KIR genes, which vary by presence and copy number, KIR3DL3 is ubiquitously present in every individual across diverse populations. No ligand or function is known for KIR3DL3, but limited knowledge of expression suggests involvement in reproduction, likely during placentation. With 157 human alleles, KIR3DL3 is also highly polymorphic and we show heterozygosity exceeds that of HLA-B in many populations. The external domains of catarrhine primate KIR3DL3 evolved as a conserved lineage distinct from other KIR. Accordingly, and in contrast to other KIR, we show the focus of natural selection does not correspond exclusively to known ligand binding sites. Instead, a strong signal for diversifying selection occurs in the D1 Ig domain at a site involved in receptor aggregation, which we show is polymorphic in humans worldwide, suggesting differential ability for receptor aggregation. Meanwhile in the cytoplasmic tail, the first of two inhibitory tyrosine motifs (ITIM) is conserved, whereas independent genomic events have mutated the second ITIM of KIR3DL3 alleles in all great apes. Together, these findings suggest that KIR3DL3 binds a conserved ligand, and a function requiring both receptor aggregation and inhibitory signal attenuation. In this model KIR3DL3 resembles other NK cell inhibitory receptors having only one ITIM, which interact with bivalent downstream signaling proteins through dimerization. Due to the extensive conservation across species, selection, and other unusual properties, we consider elucidating the ligand and function of KIR3DL3 to be a pressing question.Natural killer (NK) cell functions are modulated by polymorphic killer cell immunoglobulin-like receptors (KIR). Among 13 human  KIR genomic locus, in humans comprised of 13 KIR genes and two pseudogenes, exemplifies extreme structural, and allelic polymorphism  educate and modulate the cytotoxic and inflammatory activity of natural killer (NK) cells, which target infected and cancerous tissues –3. KIR amorphism , 7. CreaIR genes –10. SuggIR locus . BecauseKIR3DL3 is the only KIR gene present in every individual   identified in humans, and a further 45 identified in total from other catarrhine species (hominoids and old-world monkeys)  –20 as we gibbon) –24. In eiversity . KIR3DL3monkeys) , 25–29.  protein . TogetheKIR genes are organized with close correspondence between exons and the encoded functional domains , the first ITIM is conserved (VTYAQL) across all catarrhine KIR3DL3 molecules , likely indicating this Ig domain is evolving under positive selection that favors diversity  were common to all populations analyzed and a further four  were each found in seven of the eight populations. Indicating an uneven distribution of KIR3DL3 allotypes, the most frequent allotype detected was different for every population. Thus, KIR3DL3*001 is the most frequent allele in Europeans, *003 in South American Yucpa, *010 in Chinese Han, *014 in Papua New Guineans, and *038 in Khomani South Africans  release 2.7.1 (Pan troglodytes (Patr) and Pan paniscus (Papa) chimpanzee (Gorilla gorilla (Gogo) Gorilla (Pongo pygmaeus: Popy) and Sumatran (Pongo abelii: Poab) orangutans (Hoolock hoolock leuconedys: Hoho) and northern white cheeked (Nomascus leucogenys: Nole) gibbons (Macaca mulatta: Mamu) and Crab-eating (Macaca fascicularis:Mafa) macaques, African green monkey (Chlorocebus aethiops: Chae), Black-and-white Colobus monkey (Colobus guereza: cogu), Olive baboon (Papio anubis: paan) (Piliocolobus tephrosceles: Pite)  coverage Denisovan genome  as distinct alleles. Other sequence differences are discussed where relevant. Sequences corresponding to all known CDS alleles of human se 2.7.1 , and froimpanzee , Gorilla Gorilla , Borneanangutans , 18, 91, gibbons , Rhesus s: paan) , and Reds: Pite) . The seqs: Pite) ; all areKIR3DL20 . Archaicn genome  using thn genome  and adapKIR3DL3 CDS alleles obtained from the IPD, we included nine identified from southern African KhoeSan  and Papua New Guinean (N = 200) subjects, using high throughput sequencing  method . Each do*001 Ig domains was generated by homology modeling using Swissmodel, https://swissmodel.expasy.org/ . Human Genome Diversity Panel (HGDP) data  were extracted using PLINK  data  was obtang PLINK . For popPN and LL conceived and designed the experiments. LL, PN, JS, KK, ST, and CG analyzed data. KK, NN-G, and ST performed lab experiments. ZD and LG provided new data. JH, AM, SO, MC, and PP provided materials. PN and LL wrote the paper, and all authors approved the final submitted version.CG owns stock in 23andMe, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multiple myeloma (MM) is a bone marrow plasma cell neoplasm and is the second most-common hematologic malignancy. Despite advances in therapy, MM remains largely incurable. Elotuzumab is a humanized IgG1 monoclonal antibody targeting SLAMF7, which is highly expressed on myeloma cells, and the antibody is approved for the treatment of relapsed and/or refractory (RR) MM in combination with lenalidomide and dexamethasone. Elotuzumab can stimulate robust antibody-dependent cellular cytotoxicity (ADCC) through engaging with FcγRIIIA (CD16) on NK cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages. Interestingly, SLAMF7 is also expressed on cytolytic NK cells, which also express the requisite adaptor protein, EAT-2, to mediate activation signaling. Accumulating evidence indicates that antibody crosslinking of SLAMF7 on human and mouse NK cells can stimulate EAT-2-dependent activation of PLCγ, ERK, and intracellular calcium mobilization. The binding of SLAMF7 by elotuzumab can directly induce signal transduction in human NK cells, including co-stimulation of the calcium signaling triggered through other surface receptors, such as NKp46 and NKG2D. In RRMM patients, elotuzumab monotherapy did not produce objective responses, but did enhance the activity of approved standard of care therapies, including lenalidomide or bortezomib, which are known to enhance anti-tumor responses by NK cells. Taken together, these preclinical results and accumulating experience in the clinic provide compelling evidence that the mechanism of action of elotuzumab in MM patients involves the activation of NK cells through both CD16-mediated ADCC and direct co-stimulation via engagement with SLAMF7, as well as promoting ADCP by macrophages. We review the current understanding of how elotuzumab utilizes multiple mechanisms to facilitate immune-mediated attack of myeloma cells, as well as outline goals for future research. Multiple myeloma (MM) is a deadly hematopoietic cancer characterized by the expansion of monotypic plasma cells in the bone marrow, accumulation of monoclonal immunoglobulin in the serum, and end-organ damage such as anemia, lytic bone lesions, and renal dysfunction . It is eA variety of recently-developed therapies have contributed to the extended survival of MM patients, including proteasome inhibitors , immunomodulatory imide drugs , and monoclonal antibodies (daratumumab and elotuzumab). Clinical results with these therapies have been previously summarized in a variety of quality reviews –6 and wiHere, we will review current understanding of the mechanisms by which elotuzumab promotes immune responses toward MM, especially through facilitating NK cell-mediated anti-tumor activity. We further summarize clinical results from the use of elotuzumab in combination immunotherapies and discuss how the immune potentiating mechanisms may be contributing to anti-tumor responses in patients. While the CD38 targeting antibody, daratumumab, shares some mechanistic attributes with elotuzumab, we will only touch upon some aspects of the effects of daratumumab, in view of recently published reviews on the topic , 10.NK cells are believed to play important roles in immune surveillance of cancer, limiting neoplastic progression, and effectors of anti-tumor therapies , 12. TheKIR genes expressed by donor NK cells  was found highly expressed on human plasma cells and corresponding myeloma cells , 19. WhiMF7 mRNA . High exMF7 mRNA , most myMF7 mRNA . Tai et  samples . Interes samples . A recen samples . This reElotuzumab  is a humanized IgG1 monoclonal antibody that was developed to target SLAMF7. HuLuc63 was originally engineered by PDL BioPharma as a humanized version of the SLAMF7 monoclonal antibody, MuLuc63, which was originally generated in BALB/c mice , 19. HuLin vitro and in vivo  of myeloma cells by NK cells both  in vivo , 19, 38. in vivo . HuLuc63 in vivo . Further in vivo , 19, 39.in vitro studies also found minimal loss of NK cells in PBMC treated with Elo, indicating that the antibody does not induce significant NK cell fratricide upon binding to SLAMF7 on NK cells themselves. Treatment of whole blood overnight with 100 or 200 