well. Absorbance against a reagent blank was measured at 450 nm after 30 min. Trolox was used as a standard and the results are expressed as μM TE/100 g fw.The CUPRAC assay was carried out according to the procedure of Apak et al. . One milThe amino acid score (AAS) was determined by comparing the amino acid (AA) composition of each test article with the recommended FAO/WHO reference pattern, mg/g protein . Both thAnalytical determinations were performed in triplicate and the results are expressed as mean ± SD. Relevant statistical analyses of the data were performed by one-way ANOVA and a Tukey–Kramer post hoc test (α = 0.05), as described by Assaad et al. . PrincipMinerals are an important part of plant metabolism. Based on their role, they can be divided into macro- and microelements. In this regard, fruits are an essential source of minerals. In the present study, the total mineral content was evaluated. It varied from 689.74 to 1271.17 mg/kg fw .. Early-Amino acids are the building elements of proteins. They may also occur in free form or in non-protein compounds . Apart fIt is well known that most fruits do not have enough protein and the quality in terms of essential amino acid content is low . The amiThe sorbitol content in the investigated peach varieties was the lowest among all carbohydrates. Its concentration ranged from 0.2 to 0.11 g/100 g fw. In three of the peach varieties , the sorbitol concentration was the lowest—0.2 g/100 g fw. Sorbitol, together with sucrose, is the main transport sugar —a product of photosynthesis in the leaves—which is not produced in peach fruit, but is transferred from other parts of the tree by the phloem . “FilinaTotal sugar content was the highest in the “Laskava” variety (7.19/100 g fw) and the lowest in the “Gergana” variety (1.85 g/100 g fw). The total sugar values correspond well to the ones reported for other peach varieties (4.6–9.6%) . In termOverall, the total sugars did not exceed 72 g per kg fruits in the investigated peach varieties, which corresponds to the statement for early and late cultivars reported by Colarič et al. —a maximuThe energy value of each variety was calculated as well and the results show that the highest in energy was the mid-ripening variety “Laskava” (280 kJ), while “Ufo 4” (165 kJ) had the lowest energy value. “Laskava” was the variety with the highest lipid content, which considerably contributes to its energy capacity. All fruit varieties can account for a small amount of the daily energy intake, namely, between 2 and 3%. Most of the energy is at the expense of the carbohydrates contained in the studied fruit. Carbohydrates are the main energy sources for living organisms; thus, the currently investigated peaches can provide a small portion of easily accessible daily energy as well as some other important nutrients.Dietary fiber represents carbohydrates that are not digested or absorbed in the small intestine. They are thought to play an important role in preventing diseases such as cardiovascular disease, colorectal cancer, lung cancer, obesity, and type 2 diabetes . Their cThe lipid content in the analyzed peaches ranged from 0.67 g/100 fw to 2.58 g/100 g fw. The highest lipid content was detected in the “Laskava” variety, which was 3.8 times the lipid content of “Gergana”. The crude lipid contents of many edible fruits are usually lower than 2%, and vary considerably depending on the climate, variety, geographical origin, harvest year, and the methods of cultivation [Total carotenoid content varied between 2.53 and 42.24 µg/g fw and the highest values were established in the “Gergana” variety . Among tThe tocopherol content was attributed to the presence only of α-tocopherol and there was a lack of δ-tocopherol and γ-tocopherol . The conThe presence of chlorophyll is an indicator of fruit maturity. Its content decreases during the ripening of the fruit and disappears completely at full maturity at the expense of other colored compounds like carotenoids, anthocyanins, etc. . Among tP. persica varieties. Total phenolic content in the methanolic peel extracts varied between 157.97 ± 0.67 for “July Lady” and 78.19 ± 0.75 mgGAE/100 g fw for “Laskava”. This is in correspondence with the fact that the peach peels contain two to three times the concentration of total phenolic compounds compared to the flesh [Antioxidant compounds have always been an attractive research area. Phytochemicals are among the most extensively studied. In peaches and nectarines, they represent mainly phenolics and carotenoids. Polyphenols are plant secondary metabolites that have a protective role against environmental stress . Their che flesh ,62. The Flavonoids are an important group of phenolic compounds that possess antioxidant potential in addition to their anticarcinogenic, antimicrobial, and cardioprotective properties . In the Anthocyanins are a subgroup of flavonoids that are mainly responsible for the coloration in fruits . CyanidiMonilinia fructicola) resistance [Among the studied phenolic acids, hydroxycinammic acids were the most abundant. As previously reported, chlorogenic acid dominated in all samples and extracts . Besidessistance . Early-rIn fact, fruit phenolics have a role in fruit visual appearance (color), taste (astringency), and health-beneficial antioxidant properties . Tomás-B• and ABTS•+), ferric-reducing power (FRAP) and cupric ion-reducing antioxidant capacity (CUPRAC) assays  assays .. DifferFollowing the DPPH methods, significant variations between the extracts and varieties were measured—31.89 ± 0.31 to 728.98 ± 3.74 µMTE/100 g fw. “Filina” and “July Lady” showed the highest DPPH inhibitory potential regarding the methanol extracts, while “Filina” and “Gergana” in regard to the water extract .ABTS is another method that describes the capacity of neutralizing free radicals. The measured Trolox equivalents of the peel and fruit methanol extracts varied from 126.05 ± 1.70 to 1332.61 ± 6.29 µMTE/100 g fw, and from 112.10 ± 1.30 to 386.17 ± 5.03 µMTE/100 g fw in the water extracts. Both the peel and the fruit methanol extracts of “July Lady”, “Filina”, and “Laskava” showed the highest ABTS inhibitory potential. With respect to the water fruit extracts, the highest values were established again in “July Lady” and “Filina” varieties . The lowest ABTS values in both peel and fruit samples were observed for the “Ufo 4” variety.Similarly, according to the FRAP and CUPRAC assays, the highest values were established in “July Lady” methanol peel extract , followed by “Filina” and “Gergana” ones. The lowest antioxidant potential was detected in “Laskava” and “Ufo 4” water fruit extracts for both assays—under 130 µMTE/100 g fw. Briefly, a wide variation in the antioxidant potential in the studied varieties was found. Other authors observed differences related to cultivar, tissue, and ripening stage . LombardPCA has been the most frequently used clustering technique to determine how one sample is distinct from another. In the present study, principal component analysis of the obtained peach extracts was performed on 12 variables, including TFC, TPC, DPPH, ABTS, FRAP, CUPRAC, total monomeric anthocyanins, individual phenolic acids, and total phenolic acid content . The resThe present study could be assumed as the first to establish a comprehensive chemical and nutritional profile of peaches and nectarines  of different ripening stages. The heterogeneity of the study in terms of peach varieties (ripening stage and type) can better explain the differentiation of results, i.e., carbohydrates (sugars and dietary fibers), amino acid content, and lipids, as well as mineral content, fat-soluble vitamins, carotenoids and chlorophyll, polyphenolic compounds, and the corresponding antioxidant activity. Experiments confirmed that different parts of the peach  have their own distinct properties. Due to the capacity of the extracts’ solvents, the methanolic extract of the peel seemed to be richer in the studied biologically active substances compared to the fleshy part of the fruit. Peaches, the object of the current study, are a good source of several minerals and vitamins, as well as bioactive compounds of high antioxidant activity, particularly polyphenols and tocopherols. “Filina” was the variety with the richest mineral and carbohydrate content; “Gergana” had the most fiber and minerals; the most carotenoids were contained in “July Lady”, while “Laskava” possessed the most lipids. The current research results indicate many health-promoting properties of peach fruit, pinpointing the unique characteristics of each variety. Researchers can use this study to encourage the daily consumption of peaches.