TP53 mRNA splicing. In this work, we will review the different mechanisms of p53 pathway inhibition in cancer with special focus on multiple myeloma (MM), the second most common hematological malignancy, with low incidence of p53 mutations/deletions but growing evidence of indirect p53 pathway deregulation. Translational implications for MM and cancer prognosis and treatment are also reviewed.The p53 pathway is inactivated in the majority of human cancers. Although this perturbation frequently occurs through the mutation or deletion of p53 itself, there are other mechanisms that can attenuate the pathway and contribute to tumorigenesis. For example, overexpression of important p53 negative regulators, such as murine double minute 2 (MDM2) or murine double minute 4 (MDM4), epigenetic deregulation, or even alterations in  Since its discovery in 1979, the tumor suppressor protein p53 has been extensively studied. p53 is a transcription factor activated in response to several forms of cellular stress including DNA damage, hypoxia, viral infection, heat shock, and mitogenic or oncogenic stresses among others ,3. p53 pTP53 allele, and the spontaneous cancer predisposition of Trp53-null mice [Nevertheless, the importance of p53 in tumor suppression is unequivocal, as shown by its mutation in more than half of all sporadic human cancers, the susceptibility to cancer of individuals with Li–Fraumeni syndrome who inherit a mutant ull mice ,9.Interestingly, there are some tumors that inactivate the pathway almost exclusively through direct p53 mutation or deletion of its locus, like esophageal squamous carcinoma , whereasThis review summarizes the different molecular alterations that can contribute to the attenuation of p53 pathway in cancers with special focus on multiple myeloma (MM) . This caTP53 mutations are distributed mainly in coding exons with a strong predominance for exons 4–9, which encode the DNA-binding domain of the protein. In fact, 95% of the TP53 mutations stand in the core DNA-binding domain [TP53 mutations can be classified in two categories: contact and structural mutations [TP53 mutations have been detected predominantly in 6 hotspot residues located within the DNA-binding domain  [The tumor suppressor p53 is mutated in approximately 50% of human cancers ,20. TP53g domain . TP53 muutations . Contactutations ,24,25. Ind R282) ,27.TP53 mutations in hematological malignancies is generally lower than in solid neoplasms, they have been reported in Burkitt‘s lymphomas, chronic myeloid leukemia, adult T-cell leukemia, B-cell prolymphocytic leukemia and chronic lymphocytic leukemia (CLL) [TP53 mutations are uncommon at diagnosis (~8%) [TP53 mutations, two of which corresponded to hotspot residues R273 and R248 within the DNA-binding domain, and commonly mutated in other human tumors [TP53 mutations increases up to 25% in plasma cell leukemia [TP53 mutations are often acquired at relapse [Although the frequency of ia (CLL) ,13,14,15is (~8%) ,29,30, ais (~8%) ,32. Thesis (~8%) ,33. Thusleukemia . Further relapse .TP53 gene locus, is a recurrent cytogenetic abnormality in MM present in around 10% of new cases. It is one of the most powerful prognostic factors associated with unfavorable outcome [TP53 deletion has been associated with resistance to chemotherapy and early evolution to extramedullary disease [TP53 deletions remain a negative prognostic factor marker after high-dose chemotherapy plus autologous stem cell transplantation [TP53 mutations were exclusively associated with del(17p) [TP53 is more common in aggressive plasma cell neoplasms, and is frequently seen at relapse leading to rapid progression to plasma cell leukemia and/or extramedullary disease [TP53 deletion and/or mutation in the pathogenesis of MM and reinforce the necessity of new therapeutic approaches for these high-risk patients.On the other hand, deletion of the chromosomal region 17p13, containing the  outcome ,37,38,39 disease ,42,43,44antation , and eveantation . The relantation ,48. Intedel(17p) . Further disease ,49. All The most known mechanism to indirectly attenuate or disable p53 activity is through overexpression of their negative regulators. The best studied is MDM2 (murine double minute 2), which was originally identified in a double minute chromosome present in the murine tumorigenic cell line 3T3-DM . MDM2 coMDM2 seems to be a common mechanism by which human cancers abrogate the p53 pathway. MDM2 upregulation has been found in various types of cancer [MDM2 amplification, resulting from chromosome 12 diploidy, was seen in 8% of the cases, while another 8% had trisomy 12 with an equivalent increase in signals for MDM2 [MDM2 has been found in myeloma cell lines and primary samples obtained from patients with MM and plasma cell leukemia. Moreover, inhibition of MDM2 was found to trigger MM cell apoptosis [The essential role of MDM2 in the negative regulation of p53 was clearly illustrated in mouse models . In agref cancer , includif cancer . In a stfor MDM2 . Overexppoptosis .MDM2 promoter single-nucleotide polymorphisms (SNPs) are the two best-studied mechanisms for up-regulating MDM2 activity [Genetic amplification and inheritance of activity . Howeveractivity . Exampleactivity . Amplifiactivity . To our MDM4 [MDM4 is also an important negative regulator of the p53 pathway, which was identified in a screen for p53-interacting proteins in 1996 by Shvarts et al. . MDM4 seMDM4 , and eleMDM4 .Besides MDM2 and MDM4, a number of other ubiquitin ligases have been identified to regulate p53 in vitro like PIRH2, COP1, ARF-BP1, CHIP, TRIM24 or E4F1 ,65. AmplDNA methylation is a well-known epigenetic modification that decreases the expression of thousands of genes. The most investigated is methylation of the cytosines present in CpGs often grouped in CpG islands. Coding genes sequences as well as their promoters or 3′ untranslated regions (3′-UTRs) can be methylated, which abolishes gene transcription until a complex process of demethylation restores the expression . InteresTP53 reduced the expression level of a reporter gene. These results were later confirmed by Pogribny et al. [TP53 gene or its regulatory sequences have been reported in many cancers including Ewing’s sarcoma [TP53 has also been found in other diseases like cutaneous squamous cell carcinomas [TP53 may have important implications for tumor biology. In ALL, direct or indirect activation of the p53 pathway was observed after treatment with the demethylating reagent 5-aza-2′-deoxycitidine, and was accompanied by an increase in apoptosis [TP53 [The first experimental evidence for p53 regulation by DNA methylation was provided by Schroeder et al. in 1997 , who obsy et al. . Since t sarcoma , gliobla sarcoma , acute l sarcoma ,74, huma sarcoma , ovarian sarcoma , breast  sarcoma  and also sarcoma ,79. On trcinomas , sporadircinomas  and myelrcinomas . Changespoptosis  and in bis [TP53 . On the is [TP53 . Lack ofis [TP53 .TP53 methylation on protein expression. In some MM cell lines with p53 WT/− and no p53 protein expression, the second allele was found inactivated by promoter hypermethylation [TP53 promoter was hypermethylated [TP53 promoter [Few studies in MM have addressed the putative influence of hylation . Additiothylated . More repromoter .TP53 DNA methylation are complex and not well understood. In esophageal cancer cells, phospholipase C epsilon PLCE1 downregulation was linked to increased p53 level that was accompanied by reduced TP53 promoter methylation [TP53 methylation [Mechanisms involved in regulation of hylation . In MM, hylation .TP53 mutation has been shown to correlate with TP53 hypomethylation status of exons 5–8 [TP53 exons 5–8 can be increased in rat liver and colon mucosa by dietary selenomethionine, with may then influence the preservation of intact TP53 sequence [Although DNA methylation is first and foremost linked to decreased gene expression, growing body of evidence suggest other effects of this epigenetic modification. Thus, the xons 5–8 ,89. Intesequence . WhetherMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Over the past decade, the crucial role of miRNAs in the pathogenesis of many cancers has been clearly demonstrated. In particular, miRNAs are functionally deregulated in MM cells, and several studies reveal the involvement of miRNAs in MM pathophysiology. Moreover, the therapeutic potential of miRNA modulation has been investigated not only to target myeloma cells, but also to impair the functional interaction between myeloma cells and bone marrow microenvironment ,92,93,94TP53 3′-UTR site, which resulted in a decreased p53 expression in several types of cells. Similarly, other microRNAs such as miR-125b, miR-125a and miR-1285 have been described as negative regulator of TP53 [TP53, highlighting the tight regulation of p53 by miRNAs [miR-504 was the first miRNA identified and validated by Hu et al. as a negative regulator of p53 . This mi of TP53 ,105,106.TP53 directly have been validated in MM cells. Two miRNAs, miR-25 and miR-30d, which directly interact with the 3′-UTR of the human TP53 mRNA [TP53 mRNA. When these miRNAs were reintroduced into MM cells, p53 targets such as p21, BAX, PUMA and GADD45A were activated, and reduced cell apoptosis, cell cycle arrest and senescence were observed. The miR-125a-5p which has been described to be upregulated in a subset of MM patients carrying the t translocation [TP53 mRNA 3′-UTR triggering a decrease of p53 levels. On the other hand, its inhibition restored the p53 transcriptional pathway in p53 WT MM cells and also increased the level of miRNAs regulated by p53. Interestingly, inhibition of miR-125b was demonstrated to overcome dexamethasone resistance in MM cells by activation of p53 downstream targets [TP53 3′-UTR in different cellular models [Up to now, only few miRNAs targeting P53 mRNA  are downlocation , directl targets . It is ir models ,111. Forr models , miR-19br models  or miR-3r models .TP53 transcriptional network, with higher expression in WT TP53 than in TP53-mutated MM cells, after treatment with nutlin-3, a known p-53 reactivating agent [Several miRNAs have also been described to indirectly regulate p53 by affecting some of its regulators ,115. Forng agent .TP53, an increase of TP53 mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53, were observed. Taken together, the reported findings suggest an important role for miRNAs in the regulation of the p53 tumor suppression.In MM, two studies have demonstrated indirect p53 regulation through miRNAs. Pichiorri et al. observed that the ectopic expression of miRNA-192, -194 and -215 induced significant down-regulation of MDM2 that was accompanied by p53 overexpression and p21 activation . These mTP53 isoforms were later discovered in humans. Decades of research on p53 isoforms have established that a p53-mediated cell response is the sum of the intrinsic activities of the coexpressed p53 isoforms and that alterations of their expression may also trigger p53 pathway deregulation, resulting in cancer and other diseases [TP53 target genes.Although, originally, p53 was considered a single protein, an alternative C-terminally-spliced isoform was soon described in mouse . Severaldiseases . At leasdiseases ,122,123.In the last years, numerous studies have demonstrated that p53 isoforms are differentially expressed in several human cancers types: breast cancer, colon carcinoma, melanoma, renal cell carcinoma (RCC), head and neck tumors (HNSSCs), hepatic cholangiocarcinoma, acute myeloid leukemia (AML), ovarian cancer, lung tumor, and glioblastoma ,125,126.TP53 mutation status have superior prognostic value when is combined with the analysis of p53 isoform expression. Thus, the expression of p53 isoforms can modify the effects of TP53 mutations in breast cancer and determination of p53γ isoform expression in TP53 mutant breast cancer patients allows the identification of those with poor prognosis [TP53 and Δ133p53α have a better overall survival than those with mutant TP53 but no Δ133p53α isoform expression [Notably, various studies in different types of human cancers has revealed that rognosis . In seropression .TP53 is not a frequent event at diagnosis, it would be interesting to explore this mechanism of p53 regulation in MM to evaluate its potential impact on response to treatment and disease prognosis.To date, there are no studies regarding the expression of p53 isoforms in MM, neither its putative clinical implication. Since mutation of Despite, in the last years, the introduction of novel therapeutic agents with different mechanisms of action has improved considerably the median survival of patients, MM remains incurable ,132,133.TP53 gene mutations [Many different therapeutic strategies have been reported to reactivate or restore the p53 response in human cancers ,137,138.utations .MDM2 or MDM4. The first molecule identified as a potent inhibitor of the p53-MDM2 interaction was nutlin [TP53 gene and intact p53 signaling machinery [Owing to the fact that p53 protein levels is under the tight control of two negative regulators, MDM2 and MDM4, several approaches to activate p53 have tried to inhibit the association of the protein with their negative regulators. This strategy is especially interesting in those tumors overexpressing s nutlin . This moachinery ,141,142.achinery ,143,144.achinery ,146,147.achinery  and spirachinery ,150.TP53 cells in vitro and ex vivo, through the induction of p53 downstream targets like p21, MDM2, and the pro-apoptotic genes, BAK, BAX and PUMA [In MM, nutlin-3 has demonstrated potent antimyeloma activity in WT and PUMA ,143,151.and PUMA .TP53, suggesting the co-existence of an