μg/ml HuLuc63 resulted in a loss of only 20% of NK cells -human mouse xenograft model engrafted with primary myeloma cells . TreatmeNK cells . This inNK cells , 30, 43.NK cells . The groNK cells .in vitro and in vivo effect of Elo in combination with lenalidomide on NK cell activation, cytokine production and myeloma cell death  in xenograft mouse models using immunocompetent mice and syngeneic mouse tumors expressing human SLAMF7  vs. Bor/Dex alone (75 patients) for RRMM was also reported by Jakubowiak et al. . At 1 ye+ myeloma cells and consistently achieved Elo serum concentration of more than 70 ug/ml in patients, with peak concentrations of up to 1 mg/ml in serum  was carried out by Lonial et al. . In thatin serum .dimCD16+ NK cells exhibiting higher expression of the adhesion molecule, CD54 (ICAM-1), and concomitant reduction in CD45dimCD138+ myeloma cells at cycle 1 day 22, as compared to baseline  comparing treatment of 321 RRMM patients with 10 mg/kg Elo/Len/Dex and a control arm of 325 patients treated with Len/Dex alone . At 1 yeIn addition to the combined use of Elo with Lenalidomide and dexamethasone, clinical trials are underway combining Elo with related IMiD drugs, thalidomide and pomalidomide. The combination of Elo with thalidomide/dexamethasone was shown to be safe and effective in a phase II study by Mateos et al. . Furtherdim NK cells, the majority of CD56bright NK cells, many CD56+ T cells, mature dendritic cells, and small subsets of CD4+ T cells and B cells (+ monocytes (lowCD16+) and a fraction of intermediate (CD14+CD16+) monocytes (SLAMF7 (CD319) was originally discovered as a CD2-related receptor by the labs of Marco Colonna, who called it CRACC (CD2-like receptor activating cytotoxic cells) , and Por B cells , 39, 57. B cells . B cells B cells , 72. Whionocytes , 57, a monocytes .304STV) is within an immunoreceptor tyrosine-based switch motifs  and another that is embedded in a similar ITSM-like sequence (TEY284DTI). ITSM sequences are also found in most SLAM family receptors, and have capacity to switch between activating and inhibitory signaling (SH2D1A) or EWS-FLI1 activated transcript 2    to media SH2D1B) . NK cell SH2D1B) . It is c+ target cells , SLAMF7-Ig fusion protein, or exposure to SLAMF7et cells , 60, 61.et cells , 62. Enget cells . In contet cells , indicat+ T, CD8+ T, or dendritic cells -304 within the ITSM , 62. Altic cells . Interesnd EAT-2 , 76, indnd EAT-2 .EAT-2 is a small cytosolic adaptor protein that consists of an SH2 domain, which binds to Y304 on SLAMF7, and a short C-terminal sequence including a tyrosine at position 127 (Y127), which is required for SLAMF7 activating function . When EA+ target cells or have unique responsiveness to Elo compared to conventional NK cells.SLAMF7 has also been observed to exhibit some inhibitory function in NK cells from EAT-2-deficient mice or the human NK cell line, YT-S, in the absence of EAT-2 . TherefoImportantly, plasma cells and myeloma cells express high levels of SLAMF7, but lack expression of EAT-2, thereby compromising their activation signaling capacity through SLAMF7 , 67. ThiRecent intriguing work by Chen et al. established a novel mechanism for SLAMF7 signaling through physical association with the integrin receptor CD11b/CD18 (Mac-1) in the plasma membrane on the surface of macrophages . This grdim subset of NK cells, as well as intermediate and non-classical monocytes and macrophages, and can trigger potent intracellular signaling, including tyrosine phosphorylation and calcium mobilization, through physical association with the transmembrane adaptor proteins TCR-ζ and FcR-γ, which contain immunoreceptor tyrosine-based activation motifs (ITAM)  as a transmembrane receptor, designated FcγRIIIA, or (2) as a glycophosphatidylinositol-linked surface receptor, called FcγRIIIB , 79. Fcγs (ITAM) .in vitro and in vivo anti-tumor activity of Elo is mediated primarily by NK cells in a CD16-dependent manner. In vitro Elo begins to stimulate ADCC responses at concentration around 0.1 ng/ml, with peak responses in the range of 100 ng/ml  required the Fc domain on Elo, characteristic of ADCC responses  or degranulation (2 h assay) was detected on NK cells when PBMC were incubated with F(ab')2 Elo or an Fc mutant form of Elo that they showed to be incapable of binding to recombinant CD16  in vitro . Elo strin vitro . Pazina ant CD16 . Given tant CD16 . Pazina ant CD16 , Pazina ant CD16 .Despite defining the clear importance of the ADCC response, Pazina et al. also detected a novel co-stimulatory signaling effect that resulted when Elo engaged with SLAMF7 on the NK cell surface . They foin vitro ADCP capacity toward myeloma cells in the presence of Elo  in an Fcγ receptor-dependent manner . Elo sige of Elo .+ T cells were depleted in combination with Elo, thereby further implicating a cooperative role for cytotoxic T cells in the anti-tumor function of Elo , which markets elotuzumab. KC and AC have served as consultants and on scientific advisory boards for BMS.


TGFBR1/2, TGFB2/3, SMAD2/3), encoding components of the TGF-β pathway, cause LDS. Such genetic heterogeneity mirrors broad phenotypic variability with significant differences, especially in terms of the age of onset, penetrance, and severity of life-threatening vascular manifestations and multiorgan involvement, indicating the need to obtain genotype-to-phenotype correlations for personalized management and counseling. Herein, we report on a cohort of 34 LDS patients from 24 families all receiving a molecular diagnosis. Fifteen variants were novel, affecting the TGFBR1 (6), TGFBR2 (6), SMAD3 (2), and TGFB2 (1) genes. Clinical features were scored for each distinct gene and matched with literature data to strengthen genotype-phenotype correlations such as more severe vascular manifestations in TGFBR1/2-related LDS. Additional features included spontaneous pneumothorax in SMAD3-related LDS and cervical spine instability in TGFB2-related LDS. Our study broadens the clinical and molecular spectrum of LDS and indicates that a phenotypic continuum emerges as more patients are described, although genotype-phenotype correlations may still contribute to clinical management.Loeys-Dietz syndrome (LDS) is a connective tissue disorder first described in 2005 featuring aortic/arterial aneurysms, dissections, and tortuosity associated with craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations. Heterozygous mutations in 6 genes ( TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2, and TGFB3, which alter the physiological development and function of the extracellular matrix, leading to cardiovascular and multisystem abnormalities [TGFBR1 and TGFBR2) were firstly identified and an initial clinical classification of LDS type I and type II was proposed based on the presence of typical craniofacial features in LDS type I [SMAD3 displaying high frequency of osteoarthritis, prompted some authors to define the resulting phenotype as “aneurysms-osteoarthritis syndrome” or LDS type III [BGN)-associated aortic aneurysm syndrome. Additionally, in some cases further genetic testing may be indicated for the differential diagnosis with other hereditary thoracic aortic disorders caused by pathogenic variants in distinct genes including NOTCH1, ACTA2, MYH11, MYLK, PRKG1, MAT2A, FOXE3, MFAP5, and LOX [Loeys–Dietz syndrome (LDS) is a rare hereditary connective tissue disorder (HCTD) with an autosomal dominant inheritance characterized by a widespread systemic involvement. The disorder was first described in 2005 and in its most typical presentation it features aortic/arterial aneurysms and/or dissections, as well as arterial tortuosity in association with variable craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations ,2. LDS itype III . McCarri and LOX . Herein,i) the Ehlers-Danlos Syndrome and Inherited Connective Tissue Disorders Clinic at the University Hospital Spedali Civili of Brescia, (ii) the Medical Genetics Unit of the Sant’Orsola-Malpighi Hospital of Bologna, (iii) the Clinical Genetics Unit of the Regional Hospital of Bolzano, (iv) the Centre of Expertise for Marfan syndrome and Marfan-related disorders at Policlinico Tor Vergata University Hospital of Rome and (v) the Medical Genetics Unit, Department of Life, Health, and Environmental Sciences of the University of L’Aquila. Twenty-four index patients and 10 relatives with LDS, mostly of Italian origin but two from Philippines and Sri Lanka, were evaluated from 2010 to 2018 in specialized outpatient clinics for the diagnosis of HCTDs, namely: , TGFBR2 , SMAD3 , and TGFB2  were PCR amplified by using optimized genomic primers (TGFBR2 (TGFBRex2-forw: 5′-GTGGCTGTATGGTAAGAGA-3′ and TGFBR2ex4-rev: 5′-CCAGGTTGAACTCAGCTTCTG-3′) and exons 6-8 of SMAD3 (SMAD3ex6-forw: 5′-CCTAGGGCTGCTCTCCAATG-3′ and SMAD3ex8-rev: 5′-GTGCACATTCGGGTCAACTG-3′) was performed, respectively, and followed by Sanger sequencing of the RT-PCR products. All identified variants were submitted to the Leiden Open Variation Database (LOVD) [Molecular analyses were performed in the laboratory of genetic testing at the Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia. Mutational screening was achieved on genomic DNA purified from peripheral blood leukocytes of affected and unaffected family members by standard procedures. All exons and their intron-flanking regions of The medical records of 34 patients with a molecularly proven diagnosis of LDS were accessed and their phenotypes were defined using the Human Phenotype Ontology (HPO) terms by a careful review of clinical notes. Phenotypic categories were created based on the number of observations in the patient’s cohort and their relationship to LDS. In reporting the frequency of clinical features of our case series, the used denominator refers to the number of subjects for which information on a specific feature was available . We reviThis study was approved by the relevant Ethical Authorities of the: Policlinico Tor Vergata University Hospital ; University of L’Aquila, Medical Genetics Section, Department of Life, Health, and Environmental Sciences ; and local Ethical Committees. This study was performed in agreement with the principles of the 1964 Helsinki Declaration. All subjects  enrolled into the genetic study provided written informed consent.We report the clinical and genetic features of 34 individuals from 24 families. The female-to-male ratio of affected probands was 1/1. The mean age at diagnosis of the 24 unrelated probands was 26 years (range 1–51 years). Thirteen of them had a positive family history, while 11 were sporadic. Ten individuals were diagnosed upon familial segregation. The detailed clinical and molecular features of each affected individual are summarized in TGFBR1 (9/24) and TGFBR2 (10/24) genes, four families showed variants in SMAD3, and 1 patient harbored a TGFB2 variant. No variants were identified in SMAD2 and TGFB3. Fifteen variants affecting TGFBR1, TGFBR2, SMAD3, and TGFB2 were novel, while nine in TGFBR1, TGFBR2, SMAD3 were previously reported either in the literature or in the ClinVar database  located in critical and well-established functional domains without benign variation; (ii) absent in publicly available population databases, (iii) predicted as deleterious my multiple lines of computational evidence and based on the matching of the clinical phenotypes, all these variants were classified as pathogenic (class 5) according to the American College of Medical Genetics and Genomics (ACMG) guidelines [Among novel variants, 10 missense substitutions and one in-frame deletion of two highly conserved amino acid residues p.(Lys232_Ile233del) were evaluated for their putative pathogenicity through different idelines . TGFB2 was classified as pathogenic (class 5), since it leads to frameshift and formation of a premature termination codon p.(Phe160Leufs*14) that likely activates nonsense-mediated mRNA decay (NMD). Likewise, the novel c.480del variant in TGFBR2  was classified as likely pathogenic (class 4) according to the ACMG, since it creates a new splice donor site 6 bases downstream of the wild-type donor with retention of 4 nucleotides of intron 3, formation of a stop codon (p.Arg114*) and activation of NMD. The c.1009+1G>A variant in SMAD3 was classified as pathogenic (class 5), since it abolishes the canonical splice donor site of exon 7 causing in-frame exon skipping (p.Arg292_Gly337del). Lastly, although RNA was not available to investigate its effect, the c.862_871+8del pathogenic variant (class 5) variant in SMAD3, thought formally a frameshift variant, likely leads to abnormal splicing as well , as the canonical splice donor site is lost.For two out of three variants affecting splice sites, we obtained fresh blood samples and demonstrated an effect on the splicing process, corroborating their pathogenicity. In particular, the c.263+6C>T variant in TGFBR1 (MIM #190181) [TGFBR2 (MIM#190182) [SMAD2 (MIM #601366) [SMAD3 (MIM #603109) [TGFB2 (MIM #190220) [TGFB3 (MIM#190230) [SMAD2 to place this form (LDS6) in this spectrum [TGFBR1/2 may give rise to a phenotypic continuum hard to categorize in clear-cut genotype-phenotype correlations [Since the relatively recent description of LDS in 2005, six disease-causative genes have been identified, namely #601366) , SMAD3 (#603109) , TGFB2 (#190220) , and TGF#190230) . This adTGFBR1/2, SMAD3, and TGFB2 patients  and TGFBR2 (10/24) genes. While nearly all mutated patients displayed ascending/aortic root aneurysm, severe arterial involvement was recorded in half. Early aortic dissection was more rarely reported together with arterial tortuosity. Interestingly, we noticed a high number of different arteries being affected. These findings highlight the need for extended arterial imaging, as was already recommended [TGFBR2 patients. As previously described, craniofacial features may be considered to be characteristics of TGFBR1/2-LDS including dolichocephaly, hypertelorism, malar hypoplasia and highly arched palate, while abnormal palate/bifid uvula was not as frequently found as described in the literature. Recurrent skeletal anomalies included scoliosis, pes planus, long slender fingers, marfanoid habitus, and pectus deformity. Hernias were of different types  and together with joint laxity were registered in half of patients. Cutaneous findings appeared extremely frequent including translucent skin, easy bruising and striae; together with facial milia, described in half mutated patients, skin features appeared as extremely useful handle for diagnosis [TGFBR1/2 pathogenic variants, as previously assessed [According to previous studies, most of the identified mutations affected ommended . Valve aiagnosis . Overallassessed . Also, iassessed ,6. Finalassessed .SMAD3-mutated families, early osteoarthritis, considered a hallmark of this subset of patients, was described only in one, although it segregated in the proband and his father. In line with our data, despite early studies reported osteoarthritis in all SMAD3 patients [SMAD3-LDS patients displayed a less severe degree of cardiovascular complications. In fact, despite all the presence of thoracic aortic  aneurysms, dissection was reported only once, as well as arterial tortuosity and varices. Additional clinical findings overlapped with those recorded in other LDS patients, including recurrent craniofacial dysmorphism such as hypertelorism, malar hypoplasia, micrognathia and highly arched palate, cleft palate and/or uvula, cutaneous features with translucent skin, striae and facial milia, hernias and skeletal manifestation like pes planus, pectus deformity, marfanoid habitus, and scoliosis. It is worth noting that one subject had spontaneous pneumothorax, recorded in a single SMAD3 patient in the literature [Among our three patients , others patients . Interesterature ). In parTGFB2 pathogenic variant, whose cardiovascular features consisted in mitral valve prolapse and insufficiency, mild arterial tortuosity and varicose veins, in the absence of aneurysms. Additional features included bifid uvula, striae, translucent and hyperextensible skin, facial milia, hernias, joint hypermobility, pectus deformity, scoliosis, and pes planus observed in other LDS genetic subtypes. In line with previous observations, this patient presented the mildest end of LDS phenotypic spectrum [TGFB2-LDS [TGFB2, although it has been commonly encountered in LDS [We identified only one patient with a spectrum . HoweverGFB2-LDS . Furtherd in LDS . Interesd in LDS . These dSMAD2 and TGFB3. Still, limited numbers are available in literature about genotype-phenotype correlation. To date, 9 (likely) pathogenic variants in SMAD2 have now been described in 15 subjects displaying a broad range of features, including aneurysms, tortuosity of the entire arterial tree, and coronary artery dissections, even in the absence of prominent connective tissue characteristics [TGFB3, 15 different variants were reported in 56 individuals presenting with phenotypic overlap between LDS and MFS [No mutations were identified in the latest described LDS genes, namely eristics . Concern and MFS . In this work, we broadened the clinical and molecular spectrum of LDS, corroborated, and expanded previously delineated genotype-phenotype correlations, paving the way for a gene-based classification of different disease subtypes. Larger cohort screenings are needed to unravel the thorough clinical and molecular repertoires in LDS to accurately establish diagnostic criteria, define genotype-phenotype correlations, and collect natural history data for clinical prognostication.


