A growing body of research has confirmed that nanoparticle (NP) systems can enhance delivery of therapeutic and imaging agents as well as prevent potentially damaging systemic exposure to these agents by modifying the kinetics of their release. With a wide choice of NP materials possessing different properties and surface modification options with unique targeting agents, bespoke nanosystems have been developed for applications varying from cancer therapeutics and genetic modification to cell imaging. Although there remain many challenges for the clinical application of nanoparticles, including toxicity within the reproductive system, some of these may be overcome with the recent development of biodegradable nanoparticles that offer increased biocompatibility. In recognition of this potential, this review seeks to present recent NP research with a focus on the exciting possibilities posed by the application of biocompatible nanomaterials within the fields of male reproductive medicine, health, and research. The last four decades have witnessed the development of a range of nanopharmaceuticals designed for the targeted, efficacious delivery of bioavailable drugs . The genThe reproductive system functions to arouse sexual interest, produce healthy gametes, facilitate fertilization of the egg, and to provide a nurturing environment for the resultant embryo until the time of parturition. The sophistication and complexity of this system increases its vulnerability to various forms of pathology, which commonly manifest in sub-/in- fertility phenotypes. Indeed, infertility afflicts an estimated 9% of the human population of reproductive age , represein vitro study of male gametes is complicated by their relative short lifespan ex vivo and the fact that they are highly sensitive to environmental exposures as diverse as excursions in temperature and pH as well as to a plethora of xenobiotic compounds , which separates the seminiferous tubules into basal and adluminal compartments. Sertoli cells secrete a number of cytokines, growth factors and immunoregulatory molecules thus protecting the haploid germ cells from the immune system . By estaAlong with LC , the intThere are a number of cellular insults that are capable of disrupting the redox status and thereby inducing germ cell ablation within the testes. Pro-oxidants and sources of intrinsic and extrinsic oxidative stress, such as autoimmune disease, exposure to radiation , xenobioThe field of nanopharmaceuticals encompasses the development and use of nanomaterials for therapeutic, diagnostic, and theragnostic modalities. The physicochemical properties of nanomaterials overcome some of the challenges and limitations posed by more traditional therapeutic agents thus enhancing their utility for drug delivery. Such properties include the ultra-small size  , antimicQuantum dots (QDs) are semiconductor nanocrystals of ultra- small hydrodynamic radius of <10 nm and are utilised for imaging applications rather than drug delivery. QDs possess very desirable qualities such as high yield and stable fluorescence, as well as being amenable to surface modification with targeting moieties. These attributes could make them invaluable in research  as well Arguably, the most promising of the biodegradable, metal based, nanoparticles are the iron oxide NPs.Iron oxide NPs (IONPs), which have been widely researched for drug delivery and as platforms for magnetic resonance imaging (MRI) contrast reagents. IONPs are biodegraded in vivo into iron ions , which iSilica nanoparticles (SNPs) are composed of porous silica  and can in vivo to silicic acid, which is then excreted via the kidneys  constitute a large proportion of the NP formulations that have been approved by the U.S. Federal Drug Administration (FDA) and are currently being marketed . Polymerin vitro and in vivo performance of PNPs as drug delivery agents. Furthermore, there are currently six polymeric pharmaceutical products in Phase I, II, and III clinical trials .Combined, the stability, responsiveness, tunability, facility for specific functionality via surface modification, along with biocompatibility and FDA approval status afforded to NPs composed of the amphiphilic block copolymers, poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA), has identified this class of NP as the focus for intense scientific and clinical interest . As within vivo. One of these components, the cell-surface “do-not-eat-me” protein, CD47, binds to and signals phagocytes that the cell is “self” thereby inhibiting phagocytosis. in vivo clearance compared to that of PEGylated PLGA NPs  of endosomal origin formed by the fusion of multivesicular bodies with the plasma membrane  and are Exosomes possess a “homing” ability that facilities the targeting of particular recipient cell type(s). Together, their size and endowment with surface “self” proteins enable exosomes to evade recognition and removal by the immune system and bestows excellent biocompatibility . ExosomeThe current research in this use of biomimicry to produce safer and more effective EV and exosomal nanosystems for drug delivery was comprehensively reviewed by In summary, the major limitations to the wider use of exosomes as carriers include their unfavorable pharmacokinetics , lack ofCovalent attachment of targeting moieties such as peptides, antibodies or aptamers to the NP surface provides the opportunity for circulating NPs to bind to specific cell surface biomarkers. This, in turn, facilitates the delivery of the NP payload to selected cell types , 3. TargThis targeting strategy enables elevation of the local concentration of the drug whilst maintaining the systemic levels well below the maximum tolerated dose (MTD) and thus decreasing the chance of off-target effects. The targeting moieties can be coupled to PEGylated NPs via the PEG portion of the polymer before or after formation of PNPs . TargetiAdvantageously, the testes presents several uniquely expressed receptors for hormones such as the FSH, LH, and the Anti-Mullerian hormone (AMH), that are ideal for targeting using peptides or antibodies. The structure and kinetics of the FSH receptor, in particular, have been extensively studied. On binding of a ligand to its active site, both the FSH receptor and bound ligand are internalized via receptor mediated endocytosis . Indeed,There have been many recent review articles discussing the various aspects of reproductive research, health and medicine that might be addressed using specifically engineered nanosystems . Rather Cytotoxic cell ablation pharmaceuticals are currently used for a variety of scientific and medical applications to selectively ablate a single type of cell  with theAlmost all anticancer therapies rely on the enhanced permeability and retention (EPR) effect, that is, passive accumulation of therapeutics in the tumor . The advν β3 integrins over-expressed on the surface of cancer cells. When exposed to blue-light radiation, this nanosystem was able to increase the accumulation of the drug and to reverse antiapoptotic effects of survivin as well as to enhance ROS levels (Tripterygium wilfordii Hook F.) for use in ovarian cancer  payload that inhibits the translation of proteins involved in the resistance pathway. Examples of such dual-mode NPs include those described by S levels . AnotherS levels  or drugsn cancer . A recenn cancer . Therefon cancer .NPs can overcome the challenge of maximizing cytotoxicity within the tumor by incorporating all of the drugs in the one carrier and thereby delivering drug cocktails simultaneously to the cell. The use of nanotherapeutics may also obviate or reduce some of the long- and short-term side effects of chemotherapeutic drugs. For instance, the cardiotoxicity resulting from treatment with cisplatin or doxorubicin. The therapeutic potential of NP cisplatin-delivery strategies have been extensively discussed in review articles by Globally, prostate cancer (PC) is the second most commonly diagnosed cancer in men  and is rin vitro and in in vivo xenograft models of the PSMA-positive/androgen-receptor negative LNCaP cell line and the PSMA-negative/androgen-receptor positive CRPC PC-3 cell line , distribution to off-target tissues was reduced, and a significant inhibition of growth of xenograft tumors was observed.NPs may better facilitate the detection of ultra-small clusters of tumor cells by MRI than traditional contrast reagents. With this in mind,  Resovit . SimilarDespite being relatively uncommon, accounting for only 1% of all cancers, testicular cancer (TC) is the most prevalent solid malignancy in 15–30 year old males. Like PC, TC is more prevalent in developed countries where altered environmental factors and higher exposure to xenobiotics, both pre- and postnatally, are believed to be responsible for its increasing prevalence . TC is uTreatment of TC usually involves removal of the effected testis (orchiectomy) or chemotherapy followed by orchiectomy in the case of metastasis . Testis in vitro  baby in 1978, the field of assisted reproduction technology (ART) has made much progress. However, among the many challenges that remain in this field, at least some may be addressed using nanotechnology. Thus, non-human models have been used to test the efficacy and safety of nanotechnology in improving ART outcomes. The selection of good quality, healthy spermatozoa for use in artificial insemination (AI), IVF, and intracytoplasmic sperm injection (ICSI) may improve the health of the embryos, the chances of conception, and the viability of the resulting pregnancies. After undergoing a selection process, the sperm sample should show improved motility, and morphology and