Lolium perenne L.). The main objectives of the present study were to investigate the level of genetic gain and accuracy by applying GS in commercial perennial ryegrass breeding programs. Different scenarios were compared to a conventional breeding program. Simulated scenarios differed in the method of selection and structure of the breeding program. Two scenarios (Phen-Y12 and Phen) for phenotypic selection and three scenarios  were considered for genomic breeding schemes. All breeding schemes were simulated for 25 cycles. The amount of genetic gain achieved was different across scenarios. Compared to phenotypic scenarios, GS scenarios resulted in substantially larger genetic gain for the simulated traits. This was mainly due to more efficient selection of plots and single plants based on genomic estimated breeding values. Also, GS allows for reduction in waiting time for the availability of the superior genetic materials from previous cycles, which led to at least a doubling or a trebling of genetic gain compared to the traditional program. Reduction in additive genetic variance levels were higher with GS scenarios than with phenotypic selection. The results demonstrated that implementation of GS in ryegrass breeding is possible and presents an opportunity to make very significant improvements in genetic gains.Genomic selection (GS) is a potential pathway to accelerate genetic gain for perennial ryegrass ( Lolium perenne L.) is one of the most cultivated forage species in temperate grasslands, mainly farmed for its re-growth capacity after defoliation, and high value as feed for ruminants, due to palatability, digestibility, and nutritive contents  is a potential pathway to accelerate genetic gain for perennial ryegrass by reducing the length of the breeding cycle as well as increasing selection accuracy  parental individuals, selected from elite varieties, are crossed to generate Bos Taurus), for which GS has perhaps been most successful, computer simulation was first used to demonstrate the benefits of this technology  simulation of ryegrass base population and initial ryegrass varieties, (ii) simulation of conventional breeding and GS schemes in various scenarios.The QMSim software  was used−5. Mutation rates of loci were determined on the basis of the number of polymorphic loci in generation 2000 of the preliminary analysis that were necessary to obtain 1000 polymorphic SNPs and 100 QTL per chromosome. Mutations were limited to the loci in historical population. SNPs and QTL were distinct loci and were randomly drawn from segregating loci, with a minor allele frequency (MAF) higher than 0.05, in generation 2000.A genome consisting of 7 chromosomes of 100 cM with 100 segregating QTL and 1000 SNPs per chromosome was simulated . The bi-e.g., forage yield and rust resistance) in ryegrass breeding (per se) was calibrated in a way that the realized plot Four traits were simulated: Trait 1  were sampled from a multivariate normal distribution i.e., All QTL effects were assumed to be additive and phenotypes were generated by adding a random normal deviate All stages of a typical 12-yr conventional breeding scheme are shown in The conventional breeding program simulated in the present study included two rounds of multi-trait selection. First round of selection was at the stage of The proposed GS-based breeding schemes was designed to integrate with the current breeding program by replacing phenotypic selection points with GS . All steGenomic breeding strategy had two major differences with phenotypic selection. First, A key aspect of a GS is the establishment of a reference population, which is used to train prediction equations for traits in the breeding goal. The reference population for the prediction of marker effects and calculation of GEBV for Both the conventional and genomic breeding schemes were simulated for 36 years by starting a new cycle every year and 36 years of breeding program was equivalent to 25 cycles . Each yeWe examined five different scenarios . Two sceIn this scenario, all cycles for the first 11 years were started by sampling single plants from 20 initial varieties. For each of these cycles, single plants sampled from initial varieties were crossed to create  program . As new cycle 13 . FinallyIn this scenario, the 5 initial cycles were similar to the 11 initial cycles of Sc. Phen-Y12 meaning that a new cycle started by sampling and crossing single plants from the base population. However, for this scenario, it was assumed that both i.e., every year the size of reference population was growing by 250. Also, as in genomic breeding schemes, selection of SYN2 groups in each cycle were based on GEBV, the reference population for the prediction of marker effects at this stage was not limited to the SYN2 groups of the corresponding cycle. Here, once SYN2 groups were available, phenotype and genotype of each SYN2 group were also added to the same reference population that was used for the prediction of marker effects to be used in calculation of GEBV in The breeding structure of this scenario was similar to Sc. Phen-Y12 and can be considered as genomic variant of Sc. Phen-Y12. Similar to Sc. Phen-Y12, the first 11 cycles started by crossing of initial varieties as parents. Once SYN2 groups of cycle 1 were available, they were used as parents for starting cycle 12 and for starting cycle 13, SYN2 groups of both cycle 1 and 2 were available to be used as parents. Thus, in this scenario, only SYN2 groups could be used as starting material for a new cycle once they were available (cycle 12 onward). Similar to Sc. Phen-Y12, first 11 breeding cycles were running independently with no material exchange among the cycles. However, as selection at the stage of This scenario is genomic variant of Sc. Phen. Similar to Sc. Phen, the first 5 cycles started by using initial varieties as parents. For starting cycle 6, This scenario was the same as Sc. GS with one modification at the stage of SYN2. For this scenario, it was assumed that not only Bayesian ridge regression (BRR) implemented in the BGLR package was used to predict effects of SNPs . The folaa, aA and AA, respectively and Σ denotes summation over all SNPs Selections of single plants in the genomic breeding schemes at the stage of Genetic gain in all scenarios was investigated by the cumulative genetic standard deviations . To determine the decay of LD with increasing distance between SNPs, the average To evaluate the extent and magnitude of LD in the initial varieties, LD was measured by https://github.com/hadigenetic/Ryegrass-simulation). Supplemental material available at figshare: https://doi.org/10.25387/g3.1281175.All the materials used for simulation including the R scripts are available on GitHub , which led to at least doubling and trebling genetic gain compared with traditional selection. However, reduction in additive genetic variance levels were higher with GS than with phenotypic selection.The potential effects of applying GS on genetic gain in a commercial ryegrass-breeding program were investigated using simulations. GS breeding schemes resulted in a substantially larger genetic gain for the simulated traits when compared with phenotypic selection. This was mainly due to (a) GS more effectively selecting Different scenarios were compared to the conventional breeding program to investigate their potential in improvement of genetic gain. Simulated scenarios differed in the method of selection and in the structure of the breeding program. For the base scenario (Sc. Phen-Y12) limited genetic gain could be realized over 25 cycles which in practice would requires 36 years to be completed. The reason for the limited genetic gain is basically due to the fact that (a) this breeding scheme only uses SYN2 groups upon their availability as parents for a new cycle , (b) with phenotypic selection, selection of top 2 groups .An alternative for the base scenario (Sc. Phen-Y12) was Sc. Phen. In this scenario, a new cycle could use the In general, genomic breeding schemes had higher genetic gain than corresponding phenotypic based scenarios. The cumulative genetic gain in Sc. GS-Y12 for trait 3 was approximately 2 times greater than the one achieved with Sc. Phen-Y12. Similarly, Sc. GS and GS-SP realized ∼3.2 times more genetic gain than Sc. Phen . This incenarios  but alsoComparing Sc. GS and GS-SP, our results did not show any advantage of using SYN2 single plants (Sc. GS-SP) over SYN2 groups (Sc. GS) as parents for a new cycle. The reason for the similar genetic gain would be that, in both scenarios up to cycle 11, starting material for a new cycle were recruited from the To evaluate genetic gain, the cumulative genetic gain in all scenarios were standardized by the standard deviation of the The accuracy of selection in SYN2 groups was constant over the cycles with phenotypic scenarios and lower than the accuracy obtained by GS scenarios. Across the cycles, in genomic scenarios the accuracy improved for i.e., the same generation) while SYN2 single plants are two generation away from the training population  Fore.g., water soluble carbohydrate content, flowering time). Reference populations would be easier and less costly for collection from spaced plants or clonal rows, especially if phenotypic assessment could be performed with high-throughput methods , which is necessary to achieve usable prediction accuracy. Without these updates to the reference population, the accuracy of prediction would deteriorate after a few cycles. This was confirmed by several studies that have investigated the frequency of reference population updates and their effect on genetic gain  the ability to select individual plants for plot traits using GEBVs trained from a plot reference population and more effective selection of


While the loss of mental imagery following brain lesions was first described more than a century ago, the key cerebral areas involved remain elusive. Here we report neuropsychological data from an architect (PL518) who lost his ability for visual imagery following a bilateral posterior cerebral artery (PCA) stroke. We compare his profile to three other patients with bilateral PCA stroke and another architect with a large PCA lesion confined to the right hemisphere. We also compare structural images of their lesions, aiming to delineate cerebral areas selectively lesioned in acquired aphantasia. When comparing the neuropsychological profile and structural magnetic resonance imaging (MRI) for the aphantasic architect PL518 to patients with either a comparable background (an architect) or bilateral PCA lesions, we find: (1) there is a large overlap of cognitive deficits between patients, with the very notable exception of aphantasia which only occurs in PL518, and (2) there is large overlap of the patients’ lesions. The only areas of selective lesion in PL518 is a small patch in the left fusiform gyrus as well as part of the right lingual gyrus. We suggest that these areas, and perhaps in particular the region in the left fusiform gyrus, play an important role in the cerebral network involved in visual imagery. Thinking of a concept, whether it is a flower or a cat or even a unicorn, can bring up vivid, image-like experiences without external visual input. This is generally referred to as visual imagery or mental imagery, although the latter can extend to other senses . The basis of mental imagery has long been debated ,2,3 and Zeman and colleagues  gave theIt has been argued that the primary visual cortex (V1) plays a significant role in visual mental imagery ,13. SeveThus, damage to V1 appears neither necessary nor sufficient for inducing imagery deficits. A review  of case Visual imagery and perception however cannot share all mechanisms as there are patients on record with seemingly preserved mental imagery but impaired visual perception ,34,35,36Here we present patient PL518, an architect who reported almost complete loss of visual mental imagery following bilateral stroke in the areas supplied by the posterior cerebral artery (PCA). His responses on the Vividness of Visual Imagery Questionnaire  as wellPatient PL518 and four other patients participated in this specific study. All were recruited as part of a larger study of PCA stroke (the Back of the Brain (BoB) project, described in ). 