To dissect its molecular mechanism, we created a double recessive mutant line, o2o2o16o16, by introgression of the o2 and o16 alleles into the wild-type maize inbred line, by molecular marker-assisted backcross selection. The kernels (18 day after pollination (DAP), 28 DAP, and 38 DAP) of the o2o2o16o16 mutant and its parent lines were subject to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed that 59 differentially expressed genes (DEGs) were involved in lysine metabolism and 43 DEGs were involved in tryptophan metabolism. Among them, the genes encoding AK, ASADH, and Dap-F in the lysine synthesis pathway were upregulated at different stages of endosperm development, promoting the synthesis of lysine. Meanwhile, the genes encoding LKR/SDH and L-PO in the lysine degradation pathway were downregulated, inhibiting the degradation of lysine. Moreover, the genes encoding TAA and YUC in the tryptophan metabolic pathway were downregulated, restraining the degradation of tryptophan. Thus, pyramiding o2 and o16 alleles could increase the lysine and tryptophan content in maize. These above results would help to uncover the molecular mechanisms involved in the increase in lysine and the tryptophan content, through the introgression of o2 and o16 alleles into the wild-type maize.In maize, pyramiding of  Zea may L.) is the main feed and food crop, worldwide, with its quality directly affecting the healthy development and edibility by human beings, livestock, and poultry. The nutritional quality of corn grains generally refers to the nutritional components in corn grain, including protein, fat, starch, and various vitamins, as well as mineral element contents. Among them, lysine, from proteins, is a necessary amino acid for humans and monogastric animals. The content of lysine in wild-type maize is low (less than 0.3%) and cannot meet the nutritional quality requirements for human consumption and food processing (more than 0.5%). Furthermore, it cannot even meet the requirements for the content of lysine in livestock and poultry feed (0.6–0.8%) [Corn (.6–0.8%) .opaque1 (o1) and opaque2 (o2) in maize. Mertz et al. found that the mutation of o2 could significantly increase the content of lysine in the endosperm of maize seeds [O2 gene, located on chromosome 7, encodes the transcription factors of the basic leucine zipper family, recognizes the promoter of the zein gene through the O2 box (TCCACGT) and regulates the expression of 22 kDa α-zein and 15 kDa β-zein genes [floury1 (fl1) [floury2 (fl2) [floury3 (fl3) [opaque4 (o4) [opaque5 (o5) [opaque6 (o6) [opaque7 (o7) [opaque15 (o15) [proline1 (pro1) [De*-B30 [Mc [Mu transposon technology to create a new high-lysine mutation, opaque16 (o16), which has a lysine content of 0.36%. This gene is located on the long arm of chromosome 8, between molecular markers umc1141 and umc1121 [Singleton and Jones first discovered the mutation of in genes ,4. Folloy2 (fl2) , and floue4 (o4) ,9, opaquue5 (o5) ,11, opaque6 (o6) ,13, opaque7 (o7) ,15,16, o15 (o15) , proline1 (pro1) , De*-B30[De*-B30 , and Mc -B30 [Mc , amongst umc1121 .o16 and o2 genes were pyramided by a marker-assisted selection (MAS) using Qi205 (o2o2) and QCL3021 (o16o16) as parents. The content of lysine in the grain increased by 6% and 30%, compared with that of the o2 parent and o16 parent, respectively [o2o2) as the recurrent parent and QCL3021 (o16o16) as the donor parent. Marker-assisted backcross selection (MABS) were then used to introgress the o16 gene into the o2 lines, which allowed us to obtain 17 families, with increases in lysine content of 22.33% and 65.86%, compared with Tai 19 (o2o2) and QCL3021 (o16o16), respectively [o2 and o16 genes into different recurrent parents. Compared with the recurrent parents, the content of lysine and tryptophan increased by 76% and 91%, respectively [To further improve the nutritional quality of maize, the ectively . Therefoo2 gene into wild-type maize with different genetic backgrounds could cause varying degrees of changes in transcription patterns [o2 gene introgression into waxy corn could not only cause varying degrees of physiological, biochemical, and proteomic changes, but could also change the expression of endosperm proteins, amino acids, biosynthesis, starch, stress response, and signal transduction [O2 gene-regulated multiple metabolic pathways related to biological process and molecular function during waxy maize endosperm development—Zm00001d029385.1 and Zm00001d012262.1—which encode the EF-1α and LHT1, were upregulated, but the gene that encodes sulfur-rich proteins was downregulated in the o2o2wxwx lines, raising the waxy grain lysine content [o2 and o7 genes could affect amino acid metabolism; carbon metabolism; storage protein synthesis, transcription and translation; and signal transduction [o2 and o16 genes, and what the underlying molecular mechanisms are that result in the increase in lysine.The introgression of patterns . The o2 sduction . The O2 sduction . Howevero2o2o16o16), the o2 and o16 genes were introgressed into wild-type maize lines by MABS. The kernels of the o2o2o16o16 line and their parents  were subject to RNA sequencing (RNA-Seq). The transcriptional differences between the double recessive mutants (o2o2o16o16) and the wild-type parent were compared. The results provided a foundation for studying the underlying molecular mechanisms that underlay the improvement in grain nutrient quality through the introgression of the o2 and o16 genes.To dissect the molecular mechanisms behind the improved quality of the maize double recessive mutant line (o2o2o16o16) bred by our research group. Using CML530 as a recurrent parent and QCL8003_2 as a donor parent, the o2o2o16o16 mutant was converted to the CML530 background, through two backcrossing cycles, using MABS. Co-dominant SSR markers phi112 and umc1141, were used to select heterozygous (O2o2O16o16) individuals at BC1F1 and BC2F1, while selecting homozygous (o2o2o16o16) individuals at BC2F2, which was followed by several rounds of self-pollination. The bred o2o2o16o16 mutant line was named QCL8011_2. According to the 55 K SNP microarray analysis, the recovery rate of the genomic genetic background of QCL8011_2 (o2o2o16o16) was 92.62% to QCL530, which was higher than the theoretical background recovery rate (87.50%). CML530 is a maize inbred line  from CIMMYT. QCL8003_2 is a double mutant line (o2o2o16o16) in the CML530 background, were grown in an experimental field in the Guizhou Academy of Agricultural Sciences  (N26°30′14″ and E106°39′21″) during spring 2017. The maize inbred line CML530 and the endosperm mutant line QCL8011_2  and CML530 (WT) were again sampled at 28 DAP and 38 DAP. A minimum of five well-filled kernels for each genotype were sampled at 18 DAP, 28 DAP, and 38 DAP, and then immediately frozen in liquid nitrogen for RNA extraction.Generally, kernels are sampled at 18 DAP for RNA-Seq of maize endosperm, such as that done by Zhou et al. . In ordeMature kernels were harvested after physiological maturity and dried in a drying room. To minimize the effect of biological variation between ears, equal numbers of four well-filled ears were pooled and treated as one sample for nutritional evaluation.The mature and dry kernels of QCL8011_2 and CML530 were selected to observe the ear and kernel under natural light, while the transparency of the grain was observed on the light box. The kernel was cross cut by a blade and the farinaceous and keratinous degree of kernel was observed under natural light and photographed.After the peel was removed from the mature and dry kernels, a blade was used to cut the endosperm from the middle and a small piece of endosperm was taken. An ion sputtering instrument  was used to embed platinum, which was subsequently observed under scanning electron microscopy (SEM)  in the Guizhou Key Laboratory of Agricultural Biotechnology.2O. Each sample was then filtered through the membrane (0.45 μm) into the liquid chromatographic sample bottle (2 mL) and used for determination of the contents of 17 free amino acids (FAAs), which were aspartate (Asp), threonine (Thr), serine (Ser), glutamate (Glu), glycine (Gly), alanine (Ala), cysteine (Cys), valine , methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys), arginine (Arg), and proline (Pro).Amino acid contents of mature