should exhibit lower levels of DNA fragmentation, DNA oxidative damage and contamination with apoptotic sperm or other cell types  for the purpose of contraception. Additionally, imaging is an emerging application for NPs, which can be engineered to deliver any cargo including MRI contrast reagents and fluorescent tags. This may be particularly useful for such diverse clinical applications as the assessment of the ovarian reserve for family planning or to image metastatic cell clusters of reproductive origin.in vivo use of NPs will only be possible if they present with minimal toxicity/immunogenicity. In this regard, minimization of off-target effects and increased safety indices could perhaps be best addressed via functionalization of the NP surface to ensure it is engineered with the highest levels of target cell specificity and/or differential uptake efficiency into the targeted, as opposed to non-targeted cells. As additional criteria for clinical use, the NPs would benefit from an extended half-life in vivo as well as a long shelf-life.This review has sought to highlight the breadth and variety of applications to which NPs may be applied to expand and enhance existing imaging and treatment options for conditions of the male reproductive system. Besides their use in diagnostics, reproductive cancer, contraception, ART and reproductive health are all fields that stand to benefit from future developments in NP technologies. In guiding these developments, important considerations include the formulation of biocompatible, well characterized materials that are soluble or colloidal under aqueous conditions. Similarly, in vivo half-life and biocompatibility, presents many possibilities and would have a wide application in tailored cancer therapy, gene manipulation in germ cells, as well as a wide range of reproductive biology research applications.The use of exosomes, which are furnished with specific ligands targeting them to particular cell types, could be arguably among the most exciting developments in nanotechnology. Potentially, molecular engineering of the source cell to express a desired cargo or targeting peptides to then be packaged into or expressed on the membrane surface of exosomes may result in ingenious delivery agents that are customized to the patient. Innovative hybridisation of exosomes to create a new breed of “stealthy” NPs, with increased in vitro and in vivo research as well as in diagnostic imaging, offering higher yield and in vivo stability in comparison to other fluorescent tags. In reproductive biology research, possible applications for ligand-modified QDs may include gamete labelling for in vivo and in vitro tracking, as envisaged by in vivo imaging of germ cells within the testes, and particle tracking. Additionally, in vivo tumour imaging using targeted QDs may aid in cancer treatments and also detect cancer cells for early diagnosis in vivo and in ex vivo samples.The other exciting developments lie in the field of imaging. Recently, researchers have sought to synthesize quantum dots based on carbon  or silicIn the field of reproductive science, IONPs present a number of applications, including the aforementioned use in MACS-facilitated sperm selection, superparamagnetic IONPs may facilitate cell MR imaging and magnetic fluid hyperthermia for targeted cell ablation. Additionally, safer alternatives to QDs, based on IONPs may be developed. For instance, Vasquez et al. used a composite luciferase magnetic NP to label sperm which could then be detected using bioluminescent and UV-Vis-NIR microscopy . FinallyRA conceived the review topic, edited all sections, and proposed modifications to section organization and figure preparation. BF prepared the manuscript, organized all sections, and made revisions as proposed by co-authors. AP prepared all figures. JS contributed early editing of reproductive system section of manuscript. ML edited nanoparticle sections. DR contributed to co-writing the male reproductive system section. BN edited all sections and proposed modifications to section organization and figure preparation. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Small‐cell lung cancer (SCLC) is a heterogeneous disease, consisting of intratumoral and intertumoral neuroendocrine (ASCL1 and/or NEUROD1), mesenchymal‐like, and YAP‐driven transcriptional states. Lysine‐specific demethylase 1  inhibitors have recently been progressed to clinical trials in SCLC based on a promising preclinical antitumor activity. A potential clinical limitation of LSD1 inhibitors is the heterogeneous drug responses that have been observed in SCLC cell lines and patient‐derived models. Based on these observations, we studied molecular and transcriptional signatures that predict patient response to this class of drug. Employing SCLC patient‐derived transcriptional signatures, we define that SCLC cell lines sensitive to LSD1 inhibitors are enriched in neuroendocrine transcriptional markers, whereas cell lines enriched in a mesenchymal‐like transcriptional program demonstrate intrinsic resistance to LSD1 inhibitors. We have identified a reversible, adaptive resistance mechanism to LSD1 inhibitors through epigenetic reprogramming to a TEAD4‐driven mesenchymal‐like state. Our data suggest that only a segment of SCLC patients, with a defined neuroendocrine differentiation state, will likely benefit from LSD1 inhibitors. It provides novel evidence for the selection of a TEAD4‐driven mesenchymal‐like subpopulation resistant to LSD1 inhibitors in SCLC patients that may require effective drug combinations to sustain effective clinical responses. Here, we identify intratumor and intertumor heterogeneity limits the activity of LSD1 inhibitors in SCLC. Activity of LSD1 inhibitors in SCLC is confided in cells enriched in neuroendocrine transcriptional programs. Transdifferentiation of SCLC into a TEAD4‐driven mesenchymal‐like state represents an acquired resistance mechanism to LSD1 inhibitors. Drug combinations targeting YAP‐TEAD pathways with LSD1 inhibitors may be an effective strategy to target intrinsic‐ and adaptive‐resistant populations and sustain effective clinical responses. ASCL1achaete‐scute homolog 1ATAC‐seqassay for transposase‐accessible chromatin using sequencingBRD4bromodomain‐containing protein 4CREBBPcAMP response element‐binding protein‐binding proteinEMTepithelial–mesenchymal transitionEP300histone acetyltransferase p300 or E1A‐associated protein p300EZH2enhancer of zeste homolog 2GESAgene set enrichment analysisGFI1Bgrowth factor independent 1BGRPgastrin‐releasing peptideINSM1insulinoma‐associated protein 1LSD1/KDM1Alysine‐specific histone demethylase 1AMLL1/2mixed lineage leukemia protein 1/2NEUROD1neurogenic differentiation 1PDXpatient‐derived xenograftsRB1retinoblastoma protein 1scRNA‐seqsingle cell RNA sequencingSNAGSNAIL/Gfi‐1 family zinc finger proteinsTEAD4TEA domain family member 4VIMvimentinZEB1zinc finger E‐box‐binding homeobox 11in vitro and in vivo preclinical data led to clinical trials exploring the use of histone deacetylase inhibitors including romidepsin or panobinostat in relapsed SCLC. Although both agents were well‐tolerated and demonstrated some evidence of tumor shrinkage and sustained stable disease, both clinical studies failed to demonstrate a significant benefit warranting continued development . (C) Pathway analysis of genes altered in expression  by GSK690 treatment utilizing MSigDB pathway databases. Top 10 enriched pathways are shown with the p‐value cutoff of 0.05.Fig. S6. Western Blot analysis of neuroendocrine and mesenchymal protein levels using indicated antibodies in SCLC cells lines treated with DMSO or 0.3 µM GSK690 for 14 days.Fig. S7. (A) Left: cluster number selection within UMAP using resolution = 0.15. To optimize single cell clustering, we tuned the “resolution” parameter in Seurat, and selected resolution = 0.15 by visual inspection and the presence of robust differential markers between clusters. Right: cell number change in each cluster depicted as percentage of total cells in each cluster before and after GSK690 treatment. (B) Expression of ASCL1 or GRP in cluster 2 in UMAP or as violin plots following treatment with vehicle (DMSO) or 0.3 μM GSK690 for 21 days.Fig. S8. (A) Top 10 uniquely expressed genes in each single cell RNA‐seq cluster. The cluster specific genes were identified by performing DE analyses between each cluster with all the rest of clusters. DE cutoff is FDR < 5% and logFC > 0.25. (B) For differential pathway analysis, a hypergeometric test with FDR correction was applied to cluster specific differential genes using the MSigDB data base. NE (8 genes) or EMT (76 genes) gene score were calculated as the mean expression value of the detected gene sets in each single cell, and two sample t‐test was performed to calculate the significance between DMSO and treatment. Pathways analysis showed top 10 up‐regulated pathways within each cluster following treatment of 0.3 μM GSK690 for 21 days. P‐value cutoff = 0.05. Cluster 3 and 4 did not have any significant enrichment.Fig. S9. Cell viability and gene expression of neuroendocrine and mesenchymal markers in NCI‐H526 and NCI‐H69 cells pre‐treated with 0.3 µM GSK690 for 14 days and following 7‐day washout.Fig. S10. (A) Representative ATAC‐seq profiles of biological replicates from day 0, day 21 DMSO or 0.3 µM GSK690 treatment. (B) Percentage of ATAC‐seq peaks in cells treated with GSK690, separated into promoter (<±3 kb transcription start site [TSS]) and distal regions (>±3 kb TSS).Fig. S11. Long‐term proliferation assay for NCI‐H69 cells treated with T‐448 with a dose‐range up to 1.8 uM for 21 days.Click here for additional data file.