46 conPL518 suffered a bilateral PCA stroke 35 months before the current investigation. At that time, he had corrected to normal visual acuity and a slight visual field defect primarily affecting the parafovea of the upper left quadrant  or a similar background (architect) for a follow-up interview about their visual imagery. The lesion location, size, and time since injury for these patients, as well as other background characteristics, are presented in As PL 518 was the focus of the study, a long and in-depth interview was also carried out about his visual imagery before and after his stroke. In order to get more information about his ability to store visual information in his mind, he was asked to carry out the Rey–Osterrieth Complex Figure Test  at the eAll five patients were asked to complete a version of the Vividness of Visual Imagery Questionnaire , that iThe BoB project is a comprehensive neuropsychological and imaging project investigating perceptual deficits following posterior brain injury . A main This novel paradigm is designed to test immediate and delayed memory for words, objects, and faces (WOF). In the first part (delayed matching), participants were asked to decide whether two sequentially presented images varying in size are the same or not. There were 48 trials for each stimulus type and both accuracy and RTs are measured. The second part (surprise recognition) followed after a short break . Here, participants were asked to decide whether they saw the presented stimuli in the delayed matching task or not. There were 12 trials, and accuracy and RTs were measured. In total, 12 measures were derived from this task: 2 metrics (accuracy and RT) * 3 stimulus types  * 2 paradigms (delayed matching and surprise recognition). See  for a moFamiliarity decision tasks were run for faces, objects, and words. For faces, participants were asked to decide whether a presented face was famous or not . For objects, we used a 72-trial version of a well-studied object decision task , presentTests of picture naming (line drawings), face naming (famous faces) and word reading (regular words). For pictures and words, both accuracy and RTs for correctly named items are analyzed (a voice key was used for RT measurement). For famous faces, only accuracy is recorded as measuring RTs in face naming tasks is complicated by participants making other verbal responses than names .2, resolution matrix = 256 × 206 and voxels size = 0.9 × 1.7 × 0.9 mm3. Automated outlines of the area affected by stroke were generated using Seghier et al.’s modified segmentation–normalization procedure [Structural brain imaging data were acquired from all subjects. Structural scans were acquired on two 3T Phillips Achieva scanners with 32-channel head-coils and a SENSE factor of 2.5 in London and Manchester. A high-resolution T1 weighted structural scan was acquired for spatial normalization, including 260 slices covering the whole brain with TR = 8.4 ms, TE = 3.9 ms, flip angle = 8 degrees, FOV = 240 × 191 mmIn the clinical interview, PL518 reported an almost complete absence of visual imagery following his stroke. This was in stark contrast to his (in his own opinion) above average ability for visual imagery before his stroke that he had relied upon in his work as an architect. He said: “Before, my visualization abilities were pretty impressive. At my work, I could visualize and remember things that most people had not thought about. I would be sitting there and I would say, well, you can’t do X, Y and Z, because you’ve got this happening here and there. Now I have to look at the drawing and work my way through it.” During the interview, he also described how it had felt to do a mental rotation task: “I cannot do it as quickly or the same way as I would have done before my stroke. Before, bang, I would just know the answer. Now it is a much more conscious process. It’s almost as though I physically am trying to move things inside my head.” He was then asked whether his difficulty with mental rotation affected his ability to work as an architect, to which he responded: “Well I just do everything on the computer. That is one of the advantages of us using computers for these sorts of thing nowadays. You can see the stuff happen.” He also described how he is just about able to imagine very simple shapes, but this is done using something akin to motor or spatial imagery and he struggles to imagine more than one shape at a time: “If I tried to visualize shapes like a square, pyramid or sphere lined up next to each other, and I try and focus with a kind of spotlight on the corner of one shape, I can mentally trace a line around the shape. But as soon as I focus on one shape, the others disappear.” When asked if he could imagine an elephant, he seemed to mostly think of the abstract concept of an elephant: “I can think of elephants, iconic elephants like Babar or Elmer, but I can only visualize bits of them. It’s almost painful.” When asked to describe the place he stayed during his last holiday and its surroundings he provided few very vague details about a couple of the bars from the street they had lived on, and he apparently did not visually imagine himself there: “I am recalling almost like a list. I do the same when going somewhere. I have to remember a list”.PL518′s copy and retention of the Rey figure are shown in PL518′s complaints regarding his visual imagery were also clearly reflected in his responses on the VVIQ-modified where he scored 18 , corresponding to minimal imagery . None ofFor the accuracy measures, PL518 is clearly impaired with faces, and shows a deficit (performing more than two standard deviations (SDs) from the control mean) on most individual face measures. He performs within the low–normal range on the object tests and is clearly on level with controls in the tests with word stimuli. For the RT measures, PL518 shows a deficit on most face measures . He responds with latencies within the normal range on the object tests but shows elevated RTs in the lexical decision and word reading tests.Comparing the neuropsychological profile of PL518 to the other included patients, we find that one or more of them show deficits on the same tests/measures and in the cognitive domain(s) as PL518 see . The keyPL518′s lesion is most extensive on the right side, including damage to the occipital pole, the lingual gyrus, the whole fusiform gyrus and extending anteriorly to the parahippocampal region. On the left side, the lesion affects only the medial fusiform gyrus and lingual gyrus, while the left occipital pole, and lateral portions of the fusiform gyrus are spared. See First, comparing the lesions of PL518 to the architect without aphantasia (PM024) shows that PL518 has selective left hemisphere posterior medial fusiform damage extending medially and anteriorly along the collateral sulcus, and selective right hemisphere damage to the superior medial lingual gyrus. Second, comparing the lesion of PL518 to the three patients with bilateral strokes but no aphantasia shows that PL518 has selective damage in the right fusiform gyrus and a portion of the right lingual gyrus, and additional smaller areas of selective damage in PL518 are found in the left fusiform gyrus. Combined, these comparisons reveal only small areas of selective damage in PL518 in the right lingual gyrus and left posterior medial fusiform gyrus.The present study reports case PL518, an architect who lost his ability for visual imagery following a bilateral PCA stroke 35 months prior to this investigation. We compare his performance across a range of perceptual and cognitive tests and a visual imagery questionnaire with four other PCA stroke patients, an architect with a large right hemisphere lesion and three bilateral cases. PL518′s profile on the perceptual and cognitive tests was similar to other cases with the exception that PL518 reported severe visual imagery problems following his stroke. Lesion profiles were also comparable with the exception that PL518 showed selective damage in the right lingual gyrus and left medial posterior fusiform gyrus. It is tempting to suggest that these are both candidate regions for specific involvement in visual imagery.However, Bogousslavsky and colleagues  describeA seeming counterexample comes from de Gelder and colleagues . They deFitting with a role of the left fusiform gyrus in visual imagery, some developmental prosopagnosics appear to have functional abnormalities in this region ,71,72 asCompared to before his stroke, PL518 seems to make greater use of verbal strategies . If PL518 still has some mental imagery, it nonetheless mostly seems to be based on an altered strategy which could be described as motor, action-based, or spatial, such as mentally tracing a line around a shape or doing mental rotation by physically trying to move things inside the head. This is reminiscent of patient MX  who alsoThe two architects, PL518 and PM024, had similar functional deficits, including prosopagnosia, but described vastly different visual imagery . It is tempting to speculate, therefore, that the additional left hemisphere affection in PL518 contributes significantly to his disruption of imagery. In particular, the small patch in the medial left fusiform gyrus where PL518 has unique damage compared to all the four other patients presents as a good candidate for playing a critical part in the generation of visual mental imagery. While our findings indeed suggest that this region is an important node in the cerebral network underlying visual imagery, other areas, including right hemisphere ventral occipito-temporal areas, left hemisphere areas further anterior in the temporal lobe , more pThe most commonly used questionnaire to measure mental imagery is various versions of the VVIQ ,50. The It is still debated whether imagery and perception may be dissociated, or whether they depend on common networks. In one sense, the current results support the former as some patients with heavy damage to ventral stream areas and associated problems with visual cognition nonetheless appear to have intact visual imagery. Our neuropsychological approach suggests that some ventral stream regions might not be necessary for visual imagery despite containing information on imagined objects ,29,89,90It should finally be noted that individual differences in premorbid ability for imagery might play a role in the effects of stroke on these abilities. PL518 reported that his abilities for visual mental imagery had been above average before his stroke. These abilities had enabled him to visualize the spatial and visual attributes of buildings and rooms in rich details and contributed greatly to his achievements as an architect. This fits a general pattern noted by Farah  where mavisual imagery. These regions were only affected in the aphantasic architect PL518 compared to non-aphantasic patients with comparable cognitive and perceptual deficits.While several brain regions in both hemispheres are involved in different aspects of mental imagery, our results indicate that the right lingual gyrus and especially the left posterior medial fusiform gyrus are candidate regions for specific involvement in


Xiphophorus interspecies hybrids represent a valuable model system to study heritable tumorigenesis, and the only model system that exhibits both spontaneous and inducible tumors. Types of tumorigenesis depend on the specific pedigree of the parental species, X. maculatus, utilized to produce interspecies hybrids. Although the ancestors of the two currently used X. maculatus parental lines, Jp163 A and Jp163 B, were originally siblings produced by the same mother, backcross interspecies hybrid progeny between X. hellerii and X. maculatus Jp163 A develop spontaneous melanoma initiating at the dorsal fin due to segregation of an oncogene and a regulator encoded by the X. maculatus genome, while the backcross hybrid progeny with X. hellerii or X. couchianus and Jp163 B exhibit melanoma on the flanks of their bodies, especially after treatment with ultraviolet light. Therefore, dissecting the genetic differences between these two closely related lines may lead to better understanding of functional molecular differences associated with tumorigenic mechanisms. For this purpose, comparative genomic analyses were undertaken to establish genetic variants between these two X. maculatus lines. Surprisingly, given the heritage of these two fish lines, we found genetic variants are clustered together in select chromosomal regions. Among these variants are non-synonymous mutations located in 381 genes. The non-random distribution of genetic variants between these two may highlight ancestral chromosomal recombination patterns that became fixed during subsequent inbreeding. Employing comparative transcriptomics, we also determined differences in the skin transcriptional landscape between the two lines. The genetic differences observed are associated with pathways highlighting fundamental cellular functions including inter-cellular and microenvironment-cellular interactions, and DNA repair. These results collectively lead to the conclusion that diverged functional genetic baselines are present between Jp163 A and B strains. Further, disruption of these fixed genetic baselines in the hybrids may