additional, p53-independent, mechanism of action [RITA (Reactivation of p53 and Induction of Tumor Cell Apoptosis) is other investigated compound identified in a cell-based screen. In contrast to nutlin, which binds to MDM2, RITA binds to the N-terminal domain of p53. This interaction causes conformational changes in the structure of p53 protein that reduce their affinity for MDM2 ,153. Howf action ,159. A df action .TP53 gene mutations. PRIMA-1 (p53 reactivation and induction of massive apoptosis) is one the p53 mutant reactivating agents that have been developed. It reactivates mutant p53 by restoring its WT conformation and transcriptional functions, and enhances massive apoptosis in tumor cells [MET, the more potent derivative, can induce cell death independently of p53 and has been found to restore mutant p53 (R273H and R175H) function in vitro and in vivo [TP53 mutation status [The second p53-based therapeutic approach is focused on restoring the functions of p53 in cells with or cells . Lambertor cells . PRIMA-1 in vivo ,162,163. in vivo , breast  in vivo , thyroid in vivo , acute p in vivo , high-gr in vivo , acute m in vivo , epithel in vivo , soft-ti in vivo , and mul in vivo ,172,173. in vivo . Interesn status . MIRA-1 n status .Finally, it is important to underscore that although p53 restoration remains an attractive approach for certain cancers, including MM, its ultimate therapeutic value can be affected by different pitfalls. Thus, the potential clinical use of nutlin could be hindered by the observed acquisition of somatic mutations in p53 and select for p53-mutated cells after nutlin treatment . In thisTP53 locus are the most common alterations in p53 pathway, a number of other molecular mechanisms can also attenuate the p53 function. In fact, growing evidence of indirect p53 deregulation in MM through MDM2 overexpression, TP53 promoter hypermethylation and alterations in certain miRNAs that directly or indirectly affect p53 expression, such as miR-25, miR-30d, miR-125a-5p and miR-214, have been reported. However, the impact of other known p53 negative regulators or variations in isoform expression in p53 inactivation remains to be elucidated. A systematic study of p53 status that integrates all these parameters would probably increase the proportion of MM patients exhibiting a dysfunctional p53 pathway. Therefore, the determination of p53 status in clinical studies would strongly contribute to a better management of cancer and could support the rationale for clinical trials with p53-based therapy.Inactivation of the p53 pathway occurs in the majority of human cancers and usually confers resistance to therapy and poor survival. Although mutations and deletions in the


Terrimicrobium sacchariphilum strain NM-5T, a facultative anaerobic, mesophilic, fermentative bacterium belonging to the class Spartobacteria of the phylum Verrucomicrobia. The genome comprises 4,751,807 bp in three contigs and has a G+C content of 60.19%. Annotation predicted 4,175 protein-coding sequences and 54 RNAs.We report here a high-quality draft genome sequence of  Spartobacteria (subdivision 2) represent a dominant verrucomicrobial lineage in soil, and they are also ubiquitous in freshwater and marine environments. Their metabolic capacities and ecological roles remain poorly understood, however, due to the paucity of cultured representatives and sequenced genomes. Currently, only two isolates from the class Spartobacteria have been described, namely, Terrimicrobium sacchariphilum NM-5T   and soil (“Candidatus Udaeobacter copiosus”) were reported more recently  and the Nextera mate-pair library preparation kit (1- to 16-kb inserts). Sequencing was performed on the Illumina MiSeq platform using v2 chemistry (500 cycles), at coverages of 292× and 133× for the paired-end and mate-pair libraries, respectively. Raw reads were quality filtered using Trimmomatic version 0.32  of soil bacteria  with a range of carbohydrates, including arabinose, fructose, galactose, glucose, ribose, mannose, lactose, maltose, and sucrose, but not with organic acids, alcohols, amino acids, or aromatic compounds, which is consistent with its genomically encoded metabolic potential. We expect that the genome sequence reported here will contribute to improving our understanding of the metabolic capabilities and ecological roles of Spartobacteria species in the environment.The final draft assembly of T. sacchariphilum NM-5T has been deposited at DDBJ/EMBL/GenBank under accession number BDCO00000000 (BioProject PRJDB4647). The version described in this paper is the first version, BDCO01000000.The draft genome sequence of


The kidneys from six immunodeficient mice examined by Cerberus Sciences and the Animal Resources Centre, displayed karyomegaly with pale eosinophilic, intranuclear inclusions upon histopathological examination. Electron microscopy performed on kidney tissue from 5/6 mice demonstrated margination of the chromatin in large nuclei. Laboratory tests were used to detect nucleic acid of papillomaviruses, polyomaviruses, circoviruses and anelloviruses (4/6 mice), a specific PCR was used to detect murine polyomavirus (1/6), and a panel of serological tests was used to detect seroconversion to major murine pathogens (1/6). All molecular and serological tests were negative. Immunohistochemistry using polyclonal anti-bovine papillomavirus type 1 (BPV-1) L1 antibody, Camvir monoclonal anti-papillomavirus antibody (directed against the seven amino acids GFGAMDF found in human papillomavirus (HPV) 16 L1 protein), a commercially available mixture of two monoclonal antibodies, anti-BPV-1 L1/1H8 + Camvir antibodies, and a monoclonal anti-Hsc70 antibody revealed specific, positive staining of murine renal tubular epithelial intranuclear inclusions in 6/6 mice using the anti-BPV-1 L1 containing antibodies only. Methyl pyronin green, PAS and Feulgen histochemical reactions revealed that the intranuclear inclusions did not consist of RNA, DNA or carbohydrate. An immunohistochemical method now exists that can be used to confirm and evaluate suspected cases of murine inclusion body nephropathy. Veterinary pathologists examining mouse kidneys have occasionally noted the presence of large, eosinophilic intranuclear inclusions and karyomegaly in the renal tubular epithelium of immunodeficient mice . Such obHere, we report on investigations into the chemical nature of these murine renal tubular intranuclear inclusion bodies, and the serendipitous discovery of antibodies that bind to and specifically highlight them. All details about the mice in this study are included in A complete necropsy was performed on all mice by a veterinary pathologist. The kidneys were examined macroscopically and samples of fresh kidney tissue from mice A, C, D, E and F were taken for molecular tests, with a small sub-sample