Subsequently, we crossed the two lpa lines and two cultivars, and derived 19 promising lpa pea breeding lines; here we document their agronomic performance based on 10 replicated field trials in Saskatchewan. Seventeen of these lpa lines yielded greater than 95% of the check mean (associated cultivars) and 16 were above 98% of the check mean for 1000 seed weight. The 19 lpa lines showed 27 to 55% lower phytic acid concentration than the check mean. Iron concentrations were similar in all the lpa lines and cultivars, yet the Caco-2 human cell culture assay revealed 14 of the 19 lpa lines had 11 to 55% greater iron bioavailability than check means. Thus, a single round of plant breeding has allowed for closing the gap in performance of low phytic acid pea.Field pea is a pulse that delivers high protein content, slowly digestible starch and fiber, and many vitamins and minerals, including iron. Naturally occurring plant phytic acid molecules bind iron, lowering its availability for absorption during digestion. Two low phytic acid ( Pisum sativum L.) has the potential to be a high protein, low-input contributor to meeting the increasing food demands of a growing global population. In 2020, the Canadian field pea harvest was estimated at 4.6 MT, approximately 24.5% higher than the 10-year average of 3.7 MT and Saskatchewan led provincial harvest with 2.5 MT [As a pulse, field pea (lpa) crops, including: health benefits from increased bioavailability of phosphorus, potassium, calcium, zinc, and iron in digestion by humans, poultry, swine, and fish; sustainable phosphorus management that reduces fertilizer application for crops; and reduced eutrophication, i.e., lessening environmental pollution from undigested phosphorus. While micronutrient availability in lpa crops has the potential to provide health benefits, especially to pregnant women and young children [An essential first step to increasing macro and micronutrients in our food is to care for our soils and be sure nutrients are present for plants to incorporate in their tissues . Commercchildren , it shouchildren .lpa pea lines: 1-2347-144 and 1-150-81. An important aspect of genetic research in lpa lines is the identification of specific genes and pathways affected. Phytic acid biosynthesis is catalyzed by D-myo-inositol 3-phosphate synthase (MIPS) and the gene encoding this enzyme has been identified as a common target in lpa grains [lpa lines is recessive and probably allelic [MIPS was not present [lpa pea mutants in support of breeding lpa lines in other crops. When genetic pathways capable of increasing bioavailability of micronutrients exist in a crop of interest, conventional plant breeding is a sustainable approach to improving the health status of people globally ,11. Our a grains . Utilizi allelic ; howeverA focus on field pea nutrition, with their high protein content, slowly digested carbohydrates, dietary fibre, and concentration of vitamins and minerals, including iron, can be explored within the broader plant breeding vision of maintaining yield. The objective of this paper is to update the status of our ongoing breeding initiative to develop pea cultivars with lower phytic acid concentrations that maintain strong agronomic characteristics.lpa) breeding lines, 4 check cultivars , and one lpa mutant (1-2347-144)). These lines were evaluated at 10 station years from 2017 to 2020 at Kamsack, Meath Park, Rosthern, and Saskatoon locations in Saskatchewan, Canada. Yield was measured for all 10 station years; other characteristics were observed for two to nine of the station years (environment). The effects of line and environment were significant (p < 0.05) for all the reported traits, with the exception of the effect of line on Zn concentration  than the check mean, whereas stand density, lodging, maturity, and grain yield, as well as protein, iron, zinc, and phosphorus concentration were similar compared to the check cultivars , iron bioavailability (33 to 99 ng ferritin/mg protein), protein concentration (22 to 31% of the seed dry matter), grain yield , and other traits . For comultivars . Phytic Analysis of variance for station years indicated as ‘environment’ (x 2 reps each). Data in table is means. Minimum and maximum are for raw data. Agronomic data: stand density at flowering 1–9, where 1 is very poor plant stand density and 9 is full plant stand density (St Den); height as average of two random plants at maturity (HT (cm)); Lodging at maturity 1–9, where 1 is upright and 9 is completely horizontal (Lodg); plot maturity 1–3 taken at early maturity, where 1 is early and 3 is late (Mat); Yield (kg/ha); yield as % check mean ; 1000 seed weight (SDWT (g/1000)); near-infrared reflectance assessment of protein (%); element concentrations by ICP-ES and ICP-MS for phosphorus (P), zinc (Zn), and iron (Fe) (µg/g); mg phytic acid-phosphorus per g sample, as measured by Wade’s assay (mg PA-P/g sample); iron bioavailability as measured by Caco-2 bioassay (ng ferritin/mg protein).p < 0.05) (p < 0.05). Fe concentration was positively correlated with Zn concentration , whereas Fe and Zn concentrations were positively correlated with protein (p < 0.001) and phytic acid-phosphorus concentration (p < 0.001). Phytic acid-phosphorus concentration was positively correlated with protein concentration  and negatively correlated with grain yield . Stand density was positively correlated with maturity, yield, and protein, as well as Zn and Fe concentration. Stand density was negatively correlated with lodging.Pearson correlation analysis indicated phytic acid-phosphorus concentration was negatively correlated with iron bioavailability  . Iron bip < 0.01), Zn concentration (p < 0.01), phytic acid-phosphorus concentration (p < 0.001), and iron bioavailability (p < 0.05) between the 4802 and 4803 breeding lines  and 4803 (12 breeding lines). Results indicated significant differences for protein  mutants in crops, Colombo et al. [lpa mutants in wheat, maize, barley, and soybean have shown one or more of the following: suppressed germination, seed weight reduction, and susceptibility to abiotic and biotic stresses [lpa pea breeding lines of this study showed stand density, height, seed weight, and yield at the same or higher levels than cultivar parents. While we did not directly test for stress responses, the high yield in several field environments suggests the lines were not susceptible. An earlier exception to the negative effects of lpa in crops was observed in the lpa1 mutant in common bean, which was found to have equal or superior agronomic performance compared to wild type [lpa pea breeding lines of this study relative to commercial cultivars. In their review paper of low phytic acid  [lpa pea breeding lines or two control diets. Several characteristics were monitored, including body hemoglobin iron, body weight, and energetic status. Upon completion of the study, hepatic iron and ferritin, pectoral glycogen, duodenal gene expression, and cecum bacterial population analyses were conducted. The data indicated that lpa pea lines could moderately improve iron status. There were also significant effects on duodenal brush border membrane functionality and intestinal microbiota. Specifically, expression of the duodenal brush border membrane gene ferroportin (FPN) was significantly upregulated and there was an increase in abundance of beneficial gut bacteria in chicks fed lpa peas [The nutritional value of the  gallus) . Collabolpa peas .lpa pea breeding lines via the Caco-2 bioassay and a chicken feed study [lpa peas to athletic young women who are low in iron. Having documented higher iron bioavailability in ed study , the nexlpa) trait relative to cultivar controls. Two lpa pea lines (1-2347-144 and 1-150-81) were generated by non-destructive screening with Chen’s reagent following chemical mutagenization of the cultivar CDC Bronco [lpa lines were crossed with cultivars in 2011 [2 generation. Promising candidates were advanced to F5, then 400 lines from each cross were evaluated for phytic acid concentration by non-destructive seed testing. In 2016, ten F6 lines from each of four categories  to determine the effects of genotype, environment, and the genotype x environment interactions. Nutritional traits were evaluated by measuring protein, iron, and phytic acid concentration, and the Caco-2 human cell culture bioassay again assessed iron bioavailability (2018 samples).The chosen 19 breeding lines were evaluated in the field for F2. Plots were supplemented with solid core granular inoculant Rhizobium leguminosarum biovar viceae at manufacturer’s recommended rate of 3.0 kg/ha . Weeds were controlled with registered herbicide applications tailored to the site, supplemented with hand weeding. The plots were mechanically harvested in August when the majority of the plants were physiologically mature and dried by chemical desiccation.From 2017 to 2020, in agricultural fields near the communities of Meath Park, Rosthern, Kamsack, and Saskatoon in Saskatchewan, Canada , two replicates of the 24 lines were seeded in a randomized complete block design of 3.6 m × 1 m plots. Spring seeding was conducted at a rate of 90 seeds mFor the 10 station years, grain yield was measured and calculated for each plot and then converted to kg/ha. Other agronomic and nutrition data collected for station years varied but could include: stand density, plant height, lodging, maturity, 1000 seed weight, protein prediction by near infrared (NIR) analysis , and ele2O and 250 μL of modified Wade’s reagent . A 1000 µg g2 phytic acid stock  was diluted into 25, 50, 100, 200, 300, 400, 500 and 600 µg g 2as calibration standards. The samples and standards were vortexed, then 200 μL of each solution was transferred into a 96 well flat bottomed tissue culture plate and read at 490 nm using a BioRad xMark microplate spectrophotometer . Phytic acid-phosphorus (mg PA-P/g sample) concentration in seed was calculated from the absorbance reading of spectrophotometer divided by 50.Modified Wade’s reagent method  was usedTwenty grams of raw pea seed representing 96 samples (24 lines × 2 plot reps × 2 locations) grown in 2018 were placed in 15 cm diameter foil pie plates, submerged in 60 mL 18 Megaohm water, and soaked overnight at room temperature. Peas that did not imbibe were removed. To cook the peas, aluminum foil was secured as lids and the plates were autoclaved on liquid cycle at 121 °C for 30 min. The samples were cooled to room temperature, moved to a −80 °C freezer, and then freeze-dried following manufacturer instructions . The samples were ground to powder with a steel blade coffee grinder, being careful not to heat the samples. 3. 2.5 mL of pancreatic-bile solution was added, followed by adjusting pH to 6.9 to 7.0 with 1.0 M NaHCO3. Both pepsin solution and pancreatic-bile solution were purified by cation exchange resin . After digestion, the Caco-2 cells were fed 1.5 mL digested sample through a 15 kDa cutoff dialysis membrane. The cells were incubated and rocked gently for 2 h. The digested sample solution and membrane were removed and the cells were placed back in the incubator without rocking for 22 h. Finally, cells were harvested for analysis of total protein using a colorimetric assay  and for analysis of ferritin using an immunoradiometric assay  by manufacturers’ instructions. Iron bioavailability (ng ferritin/mg protein) for samples from different experimental runs was standardized by a standard lentil sample included in each run.The Caco-2 cell culture bioassay test was conducted in the laboratory of Dr. Raymond Glahn, USDA-ARS, Ithaca, NY ,36. For Analysis of variance (ANOVA) was conducted using PROC MIXED in SAS 9.4  using data from 2 to 10 station years (2017–2020). For this analysis, each station-year was considered an ‘environment’. The 19 breeding lines were considered as a fixed effect and replication as a random effect. Pearson correlation between traits was determined using PROC CORR implemented in SAS. PROC TTEST was used to evaluate differences between the two genetic backgrounds, 4802 (7 breeding lines) and 4803 (12 breeding lines).lpa pea mutants and relevant check cultivars. Current lpa pea breeding lines maintained protein concentrations and increased bioavailability of iron , without sacrificing yield or other important agronomic traits. The lpa trait has been incorporated into pea cultivars that should be ready for release to growers in the near future. Encouraging consumption of plant-based meals is an efficient and environmentally positive way to feed a growing global human population ,38. Incr