give rise to spontaneous or inducible mechanisms of tumorigenesis. Xiphophorus is similar to that of humans at the histological, transcriptome, and signaling pathway levels , age, sex, immune status, and UV exposure. Variability in genetic background in humans is known to determine susceptibility to melanoma, and possibly body sites . Howevery levels . These aXiphophorus fish, commonly known as platyfish and swordtails, comprise a genus consisting of 26 species of live-bearing fishes commonly found in Mexico, Central and South America. Xiphophorus maculatus represents a long-standing genetic model that has been adopted to study cancer etiology. The most commonly used X. maculatus lines, Jp163 A (JpA) and Jp163 B (JpB), are descendants of siblings derived from a single brood from an X. maculatus female collected in the Rio Jamapa, Veracruz, Mexico in 1939  pigmentation pattern, while JpB is characterized by a spotted side (Sp) pigmentation pattern , which is a mutant duplicate of the fish epidermal growth factor receptor (egfr) gene, and a co-evolved suppressor gene, R(Diff), that prevents the oncogene from inducing tumors in the JpA parental line  and Sp (JpB) pigment patterns, are X-linked and map to a small region of the X chromosome making recombination between xmrk and either Sp or Sd a very rare event. Therefore, Sd and Sp are hypothesized to control neural crest lineage migration to specific body compartments, wherein Sd migration is to the dorsal fin body compartment, while Sp directs neural crest cell migration to the body flanks. Under this hypothesis, the pigmentation pattern of X. maculatus is primarily a matter of inheriting either XSd–xmrk or XSp–xmrk.The species richness of ears ago . This alhellerii . This mohellerii . The tumtal line . Segregatal line  in 50% o lineage . The xmrXiphophorus is also a great system to better understand how the risk factors impact the initiation and progression of the disease, allowing studies of interaction between genome and environment. Unlike spontaneous tumorigenesis observed in interspecies hybrids between JpA and X. hellerii, a similar interspecies backcross involving JpB as the non-recurrent parent leads to hybrids displaying enhanced macromelanophore pigmentation on the flanks of the animals. Only if interspecies hybrids from the later cross  are exposed to UVB (or MNU) at juvenile stage do they exhibit induced tumorigenesis at 4 to 6 months of age , and these differences are related to, and may determine, the tumorigenic trajectory upon interspecies hybridization. Identifying inter-strain genetic variants leading to differences in tumor etiology will forward our molecular genetic understanding of spontaneous and induced melanoma. The comparative genomic and transcriptomic analyses showed that baseline genetic differences involved with inter-cellular and cell-microenvironment interactions. Most importantly, genes encoding core enzymes within base excision repair pathways are enriched with inter-strain genetic variants and differentially expressed genes.We hypothesize that fixed genetic variants between the highly inbred Animal use was approved by the Texas State University Institutional Animal Care and Use Review Board (IACUC protocol #2015107711). Fish utilized in this study were maintained in accordance with the applicable OLAW guidelines governing animal experimentation in the United States, and international legislation regulations governing animal experimentation.Xiphophorus utilized in this study were bred and maintained in the Xiphophorus Genetic Stock Center1. Xiphophorus maculatus (X. maculatus) JpA and JpB strains used for genome re-sequencing were at their 116th and 109th generation of inbreeding, respectively.For DNA isolation, 4 male JpA and 4 male JpB fish were sacrificed by over-anesthetization with MS222 (0.06%). Fish tissues were digested with Proteinase K at 37°C for 1 hr. The lysate was then used for DNA isolation and purification using DNeasy Blood & Tissue Kit (Invitrogen).n = 25), and liver samples (n = 6) were collected; For JpB, skin (n = 30) samples, and liver samples (n = 4) were collected for RNA isolation and sequencing library preparation. RNA isolation was performed following the Qiagen RNeasy RNA isolation protocol . Tissue samples harvested from fish were first homogenized using a hand-held homogenizer in a 1.5 mL microcentrifuges tube while the sample remained frozen in TRI Reagent . After homogenization, 300 μL of fresh 4°C TRI Reagent was added to the samples followed by room temperature incubation for 5 min. Chloroform extraction was performed by adding 120 μL chloroform and shaken for 15 s. Samples were centrifuged  for phase partitioning. The aqueous layer was transferred to a new 1.5 mL microcentrifuges tube and a second chloroform extraction performed . After extraction, nucleic acids in the aqueous phase were precipitated with 500 μL 70% EtOH in diethylpyrocarbonate (DEPC) treated water. The sample was then transferred to a Qiagen RNeasy mini spin column and on-column DNase treatment was performed for 15 min at 25°C. RNA samples were then washed and eluted in 100 μL RNase free water. RNA concentration was measured with a Qubit 2.0 fluorometer . To further assess the RNA quality, an RNA integrity (RIN) score was determined using an Agilent 2100 Bioanalyzer . All samples processed for RNA sequencing had a RIN score above 8.For RNA isolation, fish were anesthetized in an ice bath and upon loss of gill movement were sacrificed by cranial resection. Skin and liver tissues were dissected directly into TRI reagent  and flash frozen in an ethanol dry ice bath. For JpA, skin samples  using 150 bp paired-end (PE) sequencing strategy with an average of 33 X genome coverage. Isolated RNA samples were forwarded for Illumina High-throughput Sequencing using the Illumina TruSeq mRNA Library Prep Kit on the HiSeq 2000 platform . Each RNA sample was used to construct a single library for sequencing  using Bowtie2 “head-to-head” mode . From January 2010 to June 2019, there were a total of 203 scheme B hybrids that inherited xmrk-Sp (11 of them developed spontaneous tumors). A contingency table was tested using animal numbers for each cross scheme that developed tumors, and only exhibited benign pigment cell hyperplasia. A Chi-square test was used to test if the tumor incidences of both cross schemes are independent.Animal records for scheme A and B interspecies backcross hybrids were collected from the X. maculatus genome version 5.0 using Tophat2  ≥1, or ≤−1, False Discovery Rate (FDR) <0.05, with receiver operating characteristic (ROC) curve area under curve (auc) ≥0.8 were determined to be DEGs in skin between the two fish lines. The test was performed using JpA as the control to calculate relative gene expression between the two strains; therefore, if Log2FC ≥1, JpB showed higher-expression of a particular gene, or, if Log2FC ≤−1, JpA showed higher-expression of a gene.Processed transcriptomic sequencing reads were mapped to the  Tophat2 , and gen Tophat2 . Differe Tophat2 . The DEGXiphophorus and human were identified using blastn  for functional analyses. Bioinformatics analysis was performed using IPA for clustering and assessing the biological function of DEGs. Herein, the term “pathways” is short for canonical pathways as assigned by IPA based on input genes. Pathway analysis was performed by testing the over-representation of genes belonging to a certain pathway in the input gene list using Fisher exact test. Pathways with an enrichment -log10 score >3  were kept for further analysis.Sequence homologies between g blastn . A RecipPrinciple Component Analyses (PCA) was performed using R function prcomp. Library size normalized gene expression values were further scaled and subsequently used for PCA. Plot of the first two dimensions were made using custom R scripts.Heatmaps, dot plots and chromosomal plots were made using custom R scripts. All scripts are available upon request.X. hellerii and X. maculatus JpA or JpB  strains were first compared. From November 2008 to December 2019, there were a total of 290 scheme A hybrids at the XGSC that inherited xmrk-Sd, and 123 of them exhibited spontaneous tumorigenesis (tumor incidence = 42.4%). From January 2010 to June 2019, there were a total of 203 scheme B hybrids that inherited xmrk-Sp. 11 of them developed spontaneous tumors  . In contrast, scheme B tumor incidence does not follow Mendelian distribution . In addition, the genetic mechanism underlying both cross schemes is statistically independent  were identified to be differentially expressed in the skin (Log2FC) ≥1, or ≤−1, FDR < 0.05, ROC curve AUC ≥0.8; The inter-strain genetic variants are hypothesized to be heterozygous among the ancestral population that gave rise to both strains. These variants, shaped by evolutionary mechanisms , may lead to functional divergence of strain-specific alleles . Therefocis-effects of polymorphisms of gene expression, we found 163 polymorphisms (5%) are adjacent to 32 DEGs (7.8%) in the skin . This strategy was employed to identify pathways that may include genes exhibiting similar expression levels, but differentiated function due to codon changes, and genes that showed diverged expression patterns. Signaling pathway enrichment analyses were performed by comparing each dataset to databases consisting of common signaling pathways and genes.Next, we investigated functional differences between JpA and JpB strains using observed genomic and transcriptomic divergence between the two fish strains. Both genetic variants , and DEGs between the two strains, were used together to test over-representation of specific signaling pathways (2+ outflux (cacna2d1), neurotransmitter exocytosis (syn2 and snap25), synapse organization (thbs1 and nign1), and RAC1 mediated effect  regulated by efna2, itsn2, kalrn, and farp1 , suggesting JpA and JpB exhibited diverged DNA repair functionality or repair efficiency  (Sp or Sd) may be involved in the tumorigenic etiology of these two strains. However, differences in scheme A and B spontaneous cancer incidences support the concept that tumorigenesis in JpA and JpB backcross hybrids may occur by two different mechanisms independent of Sp/Sd  . Therefoof Sp/Sd .Sp/Sd loci that are both mapped to chromosome 21   . This de,067 bp) . ConsideThe genetic variants between the two strains define genetic background differences that may interact with the recurrent parental genomes upon interspecies hybridization. Although establishment of current genetic background differences may be due to a random ancestral event, characterization of these differences will allow us to perform genetic analyses  to identify loci/allele(s) that contribute to spontaneous versus UV-induced melanoma, and to forward our understanding of genetic and environmental contributions to melanomagenesis.xmrk-dependent as it only occurs in individuals that inherited xmrk-Sp. In addition, both JpA and JpB potentially inherited the same R(Diff) allele as there are either no genetic variants or significant transcriptional differences within the candidate R(Diff) locus (xmrk-R(Diff) interaction is further modified by additional loci. The incidence of 5.4% may suggest another two unlinked modifier genes  contribute to spontaneous tumorigenesis in scheme B hybrids. If this is true, then the fraction of hybrids possessing the particular genetic makeup is expected to be 6.25%  within the backcross hybrid cohort .Spontaneous cancer incidence in JpB established backcross hybrids  is 5.4%, compared to 42.4% within JpA established hybrids . Although significantly lower, the scheme B hybrid tumorigenesis is still f) locus . TherefoBy combining both genomic and transcriptomic differences between the two strains, we attempted to assess synergistic mutation effects, in which an altered gene product, and expression differences may play a role. DNA repair related genes encoding core enzymes in the pathway were over-represented within the dataset, and exhibited highest pathway coverage . Link is https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE157537.The datasets generated for this study can be found in the NCBI GEO (accession number: The animal study was reviewed and approved by Texas State University Institutional Animal Care and Use Review Board.YL and RW: acquisition of funds, experiment design, data analyses, and manuscript drafting. TO: data analyses and manuscript drafting. MB: experiment coordination, sample collection, and data analyses. WB: sample collection and data purification. WW: experiment design, sample collection, and data analyses. MS: experiment design, data analyses, and manuscript drafting. All authors contributed to the article and approved the submitted version.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.