fixed in glutaraldehyde for electron microscopy from these same mice. Kidneys were collected from all mice and fixed in formalin. The formalin-fixed kidney tissues were routinely processed and embedded in paraffin wax, sectioned at 3–4 μm and stained with hematoxylin and eosin, Feulgen reaction, methyl pyronin green and PAS reaction . Mycoplasma pulmonis, mouse cytomegalovirus, mouse adenovirus 1, reovirus type 3, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus 2, murine polyomavirus, Clostridium piliforme, hantavirus, Encephalitozoon cuniculi, cilia associated respiratory bacillus, K virus and mouse thymic virus. For the ELISA tests, the method of McInnes and co-workers (2011) [A whole blood sample was collected by cardiocentesis from mouse B. The blood sample was allowed to clot for 30 minutes and was then centrifuged for five minutes at 604.8g and the serum was harvested and stored at −20 °C. Serum from mouse B was tested for antibodies against the following murine pathogens using the ELISA test: mouse hepatitis virus, mouse minute virus, mouse parvovirus, mouse norovirus, rotavirus, Theiler’s murine encephalomyelitis virus, pneumonia virus of mice, Sendai virus, Kidney tissue in glutaraldehyde from mice A, C, D, E and F was postfixed in 1 % osmium tetroxides, dehydrated and embedded in resin blocks. Sections were stained en-bloc with 1% uranyl acetate and ultrathin sections were then stained with Reynold’s lead citrate and examined with a Hitachi H7500 (A) or CM 100 BioTwin (C & D) transmission electron microscope .® Tissue & Cells DNA Isolation kit ; DNEasy Tissue and Blood (Qiagen) according to the manufacturers’ instructions. For total DNA extracts from mouse A, a PCR reaction mixture contained the following components with final concentration of 20 mmol/L Tris-HCl (pH 8.5 at 25 °C), 50 mmol/L KCl, 2 mmol/L MgCl2, 200 µmol/L each dNTP , 0.5 U of Taq polymerase  and 0.2 µmol/L each primers specific for murine polyomavirus VP2 protein-coding region [Total DNA was extracted from the kidneys of mice A, C, D, E, and F with commercially available kits  [A broad-spectrum papillomavirus PCR was performed on DNA extracts from mice C, D, E, and F, according to the method described in Forslund . 1999) . Multipl. (1999) ,9.99 . MultThe antibodies used for the murine inclusion body IHC were: rabbit polyclonal anti-bovine papillomavirus L1 antibody  (dilution 1:300), Camvir monoclonal anti-papillomavirus antibody (directed against the seven amino acid linear epitope GFGAMDF found in HPV 16 L1 protein)   for 30 minutes followed by treatment with Dako Liquid DAB+ Substrate Chromogen System  for three minutes. The sections were then counterstained in Harris’s haematoxylin, dehydrated, cleared and cover-slipped.The kidneys from all six mice displayed mild to marked, focal to multifocal karyomegaly of tubular epithelial cells. Enlarged nuclei had marginated chromatin and contained pale, eosinophilic, inclusion bodies a. The reThe renal tubular epithelium of the mice examined by electron microscopy displayed multiple, enlarged nuclei within tubular epithelial cells. Chromatin was displaced peripherally within affected nuclei, around abundant electron lucent material . No recoi.e., papillomaviruses, polyomaviruses, circoviruses, anelloviruses) by multiply primed rolling circle amplification. Serology for mouse B was negative for all organisms tested.The kidneys from mouse A were negative for murine polyomavirus by PCR. Mice C, D, E and F were negative for papillomaviruses by PCR, and negative for small circular DNA genomes  is a molecular chaperone that protects cells from the damage caused by physical and chemical hazards . Hsc70 iImmunohistochemistry experiments using three different antibody preparations for detecting papillomavirus L1 protein revealed that no immunostaining was demonstrable using Camvir, which recognises the seven amino acid long linear epitope GFGAMDF of human papillomavirus (HPV) type 16 L1. Conversely, both polyclonal and monoclonal antibodies for bovine papillomavirus (BPV) type 1 L1 showed strong, specific immunostaining of the intranuclear inclusion bodies. Molecular tests and ultrastructural investigations, in this report and in previous studies, have consistently failed to demonstrate the presence of any virus within affected kidney tissue . The immThe monoclonal anti-BPV-1 L1/1H8 antibody found in the mixture with Camvir (Abcam) has not yet had its binding specificity determined. Therefore, it cannot be concluded with any certainty as to which linear or conformational epitope this antibody has bound within the intranuclear inclusion body. The binding specificities of the polyclonal against BPV1 L1 have been described , howeveret al. (2005) [et al. (2009) [There is some published evidence of extra-epithelial localization of human papillomaviruses. Bodaghi . (2005)  provided. (2009)  reportedet al. (2012) [et al. (2011) [More controversially, Roperto . (2012)  claimed . (2011)  claimed Erethizon dorsatum [Mastomys coucha [Mesocricetus auratus [Micromys minutus [Mastomys natalensis [Mus musculus [Rodent species with described papillomaviruses include dorsatum , Mastomys coucha , Mesocri auratus ,21, Micr minutus ,23, Masttalensis , Mus musmusculus ,25,26,27An alternate hypothesis is that an unknown endogenous murine protein, when concentrated within the nucleus, provides linear or conformational epitopes that antigenically resemble part of the BPV1 L1 protein closely enough, that the anti-BPV1 L1 antibodies used in these experiments cross-react to produce strong, specific positive immunostaining.scidPrkdc/J, NOD.CB17-scidPrkdc/J and B6.129S7-tm1MomRag1/J), but the immunodeficiencies of the CBySmn.CB17-scidPrkdc/J and B6.129S7-tm1MomRag1/J mice have been produced through different genetic lesions. This indicates that the propensity to develop murine renal tubular intranuclear inclusion bodies appears to be associated with severe immunodeficiency brought about by impaired adaptive immune system function, and is not a direct product of one particular genetic lesion. It is interesting to note that in the present study 5/6 mice were female and 7/8 mice described by Baze and coworkers [Previous reports have indicated that the inclusions have been noted in immunodeficient mice ,1,28,29.Known nephrotoxins include heavy metals, ochratoxin A , aminoglIn the absence of a more definitive understanding of their composition and pathogenesis, these perplexing inclusions should, for the time being, be considered part of the normal background pathology of immunodeficient mice. Regardless of the precise identity of the binding site for the immunohistochemical reaction, an immunohistochemical method now exists that can be used to confirm and evaluate the histological severity and distribution of suspected cases of murine inclusion body nephropathy.