Although research exists on the relationship between passion and engagement among employees, the mechanisms of academic passion on academic engagement among students needs to be elucidated. Guided by the broaden-and-build and situated cognition theories, we explored the positive effect of academic passion on academic engagement, the mediating effect of academic self-efficacy, and the role of teacher developmental feedback as a moderator in the relationship between academic passion and academic engagement. Based on a sample of 1,029 college students from universities in the Henan Province of China, the results showed that academic passion was positively related to academic engagement, academic self-efficacy partially mediated the relationship between academic passion and academic engagement, and teacher developmental feedback effectively moderated the relationship between academic passion and academic engagement. These findings explained the mechanism underlying the relationship between academic passion and academic engagement. Moreover, the findings highlighted important factors that promote college students' academic engagement. Academic engagement, defined as the degree of students' involvement in their studies, is indicated by the amount of energy they devote to studies  in Henan Province, China, were selected for investigation, and the research participants included undergraduates. Self-report questionnaires were completed by participants after they were assured that their responses would remain anonymous and that their participation was voluntary. The study was approved by the Ethics Committee of the Henan Normal University.To ensure the quality of data and reduce the impact of common method bias, we collected data at two time points. The survey data were collected in the form of two separate questionnaires with the help of related teachers. The survey lasted nearly a month and was conducted in two stages with an interval of 3 weeks. In the first stage, academic passion, academic self-efficacy, and demographic variables were measured. A total of 1,200 questionnaires were distributed during the first stage. There were 57 students who refused to participate in this study (4.75%). In the second stage, the participants who completed the questionnaire in the first stage were selected. In this stage, teacher developmental feedback and academic engagement data were collected. The questionnaire used a unique code matching method (required participants to provide the last six digits of their ID number) to match the two sets of questionnaires. After eliminating invalid questionnaires that were not answered carefully, 1,029 sets of valid questionnaires were successfully matched. The demographic survey results showed that the participants comprised 431 men (41.9%) and 598 women (58.1%). Their ages ranged from 18 to 24 years. There were 629 rural students (61.1%) and 400 urban students (38.9%).We measured academic passion using the scale developed by Vallerand and Houlfort . The ChiWe measured academic self-efficacy using a scale validated by Chen et al. . The scaWe measured teacher developmental feedback using a developmental feedback scale developed by Zhou . The ChiWe measured academic engagement using the shortened version scale developed by Schaufeli et al. . The ChiIn addition, as gender and birthplace have a significant impact on college students' academic engagement  did not significantly improve after adding the common method factors . The above method shows that there is no serious deviation in the general method.The Harman single-factor test was used to statistically verify the presence of common method bias. The results reported that four factors had eigenvalues >1, and the first factor explained 34.01% of the total variance. The results did not exceed a critical value of 40%. In addition, the CFA results of the method-factor approach showed that the model fit of the four-factor model , thus supporting Hypothesis 1. In addition, the interaction between academic passion and teacher developmental feedback was positively related to academic engagement . Therefore, teacher developmental feedback had a moderating effect on the relationship between academic passion and academic engagement.First, the moderating effect of teacher developmental feedback on the relationship between academic passion and academic engagement was examined. p < 0.001). Thus, Hypothesis 2 was supported. p < 0.001), and that academic self-efficacy had a significant effect on academic engagement , thus supporting Hypothesis 3. In addition, it was found that academic self-efficacy partially mediated the relationship between academic passion and academic engagement. The interaction effect between academic passion and teacher developmental feedback on academic engagement was also significant . Therefore, Hypothesis 4 was also supported. Overall, the results showed that teacher developmental feedback positively moderated the relationship between academic passion and academic engagement.Second, academic self-efficacy was included in the model. Following prior research, we further observed the direct effect of academic passion on academic engagement at two standard deviations above and below the mean of the self-construal score. Consistent with our proposition, t = 21.43, p < 0.001) than under low teacher developmental feedback , thus supporting Hypothesis 4. This showed that when students received more developmental feedback from teachers, their academic passion had a stronger positive predictive effect on academic engagement.In addition, the simple slope analysis  confirmeWhat remains to be answered is how academic passion contributes to academic engagement. Therefore, in this study, we attempted to reveal the underlying mechanism of the relationship between academic passion and academic engagement. The results showed that (a) academic passion had a positive effect on academic engagement, (b) academic self-efficacy partially mediated the relationship between academic passion and academic engagement, and (c) teacher developmental feedback moderated the relationship between academic passion and academic engagement. Thus, these findings suggested that the higher the level of developmental feedback, the stronger the positive relationship between academic passion and academic engagement.First, this study tested whether academic passion could effectively predict academic engagement. Thus, it not only effectively promoted the development of the broaden-and-build theory in the field of education, but also expanded the scope of research on the factors affecting academic engagement. This study revealed the positive effects of academic passion from the perspective of positive psychology, and answered the theoretical questions “Is there a positive relationship between academic passion and academic engagement?” and “How does academic passion affect academic engagement?” This result is consistent with an earlier study that suggested a positive relationship between academic passion and engagement (Enwereuzor et al., Second, this study found the mediating role of academic self-efficacy and revealed the internal mechanism by which academic passion affects academic engagement. This is not surprising as prior research has shown that academic self-efficacy not only helps individuals undertake and make the necessary efforts to successfully complete various tasks, but also provides them with more personal resources to achieve good academic performance (Whannell et al., Third, this study integrated teacher developmental feedback into the theoretical framework and identified the boundary condition in the relationship between academic passion and academic engagement. It was found that when teachers have more developmental feedback, the positive effect of students' academic passion on academic engagement was stronger. This can be explained as follows: teacher developmental feedback provides students with helpful information, assesses students' learning status, narrows the gap between students' understanding and performance Hattie, , satisfiFinally, this study used Chinese college students as research subjects. Therefore, in the context of Chinese culture, this study investigated the relationship between academic passion and academic engagement. Previous studies have shown that students with different learning stages and cultural backgrounds have different levels of psychological and academic engagement (Martin et al., This study explored the mechanism of academic passion's impact on academic engagement, which provided several theoretical and practical guidance; however, certain limitations were also present. First, this study only selected three universities in Henan Province, China, for investigation. Constrained by the convenience sampling method, the representativeness of the sample declined. The scope of this investigation should be further expanded. Furthermore, this study focused on the impact of college students' academic passion on academic engagement. In the future, the academic engagement of primary and secondary school students can be further studied and new research findings are expected. Second, academic engagement data were collected only by students' self-reports; thus, response bias could have influenced the results. In the future, data such as teacher evaluations should be included for more comprehensive measurements. Third, this study revealed the internal mechanism from the perspective of cognitive processes; however, the mediating paths of motivation and other factors should also be investigated in the future. In terms of boundary conditions, future researchers should examine the potential strengthening or restrictive conditions of factors, such as the learning atmosphere, for undergraduates' academic engagement.The study emphasized the role of academic passion in enhancing college students' academic engagement. In addition, the findings showed that students with high academic passion had higher academic self-efficacy, which encouraged them to maintain a higher degree of academic engagement. Furthermore, the higher the level of developmental feedback, the stronger the positive relationship between academic passion and academic engagement. It is hoped that the findings will guide the development of interventions to increase students' academic passion, thus promoting increased academic engagement.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.HZ conceived the research idea, structured and drafted the manuscript. XL and CQ collected and analyzed the data. 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.


The commonly used laboratory cell lines are the first line of experimental models to study the pathogenicity and performing antiviral assays for emerging viruses. Here, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different human cell lines, compared their growth characteristics, and performed quantitative proteomics for the susceptible cell lines. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high-to-moderate level of susceptibility to SARS-CoV-2. In Caco2 cells, the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein abundance profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3, and Huh7 cells. Together, our data show cell-type specific variability for cytopathogenicity, susceptibility, and cellular response to SARS-CoV-2 and provide important clues to guide future studies.  •Calu-3, Caco2, Huh7, and 293FT cells are differently susceptible to SARS-CoV-2•Caco2 shows high virus production without any prominent cytopathic effect•Quantitative proteomics shows cell-type specific variations in cellular response•Interferon response is commonly dysregulated in Calu-3, Caco-2, and Huh7 Virology ; Cell Biology ; Proteomics Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, is a highly pathogenic coronavirus that has created a global public health challenge . The virIn vitro studies using cell models such as Caco2, Huh7, Hek293, A549, and Vero-E6 cell lines have identified interactome of different SARS-CoV-2 proteins  organotypic cultures can mimic different organs of interest and allows to study SARS-CoV-2 infection in a more physiological context . Howeverproteins  as well proteins . Variancproteins , whereasproteins . Vero-E6proteins . Howeverproteins . Among tproteins . HoweverIn this study, we show the susceptibility of the first Swedish isolate of SARS-CoV-2 in six commonly used laboratory human cell lines. Using quantitative proteomics, we determined the changes in protein abundance caused by the virus in the susceptible cell lines, providing an overview of the signaling pathways that are altered by the SARS-CoV-2.We infected Vero-E6, Calu-3, A549, Caco2, Huh7, 293FT, and 16HBE with the first Swedish isolate of SARS-CoV-2 virus (SWE/01/2020) at a multiplicity of infection (moi) of 1 and 0.1 as previously described . Virus-iWe observed that the virus production in susceptible cell lines reached saturation by 48hpi. Therefore, we investigated the changes caused in the cell surface of Calu-3, Caco2, Huh7, and 293FT cells during virus production at 48hpi (moi 0.1) using scanning electron microscopy (SEM). We did not observe any significant changes in the morphology of the cell surface in the mock-infected cells. In SARS-CoV-2-infected Calu-3 and Caco2 cells, numerous virus-like particles corresponding to the size of SARS-CoV-2 (approx. 