maize grains were analyzed using an automatic amino acid analyzer . Approximately 120–150 mg of the dried powder of each sample was weighed and transferred to a glass tube with a cover (20 mL), before 5 mL of HCL (6 M) was added. Each tube was incubated at 110 °C, in a water bath for 24 h. After this was cooled to room temperature, the pH was adjusted to 2.0 and the solution was diluted to a volume to 100 mL using ddHTryptophan (Trp) content was measured using a spectrophotometer  by reading the absorbance at 590 nm, according to the national standard (GB7650-87 of China). Three replicates were performed for each sample.The total RNA of the whole kernels from 18 DAP, 28 DAP, and 38 DAP was isolated using a plant RNA kit (OMEGA), according to manufacturer’s protocol. The concentration and quality of each RNA sample was checked using an Agilent 2100 Bioanalyzer . The first step in the workflow involves purifying the poly(A)-containing mRNA molecules using poly(T) oligo-attached magnetic beads. After purification, the mRNA was fragmented into small pieces, using divalent cations at a high temperature. The cleaved RNA fragments were used in the synthesis of first strand cDNA, using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. A single ‘A’ base was then added to these cDNA fragments, followed by subsequent ligation of the adapter. The products were then purified and enriched using PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle, by rolling circle replication (RCR), to enlarge the fluorescent signals during the sequencing process. The DNBs were loaded into the patterned nanoarrays and they were read using a single-end read of 50 bp on the BGISEQ-500 platform, for the following data analysis study. For this step, the BGISEQ-500 platform combined the DNA nanoball-based nanoarrays and stepwise sequencing, using the combinatorial probe-anchor synthesis sequencing method. Transcriptome sequencing was carried out on a BGISEQ-500 sequencing platform by Shenzhen Huada Gene Technology Co. Two replicates of each sample were sequenced.https://github.com/BGI-flexlab/SOAPnuke) [http://bowtie-bio.sourceforge.net/Bowtie2/index.shtml) [http://deweylab.biostat.wisc.edu/RSEM) [p ≤ 0.05.In this study, low-quality reads and reads containing adapters or having an unknown base percentage >10% were removed from the original sequencing data, using SOAPnuke soft  . The resOAPnuke)  using HIOAPnuke)  and aligx.shtml) , to calcdu/RSEM) . NOISeq2du/RSEM)  was usedhttp://www.geneontology.org/), calculating gene numbers for every term, before using the hypergeometric test to find significantly enriched GO terms in the input list of DEGs. In this study, the calculated p values were subjected to the Bonferroni correction [p ≤ 0.05. Typically, functions with FDR ≤ 0.01 were considered to indicate significant enrichment.Gene Ontology (GO) enrichment analysis provided all GO terms that were significantly enriched in a list of DEGs compared to a genome background, and filtered the DEGs that corresponded to specific biological functions. This method firstly mapped all DEGs to the GO terms in the database . KEGG pathway enrichment was calculated in the same way as the GO functional enrichment analysis [p value ≤ 0.05 and FDR ≤ 0.01 were considered to be significantly enriched in DEGs.Pathway enrichment analysis of DEGs was performed with the guidance of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . Total RNA used for RNA-Seq was reversely transcribed into cDNA, using a Thermo Scientific RevertAid First Stand cDNA Synthesis . Quantitative analysis was conducted using CFX Connect Real-Time PCR System , according to the method used by Liu et al. [−ΔΔCT method was used to calculate relative changes in gene expression.A total of 28 DEGs were selected for qRT-PCR verification at 18 DAP, 28 DAP, and 38 DAP. The primers, shown in u et al. . Three rThe raw data of RNA-Seq reads were deposited in the National Center for Biotechnology Information (NCBI) database, under the accession number PRJNA 517535. Biosample accessions were as follows: SAMN10836467, SAMN10836468, SAMN10836469, SAMN10836470, SAMN10836471, SAMN10836472, SAMN10836473, SAMN10836474, SAMN10836475, SAMN10836476, SAMN10836477, and SAMN10836478. o2o2o16o16 mutant line, QCL8011_2, and its recurrent parent CML530 were observed under natural light, while the phenotypes and cross-sections of the kernels were observed under projected light. The ear of QCL8011_2 was a cylindrical type  showed that the starch granules of QCL8011_2 were mostly irregular in shape and slightly larger in size than that of CML530. There was a high density of matrix proteins that were scattered with the starch granules in the gap. The endosperm starch granules of CML530 were mostly ellipsoidal or spheroidal, with a low density of matrix proteins that encapsulated starch granules E,F.Compared with CML530, the contents of all 17 FAAs changed to different degrees in the QCL8011_2 mutant line. o2o2o16o16 line, kernels  of the QCL8011_2 and CML530 were used for the RNA-Seq analysis. Two biological replicates were used in this study. For each replicate, we generated 23,984,698 clean reads on average (Zea mays L. AGPv4.38) and 81.27% clean reads were distributed in the gene regions , cellular component (CC), and molecular function (MF). At 18 DAP, a total of 5,639 DEGs were classified into 20 terms (BP), 11 terms (CC), and 11 terms (MF) A. At 28 For further functional characterization of DEGs, we analyzed the pathway enrichment in DEGs based on the KEGG database. At 18 DAP, 28 DAP, and 38 DAP, 3,547, 608, and 1,406 DEGs were annotated into 19 pathways, respectively, and most genes were annotated as related to the metabolism. Among the DEGs related to the metabolic pathways, 297 DEGs were related to the amino acid metabolism at 18 DAP, and 46 DEGs were involved in lysine metabolism, 23 DEGs were involved in tryptophan metabolism A. A totaAccording to the KEGG annotation results, at 18 DAP, 13 DEGs were annotated into the lysine biosynthesis pathway, with five being upregulated and eight downregulated. A total of 33 DEGs were annotated into the lysine degradation pathway, where four were upregulated while 29 were downregulated. At 28 DAP, three DEGs were annotated into lysine biosynthesis pathway, where two were upregulated and one was downregulated. Five DEGs were annotated into lysine degradation pathways, where two were upregulated and three were downregulated. At 38 DAP, five DEGs were annotated into lysine biosynthesis pathway, where four were upregulated and one was downregulated. A total of 18 genes were annotated into lysine degradation, where 14 were upregulated while four were downregulated .Zm00001d050134, Zm00001d021142, Zm00001d050133, and Zm00001d005151, were downregulated at 18 DAP. Furthermore, Zm00001d042471 and Zm00001d023278 were upregulated while Zm00001d005535 gene was upregulated at 38 DAP. A total of seven DEGs encoded the aspartate-semialdehyde dehydrogenase , involved in the conversion of L-aspartyl-4-phosphate to L-asparte-4-semialdehyde in the lysine biosynthesis pathway, and Zm00001d042275 was upregulated at 18 DAP, while Zm00001d033836 and Zm00001d052231 were downregulated. At 28 DAP, Zm00001d012847 was downregulated, while Zm00001d034765 was upregulated. Zm00001d009968 and Zm00001d033837 were upregulated at 38 DAP. Zm00001d008615 and Zm00001d028165, encoding L,L-diaminopimelate aminotransferase  involved in the conversion of -4-hydroxy-2,3,4,5-tetrahydrodipicolinate to (S)-2,3,4,5-tetrahydrodipicolinate, were downregulated at 28 DAP and 38 DAP, respectively. Zm00001d030677 and Zm00001d041575, encoding diaminopimelate epimerase  involved in the conversion of L,L-2,6-diaminopimelate to meso-diaminopimelate, were upregulated at 18 DAP.Under the lysine biosynthesis metabolism, seven DEGs encode aspartate kinase , involved in the conversion of L-aspartate to L-aspartyl-4-phosphate, in the first step of lysine biosynthesis pathway; of these, Zm00001d052079, encoding saccharopine dehydrogenase  involved in the conversion of lysine to (S)-2-aminoadipate-6-semialdehyde in the lysine degradation pathway, was downregulated at 18 DAP and 28 DAP, while Zm00001d020984 encoding L-pipecolate oxidase  was downregulated at 18 DAP, 28 DAP, and 38 DAP. Zm00001d050495 encoding aldehyde dehydrogenase family seven