Scientific Reports7: Article number: 42197; 10.1038/srep42197 published online: 02142017; updated: 01172018.Due to a typesetting error, the original version of this Article contained errors in the Reference list and in-text citations. References 39 to 102 were incorrectly listed as 40 to 103. In addition, reference 103 was incorrectly listed as 39. In the Discussion section,9,36,37,38,39, additionally, the magnetic information is presented close to real time and shares the reference frame of the eyes, ears and vestibular equilibration, favoring temporal and spatial integration39,40,41,42.”“Contrary to tactile waist-stimulation approachesnow reads:9,36,37,38,103, additionally, the magnetic information is presented close to real time and shares the reference frame of the eyes, ears and vestibular equilibration, favoring temporal and spatial integration39,40,41,42.”“Contrary to tactile waist-stimulation approaches31,56–58.”“The perception of self-rotation has multiple, less-well known cortically-based underlying mechanisms that can be recalibrated independently of the VOR and have been shown to integrate signals from multiple sourcesnow reads:32,56–58.”“The perception of self-rotation has multiple, less-well known cortically-based underlying mechanisms that can be recalibrated independently of the VOR and have been shown to integrate signals from multiple sources82,83 or hand7, or magnetic orientation9,36,37,38,39 or path information for wayfinding84,85,86,87,88 using waist-stimulating belts.”“In particular, tactile devices for sensory augmentation have been proposed signaling for instance whisker-like distance information to the headnow reads:82,83 or hand7, or magnetic orientation9,36,37,38,103 or path information for wayfinding84,85,86,87,88 using waist-stimulating belts.”“In particular, tactile devices for sensory augmentation have been proposed signaling for instance whisker-like distance information to the head29,93, according to which conscious perceptual experience is constituted by knowledge and mastery of systematic changes in sensory input resulting from action, termed sensorimotor contingencies (SMCs).”“The idea of contingency mimetic sensory augmentation is inspired by the sensorimotor theory of conscious perceptionnow reads:28,93, according to which conscious perceptual experience is constituted by knowledge and mastery of systematic changes in sensory input resulting from action, termed sensorimotor contingencies (SMCs).”“The idea of contingency mimetic sensory augmentation is inspired by the sensorimotor theory of conscious perceptionFurthermore in the Methods section under the subheading ‘Angular Pointer’,“Participants indicated the perceived size of self-rotation using a custom made potentiometer-based angular pointer .”now reads:“Participants indicated the perceived size of self-rotation using a custom made potentiometer-based angular pointer .”In the Methods section under the subheading ‘Self-Rotation Experiment’:“Before and after the training phase, we tested participants’ vestibular perception of self-rotation using a vestibular adaptation experiment similar to the one developed by refs 34, 102.”now reads:“Before and after the training phase, we tested participants’ vestibular perception of self-rotation using a vestibular adaptation experiment similar to the one developed by refs 35, 102.”In addition, this Article contained errors in the following equations:In Equation 1:now reads:In Equation 2,now reads:These errors have now been corrected in the PDF and HTML versions of the Article.


The infant eventually succumbed to respiratory failure. Bacterial and viral cultures/studies were all no growth/negative. Pulmonary capillaritis and vasculitis were noted at autopsy. This report presents a new case of Noonan syndrome with unusual associated disorders and a review of the literature.We report a term male neonate with congenital myeloproliferative disorder, thrombocytopenia, a horseshoe kidney, feeding difficulty secondary to dysphagia/foregut dysmotility, and respiratory failure. Prenatal molecular genetic analysis revealed a fetus carrying PTPN11, SOS1, SOS2, KRAS, NRAS, RAF1, SHOC2, CBL, RRAS, RIT1, RASA2, MAP3K8, SPRY1, MYST4, LZTR1, A2ML1, and PP1CB) that underlie this disorder or clinically related phenotypes [Noonan syndrome (NS) is transmitted as an autosomal dominant trait and is genetically heterogeneous. So far, heterozygous pathogenic variants have been documented in seventeen genes (Z score), length 44 cm (<−2 Z score), and head circumference 34 cm (−0.25 Z score). Multiple anomalies were noted: hypertelorism, low set ears, short philtrum, high arched palate, short neck, nuchal edema, and widely spaced nipples. Transient thrombocytopenia was noted during the first week of life; the lowest platelet count was 62,000/mm3. Leukocytosis (32–45 × 109/L), monocytosis (8–28%), and blasts (2%) were noted throughout his 76 days of hospitalization. He had feeding difficulties (poor suck) and gastrointestinal symptoms  requiring gavage feeding since the first week of life. A contrast upper GI and small bowel follow-through study at 3 weeks of age showed delayed gastric emptying and pylorospasm. The baby did not thrive despite feeding with high calorie formula/breast milk. Abdominal and renal ultrasound showed a horseshoe kidney and a small splenic cyst (5 × 4 mm).A 3295 g male newborn was delivered at 38 weeks' gestation to a 34-year-old gravida 5 para 2 (2 miscarriages) Mexican mother by cesarean section secondary to prenatal diagnosis of NS fetus with possible thrombocytopenia. Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. Pregnancy was complicated by a prenatal diagnosis of a large cystic hygroma at 16 weeks of gestation; amniocentesis was performed. The karyotype was 46, XY, and the single nucleotide polymorphism (SNP) microarray analysis was normal. Prenatal diagnosis of Noonan syndrome was made based on prenatal ultrasound findings and molecular genetic testing. There was no polyhydramnios noted on prenatal ultrasound. Family history was negative for congenital anomalies and consanguinity. There was no in utero exposure to known teratogens. Physical examination revealed a weight of 3295 grams . At 7 weeks of age, while waiting for cardiac catheterization to delineate the cause of PH, he developed respiratory failure that required extracorporeal membranous oxygenation (ECMO) for more than 3 weeks. Bacterial and viral cultures/studies were all unremarkable. At 11 weeks of age, cranial image revealed severe ischemic changes and parents agreed to withdraw life supporting measures. BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, and SHOC2. The pathogenic variant NS gene PTPN11 (NM 002834.3) was identified, genomic position: Chr12 (GRCh37): g.112888168, cDNA change: c.184T>G, amino acid change: p.Tyr62Asp.The genes that were evaluated wereExternal examination revealed dysmorphic features in keeping with Noonan syndrome. Internal examination confirmed a horseshoe kidney  with extIn 1963, Jacqueline Noonan first described nine NS patients with pulmonary valve stenosis, small stature, hypertelorism, mild intellectual disability, ptosis, undescended testes, and skeletal malformations . Clinica (PTPN11) as the first molecular causes