70nM) were observed to be attached to the cell surface or cellular projections A. IntereAngiotensin-converting enzyme 2 (ACE2) receptor and transmembrane serine protease 2 (TMPRSS2) activity have been shown to be critical for SARS-CoV-2 entry into the cell . To corrTo compare the tropism of the Swedish SARS-CoV-2 virus with the other globally isolated strains, we performed a literature survey to determine the susceptibility and cytopathogenicity in all the six cell lines as noted above. We have included virus isolates from Germany (FFM1/2020)  and Mun, France Because we observed differential susceptibility to SARS-CoV-2 infection in different cell lines originating from the human lung , intestine (Caco2), liver (Huh7), and kidney (293FT), we investigated how the cellular proteins are regulated during infection in these cell lines. To this end, we either infected or mock-infected Calu-3 cells (polarized), Caco2 cells, Huh7 cells, and 293FT cells with SARS-CoV-2 (moi 1) in triplicates. For proteomics, we used 24 h of infection because at this time point despite maintaining high viability all the cell lines showed exponential virus production, that plateaued after 24hpi. The cells were harvested at 24hpi, lysed, and equal amounts of the proteins were used to perform quantitative proteomics using a TMT-labeling strategy as previously described . The unpBecause Calu-3 and Caco2 were the only cell lines that showed substantial protein regulation upon infection at 24 h, we compared the significantly regulated proteins in those two cell lines. As shown in the Venn diagram in Type-I IFN, like IFN-β, plays a major role in host defenses against various viruses, and in our proteomics data we observed dysregulation of type-I-IFN-response-associated proteins including ISGs; therefore, we assessed the mRNA expression of IFN-β and the downstream ISGs  using qPCR. In line with our proteomics findings, the mRNA levels of IFN-β and all the tested ISGs significantly increased upon SARS-CoV-2 infection in Calu-3 and Caco2 cells. 293FT and Huh7 cells did not show any significant variations at 24hpi A. IFN-β 48h) compared with Calu-3 and Caco2 cells, we extracted the proteomics dataset from our earlier study (48h (no change). IFI35 was upregulated in both Calu-3 and Caco2 cells but was downregulated in Huh748h.Previous proteomics-based studies have observed that SARS-CoV-2 causes global proteomic changes after 48hpi specifically in pathways related to ErbB, HIF-1, mTOR, and TNF signaling ; complemer study . Next, wer study A, showiner study . We empler study B. We loo48h cells, we identified the proteins that were significantly uniquely altered by infection in each of the three cell lines. There were 3,897 proteins in Calu-3, 1,347 proteins in Huh748h, and 38 proteins in Caco2 that uniquely changed  via the PRIDE partner repository with the accession number PXD023760. All the generated codes are deposited in GitHub (https://github.com/neogilab/COVID_cell_lines). All the unprocessed and processed proteomics raw data are uploaded as supplementary tables  have been deposited to the ProteomeXchange Consortium (y tables , and S11All methods can be found in the accompanying


Amyotrophic lateral sclerosis (ALS) is a devasting neurodegenerative disorder for which no successful therapeutics are available. Valproic acid (VPA), a monocarboxylate derivative, is a known antiepileptic drug and a histone deacetylase inhibitor.To investigate whether monocarboxylate transporter 1 (MCT1) and sodium-coupled MCT1 (SMCT1) are altered in ALS cell and mouse models, a cellular uptake study, quantitative real time polymerase chain reaction and western blot parameters were used. Similarly, whether VPA provides a neuroprotective effect in the wild-type  and ALS mutant-type  NSC-34 motor neuron-like cell lines was determined through the cell viability assay.3H]VPA uptake was dependent on time, pH, sodium and concentration, and the uptake rate was significantly lower in the MT cell line than the WT cell line. Interestingly, two VPA transport systems were expressed, and the VPA uptake was modulated by SMCT substrates/inhibitors in both cell lines. Furthermore, MCT1 and SMCT1 expression was significantly lower in motor neurons of ALS (G93A) model mice than in those of WT mice. Notably, VPA ameliorated glutamate- and hydrogen peroxide-induced neurotoxicity in both the WT and MT ALS cell lines.[Together, the current findings demonstrate that VPA exhibits a neuroprotective effect regardless of the dysfunction of an MCT in ALS, which could help develop useful therapeutic strategies for ALS. SOD1), fused in sarcoma (FUS), TAR DNA-binding protein (TDP), and chromosome 9 open reading frame 72 (C9ORF72) Valproic acid  was purchased from American Radiolabeled Chemicals Inc. . All other chemicals and reagents were purchased from Sigma-Aldrich  and are labelled as a high-grade commercial product.[Male transgenic ALS mice of the mSOD1 (G93A) H1 high-expresser strain  were bred with females with a similar background (B6/SJLF1). Postmortem spinal cord tissue sections were used in this study. The animal study protocol has been approved by the institutional animal care and use committee of the Korea Institute of Science and Technology .WT)] and mutant-type  cell lines were cultured following previously described methods VPA was dissolved in the ECF buffer in the presence or absence of specific inhibitors or substrates. Then, the uptake experiment was carried out on the NSC-34 cell lines following the designated time and pH. The uptake rate [cell-to-medium ratio (µL/mg protein)] was calculated using the following formula , respectively [+ free buffer was created to assess the effect of ions (Na+) on the [3H]VPA transport into the NSC-34 cell lines. Similarly, to observe the effect of pH, the ECF buffer was adjusted to pH 6.0–7.4 using HCl or NaOH following a previously described experimental procedure VPA uptake for 10 s at pH 7.4. Then, the kinetic parameters were calculated and fitted to the Michaelis–Menten constant (Km) and maximum uptake rate (Vmax) through the non-linear least-squares regression analysis using Eq.  values were calculated by converting the net [3H]VPA uptake to percentage of inhibition in the presence of edaravone, ibuprofen, and salicylic acid. The obtained values were plotted using Sigma plot version 12, and were subjected to nonlinear regression .Similarly, half maximal inhibitory concentration . Then, 25 µg of protein was subjected to SDS/10% PAGE and blotted with primary antibodies  [MCT1 (SLC16A1)  and SMCT1 (SLC5A8/AIT) ]. β-Actin (ACTB) was used as a protein loading control   on the same membrane.To determine MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT) immunoreactivity changes, WT littermate (n = 5) and SOD1 (G93A) mice (n = 5) at 120–150 days of age were perfused using 4% buffered paraformaldehyde in PBS. Their spinal cords were extracted and serially sectioned at 30 µm thickness using a cryostat. Lumbar spinal cord sections  were used for immunostaining to detect MCT1 and SMCT1 levels. First, the tissue sections were incubated for 1.5 h in a blocking solution  and were further incubated with primary antibodies MCT1(SLC16A1) (1:200 dilution)  and SMCT1 (SLC5A8/AIT). (1:200 dilution) , BAX  (1:100 dilution), and BCL2  (1:100 dilution) in a blocking solution at 4 °C for 24 h. After washing thrice with PBS, the tissue slides were processed with a Vector ABC Kit . The MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT) signals were developed using the DAB chromogen . Images were then analyzed using an Olympus microscope system . The semi-quantification of immunoreactivity was analyzed using ImageJ. In particular, the intensity of MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT), BAX, and BCL2 signals was counted from five to 10 motor neurons per section. A total of five sections (section/case) were used for the semi-quantification.3H]VPA in NSC-34 cell lines, mMCT1 (SLC16A1), mSMCT1 (SLC5A8), and SMCT2 (SLC5A12) transporters and the non-targeting pool (control) siRNAs  with Lipofectamine® 2000  were selected at a concentration of 200 nM. Using the siRNAs, NSC-34 WT and MT cell lines were transfected and incubated at 37 °C. Following the transfection, [3H]VPA uptake was performed for 10 s under physiological pH. Western blot analysis was also done using both the NSC-34 WT and MT cell lines  solution (5 mg/mL) was added and the cells were stored in the dark at 37 °C until the appearance of a purple precipitate (formazan) VPA transport characteristics, a time course of uptake was performed in WT and MT ALS cell lines for 5 to 30 s at an acidic pH (6.0) at 37 °C. The VPA uptake was demonstrated to be time dependent, with the transport rate rapidly and linearly increasing up to 10 s in both WT and MT ALS cell lines  to physiological (7.4). The VPA transport system was shown to be pH sensitive. The uptake rate decreased with the pH increase from acidic to physiological in both cell lines  and the maximum transport velocity (Vmax) at two sites. At the high-affinity sites, the obtained Km1 values for WT and MT ALS cell line was 37.8 ± 3.0 and 19.1 ± 0.7 µM, respectively. The Vmax1 values for the WT and MT ALS cell lines were 0.0083 ± 0.0044 and 0.0232 ± 0.0007 nmol/mg protein/10 s, respectively. At the low-affinity sites, the Km2 and Vmax2 values were 4.59 ± 0.62 and 1.14 ± 0.14 mM and 1.86 ± 0.24 and 0.231 ± 0.015 nmol/mg protein/10 s for the WT and MT ALS cell lines, respectively , SA, 4-phenylbutyric acid (PBA), and α-cyano-4-hydroxycinnamate (CHC) at concentrations of 1 and 10 mM strongly inhibited the VPA uptake in both WT and MT ALS cell lines compared with that in the respective controls. In addition, the organic anion transporter (OAT) substrate/inhibitor para-aminohippuric acid (PAH) significantly reduced the VPA uptake. However, a tricarboxylate (citric acid) did not have a significant effect on the uptake of VPA in both WT and MT ALS cell lines  the VPA uptake by both the WT and MT ALS cell lines at concentrations of 1 and 10 mM. The OAT substrates edaravone (drug for ALS) and estrone 3-sulfate (E-3S) also significantly reduced the VPA uptake rate. Moreover, furosemide, a loop diuretic, and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an anion exchange inhibitor, caused noticeable depletion (approximately 40%) in VPA transport at a concentration of 1 mM both in WT and MT ALS cell lines  cell lines. The protein expression pattern of MCT1 and SMCT1 were depleted in the MT cell lines than in the WT (control) cell lines  ALS mice Fig. .Fig. 4PrImmunoreactivity of MCT1 and SMCT1 was mainly observed in motor neurons, and their levels in the spinal cord gray matter were lower in ALS G93A) mice than in WT littermate mice Fig. a–d, whicA mice thSMCT1 and MCT1 siRNA transfection affected the protein expression levels in the NSC-34 cell lines. After 48 h of siRNA transfection, the protein expression pattern was evaluated through Western blot analysis  and Smct2 (Slc5a12) siRNAs. Afterward, [3H]VPA uptake was performed for 10 s at the physiological pH. Both SMCTs were selected because VPA is a monocarboxylate and its uptake was shown to be sodium dependent in both WT and MT cell lines .VPA is a primary substrate in the mitochondrial fatty acid β-oxidation pathway . It has 3H]VPA uptake by motor neurons was time, pH, and sodium dependent  cells and in the biomedical model Dictyostelium was shown to be time and pH sensitive VPA transport into the WT cell lines, but SMCT1 primarily facilitated the transport into the MT ALS cell lines  of drugs such as ibuprofen, SA, and edaravone were needed to block the biological process of the [ine Fig.  SA is a ubstrate . Accordi-5 cells , 37. It 2O2-induced neurotoxicity in both the WT and MT ALS cell lines 2O2-inducdel Fig. .In conclusion, the expression of MCTs (MCT1 and SMCT1) and the function of VPA transport systems were altered in ALS cellular and animal models, indicating that MCT1 and SMCT1 could be pathological markers for ALS. SMCT1 primarily facilitated the VPA transport in the ALS cell line model. VPA showed a neuroprotective role against oxidative stress and excitotoxins in the ALS cell line model. Together, VPA could be used to treat the motor neuron disease, ALS.