Sitobion avenae (Fabricius), Rhopalosiphum padi (Linnaeus), Schizaphis graminum (Rondani), and Metopolophium dirhodum  (Hemiptera: Aphididae) are important pests of wheat and other cereals worldwide. In this study, the susceptibilities of four wheat aphid species to seven insecticides were assessed. Furthermore, the activities of carboxylesterase (CarE), glutathione S-transferase (GSTs), and cytochrome P450 monooxygenase (P450s) were determined in imidacloprid treated and untreated aphids. The results showed that the susceptibilities of four wheat aphid species to tested insecticides are different and M. dirhodum has shown higher tolerance to most insecticides. Relatively higher CarE and GST activities were observed in M. dirhodum, and P450s activities increased significantly in response to imidacloprid treatment. Moreover, susceptibility to imidacloprid were increased by the oxidase inhibitor piperonyl butoxide in M. dirhodum (20-fold). The results we have obtained imply that P450s may play an important role in imidacloprid metabolic process in M. dirhodum. We suggest that a highly species-specific approach is essential for managing M. dirhodum. Sitobion avenae, bird cherry-oat aphid Rhopalosiphum padi, greenbug aphid Schizaphis graminum, and Metopolophium dirhodum (Homoptera: Aphididae) are important pests of wheat and other cereals worldwide  insecticides parathion and disulfoton were commonly used for controlling he 1990s . Pyrethrhe 1990s . Neonicohe 1990s , and it he 1990s . Howeverhe 1990s . New typS. graminum  activities in four wheat aphids were analyzed, focusing on imidacloprid treated and untreated aphids. The effects of three synergists piperonyl butoxide (PBO), triphenyl phosphate (TPP), and diethyl maleate (DEM) on imidacloprid against four wheat aphids were also detected. Furthermore, the susceptibilities of four wheat aphids to the other six frequently used insecticides, including thiamethoxam, avermectin, beta-cypermethrin, omethoate, matrine, and rotenone belong to class of neonicotinoid, macrolide, pyrethroid, OP and botanical were determined, following an application of leaf dipping method.We found that the field occurrence of al years . An earlrugineus . To uncoSitobion avenae, Rhopalosiphum padi, Schizaphis graminum, and Metopolophium dirhodum with no exposure to insecticides since collected from Langfang  in 2013  and reared on wheat seedlings under standard conditions (20 ± 1°C and 60 ± 10% relative humidity with a 16-h light:8-h dark photoperiod).The population of The insecticides used for bioassays included imidacloprid , thiamethoxam , beta-cypermethrin , omethoate , matrine , rotenone , and avermectin . Synergists piperonyl butoxide , triphenyl phosphate , and diethyl maleate  were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd., United States.Fast blue B salt, α-naphthyl acetate (α-NA), eserine, reduced glutathione (GSH), coomassie brilliant blue G250, 1-chloro-2,4-dinitrochlorobenzene (CDNB), phenylmethylsulfonyl fluoride (PMSF), n-phenylthiourea (PTU), albumin bovine (BSA), ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT) were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd., United States.A leaf-based insecticide bioassay method was performed as previously described . Serial The activities of enzymes were measured as previously described  with som–1 Fast blue B salt), 50 μL of PBS and 50 μL of the enzyme source. Absorbance was measured at 600 nm and 30°C using the kinetic model for 5 min continuously in a microplate reader .Carboxylesterase activity was determined using the α-naphthyl acetate (α-NA) as described earlier with some modifications . The totThe activity of GSTs was determined according to a slightly modified published method involving CDNB . BrieflyR2 = 0.9973), the results are expressed as IU/L.The monooxygenase enzyme activity of four aphids were measured by an insect function oxidase ELISA kit  as previously described  with sliThe protein contents of the enzyme solutions were determined by the Bradford method. A serial albumin bovine (BSA) concentration solutions and samples were measured together and the protein content of samples were calculated by a standard curve based on BSA solutions. Three replicates of 10 μL diluted enzyme solutions were mixed with 200 μL protein assay dye reagent. After a 5 min incubation, the absorbance at 595 nm was measured.50 values that 62.21 mg L–1 for R. padi, 6.79 mg L–1 for S. avenae, 41.11 mg L–1 for S. graminum and 261.91 mg L–1 for M. dirhodum, respectively, then selected to petri dishes containing moistened filter paper after air-dried at room temperature for 30 min. The aphids still live after 24 h were collected for enzyme activities analysis, 120 aphids were collected for each aphid species.Wheat seedlings (about 15 cm high) were immersed in imidacloprid solutions for 5 min, the concentrations were LCThe effects of three synergists PBO, TPP, and DEM on imidacloprid against four wheat aphids were tested. Stocks of insecticides with synergists were prepared and adjusted to final concentrations by serial dilution with distilled water containing 0.1% (v/v) Tween-80 for the bioassays. The highest concentration of PBO, DEF and DEM used were 625 mg/L and the mortality as low as control (<10%). The bioassay method was performed as described in section “Bioassay.”50 values, 95% confidence intervals and slopes were calculated . Toxicity difference ratio (TDR) indicates the tolerance difference among pests. The TDR of tested insecticides to M. dirhodum, S. graminum, R. padi, and S. avenae were calculated based on the LC50 values. Differences in the enzyme activities among aphid species were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test and Student’s t-test was used to separate the means between control and imidacloprid treatments (p < 0.05) .The mortality data were corrected based on the control mortality with Abbott’s formula. The LCM. dirhodum and the toxicity difference ratio (TDR) were greater than 2.0, except matrine. Imidacloprid and avermectin showed highest toxicity to S. avenae, and the TDR were ranged from 0.10 to 4.02, and 0.39 to 1.15; thiamethoxam has highest toxicity to S. graminum, the TDR was ranged from 0.49 to 11.81; omethoate, beta-cypermethrin, matrine, and rotenone showed highest toxicity to R. padi. The toxicity of avermectin to four aphid species is roughly comparable with LC50 ranged from 3.92 to 11.48 mg L–1  and S. graminum (2.47-fold) (M. dirhodum at the same level as R. padi and P450s activity in S. graminum was significantly lower than S. avenae (3.05-fold)  . However05-fold) .M. dirhodum and S. graminum were significantly higher in imidacloprid-treated aphids than untreated by 5.37 and 5.39-fold , which consistent with the enzyme activities induced by imidacloprid. The susceptibility of R. padi to imidacloprid was increased by TPP (6.88-fold) and DEM (8.32-fold). In contrast, TPP, DEM, and PBO had inconsequential effects on imidacloprid against S. avenae.The effects of PBO, TPP, and DEM on imidacloprid activities against four wheat aphids was presented in M. dirhodum than other three wheat aphids. This means that M. dirhodum has the highest insecticide tolerance , whereas R. padi seems more susceptible to most of insecticides that we tested in this study. These results are consistent with the result that M. dirhodum has potential becoming a dominant aphid species in fields . These results imply that the P450 activity induced by imidacloprid may be related to the insensitivity of M. dirhodum to imidacloprid. Expression induction of P450 genes by imidacloprid in N. lugens showed some P450 genes  were up-regulated genes . Similarly, a previous study demonstrated that TPP suppressed imidacloprid activity by 2.45-fold in an imidacloprid-resistant R. padi strain (R. padi. Curiously, the activity of P450s in S. avenae with imidacloprid treated was significantly lower than control, both PBO and TPP show significant antagonism of imidacloprid in S. avenae which need further study, but PBO showed antagonism with some insecticides that have been reported (The accumulation of P450 is the main mechanism underlying the imidacloprid resistance in . lugens . But theed genes . CYP6 alnogaster , Bemisiaa tabaci , Myzus ppersicae . The deti strain  and anoti strain  suggestireported .M. dirhodum to imidacloprid may related to an inducible increase activity of P450.In conclusion, this study revealed the difference in susceptibilities of four aphid species in wheat to various insecticides. The insensitivity of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.PG, DC, and XZ conceived and designed the research. PG, CW, and ML conducted the experiments. PG and XAL analyzed the data. PG and XZ wrote the manuscript. YZ and XRL revised the manuscript. All authors have read and approved the 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.