of NS. This discovery was consolidated by the observations that PTPN11 resided within the NS1 critical region and the studies with a mouse model of PTPN11 deficiency revealed that its protein product, SHP-2, was critical for the embryologic development of the semilunar cardiac valves [In 1994, linkage analysis of a large family inheriting NS established definitive linkage to the first NS locus, defined as chromosomal bands 12q22-qter and named NS. Genetic heterogeneity was subsequently established . In 2001PTPN11 pathogenic variants have been found in approximately 50% cases with NS and are also frequently positively associated with familial inheritance, pulmonary stenosis, ASD, HCM, short stature, sternal deformity, bleeding diathesis, juvenile myelomonocytic leukemia (JMML), and NS/MPD [9/L. A monocytic proliferation disorder of NS that was noted in the early neonatal period may resolve spontaneously, persist with or without hepatosplenomegaly, or manifest as chronic myelomonocytic leukemia (CMML) [d NS/MPD , 6, 10. d NS/MPD . The diaa (CMML) . Vasculia (CMML) . LeukocyFeeding difficulties occur frequently but are often unrecognized and cause major management difficulties in patients with Noonan syndrome. Feeding problems appear to be the result of delayed gastrointestinal motor development. However, the underlying etiology is poorly understood. The reported causes of feeding difficulties in NS patients were gastroesophageal reflux, delayed gastric emptying, and pseudo-obstruction syndrome . The finThe prevalence of horseshoe kidney (HK) is approximately 0.25% in general population and 15% in females with Turner syndrome . There w c.184T>G (p.Tyr62Asp) variant in PTPN11. The patient succumbed to complications of acute respiratory failure with pulmonary capillaritis and vasculitis. The pulmonary vasculitis and horseshoe kidney have not been previously reported in NS.In summary, we report a neonate with severe NS phenotypes including myeloproliferative disorder, transient thrombocytopenia, a secundum atrial septal defect, moderate perimembranous ventricular septal defect, dysplastic mitral and tricuspid valves, bicuspid aortic valve and mild hypoplastic aortic arch, pylorospasm, splenic cyst, and a horseshoe kidney. The molecular genetic analysis revealed a neonate carrying


Arabidopsis give rise to new paradigms. Given the comprehensive nitrate transport, sensing, signaling and regulations at the level of the cell and organism, nitrate itself is a local and long-distance signal molecule, conveying N status at the whole-plant level. A direct molecular link between nitrate signaling and cell cycle progression was revealed with TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) – NIN-LIKE PROTEIN 6/7 (NLP6/7) regulatory nexus. NLPs are key regulators of nitrogen responses in plants. TCPs function as the main regulators of plant morphology and architecture, with the emerging role as integrators of plant developmental responses to the environment. By analogy with auxin being proposed as a plant morphogen, nitrate may be an environmental morphogen. The morphogen-gradient-dependent and cell-autonomous mechanisms of nitrate signaling and regulation are an integral part of cell growth and cell identification. This is especially true in root meristem growth that is regulated by intertwined nitrate, phytohormones, and glucose-TOR signaling pathways. Furthermore, the nitrate transcriptional hierarchy is emerging. Nitrate regulators in primary nitrate signaling can individually and combinatorially control downstream transcriptional networks and hormonal pathways for signal propagation and amplification. Under the new paradigms, nitrate-induced hormone metabolism and signaling deserve fresh examination. The close interplay and convergent regulation of nitrate and hormonal signaling at morphological, physiological, and molecular levels have significant effects on important agronomic traits, especially nutrient-dependent adaptive root system growth and architecture.In nature and agriculture, nitrate availability is a main environmental cue for plant growth, development and stress responses. Nitrate signaling and regulation are hence at the center of communications between plant intrinsic programs and the environment. It is also well known that endogenous phytohormones play numerous critical roles in integrating extrinsic cues and intrinsic responses, regulating and refining almost all aspects of plant growth, development and stress responses. Therefore, interaction between nitrate and phytohormones, such as auxins, cytokinins, abscisic acid, gibberellins, and ethylene, is prevalent. The growing evidence indicates that biosynthesis, de-conjugation, transport, and signaling of hormones are partly controlled by nitrate signaling. Recent advances with nitrate signaling and transcriptional regulation in  As a constituent of amino acids and nucleotides, nitrogen (N) is an essential building block for all forms of life. Not surprisingly, the mineral nutrient needed in greatest abundance by plants is N . N availPlants are sessile organisms that always face spatiotemporal fluctuations of nitrate concentrations in soil solution by up to four orders of magnitude due to leaching and microbial activity . In inteArabidopsis thaliana as a model. The accumulating evidence indicates that biosynthesis, de-conjugation, degradation, transport, and signaling of hormones are partly controlled by nitrate signaling, so that the environmental and internal signaling pathways are seamlessly integrated.Furthermore, nitrate and hormonal signaling and their interaction are of fundamental importance, underlying a plethora of plant physiological, morphological, and developmental processes in plants . Much ofMuch of the literature on the interaction between nitrate and hormonal signaling pathways has been focused on hormonal control of nitrate metabolism and signaling , while tA. thaliana, two NR enzymes, NIA1 and NIA2, are responsible for 10 and 90% of the total NR activity in seedlings, respectively , reducing equivalents, and C skeletons (Nitrate is taken up by roots then transported into root and shoot cells mainly via the NITRATE TRANSPORTER 1 (NRT1) and NITRATE TRANSPORTER 2 (NRT2) family of nitrate transporters . Once inectively , 1993. Nectively . Notablyectively . In addikeletons . Therefonia1 nia2) mutants, numerous genes, including the key nitrate assimilatory genes, directly respond to nitrate independent of nitrate reduction  plants. Among those genes, 595 genes in both roots and shoots directly responded to nitrate, which is confirmed in the NR-null mutant (nia1 nia2)  . The PNRa1 nia2) .Indeed, the nitrate response is a whole-plant response and nitrate itself can function as both a local and long-distance systemic signal . The pheNitrate signaling and regulation underlie the genome-wide expression reprogramming in nitrate responses, which leads to activation and adaptation of N-regulated metabolism and development. The process involves membrane and cytosol sensing, signal transduction, transcription factors (TFs), the interactions of TFs, and nitrate-responsive DNA regulatory elements . On the In plants, the first identified and characterized nitrate transporter is known as chlorate