In addition, our high-throughput transcriptomic studies provide information on the genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients.In the present study, we define the expression of 15 selected circular RNAs, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of tumors and consisting of three distinct groups according to the mesenchymal/epithelial phenotype. We identify DOCK1 gene (hsa_circ_0020397) shows low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines, independent of estrogen receptor or HER2 positivity. RNA-sequencing experiments performed on the triple-negative/mesenchymal MDA-MB-231 and MDA-MB-157 cell lines engineered to overexpress hsa_circ_0020397 demonstrate that the circRNA influences the expression of 110 common genes. Pathway analysis of these genes indicates that overexpression of the circular RNA differentiates the two mesenchymal cell lines along the epithelial pathway and increases cell-to-cell adhesion. This is accompanied by growth inhibition and a reduction in the random/directional motility of the cell lines. The upregulated AGR2, ENPP1, and PPP1R9A genes as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 genes show an opposite regulation by hsa_circ_0020397 silencing in luminal CAMA1 cells. The results provide novel insights into the role played by specific circular RNAs in the generation/progression of breast cancer.Circular RNAs are regulatory molecules involved in numerous cellular processes and may be involved in tumour growth and diffusion. Here, we define the expression of 15 selected circular RNAs, which may control the process of epithelial-to-mesenchymal transition, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of these tumours and consisting of three groups according to the mesenchymal/epithelial phenotype. A circular RNA from the  Circular RNAs are covalent circles of single-stranded RNA that are generated from non-canonical back-splicing of pre-mRNAs and non-coding RNAs ,2,3. CirDOCK1 gene  shows the most interesting profile, as it is expressed at low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines. The whole-genome RNA-sequencing (RNA-seq) data obtained from two triple-negative mesenchymal cell lines engineered to overexpress circDOCK1-1 indicate that this circular RNA controls the expression of genes involved in epithelial differentiation and cell adhesion. Among these genes, the upregulated AGR2, ENPP1, and PPP1R9A as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 are of particular interest. In fact, the seven abovementioned genes show an opposite behaviours upon circDOCK1 silencing in luminal CAMA1 cells. From a functional point of view, forced expression of circDOCK1-1 in two mesenchymal breast cancer cell lines causes a growth inhibitory effect and a significant reduction in the random/directional motility of both cell lines.In the present study, we focus our interest on a small set of circular RNAs that may be involved in the process of epithelial-to-mesenchymal transition (EMT)  and we dhttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga, accessed on 12 May 2021) and the CCLE (Cancer Cell Line Encyclopedia) of the Broad Institute , as indicated in the appropriate sections of the manuscript.All the reagents, the list of the cell lines, and their phenotypic characteristics/source are available in MDA-MB-231 and MDA-MB-157 cells characterized by forced expression of circDOCK1-1 and the CAMA1 cells genetically engineered for circDOCK1-1 silencing are described in the The methodologies used for the generation of the Total RNA was extracted using the miRNeasy Mini Kit . The RNA was reverse transcribed using the Maxima H Minus cDNA Synthesis Master Mix  and it was amplified with a Taqman Universal Master Mix , using specific Taqman probes (Applied Biosystems). Some of the mRNAs were determined with the SYBR Green PCR Kit (Qiagen) using oligonucleotides as primers. circRNAs were reverse transcribed with the Quantitect Reverse Transcription Kit (Qiagen). The corresponding cDNAs were amplified using the SYBR Green PCR Kit (Qiagen). A list of the Taqman probes and the structure of specific oligonucleotide primers is available in the Whole-cell protein extracts were prepared according to previously described methods . AliquotThe growth rate of cells was determined with the sulforhodamine B assay. Single-cell motility was examined with a previously described method , while wRNA-seq analysis of the circDOCK1-overexpressing MDA-MB-231 and MDA-MB-157 cells and the respective pVOID controls, three biological replicates of each cell line were grown in the presence of G418. For the silencing experiments, three biological replicates of cells were treated for 4 days in the presence/absence of doxycycline. Total RNA was extracted with the miRNeasy Mini Kit (Qiagen). cDNA libraries were prepared with the Illumina TruSeq RNAlibrary kit  and sequenced on the Illumina NextSeq500 with paired-end 150 base-pair-long reads. The RNA-seq data were analysed as described [GRCh38) were performed with the “Spliced Transcripts Alignment to a Reference” (STAR) software (v.2.5.2a). Gene expression was quantitated with the use of the annotations available in Gencode (version 27). Raw counts were processed in the R-statistical environment and differential expression analysis was conducted with the DESeq2 pipeline [For the pipeline . Genes eRNA-seq data are detailed in the above RNA-Sequencing Studies paragraph.The details regarding the statistical tests for the analysis of the quantitative data presented are available in the text or figure legends. All the analyses were performed using two-tailed statistical tests. The statistical treatments performed on the RNA-seq studies in HMLE  cells subjected to EMT by exposure to TGFβ (mesHMLE) [HMLE counterparts, mesHMLE cells showed upregulation of 11 circRNAs and downregulation of 4 circRNAs  . RelativcircRNAs .RNA-seq  oestrogen receptor-positive/progesterone receptor-negative/HER2-positive MDA-MB-361 cells; (c) oestrogen receptor-positive/progesterone receptor-negative/HER2-negative MDA-MB-175VII and ZR75.1 cells; and (d) oestrogen receptor-positive/progesterone receptor-positive/HER2-negative CAMA1 and HCC-1500 cells. The basal gene expression profiles separated the remaining cell lines, which are characterized by a triple-negative phenotype into two sub-groups, showing an epithelial  and a mesenchymal  morphology  of the transcriptomic data obtained in each cell line by RNA-seq . The Lumhenotype : (a) oesrphology . ssGSEA terparts . mesHMLE cells , did not show any difference in Lum, TNBC, and TNBC-mes cells  gene , though low, correlation between the expression levels of circDOCK1-1 RNA and the corresponding linear transcript in the various cell lines was observed  as a negative control. Following selection in G418, we isolated three independent empty vector and circDOCK1-1-overexpressing cell clones  for each cell line. As expected, the pVOID-transfected MDA-MB-231 and MDA-MB-157 cells did not express detectable amounts of circDOCK1-1  and two downregulated  genes using PCR (circDOCK1-1 overexpression upregulates 54 (including DOCK1) and downregulates 56 of these mRNAs in both MDA-MB-231 and MDA-MB-157 , we defined 10 “protein-interaction-networks”, five of which contain gene products involved in cell adhesion and cell–cell interactions  (As the ssGSEA results further indicate that the two top enriched pathways (GO:0010769 and GO:0022604) control cell morphogenesis and differentiation, we evaluated whether t cancer . circDOCnduction D of vari SPOCK1) D.TNBC-mes/TNBC/Lum cell lines and established correlations between the levels of these mRNAs and circDOCK1-1 in each cell line. As for the upregulated transcripts, the levels of 20 mRNAs, including AGR2, ARAP2, CLDN4, KRT7, and RBM47, tended to be lower in TNBC-mes than Lum cell lines (2 > 0.1) with circDOCK1 expression in TNBC-mes, TNBC, and Lum cell lines . Finally, the amounts of the 11 mRNAs tend to be larger in TNBC-mes than Lum cell lines  are devoid of a significant inverse correlation with circDOCK1 (2 < 0.1). In contrast, the levels of six mRNAs tend to be lower in TNBC-mes than Lum cells -inducible shRNAs targeting circDOCK1-1. Following infection, we isolated two puromycin-resistant CAMA-1 cell populations (circDOCK-sh1 and circDOCK-sh2) expressing each construct  and DOX (+DOX). We did not observe any significant difference in the expression levels of the DOCK1 transcript . These data support the idea that circDOCK1-1 knockdown does not lead to significant off-target effects.The two A1 cells C. Indeedelopment . This iscircDOCK1-1 is involved in the generation/maintenance of the epithelial phenotype, which is characterized by a decrease in the proliferation rate and motility as well as an increase in cell–cell interactions relative to the mesenchymal counterpart. Thus, we evaluated the effects of circDOCK1-1 in our genetically engineered MDA-MB-231 cells. In basal conditions, control pVOID1/pVOID2 and parental MDA-MB-231 cells show similar growth rates  and empty (pVOID3) vectors  that originate from the DOCK1 gene  as a positive regulator of the epithelial differentiation of mammary tumour cells. Indeed, the data obtained in a large panel of cell lines, recapitulating the heterogeneity of breast cancer, indicate that circDOCK1-1 is undetectable in cultures of TNBC cells with a mesenchymal phenotype (TNBC-mes), while it is highly expressed in Lum and TNBC cells characterized by an epithelial phenotype.In the present study, we identified one of the 126 circular RNAs  caused the upregulation of genes encoding epithelial proteins and the downregulation of genes encoding mesenchymal proteins. Our RNA-seq data indicate that circDOCK1-1 induces and maintains certain traits of the epithelial phenotype in breast cancer cells via the direct or indirect regulation of approximately 100 target genes.Forced expression of circDOCK1-1 in MDA-MB-231 and MDA-MB-157 cells code for proteins that control cell-to-cell contacts and cell adhesion, two parameters that are generally increased in luminal/epithelial relative to mesenchymal breast cancer cells. Among the genes upregulated by circDOCK1 in the two TNBC-mes cells and belonging to the protein-interactome networks identified  is of particular relevance. EPHB3 is an ephrin-binding membrane receptor, which facilitates cell adhesion [circDOCK1-1-induced epithelial differentiation, we performed silencing experiments in a Lum cell-line (CAMA-1) characterized by high levels of circDOCK1-1 expression. The RNA-seq studies performed in this model resulted in the identification