member A1  in the conversion of 2-aminoadipate-6-semialdehyde to L-2-aminoadipate was downregulated at 18 DAP. Zm00001d023552 encoding 2-oxoglutarate dehydrogenase  in the conversion of 2-oxoadipate to S-glutaryl-dihydrolipoamide was upregulated at 18 DAP and 38 DAP, while Zm00001d021889 was downregulated at 18 DAP. Zm00001d003923 and Zm00001d025258 encoding dihydrolipoamide succinyltransferase  in the conversion of S-glutaryl-dihydrolipoamide to glutaryl-CoA were upregulated.Under the lysine degradation metabolism, According to the KEGG annotation results, at 18 DAP, 23 DEGs were annotated into the tryptophan metabolism pathway, where 13 were upregulated and 10 DEGs were downregulated. At 28 DAP, 10 DEGs were annotated into the tryptophan metabolism pathway, which were all downregulated. At 38 DAP, 24 DEGs were annotated into tryptophan metabolism pathway, where one DEG was upregulated and 23 DEGs were downregulated .Zm00001d037674, Zm00001d043650, Zm00001d037498, and Zm00001d008700, were upregulated at 18 DAP, while Zm00001d012728 and Zm00001d008708 were downregulated. Zm00001d011562 and Zm00001d037674 were downregulated at 28 DAP. Zm00001d043651 was downregulated at 38 DAP. A total of four DEGs encoding aldehyde dehydrogenase , involved in the conversion of indole-3-acetaldehyde to indoleacetate, Zm00001d023580 and Zm00001d025958, were upregulated at 18 DAP, while Zm00001d050495 was downregulated. Zm00001d004731 was upregulated at 18 DAP but downregulated at 28 DAP and 38 DAP. Zm00001d034388, encoding indole-3-acetaldehyde oxidase  involved in the conversion of indole-3-acetaldehyde to indoleacetate, was downregulated at 18 DAP. The 8 DEGs encoding indole-3-pyruvate monooxygenase , involved in the conversion of indolepyruvate to indoleacetate, were downregulated at 38 DAP. Zm00001d043165 encoding N-hydroxythioamide S-beta-glucosyltransferase , involved in the conversion of indole-3-thiohydroximate to indolylmethyldesulfo-glucosinolate, was upregulated, while Zm00001d00424 was downregulated, at 28 DAP and 38 DAP. Zm00001d039669 encoding aromatic desulfoglucosinolate sulfotransferase , involved in the conversion of indolylmethyldesulfo-glucosinolate to glucobrassicin, was downregulated at 38 DAP. Under tryptophan metabolism, eight DEGs encoding L-tryptophan-pyruvate aminotransferase , involved in the conversion of tryptophan to indolepyruvate, 2, between the relative expression levels of qRT-PCR and RNA-Seq, was 0.7559, 0.7402, and 0.6426 at 18 DAP, 28 DAP, and 38 DAP, respectively . The lysine content of this line was increased by 56.08%, compared with wild-type line, CML530. By SNP chip scanning, the genetic background recovery rate of QCL8011_2 was 92.62%, which was higher than the theoretical background recovery rate of 87.50%. RNA-Seq analysis showed that, 46, 8, and 23 DEGs were involved in lysine metabolism at 18 DAP, 28 DAP, and 38 DAP, respectively, while 23, 10, and 25 DEGs were mainly involved in the tryptophan metabolism. These could help uncover the transcriptional regulation mechanism in the increase in lysine and tryptophan content, through the introgression of o2 and o16 alleles into the wild-type maize.In the present study, we introgressed the o2o2o7o7 seeds, compared with the recurrent parent A69Y [o2o2o16o16 were all increased to varying extents, which was consistent with the results tested by Hartings et al. However, the content of Glu, Val, Met, Ile, Leu, and Phe decreased, which was different from the results of Hartings et al. This difference may be related to the introgression of the o16 allele and the different genetic background (A69Y vs CML530).Hartings et al. determined that Asp, Glu, Gly, Val, Met, Ile, Leu, Phe, Lys, His, and Arg were increased, to varying degrees, in double mutant ent A69Y . In our o2o7 endosperm showed a reduced transcription level of the 19 and 22 kDa α-zein, and the 10, 27, and 50 kDa γ-zein. Li et al. [o2o2o16o16 mutant as compared to the wild-type (GRMZM2G044152 (Zm00001d048813) gene was also detected by Li et al. These α-zein genes were downregulated in the o2o2o16o16 mutant, resulting in a decrease of the zein synthesis and increase in the lysine content. Aspartate kinase  was committed in the first step of the Asp-derived amino acid pathway, in microorganisms and plants, leading to the biosynthesis of essential amino acids, such as lysine, threonine, methionine, and isoleucine [o2 mutation in maize [Zm00001d009968 and Zm00001d034765, encoding ASADH involved in the conversion of L-aspartyl-4-phosphateto L-asparte-4-semialdehyde, were upregulated at 28 DAP and 38 DAP in QCL8011_2. Zm00001d030677 and Zm00001d041575, encoding Dap-F involved in the conversion of L,L-2,6-diaminopimelateto meso-diaminopimelate, were upregulated at 18 DAP. These changes might promote lysine synthesis and increase the grain lysine content of the QCL8011_2 lines.Hartings et al.  reportedi et al.  discoveri et al.  discoverild-type . Fourteeoleucine ,40,41,42oleucine . The genin maize . In thisGRMZM2G181362), which was involved in lysine catabolism, was downregulated by 4.57-fold in the o2 endosperm [o2 mutants was decreased by more than 90% and that the enzyme activity was significantly decreased, which reduced the degradation of lysine [lkrsdh1 encoding LKR/SDH, involved in the conversion of lysine to 2-aminoadipate-6-semialdehyde, was downregulated at 18 DAP and 28 DAP in QCL8011_2. Meanwhile, Zm00001d020984, encoding LOP, was downregulated at 18 DAP, 28 DAP, and 38 DAP. These changes inhibited lysine degradation and increased the grain lysine content of the QCL8011_2.Lysine α-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), which was a bifunctional enzyme involved in lysine catabolism, was also regulated by O2 . The O2-ndosperm . Kemper f lysine . In thisZm00001d011562 and Zm00001d037674, which encode the TAA involved in the conversion of tryptophan to indolepyruvate, we found that Zm00001d011562 was downregulated at 28 DAP and Zm00001d037674 was downregulated at 38 DAP in QCL8011_2. A total of eight DEGs encoding IPMO, involved in the conversion of indolepyruvate to indoleacetate, were downregulated at 38 DAP. The aldehyde dehydrogenase (ALDH) superfamily is composed of a wide variety of enzymes involved in endogenous and exogenous aldehyde metabolism. ALDH enzymes use either NAD+ or NADP+ as a cofactor to convert aldehydes to their corresponding carboxylic acids and NADH or NADPH [Zm00001d050495 and Zm00001d004731, which encode ALDH in the conversion of indole-3-acetaldehyde to indoleacetate in tryptophan metabolism, we found that Zm00001d050495 was downregulated at 18 DAP and Zm00001d004731 was downregulated at 28 DAP and 38 DAP. Aldehyde oxidase  could oxidize indole-3-acetaldehyde into indole-3-acetic acid [Zm00001d034388 encoding IAAO was downregulated at 18 DAP. These changes inhibited the tryptophan degradation and increased the grain tryptophan content of the QCL8011_2.Trp was first converted to indole-3-pyruvate (IPA) by the TAA family of aminotransferases and, subsequently, indol-3-acetic acid (IAA) was produced from IPA by the YUCCA (YUC) family of flavin monooxygenases. The two-step conversion of Trp to IAA was the main auxin biosynthesis pathway that played an essential role in many developmental processes ,47. Whenor NADPH . When weo2 and o16 alleles into the wild-type maize were identified at the transcriptional level. Among them, genes encoding AK, ASADH, and Dap-F, in the lysine synthesis pathway, were upregulated at different stages of endosperm development , which promoted the synthesis of lysine. Meanwhile, genes encoding LKR/SDH and L-PO in the lysine degradation pathway were downregulated, which inhibited the degradation of lysine. In addition, genes encoding TAA and YUC in the tryptophan metabolic pathway were downregulated, which inhibited the degradation of tryptophan. These would help to uncover the transcriptional regulation mechanisms involved in the increase in lysine and tryptophan content, through the introgression of o2 and o16 alleles into wild-type maize.In this study, the key genes responsible for the increase in lysine and tryptophan content after introgression of o2 and o16 alleles were introgressd into the wild-type inbred line CML530 by MABS, and a double recessive mutation QCL8011_2 (o2o2o16o16) was acquired. The lysine content of QCL8011_2 was improved, compared with that of its recurrent parent. Kernels from QCL8011_2 and CML530  were subject to the RNA-Seq. The RNA-Seq analysis revealed that 59 DEGs were involved in lysine metabolism, and 43 DEGs were involved in tryptophan metabolism. Among them, the genes encoding AK, ASADH, and Dap-F in the lysine synthesis pathway were upregulated at different stages of the endosperm development , which promoted the synthesis of lysine. Meanwhile, the genes encoding LKR/SDH and L-PO in the lysine degradation pathway were downregulated, which inhibited the degradation of lysine. In addition, genes encoding TAA and YUC in the tryptophan metabolic pathway were downregulated, which inhibited the degradation of tryptophan. These results could help uncover the molecular mechanism in the increase in lysine and tryptophan content, through the introgression of the o2 and o16 alleles into the wild-type maize.In this study, the