resistant 1 (CHL1) or AtNRT1.1 or AtNPF6.3 . It is aFigure 1). All transport proteins are localized at the plasma membrane, except that NRT2.7 and CLCa/b are localized at the tonoplast , NRT1.2 (NPF4.6/AIT1), NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in nitrate uptake from soil ; NAXT1 ; NRT1.5 onoplast .Arabidopsis roots, LATS involves NRT1.1 (CHL1/NPF6.3) and NRT1.2 (NPF4.6/AIT1)  gradients, which potentially provides required energy to both LATS and HATS for co-transporting two or more protons per nitrate into the cell, a process also involving membrane depolarization. Both nitrate influx and efflux could be mediated by the proton-coupled mechanism  for high external nitrate concentration (>0.5 mM) and high-affinity transport system (HATS) for low nitrate concentration (<0.5 mM), into which most nitrate transporters in roots and shoots can be categorized . All kno.6/AIT1) , and HAT.6/AIT1) . The NRT.6/AIT1) . When roechanism .Arabidopsis, NRT1.1 (CHL1/NPF6.3) is the only dual-affinity transporter , NRT1.1m sensor . Throughm sensor . NRT1.1 m sensor . It is am sensor . Neverthm sensor . NRT2.1 m sensor .per se, subgroup III calcium-dependent protein kinases (CPKs), CPK10, CPK30, and CPK32 are also required for rapid nitrate-induced cellular and metabolic responses, and nitrate-regulated root and shoot growth , NLP6, NLP7, LOB DOMAIN-CONTAINING PROTEIN 37/38/39 (LBD37/LBD38/LBD39), SQUAMOSA PROMOTER BINDING PROTEINLIKE 9 (SPL9), HIGH NITROGEN INSENSITIVE 9 (HNI9), NAC DOMAIN-CONTAINING PROTEIN 4 (NAC4), BASIC LEUCINE-ZIPPER 1 (bZIP1), TGACG MOTIF-BINDING FACTOR 1/4 (TGA1/TGA4), TCP20, HYPERSENSITIVE TO LOW PI-ELICITED PRIMARY ROOT SHORTENING 1 (HRS1), NITRATE REGULATORY GENE2 (NRG2), BRIC-A-BRAC/TRAMTRACK/BROAD-COMPLEX 1/2 (BT1/BT2), and NLP8; -calcium signaling pathway downstream of NRT1.1 (CHL1/NPF6.3)  . In resp/NPF6.3) .Figure 2) , NRT2.1 and NIA1 at different nitrate concentrations   . Moreovetrations . In conttrations . It is wtrations . The inttrations . Most retrations . Centereigure 2) .TCP20 and NLP6/7 belong to two ancient gene families, the protein sequences of which contain multiple, deeply conserved motifs in plants  mutants are comparable. Putative binding sites of NLP6 and NLP7 were found in the CYCB1;1 promoter region. The defective CYCB1;1 expression was also observed in nlp6 nlp7 double mutants, which, however, did not satisfy the significance test when total roots were measured, but would likely pass the test if measuring only root tips. Under N starvation, TCP20-NLP6/7 heterodimers accumulate in the nucleus. The transcriptional complexes not only bind to and upregulate sentinel nitrate-responsive genes for transport, assimilation and signaling, but also bind to and downregulate CYCB1;1, a division marker of apical meristems, for the control of G2/M transition in cell cycle progression, supporting root apical meristem (RAM) growth .TCP20 and NLP6/NLP7 bind to adjacent sites in the upstream promoter region of the NR gene, lability . The reglability . In the lability . The sev) growth , 2017. T and ORC . TherefoCYCB1;1 and putatively ribosomal protein genes by binding to the GCCCR motif in their promoters in vitro and in vivo, which was proposed to be a mechanism for regulation of the cell cycle and cell growth at a synchronized rate , which is at the interface of growth and nutrient availability in unicellular organisms and through acquiring additional roles, becomes a central controller of organism growth, and energy and nutrient homeostasis in multicellular organisms . (m)TORCzing NUE . Intriguzing NUE . Moreovezing NUE . The groInterestingly, the TCP20-NLP6/7 regulatory nexus employs the type I/II Phox and Bem1p (PB1) domains of NLP6/7, a protein-interaction module conserved in animals, fungi, amoebas, and plants . In animArabidopsis, RAM growth is under the antagonistic effects of auxin and CK, which mediate cell division at the apical meristem and cell differentiation at the transition zone, respectively  in WT (in both cases TOR activity is inhibited) and in occurred . Under Nansition . Auxin aansition . The accArabidopsis seedlings  that gave rise to all currently known living eukaryotic species . Althougeedlings . Could TThe glucose-TOR signaling imposes an overall limit on plant organ growth, specifically on organ size . By contArabidopsis roots, defective localized auxin biosynthesis cannot be replenished by auxin transported from shoots, indicating that shoot-derived auxin alone is not sufficient for supporting root elongation and root gravitropic responses  were compared with those under low N conditions (0.1 mM NH4NO3) after 7 days treatment  are among the top NLP7-activated genes, together with nitrate assimilation genes such as NiR, NIA1, FNR2, and NRT2.1 .However, with ammonium in the media, it was difficult to identify which N source, nitrate or ammonium, or both of them, could be responsible for the gene expression regulation. The recent genome-wide transcriptional profiling showed that d NRT2.1 . Plasma d NRT2.1 . This evXenopus oocytes. It is also responsible for modulation of auxin gradient in LR primordium  exposure, and the nitrate induction of miR393 peaked at 2 h, strikingly coinciding with the fast declining of the already peaked expression of AFB3. This miR393-dependent repression was not observed in NR-null mutants, correlating with the absence of miR393 expression. Further evidence support that nitrate signal alone is responsible for the transcriptional induction of AFB3 in root tips, which can be subsequently post-transcriptionally repressed by miR393 induced by unidentified N metabolite(s) downstream of nitrate reduction. Such a mechanism agrees with the type I incoherent feed-forward loop (FFL) motif featured in transcriptional controls in yeast, bacteria, and mammals  that allows auxin-responsive transcription  in regulating nitrate response of AFB3 and NAC4 were tested , only the transport function of the NRT1.1 (CHL1/NPF6.3), not those of NRT2.1, NRT1.2 (NPF4.6/AIT1), and NRT2.2, matters on the expression levels of AFB3 and NAC4. Moreover, the nitrate induction of AFB3 and NAC4 was independent of affinity mode of NRT1.1 (CHL1/NPF6.3). Notably, the NRT1.1T101D was demonstrated to modulate auxin gradient in LRP as WT NRT1.1 (CHL1/NPF6.3) in the absence of nitrate, which excludes the possible involvement of auxin transport of NRT1.1 (CHL1/NPF6.3) in the nitrate induction of AFB3 and NAC4 (under 5 mM nitrate treatment). It was suggested that an unidentified signaling pathway independent from the signaling via NRT1.1 (CHL1/NPF6.3) phosphorylation was triggered by NRT1.1 (CHL1/NPF6.3) transport of nitrate  mutants: e tested . Specifiity mode . Interes nitrate . It furtdination .de novo CK synthesis and accumulation in the roots of barley, maize, and Arabidopsis (adenosine phosphate-isopentenyltransferases (IPTs) are expressed. IPTs are key enzymes that catalyze the first and rate-limiting step of CK biosynthesis, i.e., prenylation of adenosine 5′ phosphates, such as ATP and ADP, at the N6-terminus with dimethylallyl diphosphate (DMAPP) , is induced by nitrate during the PNR  (3) was re-supplied to nitrogen-limited seedlings, the kinetics of IPT3 and NIA1 transcripts that were rapidly accumulated within 