of seven genes: AGR2, ENPP1, PPP1R9A, APOE, AQP3, CD99L2, and IGFBP4, whose expression pattern is opposite to that observed upon circDOCK1-1 overexpression. circDOCK1-1 knockdown increases the levels of the AGR2, ENPP1, and PPP1R9A transcripts, while it decreases the amounts of the APOE, AQP3, CD99L2, and IGFBP4 mRNAs. AGR2, APOE, AQP3, and CD99L2 may be of particular interest for the epithelial differentiating action exerted by circDOCK1-1 in TNBC-mes breast cancer cells. AGR2 is a protein disulfide isomerase [AGR2 is believed to promote lobulo-alveolar development, which is consistent with the role exerted in the intestine, where the protein is implicated in maintaining the epithelial barrier [APOE controls the differentiation and morphogenesis of neuronal cells [AQP3 may have a role in kidney development [CD99L2 is encoded by one of the six mesenchymal genes, which are part of an EMT signature in ovarian carcinoma [CD99L2 causes colony compaction, indicating that the protein plays a negative role in cell-to-cell adhesion.A significant proportion of the genes up- and downregulated by entified D, ARAP2 dhesions . ARAP2 uer cells ,40. Amonadhesion , enhanciadhesion . In addiadhesion . To restsomerase  and it isomerase , where i barrier . APOE coal cells , while Aelopment . CD99L2 arcinoma . In mesecircDOCK1-1 on the expression of a large number of target genes identified in our study, protein and microRNA sponging effects may be considered. Indeed, one of the hypothesized mechanisms underlying the gene regulatory action of circular RNAs is related to the ability of these molecules to act as sponges for specific proteins and microRNAs [AGR2, ENPP1, and PPP1R9A genes, which are upregulated by forced expression and silencing of circDOCK1-1. In fact, our data are consistent with the idea that circDOCK1-1 may be a direct regulator of these genes in breast cancer cells. To predict microRNAs that may bind each of the three mRNAs mentioned above and circDOCK1-1 contemporaneously, we used an in silico approach . The results of this analysis (AGR2, ENPP1, and PPP1R9A mRNAs have the potential to bind four, eight, and four microRNAs, which can interact with the circDOCK1-1 RNA as well. Noticeably, the transcriptomic results available in the CCLE and TCGA databases support the idea that the only microRNAs for which there is evidence of expression in breast cancer cell lines or tissues are hsa-mir-539-3p and hsa-miR-545-3p. Thus, circDOCK1-1 may increase the expression of AGR2, ENPP1, and PPP1R9A transcripts via a sponging effect exerted on hsa-mir-539-3p, hsa-miR-545-3p, and/or some of the other microRNAs identified in circDOCK1-1 in TNBC-mes breast cancer cells.As for the potential mechanisms underlying the regulatory action of icroRNAs ,51. To oanalysis  indicatecircDOCK1-1 is endowed with antioncogenic properties in breast cancer, as it seems to favour epithelial differentiation and reduce the proliferation as well as the motility/invasiveness of tumour cells characterized by a mesenchymal phenotype. To the best of our knowledge, no data are available on the expression levels or functional activity of any of the 126 circDOCK1 RNAs, including circDOCK1-1, in triple-negative breast cancer. However, a certain amount of relevant data on the functional role played by circDOCK1 RNAs in other types of tumours is present in the scientific literature. As an example, Zhang et al. recently reported that circDOCK1-1 expression is high in colorectal cancer cells and forced expression of the circular RNA increased the viability and invasive properties of cells derived from this tumour, suggesting a pro-oncogenic action [circDOCK1-1 in regulating the homeostasis of cancer cells is tumour type specific. To further complicate the issue, it should be noticed that another member of the DOCK1 family of circular RNAs, i.e., hsa_circ_0020394 (see SOX5 signalling pathway [circDOCK1-1 and other members of this circular-RNA family in breast cancer and other tumour types.Taken together, the results obtained in this study suggest that c action . This ma0394 see , has bee pathway . All thicircDOCK1-1 as an important regulator of the epithelial differentiation of mammary tumour cells. In addition, the high-throughput transcriptomic studies performed provide information on the number and type of genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients.The results of the study are of basic interest, as they provide novel insights into the role played by specific circRNAs in the generation and progression of breast cancer. In fact, our data identified


The prevalence of breast cancer in young women (YWBC) has increased alarmingly. Significant efforts are being made to elucidate the biological mechanisms concerning the development, prognosis, and pathological response in early-onset breast cancer (BC) patients. Dysfunctional DNA repair proteins are implied in BC predisposition, progression, and therapy response, underscoring the need for further analyses on DNA repair genes. Public databases of large patient datasets such as METABRIC, TCGA, COSMIC, and cancer cell lines allow the identification of variants in DNA repair genes and possible precision drug candidates. This study aimed at identifying variants and drug candidates that may benefit Latin American (LA) YWBC. We analyzed pathogenic variants in 90 genes involved in DNA repair in public BC datasets from METABRIC, TCGA, COSMIC, CCLE, and COSMIC Cell Lines Project. Results showed that reported DNA repair germline variants in the LA dataset are underrepresented in large databases, in contrast to other populations. Additionally, only six gene repair variants in women under 50 years old from the study population were reported in BC cell lines. Therefore, there is a need for new approaches to study DNA repair variants reported in young women from LA. Breast cancer (BC) is one of the main causes of death in women around the world , despiteBRCA1/2 genes account for 50–60% and the remaining variants, to non-BRCA genes of moderate and low penetrance, including ATM, PALB2, RAD51, and BARD1, all involved in double-strand break repair pathways .,34.BRCA1For this analysis, the GDSC2 dataset from the Genomics of Drug Sensitivity in Cancer repository was evaluated. A total of 30 cancer types, 192 drugs, and 135,242 entries were retrieved. No PARP inhibitors were tested in these drug databases for the selected cell lines. Later, different filters were applied to obtain specific information. First, the BC filter displayed seven different BC cell lines. For this analysis, the concentration range selected was ≤1 μM for all drugs. Followed by a selection of drugs that displayed an area under the curve (AUC) lower than 0.6. With this information, LA variants from young women previously selected in this study were compared and drugs with the best performance in cell line assays were retrieved. Six BC cell lines were retrieved: AU565, HCC1143, HCC1395, HCC1937, HCC70, and MDA-MB-468. A total of 27 candidate drugs were collected from this analysis, including drugs impacting different pathways such as apoptosis regulation, cell cycle, DNA replication, IGF1R signaling, mitosis, protein stability and degradation, and receptor tyrosine kinase (RTK) signaling .Further, Herein Databases serve as preliminary approaches in preclinical in vitro and in vivo assays to address inquiries regarding pathogenicity and drug candidates. For this study, the TCGA and METABRIC databases were analyzed to identify frequent variants in DNA repair genes in YWBC. In addition, using these tools we were able to compare reported variants in different age groups from LA countries.On the other hand, in vitro studies with cancer cell lines remain relevant for advancing clinical cancer research because they resemble molecular characteristics and allow analyses on therapy response in drug evaluation. In addition, they are an important tool to perform large-scale drug sensitivity assays and interactions among drugs and genes, which are fundamental in Precision Medicine. These studies are unbiased for exploring alleged factors of drug sensitivity . Cancer TP53, ATR, and FANCA.The analysis performed in the METABRIC and TCGA databases shows that there is a significant presence of young women <50 years of age in both databases. It is observed that different subtypes of BC predominate in each age group, such as basal and luminal A, in patients in the 25–30-year-old group. Remarkably, survival analysis shows that young women between the ages of 25 and 35 have the lowest survival rates. This group includes a 21-year-old woman registered in the METABRIC database. DNA repair genes that are frequently altered in both METABRIC and TCGA include As expected, mutational load analysis shows accumulation of mutations as a function of aging. Unfortunately, there is an underrepresentation of YWBC from LA in the METABRIC (no patients) and TCGA (11 patients) studies to corroborate this assertion. According to Wojtyla et al., BC death rates are not decreasing in LA countries. For women aged 20–49 years, low mortality rates are observed only in Chile and Cuba, while an increase in mortality is observed in the rest of the LA countries . AlthougAnother observation from this study is that the mutational profile of many populations from LA is limited, as concluded by Ren at al. . We obseTP53, since this variant has been correlated with worse overall survival in BC patients in contrast to wild-type [In this study, 27 drugs were identified with effect in six BC cell lines . In addition, according to CIVIC database there are no FDA-approved or NCCN-compendium register treatments especially for patients that carry some of the LA reported variants such as R248Q variant for ild-type ,42.SIK1 and FOSB (tumor suppressors), KDM6B (histone methylation), and NOCT, PER, BHLHe40, and NFIL (circadian rhythm). Conversely, downregulation of GUSBP3 (glucuronidase), some micro-RNAs, and DNA damage repair players like CENPI, POLQ, RAD54B was also observed. The most affected pathways were ATM/FANC  and ATR [Some drugs are being tested in clinical trials, others are still being assessed in preclinical models, particularly in cultured lines. Information for some drugs was either scarce or null. Among these, sepantronium bromide (YM-155) targets the survivin protein inducing anti-apoptotic capacity. Preclinical and phase I clinical trials reported promising results, nonetheless, a poor performance was observed in phase II clinical trials for different cancer types. Wani et al. developed a BC cell line (MCF-7) expressing resistance to YM-155 and observed that