The current interest in mental imagery in fields such as sport and physical training, health, education, underscore the need for designing general measures of imagery vividness that include different sensorial modalities such as the Plymouth Sensory Imagery Questionnaire Psi-Q; . The PsiThe questionnaire was back-translated, and administered to 394 fine arts undergraduates. Moreover, this test was compared to other questionnaires measuring different types de imagery.p < 0.01) with the total of the Betts’ QMI subscales, and the VVIQ, with the highest significance observed between the Psi-Q Touch and Betts’ QMI Cutaneous (r = −0.57), and between the Psi-Q Olfactory and Betts’ QMI Smell (r = −0.56). Owing to its novelty, the high correlation and significance (p < 0.01) between Psi-Q Vision and the OSIVQ Object (r = 0.36) is worth noting.A confirmatory factor analysis found that the Psi-Q had seven factors  with results similar to the original test. Values suggested a better fit for the model of the short version. The internal consistency values were 0.93 for the long and 0.89 for the short test. The Psi-Q subscales correlated significantly (The Spanish version of the Psi-Q was an adequate measure for evaluating different sensorial modalities of imagery vividness, and exhibited similar psychometric properties to those of the original version. The growing interest in mental imagery in different fields of application justifies the need for adapting the Psi-Q for the Spanish speaking population. This questionnaire is a valuable tool for the understanding of imagery as a psychological process, and as a variable influencing other processes. Information can be stored and processed by mental imagery. These mental images can be subsequently used in an array of cognitive activities such as thinking, recalling, problem-solving, and daydreaming . AccessiIn the pioneering work of The Plymouth Sensory Imagery Questionnaire Psi-Q;  was deveThe use of mental imagery in fields such as sports and physical training has led to the proliferation of studies covering a broad range of topics such as first-person) or observer (third-person) perspective. These authors found that higher levels of anxiety were associated with obsessive images experienced in the first person. Moreover, the use of mental images is increasingly playing a role in physical healthcare. In their study, An example of the implications of images in education is The ongoing research in mental imagery highlights the need to design general measures of imagery vividness that include different sensorial modalities such as the Psi-Q. The review of applied research methods in mental imagery by Furthermore, the Psi-Q has been used in several recent studies to measure depression , as a meThe mounting interest in mental imagery in different fields of application justifies the adaptation of the Psi-Q for the Spanish speaking population by developing a Spanish version of the test. Thus, the aim of the present study was to translate the Psi-Q into Spanish and to assess the structure and stability of the Spanish version of the Plymouth Sensory Imagery Questionnaire Psi-Q; , providiM = 21.01, SD = 3.19). The majority (51%) of the students were sophomores, some were freshmen (23.4%), and others were juniors (12.9%) and seniors (12.7%).Our sample consisted of 394 undergraduate students from Spain (293 women and 101 men) between 18 and 30 years of age (no imagery) and 7 . The visual modality refers to appearance  The auditory modality begins with “imagine the sound of.”  The olfactory modality begins with “imagine the smell of…”  The gustatory modality begins with “imagine the taste of…”  The cutaneous modality begins with “imagine touching…”  The corporal sensation modality begins with “imagine the bodily sensation of…”  Finally, the emotions modality begins with “imagine feeling”  The total score is calculated by summing all of the items.The Plymouth Sensory Imagery Questionnaire Psi-Q;  has two The Cronbach’s alpha calculated for the long version of the Psi-Q was 0.92, and it was 0.88 for the short version. The internal consistency for each of the subscales of the Psi-Q was vision = 0.68, sound = 0.77, smell = 0.72, taste = 0.75, touch = 0.75, body = 0.68, and emotions = 0.72. The Cronbach’s alphas of both the long and the short versions of the Psi-Q were marginally lower than those obtained in the original test 0.96 and 0.94 , and theperfectly clear imagery that is as vivid as the real experience) and 5 . The total score is calculated by summing all of the items. Thus, high scores on the VVIQ indicate low imagery vividness. The Cronbach’s alpha for the VVIQ was 0.93, a good internal consistency and similar to the Cronbach’s alpha of 0.88 obtained by The Spanish version  of the VPerfectly clear image that is as vivid as the actual experience) and 7 . The total score is calculated by summing all of the items. An example of an item is: “Think of some relative or friend whom you frequently see. The exact contour of their face, head, shoulders, and body.” The higher the scores, the lower the imagery ability. The Cronbach’s alpha for this sample was 0.92, which is on par with the reliability reported by The Spanish version  of the Be.g., “My images are very vivid and photographic”), 15 items correspond to a visual-spatial cognitive style , and 15 items correspond to a verbal cognitive style . Each item is anchored on a five-point Likert-type scale ranging between 1  and 5 . The total score is calculated by summing all of the items.The Spanish version  of the OThe Cronbach’s alpha for the scales of the OSIVIQ were verbal = 0.77, object = 0.81, spatial = 0.76, and overall = 0.79. The results were similar to those reported in previous studies. The study was conducted in accordance with the ethical rules contained in the Declaration of Helsinki of 2000 and was approved by the ethics committee of our University. Undergraduate students volunteered to participate in the study.The translation process of the Psi-Q  was perfThe Psi-Q, VVIQ, Betts’ QMI, and OSIVQ questionnaires were administered to participants in groups of approximately 20 undergraduates in their usual classrooms. The order of tests was counterbalanced.p (p + 2), where p is the number of variables observed.Statistical analysis was performed using SPSS 25.0 software and IBM SPSS Amos 25. The univariate normality was assessed with the skewness and kurtosis, where indexes close to zero and less than 2 indicate similarity with the normal curve of univariate data . Mardia’2 to degrees of freedom (df) ratio—because this index alone is very sensitive to sample size  was performed using SPSS Amos software, 25 version (IBM), which gives model-fitting indicators , 1999. Tple size —, the gogood fit . GFI valgood fit . Values good fit . FinallyAs for univariate normality, the skewness and Kurtosis indexes of the questionnaires were near zero and below the value of 2. In addition, the univariate normality was calculated by the skewness and kurtosis of each item of the Psi-Q, obtaining values that in most items were near zero and less than the 2, except in the kurtosis index for the values of items 4 (2.71), 5 (2.82), 19 (2.04), and 21 (2.34), which were above the recommended values . Therefop < 0.001). As estimated by the model, the correlations among the seven factors ranged from 0.53 for vision and touch to 0.80 for touch and body. All values were statistically significant (p < 0.01).2(733.95), df (413), and their ratio 1.78 (p < 0.001). Index values were: GFI (0.89), CFI (0.92), and NNFI (0.91). RMSEA and SRMR values were 0.04 and 0.05.The values obtained for the long version suggested an adequate fit for the model with χp < 0.001). As estimated by the model, the correlation among the seven factors ranged from 0.42 for sound and taste to 0.74 for touch and body. All values were statistically significant (p < 0.01).2(216.77), df(149), and their ratio 1.46 (p < 0.001). Index values were: GFI (0.95), CFI (0.97), and NNFI (0.96). RMSEA and SRMR values were 0.03 and 0.04.The results of the short version suggested a better fit for the model of the short version with χp < 0.01) with the total of the Betts’ QMI subscales and the VVIQ, with the highest significance observed between Psi-Q Touch and Betts’ QMI Cutaneous (r = −0.55), as well as between Psi-Q Olfactory and Betts’ QMI Smell (r = −0.56). The significant correlations (p < 0.01) between OSIVQ Object and all Psi-Q scales were also worth noting The need for multidimensional measures of mental imagery is derived from previous studies  This would explain the significant correlations between Psi-Q Body and OSIVQ Spatial. Likewise, OSIVQ Verbal examined preferences in verbal instructions to describe an object or person , which would explain the correlations between Psi-Q Body and OSIVQ Verbal. In short, the significant correlations were in agreement with the content of the tests and confirmed the appropriateness of this innovative multisensory questionnaire.The correlations between Psi-Q Body, OSIVQ Spatial, and OSIVQ Verbal were significant but not high. Spatial ability is related to body position and movement in space, and several items on the Body scale of the Psi-Q referred to these situations (see in particular items 27 and 28, The role of imagery and its significance as a variable in different fields of research is expanding, and this promotes research focused on mental imagery (for a review, see Hence, this study has shown that the Spanish versions of the Psi-Q are an adequate measure for evaluating different sensorial modalities of imagery vividness and are valuable tools for the understanding of imagery as a psychological process or as a variable influencing other processes . The maiIn short, the Spanish versions of the Psi-Q provide a multimodal measure of imagery intensity that is sensitive and valid for cognitive, neuroscientific, and clinical research, as well as work on imagery. The Psi-Q scores provided evaluations of general and/or specific image vividness for each sensorial modality. As The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by University of Vigo. The patients/participants provided their written informed consent to participate in this study.MP-F and AC contributed conception and design of the study. MP-F organized the database and wrote the first draft of the manuscript. AC performed the statistical analysis and wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