Acute kidney injury (AKI) is a serious complication of methotrexate (MTX). This study explored the protective effect of the isoflavone formononetin (FN) against MTX nephrotoxicity with an emphasis on oxidative stress, inflammation, and nuclear factor (erythroid-derived 2)-like 2/heme oxygenase 1 (Nrf2/HO-1) signaling. Rats received FN  for 10 days and a single dose of MTX on day 7. MTX induced kidney injury was characterized by increased serum creatinine and urea, kidney injury molecule-1 (Kim-1), and several histological alterations. FN ameliorated kidney function and inhibited the renal tissue injury induced by MTX. Reactive oxygen species (ROS), lipid peroxidation (LPO), nitric oxide, and 8-Oxo-2′-deoxyguanosine were increased, whereas antioxidant defenses were diminished in the kidney of MTX-administered rats. In addition, MTX upregulated renal iNOS, COX-2, TNF-α, IL-1β, Bax, caspase-9, and caspase-3, and decreased Bcl-2, Nrf2, and HO-1. FN suppressed oxidative stress, LPO, DNA damage, iNOS, COX-2, proinflammatory cytokines, and apoptosis, and boosted Bcl-2, antioxidants, and Nrf2/HO-1 signaling in MTX-administered rats. In conclusion, FN prevents MTX-induced AKI by activating Nrf2/HO-1 signaling and attenuates oxidative damage and inflammation. Thus, FN may represent an effective adjuvant that can prevent MTX nephrotoxicity, pending further mechanistic studies. Methotrexate (MTX), a folic acid antagonist, is a potent chemotherapeutic agent used in the treatment of malignancies and inflammatory diseases . HoweverOne of the untoward effects of MTX is its ability to promote ROS production, leading to inflammation and cell death. As reported previously, MTX has the ability to increase the production of ROS through provoking nicotinamide adenine dinucleotide phosphate (NADPH) depletion , neutropTrifolium pratense) and Astragalus membranaceus [Several studies have demonstrated the renoprotective effects of various antioxidants in animal models of AKI ,7,22. Foranaceus . It has ranaceus ,24,25,26ranaceus , and attranaceus . A recenranaceus . Furtherranaceus . DespiteThirty-six male Wistar rats weighing 160 to 180g were used to investigate the nephroprotective effect of FN. The animals, obtained from VACSERA , were housed under standard conditions and allowed free access to food and water. Protocols involving the use of animals were performed in line with the guidelines of the National Institutes of Health , and approved by the local ethical committee (2019/198).n = 6 rats in each group) were employed to study the renoprotective potential of FN as follows:According to the study protocol, six groups of rats (Group I (Control): Rats received 0.5% carboxymethyl cellulose (CMC) orally for 10 consecutive days.Group II (FN): Rats received FN (40 mg/kg)  orally fGroup III (MTX): Rats received 0.5% CMC orally for 10 days and MTX at day 7.Group IV (MTX + 10 mg FN): Rats received 10 mg/kg FN  orally fGroup V (MTX + 20 mg FN): Rats received 20 mg/kg FN  orally fGroup VI (MTX + 40 mg FN): Rats received 40 mg/kg FN  orally fth day, the animals were sacrificed, and blood and kidney samples were harvested for further investigations. Serum was prepared from the blood samples, and the kidneys were washed in cold phosphate buffered saline (PBS). Specimens from the kidneys were fixed in 10% neutral buffered formalin while others were kept frozen at −80°C. Other samples were homogenized (10% w/v) in PBS supplemented with 0.5 mM EDTA and proteinase inhibitors, centrifuged, and the clear supernatant was collected.MTX  was dissolved in saline and injected intraperitoneally (i.p.) at 20 mg/kg dose . Groups Serum creatinine  and ureaKidney injury molecule-1 (Kim-1), caspase-3, and caspase-9 were assayed using ELISA kits purchased from Cusabio . Tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β were assayed using R&D Systems ELISA kits .ROS were measured immediately in the tissue homogenates using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), as previously described . ThiobarTotal HO-1 activity was measured following the method of Abraham et al. . BrieflyAdenosine triphosphate (ATP) content in the renal homogenate was measured using a kit supplied by Sigma . In this test, ATP is determined by phosphorylating glycerol, and the product is proportional to the amount of ATP in the sample and is determined calorimetrically at 570 nm.Specimens from kidney fixed in 10% buffered formalin were dehydrated, embedded in paraffin wax and cut into 5-μm sections. Following deparaffinization and rehydration, the sections were processed for hematoxylin and eosin (H&E) staining and then examined. iNOS), BAX, BCL-2, cyclooxygenase-2 (COX-2), IL-1β, TNF-α, Nrf2, HO-1, and caspase-3 was quantified using qRT-PCR, as previously reported [−ΔΔCt method [The effect of MTX and FN on the mRNA expression levels of inducible nitric oxide synthase  buffer containing protease inhibitors, centrifuged, and protein content was determined in the supernatant using Bradford reagent. Forty micrograms of proteins were subjected to 10% SDS-PAGE followed by electrotransfer to a nitrocellulose membrane, which was blocked and probed with anti-Nrf2 and anti-β-actin. After overnight incubation at 4 °C and washing, the secondary antibodies were added, and the blots were developed. The obtained bands were scanned, and intensity was quantified using ImageJ . The results were normalized to β-actin and presented as a percent of control. All antibodies were provided by Novus Biologicals .2. Upon confluency, the cells were trypsanized and seeded in 96-well plates (104 cells/well). The cells were treated with different doses of FN for 24 h, followed by MTX for 48 h. The cells were stained with 5 mg/mL MTT, incubated for 2 h at 37°C. The medium was replaced by 100 µl DMSO, and the absorbance was read at 570 nm after 10 min.HepG2 cells were grown in PRMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin (100 U/mL) at 37 °C and 5% COp-value < 0.05 was considered significant.The significance value of the obtained data was analyzed by one-way (ANOVA) followed by Tukey’s test using GraphPad Prism 7 . All results were presented as mean ± standard error of the mean (SEM). A p < 0.001) as depicted in MTX increased creatinine, urea, and Kim-1 significantly  in renal ROS  and FN treatment prior to MTX restored mitochondrial function (Owing to the role of MTX in inducing mitochondrial dysfunction, we investigated the effect of MTX and FN on ATP content in the kidney. MTX diminished renal ATP (function . FN alonAnalysis of the mRNA expression in the kidney of rats which received MTX revealed significant upregulation of COX-2, iNOS, TNF-α, and IL-1β . The infp < 0.001; Oxidative damage, mitochondrial dysfunction, and inflammation can drive cell death. Given that MTX administration was associated with these processes, we determined its effect as well as the ameliorative potential of FN on apoptosis. MTX-mediated apoptosis was evidenced by the increased mRNA abundance of Bax (To assess the effects of FN on the antitumor properties of MTX, we investigated the cytotoxic effects of MTX alone and in combination with FN in HepG2 cells. Treatment of HepG2 cells with either MTX or FN resulted in cytotoxicity. Per-treatment of HepG2 cells with FN did not interfere with the cytotoxic action of MTX .MTX is an effective anticancer and immunosuppressive agent; however, its serious side effects