continuous treatment with YM155 resulted in a low expression of survivin. This compound confers its chemotherapeutic effect by causing oxidative stress-mediated DNA damage. Therefore, DNA damage-response pathway proteins will be appropriate predictive biomarkers of YM155 response in these cells . Mazzio  and ATR . Moreover, for the acetalax drug, Rajapaske et al. propose repositioning acetalax as an anticancer drug candidate for triple-negative BC based on their cytotoxic and antiproliferative activities in BC cell lines . Similarα combination treatment [A study conducted by Hennessy et al. with AZD5582 showed apoptosis induction in BC MDA-MB-231 cell line at subnanomolar concentrations characterized by cIAP1 degradation. These observations were corroborated in xenograft-bearing mice implanted with MDA-MB-231, in which tumor regression was achieved and molecular analyses confirmed the degradation of cIAP1 this compound is being considered as a candidate for clinical trials . Similarreatment .AZD7762 (DN10764), a selective inhibitor of checkpoint kinases 1 and 2, has been reported as useful to suppress BC metastasis. Park et al. observed that this compound inhibited cell proliferation and GAS6-mediated AXL signaling, resulting in reduced invasion and migration capabilities. It has been shown that this drug promotes caspase 3/7 apoptosis in BC cells. Similar results were observed in in vivo metastasis models. This study suggests that combined therapy strategies targeting AXL, like AZD7763, could improve responses in drug-resistant solid tumors where AXL is involved, such as NSCLC, BC, ovarian cancer, and others . MoreoveAlsamman et al. examined an approach to overcome cisplatin resistance using staurosporine in breast, colon, and ovarian cancer cell lines by evaluating proliferation, morphology, and p62 levels after one of three treatments . Results showed an elevation of p62 levels after cisplatin treatment. On the contrary, a reduction of p62 level was reported after staurosporine treatment. These results propose that cancer cells could be sensitized to cisplatin using staurosporine by downregulation of p62 .p < 0.05) of IC50 and cell viability of cell lines A549 (NSCLC) and MDA-MB-231 (BC) was observed compared with single drug treatments. This observation was corroborated in in vivo studies showing enhanced tumor reduction after vincristine plus doxorubicin therapy [Some combination therapies are studied and used in selected cancer types such as TNBC and NSCLC. These therapies show benefits and promising results, yet the disadvantages of treatments like doxorubicin against TNBC and NSCLC are toxicity and resistance. Studies conducted by Ghosh et al. explored a combined therapy with doxorubicin and vincristine for these cancer types, optimizing a single PEGylated liposomal formulation. A significant reduction , a selective Aurora A Kinase (AAK) inhibitor, shows good antitumor and AAK inhibitor activity in xenograft models consisting of different tumor types. This compound exhibited antitumor activity at an oral dose of 40 mg twice daily plus weekly paclitaxel 60 mg/mexpected . Alisertexpected . It has expected .In a study conducted by Li et al., docetaxel was evaluated in combination with lobaplatin versus docetaxel combined with gemcitabine for treatment in 26 patients with recurrent metastatic BC for each treatment group. Lobaplatin is a dual inhibitor targeting EGFR and HER2 which do not show cross-resistance with cisplatin and this combination is effective in BC. Gemcitabine has also shown certain effects in recurrent metastatic BC. Complete remission was observed in five patients (11.6%), three patients with docetaxel and lobaplatin and two patients with combined therapy of docetaxel and gemcitabine, and partial response in 16 patients (37.2%). Reported response rates of the groups were comparable . Median survival times after relapse and metastasis of the docetaxel and gemcitabine group were 25 months, whereas the docetaxel and lobaplatin group reported 18 months. Similar results were observed for median progression-free survival after relapse and metastasis. Therefore, researchers concluded that these combinations are effective and tolerable for advanced BC . SimilarA study conducted by Lin et al.  in lung ERα/ESR1) and the BCL2 genes [Sinha and collaborators evaluated topotecan effects in MCF-7 BC cells. The study reported that reactive free radical species are key players in cancer cell death. Topotecan significantly downregulates the estrogen receptor alpha  1/2/5/9 inhibitor, has shown encouraging results in preclinical studies and is being evaluated in phase I clinical trials for BC treatment . In a stFurthermore, one limitation of this study is that there are few studies reporting DNA repair variants in YWBC in LA countries, therefore variants representing this population are unknown. Another limitation of this study is that not all the variants reported are totally validated in public databases, some of them are reported as variants of unknown significance (VUS), hence, the effect of these variants needs to be studied. In addition, there are few BC cell lines that represent variants reported in LA.For this study, 90 selected DNA repair genes were analyzed in BC databases such as TCGA, METABRIC, CCLE, and COSMIC Cell Lines. All this information was specific for women under 50 years old (y.o.), tumor sample data was analyzed for METABRIC and TCGA. For Latin American women, germline variants were considered. In addition, drug analyses were studied specifically for BC cell lines that represented DNA repair variants reported in LA YWBC .For this study a selection of 90 genes were analyzed in different databases . For thihttps://www.cbioportal.org/ accessed on 23 March 2021, selecting patients under 50 years old at diagnosis for further analysis. A total of 2509 samples were obtained from METABRIC, for the purpose of this study only samples of patients under 50 years were selected. A total of 567 samples from METABRIC were used to complete further analyses. From the TCGA database, a total of 1084 samples were retrieved. Considering the selection criteria of women under 50 years, 292 samples were chosen for further analyses. Later, a search for 90 DNA repair genes was made to obtain data of frequently altered genes and information concerning age at diagnosis and other clinical data such as BC type and survival from these samples.Data was retrieved from TCGA and METABRIC from https://cancer.sanger.ac.uk/cell_lines/download (last accessed on 21 November 2021), data file was filtered by cancer type, BC and analyzed. For CCLE, https://portals.broadinstitute.org/ccle (last accessed on 21 November 2021), the CCLE_DepMap_18q3_maf_20180718.txt/merged mutation calls  data was analyzed for this study. A total of 227,855 mutations were retrieved from COSMIC BC cell lines. Following, this data was filtered searching for the 90 DNA repair genes selected, to identify all the cell lines harboring mutations in one or more of these genes obtaining 2335 reported mutations in 52 BC cell lines for 84 DNA repair genes. In addition, for CCLE, a total of 35,590 mutations were obtained for BC cell lines. After selecting mutations for the 90 DNA repair genes 274 mutations remained for 74 genes in 65 cell lines. Furthermore, an analysis was performed to identify cell lines that presented frequent DNA repair variants observed in young women with BC. This selection will be useful to implement further in vitro analysis of candidate cell lines with altered DNA repair genes and evaluate drug sensitivity.Data for a panel of 52 human BC cell lines was obtained from The Catalog of Somatic Mutations in Cancer (COSMIC) Cell Line Project database, and 69 BC cell lines from The Cancer Cell Line Encyclopedia (CCLE). In http://www.dgidb.org/search_interactions, last accessed on 21 November 2021) [https://www.cancerrxgene.org/, last accessed on 21 November 2021), a database to find drug response and genomic markers of sensitivity. In addition, Clinical Interpretation of Variants in Cancer (CIVIC)  and Drugbank  databases were used to gather supporting data for reported variants with drug sensitivity in previously mentioned databases. The Drugbank database is a bioinformatics resource that provides drug data combined with target information.Data from databases was analyzed aiming to spot drug sensitivity, half maximal inhibitory concentration (IC50), in BC cell lines with altered DNA repair genes. Information for drug–gene interaction was obtained from The Drug Interaction DataBase (DGIdb) (er 2021)  and The A comparative analysis took place between frequent reported variants observed in YWBC from LA countries ,78 and rThere is vast information supporting the participation of DNA repair genes in BC and their possible implication in predisposition, development, outcome, and therapy response. Even though there is a wide range of publicly databases and studies addressing DNA repair variants, the representation for populations such as the Latin-American is minor and in contrast with other regions of the world reported data is limited. For the METABRIC and TCGA study there was an underrepresentation of YWBC from LA countries. Added to that, BC cell lines do not represent frequent DNA repair variants reported in countries from our region. Therefore, there is an opportunity area to address this matter.BRCA1 and TP53 genes. In addition, from the 15 DNA repair genes identified in young women <50 y.o. in LA, there are no BC cell lines that carry variants for 13 of those genes. Therefore, it represents a challenge to study these variants reported in the LA region and their research focusing on preliminary drug assays.The use of cell lines is fundamental for drug analysis, along with cell lines databases for preliminary studies. Herein, comparing LA-reported DNA repair variants from women <50 y.o. with BC cell lines resulted in a minor representation of these variants observing only seven BC cell lines for Of our selection of 90 genes participating in DNA repair mechanisms, no representativeness of variants reported in LA was observed for YWBC <50 y.o., compared to variants from other populations.Cell line database analyses resulted in few variants representation of YWBC from LA countries in BC cell lines.BRCA1, BRCA2, and TP53 to generate cell lines that represent variants for this population.More studies for LA population are needed to approach DNA repair genes and mechanisms for YWBC besides Our analyses resulted in different candidate drugs, nevertheless, not all of these drugs are validated in clinical practice.