Lactobacillus plantarum HY7712, Lactobacillus plantarum HY7715, Lactobacillus helveticus HY7801, and Bifidobacterium animalis ssp. lactis HY8002. A total of 41 metabolites, 24 lipids, and 4 carotenoids were detected in the fermented and non-fermented VJ (control). The lycopene, α-carotene, and β-carotene levels were higher in VJ fermented with L. plantarum strains (HY7712 and HY7715) than in the control. Proline content was also elevated in VJ fermented with HY7715. Uracil, succinic acid, and α-carotene concentration was increased in VJ fermented with HY7801, while glycine and lycopene levels were raised in VJ fermented with HY8002. This study confirmed that each probiotic strain has distinctive characteristics and produces unique changes to metabolic profiles of VJ during fermentation. Our results suggest that probiotic-fermented VJ is a promising functional beverage that contains more beneficial metabolites and carotenoids than commercial non-fermented VJ.Fermented vegetable juices have gained attention due to their various beneficial effects on human health. In this study, we employed gas chromatography–mass spectrometry, direct infusion-mass spectrometry, and liquid chromatography–mass spectrometry to identify useful metabolites, lipids, and carotenoids in vegetable juice (VJ) fermented with  Lactobacillus acidophilus, B. lactis, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, and Streptococcus thermophilus are probiotic strains that produce lactic acid. Bacillus and Saccharomyces strains, which do not produce lactic acid, are also considered probiotics [The World Health Organization (WHO) defines probiotics as living microorganisms that, when consumed at a sufficient level, promote host health . Represeobiotics . Traditiobiotics . Consumiobiotics .Lactobacillus, Lactococcus, and Enterococcus strains are present in traditional fermented health foods, such as sauerkraut, pickled cucumbers, and kimchi [Lactobacillus, Lactococcus, Leuconostoc, and Saccharomyces display antioxidant capacity compared to non-fermented vegetable juices [Bifidobacterium strains [Lactobacillus strains is abundant in calcium, phosphorus, and magnesium, and those fermented with L. acidophilus IFO 3025 and L. brevis FSB-1 display improved nutritional and mineral composition, respectively [Vegetable juices (VJs) have gained widespread popularity as an alternative to raw vegetables and fruits to supply micronutrients, phenolic compounds, carotenoids, and fiber ,6. A died kimchi , and cond kimchi . Strawbee juices . The antectively . The colectively .Lactobacillus strains [Metabolomics and lipidomics can be used to investigate the metabolic and lipidomic changes in vegetables and VJ during fermentation. Recently, it was reported that nuclear magnetic resonance spectrometry (NMR) and liquid chromatography–mass spectrometry (LC-MS) can rapidly discriminate the metabolic profiles of vegetable juice and medium fermented with different  strains ,15. This strains . Tomita  strains . In anot strains . GinsengLactobacillus plantarum HY7712, Lactobacillus plantarum HY7715, Lactobacillus helveticus HY7801, and Bifidobacterium animalis ssp. lactis HY8002. Several beneficial metabolites and lipid species, including carotenoids, were discovered in fermented VJ, which could have practical implications for improving public health.In this study, we hypothesized that each probiotic strain is able to generate distinct metabolic and lipidomic profiles during fermentation of VJ. To test this hypothesis, we used GC-MS, direct infusion-mass spectrometry (DI-MS), and LC-MS to assess the relative levels of various metabolites and intact lipid species in VJs fermented by four different probiotic strains: Lactobacillus strains HY7712, HY7715, and HY7801, and Bifidobacterium HY8002, from the Korea Yakult Probiotics Library , were cultured in De Man, Rogosa and Sharpe (MRS) media  before each strain (>107 CFU/mL) was inoculated into a sample of the sterilized VJ . Non-inoculated VJ (pH 4.8) was considered as the control. The inoculated juices were fermented at 37 °C for 24 h , then stored at −70 °C until analysis.The VJ and fermented VJs were provided by Korea Yakult Co., Ltd. . The juice consisted of tap water, 18–24% organic carrot juice concentrate , 7–10% organic tomato paste , 1–3% organic mixed VJ 1  , 0.01–1% organic broccoli juice , 0.01–1% organic zucchini and pumpkin juice concentrate , and 0.01–1% organic mixed VJ 2  . The juice composition was based on “Haru Yache Original” from Korea Yakult Co., Ltd. g for 3 min at 4 °C, and the supernatant was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) syringe filter . Of this, 200 µL were transferred to GC vials and dried under nitrogen for 20 min. The derivatization and GC-MS analysis of each sample were conducted according to previously reported methods [The fermented and non-fermented juices (100 µL) were transferred to separate microfuge tubes , and extracted with 1 mL of methanol . The samples were briefly vortexed and sonicated for 30 min at 40 kHz to improve extraction yields . After sg for 10 min. The upper phase was collected and dried under nitrogen gas. The dried lipid extract was dissolved in 300 µL of chloroform/methanol  solution. For DI-MS analysis, methanol/chloroform  containing 7.5 mM ammonium acetate solution was added to each lipid extract. DI-MS analysis of each sample was performed as previously reported [m/z 400–1200 and 500–1300 in positive and negative mode, respectively. Mass spectra were acquired in both positive mode  and negative mode . Tendem MS spectra was obtained to pooled samples from each group to identify lipid species. Lipid species were identified by comparing LipidBlast database by Kind et al. [The fermented and non-fermented juices (50 µL) were transferred to separate microfuge tubes, and intact lipid species were extracted using the modified Matyash methyl tert-butyl ether (MTBE) method ,22. Bried et al. . In addid et al.  were useg for 10 min at 4 °C. After centrifugation, the supernatant was collected into microfuge tubes, and the residue re-extracted with 360 μL of acetone and 540 μL of hexane. The mixture was vortexed, sonicated, and centrifuged as described above. The supernatant was collected into the microfuge tubes used for the first extraction, and 200 μL of water  with 0.1% BHT was added for phase separation. The top phase was collected and filtered through a 0.2 μm PTFE syringe filter (Whatman). The filtrate was transferred into an amber vial, and 2 μL of β-apo-8′-carotenal was added , with 100 μg/mL used as an internal standard. Each sample was dried under nitrogen for 20 min and resuspended in 100 μL of acetonitrile  and methanol solution . The fermented and non-fermented juices (50 µL) were transferred to separate microfuge tubes and extracted with 360 μL of acetone  containing 0.1% butylated hydroxytoluene  and 540 μL of hexane  containing 0.1% BHT. The mixture was briefly vortexed, sonicated for 5 min at 4 °C, then centrifuged at 1000× v/v). LC-MS analysis of each sample was conducted as previously reported, using an Accela LC  equipped with a degasser, Accela 600 pump, linear ion-trap mass spectrometer , and Accela AS autosampler [To increase the stability of the carotenoid standard solutions, a 100 μg/mL stock solution was prepared in hexane with 0.1% BHT . The staosampler . A 1.9 µp < 0.05 were considered statistically significant. For PCA and PLS-DA, all data were mean-centered and scaled to unit variance in SIMCA-P+ software . Pathway analysis was performed in the web-based software tool MetaboAnalyst (version 4.0) [The GC-MS, DI-MS, and LC-MS data were collected in Microsoft Office Excel  and used for principal component analysis (PCA), partial least squares–discriminant analysis (PLS-DA), and pathway analysis. The differences in the relative levels of metabolites, lipids, and carotenoids were evaluated by Mann–Whitney test in SPSS software , and those with ion 4.0) .L. plantarum HY7712, the levels of one fatty acid (stearic acid), two organic acids (lactic acid and succinic acid), and one alcohol (glycerol) were significantly higher, whereas those of other 28 metabolites were significantly lower, than in the control. In VJ fermented with L. plantarum HY7715, the concentrations of one amino acid (proline), two organic acids  and one sugar alcohol (glycerol) were significantly higher, whereas those of 23 other metabolites were significantly lower than in the control. Compared to non-fermented juice, VJ fermented with L. helveticus HY7801 had a higher content of two organic acids (lactic acid and succinic acid) and uracil and a lower content of 26 other metabolites. In VJ fermented with B. lactis HY8002, the levels of one amino acid (glycine), two organic acids (lactic acid and succinic acid), two sugars (glucose-6-phosphate and xylose), and one sugar alcohol (glycerol) were significantly higher, whereas those of 13 other metabolites were significantly lower, than in the control.Comprehensive GC-MS analysis of the fermented and non-fermented VJs identified 41 metabolites: 13 amino acids , 4 fatty acids , 8 organic acids , 7 sugars , 2 sugar acids (glyceric acid and threonic acid), 5 sugar alcohols , phosphoric acid, and uracil . The levL. plantarum HY7712, the concentration of PE 18:2/18:2, PG 16:0/18:2, PS 18:2/22:0, and PI 16:0/18:1 was significantly higher, whereas that of PE 18:0/18:2 was significantly lower, than in the control. In VJ inoculated with L. plantarum HY7715, the levels of PE 18:2/18:2, PS 18:2/22:0, and PI 16:0/18:1 were significantly higher, while those of PE 16:0/18:2 and 18:0/18:2 were significantly lower, than in the control. In VJ fermented with L. helveticus HY7801, the levels of PE 18:2/18:2, PE 18:1/18:2, PG 16:0/18:2, PS 18:2/22:0, and PI 16:0/18:1 were significantly higher, while those of PE 18:0/18:2 and PS 18:2/20:0 were significantly lower, than in non-fermented juice. VJ fermented with B. lactis HY8002 had a significantly higher content of PE 18:2/18:2, PS 18:2/22:0, and PI 16:0/18:1 and a significantly lower content of Lyso-PC 18:2 and PE 18:0/18:2 than in the control.DI-MS analysis detected the following intact lipid species in the fermented and non-fermented VJs: three monogalactosyldiacylglycerols , three lysophosphatidylcholines , one phosphatidylcholine , one phosphatidylethanolamine , and five triacylglycerides  in positive ion mode, and two phosphatidic acids , four phosphatidylethanolamines , one phosphatidylglycerol , two phosphatidylserines , and two phosphatidylinositols  in negative ion mode . In VJ fL. plantarum HY7712 and HY7715 than in the control. VJ fermented with L. helveticus HY7801 had lower levels of lutein and higher levels of α-carotene, while VJ fermented with B. lactis HY8002 had significantly higher levels of lycopene, than in the control. Relative levels of carotenoids in the different VJ samples are listed in L. plantarum strains (HY7712 and HY7715) were similar and clearly distinguishable from those of VJ fermented by L. helveticus HY7801. Moreover, the data for B. lactis HY8002-fermented juice differed from those for Lactobacillus HY7712, HY7715, and HY7801-fermented juices. The metabolic and lipidomic data for the different juices were clearly distinguished in the PCA and PLS-DA score plots Our findings confirm that the metabolites and lipids present in fermented VJ differ depending on which probiotic strain was used to produce it. It follows that different probiotics might utilize different nutritional compounds of the juice during fermentation. A metabolic pathway