limit its clinical applications. The mechanism of MTX-induced nephrotoxicity involves oxidative injury and inflammation ,7. DespiAKI in MTX-administered rats was evidenced by the elevated serum creatinine and urea, and renal Kim-1. Creatinine and urea are commonly measured as indices of glomerular function ,45. Kim-Oxidative stress is one of the main mechanisms through which MTX induces tissue damage ,7,11,49.FN has been reported to exert antioxidant actions in several preclinical models of a variety of pathological conditions associated with oxidative stress ,26,29. HIn addition to oxidative stress, MTX-induced ROS generation can elicit various stress signaling, such as NF-κB, which promotes the expression of TNF-α, IL-1β, COX-2, and iNOS ,7,8,49. c release from the mitochondria and consequent activation of caspases. In addition, excessive mitochondrial ROS production during MTX metabolism can damage the mitochondrial membrane, resulting in the loss of membrane potential and consequently the release of cytochrome c which ultimately culminates in renal apoptosis by activating caspase-3 [Accumulating evidence has pointed to the role of oxidative injury and inflammation in provoking apoptosis in MTX-induced AKI ,7,8,49. aspase-3 . Thus, iaspase-3  and cispaspase-3  in rodenaspase-3 .To gain more insight into the potential underlying mechanism of the renoprotective effect of FN, we investigated the role of Nrf2/HO-1 signaling in mediating the effects of FN. Extensive evidence indicated that Nrf2 signaling plays comprehensive cytoprotective roles through activating the expression of several genes encoding detoxification, antioxidant, and anti-inflammatory proteins ,58,59,60FN upregulated renal Nrf2 and consequent induction of HO-1, CAT, and SOD in rats challenged with MTX. Interestingly, FN increased HO-1 mRNA and activity in normal rats. Upregulated Nrf2 signaling by FN resulted in enhanced antioxidants and diminished ROS and oxidative damage. Furthermore, activation of Nrf2 had a key role in the anti-inflammatory and antiapoptotic effects of FN. Nrf2 and HO-1 can directly inhibit NF-κB signaling and proinflammatory cytokines, and activate the anti-inflammatory cytokines, thereby regulating the inflammatory cascade . In thisTo further investigate whether the above-mentioned beneficial effects of FN would interfere with the antitumor activity of MTX, we evaluated its effect on MTX-induced cytotoxicity in HepG2 cells. Per-treatment of HepG2 cells with FN did not interfere with the cytotoxic action of MTX. Indeed, several molecular mechanisms have been involved in the anticancer activity of MTX; however, oxidative/nitrative stress does not seem to be implicated. Efficient antioxidant strategies, which counteract the toxicity of chemotherapies, do not interfere with their antitumor action. For example, the cardioprotective agent dexrazoxane that reduces doxorubicin cardiotoxicity is a potent antioxidant . Here, tOur findings indicate that the natural isoflavone FN prevented AKI induced by MTX. FN suppressed excess ROS, oxidative injury, and improved mitochondrial function. Consequently, FN attenuated inflammation and inhibited cell death in the kidney of rats and hence, possesses a therapeutic benefit against MTX toxicity. Activation of Nrf2/HO-1 signaling and enhancement of the antioxidant defenses represent the main mechanism underlying the nephrotprotective effect of FN . These r


Autophagy is an evolutionarily conserved process in eukaryotes to maintain cellular homeostasis under environmental stress. Intracellular control is exerted to produce energy or maintain intracellular protein quality controls. Autophagy plays an important role in embryogenesis, implantation, and maintenance of pregnancy. This role includes supporting extravillous trophoblasts (EVTs) that invade the decidua (endometrium) until the first third of uterine myometrium and migrate along the lumina of spiral arterioles under hypoxic and low-nutrient conditions in early pregnancy. In addition, autophagy inhibition has been linked to poor placentation—a feature of preeclamptic placentas—in a placenta-specific autophagy knockout mouse model. Studies of autophagy in human placentas have revealed controversial results, especially with regard to preeclampsia and gestational diabetes mellitus (GDM). Without precise estimation of autophagy flux, wrong interpretation would lead to fixed tissues. This paper presents a review of the role of autophagy in pregnancy and elaborates on the interpretation of autophagy in human placental tissues. Cellular homeostasis is maintained through protein quality controls that balance synthesis and degradation. Although turnover rate varies in each cellular component, eukaryotic cells degrade proteins using two intracellular degradation systems—the autophagy-lysosomal system and the ubiquitin-proteasome system. Proteasomal degradation selectively recognizes ubiquitinated proteins, which mainly consist of short-lived proteins. Lysosomal-mediated degradation targets long-lived proteins in a complex process ,2,3: cytThere are several types of selective autophagy that behave like a vacuum cleaner in cells . ImpaireThere are three types of autophagy: macroautophagy, microautophagy, and CMA . MacroauMultiple autophagy-related (Atg) proteins intertwine to form autophagosomes after induction. The ULK1 complex, which includes Atg13, Atg101, and FAK family kinase-interacting protein of 200 kDa (FIP200), translocates to the ER regulating class III phosphatidylinositol 3-kinase complex (PI3K), which is involved in the early stage of autophagosome formation. Next, pro-MAP1LC3 (Microtubule associated protein 1 light chain 3) is converted to MAP1LC3-I by Atg4B proteins, a cysteine protease . ConversFunctions of oocytes, including ovulation, fertilization, and implantation, were not affected by autophagy inhibition in the ovary-specific Beclin1 knockout mouse model . Atg7 waC74A, an inactive mutant of Atg4B that inhibits autophagic degradation and lipidation of MAP1LC3B paralogs in hypoxia [The MAP1LC3 protein families: MAP1LC3A, MAP1LC3B, and MAP1LC3C, are expressed in both the labyrinth zone and decidua basalis in mouse models. Expression of MAP1LC3A and MAP1LC3B were higher in the decidua basalis than in the labyrinth layer . Autopha hypoxia ,25. Phys hypoxia ,26. Alth hypoxia ,28,29. TTrophoblastic stem cells differentiate to syncytiotrophoblasts as well as EVTs. Autophagy regulates the differentiation of invasive trophoblasts via reduction of galectin-4, which is required for normal placental development, as seen in a rat model ,31. AutoHIF1α is a key factor for EVT invasion. HIF1α expression, induced by hypoxia, was not affected by autophagy suppression in trophoblast cells . InteresKip2 mice, which develop hypertension and proteinuria in dams [It has been reported that the expression of BECN1, involved in autophagosome formation in mammalian placentas, is higher in the presence of FGR without preeclampsia, but not when preeclampsia is present ,39. Howe in dams . The inc in dams . In addiAutophagic vacuoles are more likely to be present in the syncytiotrophoblast layer of human FGR placentas, which indicates autophagy activation ,53. HighINK4a pathway, the p53 pathway, and the autophagy-mediated GATA Binding Protein 4 (GATA4) pathway. Increased expression of p53, p21, and p16INK4a proteins has been reported in preeclampsia [Protein aggregation caused by autophagy suppression has been reported in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease ,57. Receclampsia ,65. GATAclampsia ,67. TherGestational diabetes mellitus (GDM) is a type of diabetes that develops during pregnancy and affects 3–30% of pregnant women ,69,70. GAtg16L1 is essential for forming autophagosomes with the Atg5-Atg12 complex, which is associated with Crohn’s disease . Atg16L1C74A mutation, and EVTs in biopsies taken from the placental bed of women with preeclampsia [The importance of estimating autophagy is to precisely calculate the velocity of autophagy flow. Estimating autophagy using a single method is impossible, and is more difficult in vivo—and in humans—than in vitro, or in animal models ,83. In wclampsia . This woclampsia . Taken tAutophagy mediates a variety of life process, including cancer development, immune response, and aging pathophysiology . Autopha