analysis of the 41 metabolites identified by GC-MS showed that the following processes were activated after probiotic fermentation of VJ: alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; the citrate cycle; aminoacyl-tRNA biosynthesis; starch and sucrose metabolism; and arginine and proline metabolism . The maiBifidobacteria [Lactobacillus strains [L. plantarum convert l-malate to l-lactate [L. rhamnosus and L. sakei is associated with a number of enzymes, including α-d-glucosidase, β-d-glucuronidase, and α-phosphoglucomutase [We hypothesized that the change in the levels of metabolites, lipids, and carotenoids in VJ might be affected by the activities of probiotic enzymes. Galactosidases, glucosidase, lipase, and leucine aminopeptidase are present in human bacteria , while β strains . Malolac-lactate . In addicomutase ,32.Orostachys japonicus A. Berger, commonly called rock pine, fermented with L. plantarum displays 4.73 times the uracil content of non-fermented Orostachys japonicus A. Berger [L. plantarum show antihypertensive effects [L. helveticus HY7801. Research in animal models has shown L. helveticus HY7801 to have a number of anti-inflammatory effects. Orally administered HY7801 regulates immune biomarkers, including TNF-α, IFN-γ, IL-17A, IL-10, and IL-12, and might be useful in the treatment of rheumatoid arthritis [Candida albicans and down-regulating TNF-α, COX-2, iNOS, and IL-1β levels [Our results suggest that the production of certain metabolites is markedly enhanced because of the activation of multiple metabolic pathways . In HY78. Berger , and it . Berger . Uracil  effects . Moreove effects . Kim et  effects  suggeste effects  revealedrthritis . In addiβ levels . Accordiβ levels , adminisL. helveticus produces succinic acids through citric acid metabolism, which are thought to contribute to the flavor profiles of Emmental and cheddar cheese [Streptococcus lactis (recently Lactococcus lactis) converts fumaric acid to succinic acid [In HY7801-fermented VJ, the level of succinic acid was also higher by 36.67 times than in the control. Succinic acid is an intermediate of the tricarboxylic acid (TCA) cycle and the end-product of anaerobic fermentation  and is wr cheese . In addinic acid . Succininic acid . Therefonic acid  and is tnic acid . Furthernic acid  and mighnic acid .Saccharomyces cerevisiae might be a response to nutrient exhaustion and entry into the stationary phase [Saccharomyces strains is affected by increased PI levels [In addition, in HY7801-fermented VJ, the level of PI 16:0/18:1 was higher by 3.54 times than in the control. PI is produced from CDP-diacylglycerol and plays important roles in cell signaling, cell wall structure, and protein metabolism ,54. Incrry phase . EthanolI levels . We specI levels . KüllenbI levels  suggesteI levels .Saccharomyces cerevisiae might synthesize it to adapt to stress during fermentation [Kurthia catenaforma fermentation [Moreover, proline content of HY7715-fermented VJ was 1.66 times higher than that of non-fermented juice. Proline is an amino acid that participates in protein synthesis ,60,61. Ientation . Prolineentation . Prolineentation  and is uL. salivarius and P. acidilactici co-culture [Glycine content of HY8002-fermented VJ was also 1.76 times higher than that of non-fermented juice. Glycine is essential for the formation of secondary protein structure . Moreove-culture . Glycine-culture  and prev-culture .L. plantarum HY7712 and HY7715-fermented juices than in non-fermented juice  than in the control. Particularly, the levels of uracil and succinic acid were increased in HY7801 fermented VJ, while Proline was also elevated in HY7715. In addition, glycine was increased in VJ fermented with HY8002. We also revealed that a number of metabolic pathways were activated in probiotics during the fermentation process, including the citrate cycle; alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; glycolysis; and carotenoid pathways. Compared to previous studies [Lactobacillus strains and one Bifidobacterium strain were also investigated. Thus, we confirmed that metabolomics and lipidomics are the effective approach to provide more scientific evidence for discovering new beneficial effects of probiotics. Furthermore, we advanced the understanding of how fermented foods mediate their health benefits by revealing the metabolic changes that occur during probiotic fermentation.In this study, VJ was fermented with four probiotic strains,  studies ,17,18, w


The extensive breeding of commercial chickens has led to a sharp decrease in the resources of many indigenous chickens, especially the indigenous chickens in the southeastern coastal region, which are on the verge of extinction, and the indigenous chickens in the northwestern region of China, which are also at risk. However, there are few reports on the evaluation of genetic diversity and conservation of genetic resources of indigenous chickens in remote areas in the Northwest of China.In the present study, the genetic diversity and phylogenetic relationship of six indigenous chickens from different regions were studied based on variation in mitochondrial DNA control region (D-loop), and the degree of introgression from commercial breeds into these chickens was determined by the amount of haplotype sharing between indigenous and commercial breeds.Twenty-five polymorphic sites and 25 haplotypes were detected in 206 individuals. Principal component analysis showed that the Jingning chicken had the highest genetic diversity among the six indigenous chickens. According to the degree of introgression, the six indigenous breeds may be involved in haplotype sharing with commercial breeds, and the introgression from commercial chickens into the Haidong chicken is the most serious.The genetic uniqueness of indigenous chickens has been eroded, so it is necessary to consider the protection of their genetic resources. Phylogenetic analysis suggests that the six indigenous chickens have two major matrilineal origins: one from Yunnan or its surrounding areas in China and the other from the Indian subcontinent. Domestic animals have evolved genetic adaptations to a new environment, the farm, and have been subjected to strong human-driven selection, leading to remarkable phenotypic changes in morphology, physiology and behavior. Identifying the genetic changes underlying these developments provides new insight into the general mechanisms by which genetic variation shapes phenotypic diversity . In the All studies involving animal were carried out in accordance with the regulations for the Administration of Affairs Concerning Experimental Animal . Blood samples collection was approved by the ethics committee of Agricultural University. Sample collection were conducted according to the guidelines established by the Ethics Committee for the Care and Use of Laboratory Animals at Gansu Agricultural University.Blood samples were collected on the Flinders Technology Associates cards (FTA)  from six indigenous breeds 175 birds) of China: Jingning chicken (JN), Huining chicken (HN), Minqin chicken (MQ), Taiping chicken (TP), Beijing fatty chicken (BY), and Haidong chicken (HD) . Within  birds of2, 1 μL dNTP (2.5 mM), 1 μL of each primer, 0.3 μL Taq-polymerase, 2.0 μL Template DNA, and 21.7 μL ddH2O. The PCR cycle included the initial denaturation at 94°C for 10 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 61°C for 30 s and extension at 72°C for 30 s with a final extension at 72°C for 10 min using GenAmp 9700 . The PCR products were purified with Tiangen PCR purification kit  according to the manufacturer’s instructions. Sequencing reaction was performed by using Big Dye Terminator Cycle Sequencing Ready Reaction Kit  and electrophoresis was done by a ABI 3130XL DNA Genetic Analyzer .The D-loop region was amplified directly from the genomic DNA by polymerase chain reaction (PCR). The primer pair, L16750 (5′-AGGACTACGGCTTGAAAAGC-3′) and H547 (5′-ATGTGCCTGACCGAGGAACCAG-3′), described by Niu et al [http://www.igbmc.ustrasbg.fr/pub/ClustalX), and identical sequences were considered to be the same haplotype. The diversity parameters were estimated using DnaSP 5.0 [The mtDNA nucleotide sequences obtained in this study were aligned by using the ClustalX program (naSP 5.0 . CorrelanaSP 5.0 . AnalysinaSP 5.0 . A haplonaSP 5.0 . A neighnaSP 5.0 . For connaSP 5.0 , in whicAnalysis of the mtDNA D-loop sequences from the 206 samples showed a total of 25 nucleotide changes, which were grouped into 25 haplotypes . The fouThe genetic diversity calculated for the six indigenous chickens and commercial chickens is shown in To comprehensively evaluate the genetic diversity of the indigenous chickens, correlation analysis and PCA were performed on Pi, K, and Hd , 3. CorrTwenty-five haplotypes were identified in 206 individuals of the indigenous and commercial breeds. The commercial breeds were distributed among 6 haplotypes. The haplotypes of commercial breeds were shared with all indigenous breeds except for the HN chicken. As shown in Phylogenetic trees were constructed based on nucleotide sequences from the six indigenous breeds, and median-joining network profiles were constructed based on 25 haplotypes and 11 reference sequences . As showThe Hd, Pi, and K reflect different aspects of genetic diversity, while PCA can comprehensively evaluate genetic diversity. PCA is a statistical process that reduces the dimensionality of a data set by transforming the data into a new set of variables . TherefoThe genetic diversity of the six indigenous chickens is shown in Due to the large-scale production and promotion of commercial breeds, the indigenous chicken breeds with relatively low egg and meat production are threatened, and the amount of breeding is gradually reduced or even eliminated. Such commercial breeding reduces the genetic diversity of indigenous chicken breeds , and theG. g. gallus, G. g. jabouillei, and G. g. spadiceus distributed in Yunnan Province and Laos may have originated from red jungle fowl subspecies in Yunnan, Laos, and Vietnam and its surrounding areas. Gong et al [According to Ohno , half-sing et al  reportedng et al . Haplogrng et al ,5. BasedOur results reveal high genetic diversity among the six indigenous chickens, especially the JN chicken, which may be related to their distribution in remote rural areas. Therefore, it is necessary to consider the protection of their genetic resources. According to the degree of introgression from commercial breeds into indigenous chickens, the six indigenous breeds may be involved in commercial breeding, and the introgression from commercial chickens into HD chickens is the most serious. In contrast, no traces of commercial chickens were found in the HN chicken, so full population protection should be considered. Phylogenetic analysis suggested that these six indigenous chickens have two major maternal origins: one from the Yunnan region of China or surrounding areas and the other from the Indian subcontinent.


All subjects underwent ELISA serological screening twice, at the same time as the cell-ELISA (T2) as well as 35–60 days earlier (T1). Cryopreserved PBMCs of healthy donors obtained years before the COVID-19 pandemic were also included in the analysis. The preliminary results presented here show that out of 45 tested subjects, 16 individuals (35.5%) were positive to the cell-ELISA, 11 (24.5%) were concomitantly positive in the serological screening (T1 and/or T2), and only one person was exclusively positive in ELISA (T1) and negative in cell-ELISA, though values were close to the cutoff. Of note, five individuals (11.2%) tested negative in ELISA but positive in cell-ELISA and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum, which challenges the negative results of the serological screening. The relative level of in vitro secreted IgG was measurable in positive subjects, ranging from 7 to 50 ng/well. Accordingly, all anti-SARS-CoV-2 antibody-positive subjects previously reported moderate to severe symptoms attributable to COVID-19, even though the RT-PCR data were rarely available to confirm viral infection. Overall, the described cell-ELISA might be an effective method for detecting subjects who encountered the virus in the past, and thus helpful to improve serological ELISA tests in the case of undetectable/equivocal circulating IgG levels, and a suitable and improved tool to better evaluate SARS-CoV-2-specific humoral immunity in the COVID-19 pandemic.Knowledge of the antibody-mediated immune response to SARS-CoV-2 is crucial to understand virus immunogenicity, establish seroprevalence, and determine whether subjects or recovered patients are at risk for infection/reinfection and would therefore benefit from vaccination. Here, we describe a novel and simple cell-ELISA specifically designed to measure viral spike S1-specific IgG produced in vitro by B cells in peripheral blood mononuclear cell (PBMC) cultures from a cohort of 45 asymptomatic ( Memory B cells and/or circulating plasma cells can be identified by their capability to secrete antigen-specific antibodies long after first encountering an antigen ; thus, tA cell-based ELISA test adapted for COVID-19 would be useful to measure IgG specific for the S1 protein of SARS-CoV-2 produced in vitro by B cells, even when circulating antibodies become less detectable over time. Furthermore, by combining this assay with ELISA, it is possible to simultaneously detect the level of S1-specific plasma IgG and the presence of IgG-producing B cells in peripheral blood mononuclear cells (PBMCs) in plasma from the same donor. Such an approach may be helpful to improve serological analysis and determine the immunization status for SARS-CoV-2.In this brief communication, we describe a new cell-ELISA, performed in combination with a commercially available COVID-19 serological test, in a cohort of 45 subjects. This assay is used to detect in vitro cell-produced IgG directed against the SARS-CoV-2 S1 protein. Although further work is required to confirm our preliminary results, we propose this cell-ELISA as a potential supplementary diagnostic tool useful for ruling out individuals who have very low serum IgG levels undetectable by ELISA, and thus improving the assessment of past infection and immune response in subjects during the COVID-19 pandemic.Forty-five consecutive subjects  of different ages (ranging from 8 to 99 years) undergoing COVID-19 serological analysis in an authorized diagnostic center  were considered appropriate for enrollment in this study. The subjects of the study were consecutively enrolled in the study from the end of April to June 2020. The enrolled volunteers, who were asked to undergo serological tests and agreed to participate in the research project by signing an informed consent, were either asymptomatic or with mild to severe symptoms ascribable to COVID-19. The included subjects asked for ELISA (timepoint ELISA 1), either independently or after suggestion from their physician, in order to have a serology assessment of their infection status. All specimen and handling procedures were conducted taking precautions to provide a barrier between the specimen and personnel and using a class II biosafety cabinet (BSC). The following data were collected from each patient: age, sex, previous serology IgG screening, quantitative RT-PCR screening  when available, and presence of COVID-19 symptoms, as described in The blood was collected twice from each donor, at baseline (ELISA 1) and after 35–60 days (ELISA 2). In addition, pre-COVID-19 frozen PBMCs, used as controls, were obtained in 2017 from the blood of four healthy donors  and cryopreserved in liquid nitrogen at Fondazione Santa Lucia LNS Cell Bank.The study was approved by the Regional Ethical Board in Ospedale L. Spallanzani, Roma,  and, in accordance with the Helsinki Declaration, written informed consent was obtained from all subjects.g for 20 min at room temperature. The PBMC layer was collected with a Pasteur pipette, and cells were washed with 10 mL of saline by centrifuging at 450× g for 10 min at room temperature. After washing, PBMCs were resuspended in 5 mL of RPMI medium containing 10% foetal calf serum FCS and antibiotics, and cells were counted in a hemocytometer with trypan blue. PBMCs collected in 2017 from healthy donors and cryoconserved in liquid nitrogen were thawed, resuspended in complete RPMI medium, and counted in trypan blue. The cell concentration was adjusted to 9 × 106/mL, and 100 μL cell dilutions were plated into polystyrene wells pre-coated with recombinant spike S1 protein as antigen, supplied from a commercial ELISA kit . As controls, PBMCs were added to 10 μg/mL of cycloheximide  to block protein synthesis, or plated in complete RPMI (110 μL/well) into a 96-well culture plate  to assess spontaneous release of IgG from PBMCs. Each experiment was conducted in duplicate. Cells were incubated for 48 h at 37 °C in a humidified incubator with occasional gentle shaking, and after incubation the cells were removed, and the wells washed three times with 150 μL of saline. The RPMI medium from the cells plated in the absence of antigen (spontaneous releases) was collected after centrifuging the plate at 450× g for 5 min and added (100 μL) to ELISA wells coated with antigen (Dia.Pro). The polystyrene wells were incubated for one hour at 25 °C and processed following the ELISA kit instructions and materials as described above. Briefly, the wells were washed three times with 150 μL of saline and incubated for one hour at 25 °C with a peroxidated anti-human IgG dilution contained in the kit, washed again as above, and enzyme reaction was visualized with a tetramethylbenzidine solution kit for 15 min at 25 °C. The reaction was then stopped with 3N sulfuric acid, and optical density (OD) was read at 450 nm in a plate reader. To evaluate the amount of IgG produced in vitro by cell-ELISA, human γ-globulins (Sigma G4386-5G) were dissolved in 50 mM carbonate-bicarbonate buffer  and were adsorbed overnight at 100 μL/well in duplicate wells of a polystyrene ELISA plate. The wells were then washed with Tris–HCl  containing 0.05% Tween-20 and 0.15 M NaCl (TTN). Binding sites were saturated with 3% bovine serum albumin in TTN for 30 min. After three washes with TTN, the wells were incubated with a peroxidated anti-human IgG antibody solution kit and processed as above.The protocol was adapted from a previous study . PBMCs cg for 10 min. The levels of IgG antibody specific for the S1 protein of SARS-CoV-2 were measured in 10 μL of plasma diluted in 200 μL of binding solution using a commercial ELISA kit that employs the recombinant S1 peptide as adsorbed antigen (Dia.Pro). Specific anti-S1 IgGs were detected by incubation at 25 °C with a peroxidated goat anti-human IgG in 10 mM Tris-HCl (pH 6.8) for one hour, according to the manufacturer’s protocol. After washing with saline and adding the enzyme substrate (tetramethylbenzidine), color development proceeded for 15 min. The reaction was then stopped with 3 N sulfuric acid, and OD was read at 450 nm in a microplate reader. The ELISA kit contained an internal negative control (1% goat serum in 10 mM citrate buffer pH 6.0) and a positive control (IgG specific for SARS-CoV-2 in 10 mM citrate buffer pH 6.0 and 1% goat serum).Peripheral blood (5 mL) was collected in heparinized tubes from enrolled volunteers, and plasma was obtained by centrifugation at 490× p-values and T-values of <0.05 and >2.5, respectively.Numerical data between different treatment groups were processed for their statistical significance by one-way ANOVA and two-tailed test of significance, with accepted 5 cells/well) from 45 subjects were tested by cell-ELISA, obtaining OD450 values ranging from a minimum of 0.004 to a maximum of 0.23. Net values were calculated for each sample by subtracting values obtained from adding the protein synthesis inhibitor CXM to cells. Spontaneous IgG release for each sample reached lower OD values than those obtained in the presence of CXM (not shown). As additional control, thawed PBMCs from four healthy control subjects, cryopreserved in 2017 (trypan blue viability > 90%), showed a mean OD450 value of 0.014 ± 0.01 when tested by cell-ELISA. A cutoff value for negative samples was arbitrarily established at an OD450 value of 0.065, calculated by adding two SDs on top of the mean OD450 of CXM samples . With this cutoff value, N = 16 samples were positive (with OD450 > 0.065), and N = 29 were negative.PBMCs  to 0.047 ± 0.02 (3 × 105 cells) and 0.013 ± 0.07 (1 × 105 cells). With respect to the control PBMC cultures incubated with CXM, or to control cultures incubated without the antigen, the OD450 values were statistically significant  only when using the higher cell number of 9 × 105 PBMC/well.In vitro production of IgG anti-SARS-CoV-2 was determined in a subgroup of subjects (N = 10), where the optimal conditions for cell-ELISA in terms of PBMC number/well were set, as shown in To obtain information about the relationship between IgGs specific for the SARS-CoV-2 S1 protein present in the serum and those produced in vitro by memory B cells or plasma cells, we compared the data obtained by cell-ELISA with those by ELISA, and the results are shown in 5 cells).ELISA data are expressed as the ratio between the OD450 nm and cutoff values, as reported in the ELISA kit procedure (Dia.Pro). Cell-ELISA data are expressed as net OD450 values. The table shows the donors testing as negative (N = 5), equivocal (N = 1), and positive (N = 10) by ELISA 1 at day 0; the same donors tested as negative (N = 9), equivocal (N = 2), and positive (N = 5) by ELISA 2 after 35–60 days. By comparing serological and cell-ELISA data, Accurate tests able to identify the SARS-CoV-2 virus and monitor the presence of antiviral antibodies are key in detecting those who have had an immune response to the virus, to help the management and surveillance of the virus, and thus to fight the COVID-19 pandemic. Diagnosis for SARS-CoV-2 is currently performed after running qRT-PCR of selected viral genes from nasal/respiratory tract swabs, monitoring the immune humoral response by serological assays, and/or measuring circulating IgM/IgG antibodies using various immunoenzymatic ELISA platforms available on the market. Previous studies have reported that serological testing may be useful to identify asymptomatic or subclinical infection with SARS-CoV-2 among medical workers in close contact with patients with COVID-19 . HoweverCell-ELISA positivity was assessed, using the arbitrary threshold of an OD450 value of 0.065 (as described above), on data obtained with PBMCs incubated with CXM, on spontaneous releases and from PBMCs cryoconserved before the COVID-19 pandemic.Cell-ELISA data obtained for all tested samples, reported in Although no conclusive statements can be drawn because of the relatively small size of the analyzed population, five individuals out of 45 (11.2%) tested negative by both ELISA1 and ELISA2 but positive by cell-ELISA, and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum. This challenges the negative results from serological screening and suggests that the cell-ELISA may detect S1-specific IgG secreted by B cells even when a serological ELISA does not. This assumption is reinforced by the observation that all 16 subjects tested positive by cell-ELISA, including those who ELISA tested as negative and claimed to have had symptoms attributable to COVID-19.This finding confirms previous observations describing that circulating antigen-specific memory B cells can be present and persist after immunization, even in the absence of detectable serum IgG . In addi6/mL PBMCs to gain reliable cell-ELISA data.To confirm effective IgG production by B cells, in vitro cultures of PBMC were also evaluated in the presence of CXM, an antibiotic drug known to block initial phases of protein elongation in ribosomes, which have already been employed in a cell-ELISA animal model . The datEven though the results we obtained by cell-ELISA appeared specific, as demonstrated by the CXM control, they were mainly based on low OD450 values. This probably is due to the small number of antibody-secreting cells present in peripheral blood and/or to the low sensitivity of reagents in the kit, which are calibrated for plasma/serum IgG. However, we choose to use a commercial kit to measure cell-secreted antiviral antibodies in order to support the enforceability of the assay in standard diagnostic labs. In addition, it is possible to select the antibody class to be tested by the secondary antibody employed.To understand the possible levels of IgG secreted by PBMCs and the sensitivity threshold of the assay, the OD450 values of positive samples were compared to those from reference concentrations of human γ-globulin determined by ELISA. Obtained data suggest a minimal assay sensitivity of 7 ng IgG/well, which increased up to 50 ng/well of secreted IgG in donors. Interestingly, the donor whose PBMCs secreted the highest level of anti-S1 IgG in vitro tested negative in both serological ELISA 1 and ELISA 2 and declared no symptoms.In conclusion, we propose here a modified ELISPOT platform, which was converted to a simpler cell-ELISA adapted to detect the S1 spike protein of SARS-CoV-2 and able to monitor the presence of memory B and plasma cells in the blood.The proposed cell-ELISA, although needing more work addressed at increasing the number of donors and sampling over time, may improve upon conventional plasma or serum-based ELISAs, especially in the case of undetectable IgGs in serological analysis. Importantly, the assay could be of help in assessing the immunization status via S1 protein detection, and thus, providing a clearer picture of infection progression, and can readily be applied in clinical trials. Such important information may prove crucial in establishing seroprevalence in a more accurate way, in determining which individuals will benefit more than others from vaccination, and eventually in defining the efficacy of new vaccines.