1 h. resembled each other  CK in roots, while it downregulates that of LOG5. The type-A ARR genes ARR3, ARR5, and ARR7, like the CK metabolism genes, were shown to respond to nitrate but not to ammonium (CYTOKININ RESPONSE FACTORS (CRFs)  are a class of phytohormones known for promoting cell division and differentiation . CKs canbidopsis . CK bios (DMAPP) . IPT exp the PNR . IPT3 wa/NPF6.3) , 2009. Wch other . NIA1 is the PNR ; therefo in soil . More reammonium . Also inS (CRFs) , which iS (CRFs) .IPTs, IPT3 is mainly expressed in phloem tissue throughout the whole plant, specifically found in phloem companion cells, and IPT5 is in the LR primordium and pericycle, which are consistent with where CK biosynthesis is suggested to occur. The spatial differentiation of IPTs transcript also suggests that in terms of CK production, IPT5 and IPT3 could contribute most in roots, while IPT3 is most dominant in shoots. IPT5 was not responsive to either nitrate or ammonium under a short-term hour-long treatment, however, was demonstrated to be a “long-term” or systemic N status-responsive gene, with its transcript abundance being responsive to both nitrate and ammonium media at different concentrations over a long course of observation, 11 days. By contrast, IPT3 expression pattern (strongly in roots and weakly in shoots) is quickly induced during the PNR, a frequently occurring whole-plant nitrate response due to dramatic fluctuations of nitrate in soil , CK signaling has a relatively delayed (within 4 h) feedback control over most the IPTs, IPT1,3,5,7 by downregulating them in roots, where IPT5 and IPT7 can be upregulated concurrently by auxin  was globally up-regulated by nitrate in roots and shoots at a later time  compared with the much quicker expression of IPT (within 1 h)  . The res of IPTs . Since bre rapid , the muc nitrate  suggest tcp20 mutants strongly suppress the preferential growth of LRs by equalizing growth across heterogeneous nitrate environments, mainly through increasing the LR growth on low-nitrate media of split-root plates as if the plants were impervious to any systemic signal. An earlier study showed that the main class of AtTCP20::EAR-repressed genes include ARR4,6,7 and AUX/IAA13,16,27 that repress ARFs in auxin signaling  corresponding to steady-state cytosolic nitrate concentrations (4–6 mM) , additioR growth .Arabidopsis due to very high nitrate (30 mM) resembled the repressed growth of LR treated by 30 mM KCl or 60 mM mannitol. All the treatments, including high nitrate (>30 mM), could impose osmotic stress  (Arabidopsis. And notably the high nitrate-ABA pathway is independent of auxin (The accumulation of ABA was detected mainly in the endodermis and quiescent center of cylinder . A three(ABA-GE) . In para(ABA-GE) . All sugof auxin .Arabidopsis  is constitutively expressed before and after nitrate exposure (ABA-IMPORTING TRANSPORTER (AIT) 1, also characterized as the constitutive, low-affinity nitrate transporter NRT1.2 (NPF4.6), mediates cellular ABA uptakes during seed germination and post-germination growth of bidopsis . In respsistance . The twosistance . With thexposure .2+-sensor moiety in the associated CBL  in the ABA signal-transduction cascade  has been identified as a potential interacting protein of the CBL1-CIPK23 complex, which like CBL9-CIPK23 has inhibitory effects on nitrate transport of NRT1.1 (CHL1/NPF6.3), under >30 mM nitrate . The CBLated CBL . During esponses . It sugg cascade . The NO  cascade .CYP707A2, which is indispensable for nitrate-induced seed germination. This activation appears to occur directly, through NLP8 binding to the promoter of CYP707A2, which encodes ABA 8′-hydroxylase, a key ABA catabolic enzyme , which are catalyzed by ACC synthase (ACS) and ACC oxidase (ACO)  . The nitChl1-5 and nrt2.1-1 mutants and ethylene-insensitive mutants, etr1-3 and ein2-1, ethylene was demonstrated as an important modulator in the regulation of nitrate-dependent expression of the two main transporters, NRT1.1 (CHL1/NPF6.3) and NRT2.1 , is repressed and activators of flowering, FLOWERING LOCUS T (FT), LEAFY (LFY), and APETALA1 (AP1), are induced in low-nitrate conditions. Interacting with photoperiod- and temperature- and GA-signaling pathways, nitrate regulates floral induction by communicating nutrient availability , an integrator of the GA-dependent flowering pathway which coordinates all the endogenous pathways: GA, vernalization, autonomous, and photoperiod (Arabidopsis seedlings in response to nitrate re-addition (3 or 5 mM KNO3) also showed the repressed expression of GID1B, GA receptor and the induced expression of GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED (GNC) and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 (GNL/CGA1) TFs, negative regulators of GA signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS (PIFs)  .NPF3.1 expression in endodermis were found to be transcriptionally repressed by GA and promoted by ABA  being an environmental morphogen in embryonic development and stem cell function in animals .Why does nitrate signaling critically regulate so many types of phytohormones at so many levels? The localized hormone biosynthesis, deconjugation and degradation seem to be the primary connection between nitrate and hormones, for which solid molecular evidence has been increasingly found. Being an environmental cue and once being absorbed into plants, also becoming an environmental morphogen, nitrate transcriptionally regulates the metabolism and signaling of hormones at the whole plant level. Indeed, this regulatory process is highly context-dependent and spatiotemporal. Conversely, the hormones from nitrate-induced production, deconjugation and degradation, could act as mediators and/or modulators in N-dependent signaling and regulation and provide feedback controls at the regional level. Notably, numerous hormone signaling and regulatory components were found to be transcriptionally activated by nitrate in certain contexts, suggesting that the signaling of the two morphogens is intertwined at multiple levels, if not all levels. The genome-wide transcriptome further revealed that the molecular conductors at the top-level of nitrate regulatory hierarchy could exert direct controls over hormonal pathways , so thatDictyostelium discoideum (Intriguingly, cell-autonomous regulation by N in determining cell growth and fate is strongly indicated by TCP20-NLP6/7 regulatory nexus, which is involved in sensing nutrient status and transcriptional control of G2/M transition in cell cycle progression . Growingscoideum .Hormones have long been regarded to provide an indispensable link between N and plant growth and development. However, among the most deeply conserved in plants, nitrate signaling and regulation with a highly organized transcriptional hierarchy are as crucial as hormonal signaling and regulation in growth, development and stress responses. The novel model that underlies substantial plant development and adaptive responses could involve other TCPs because of functional redundancy between TCP20 and its homologs. The classes I and II TCPs exert either coordinate or competitive regulation of transcription that could be essential for defining growth rate and organ development . The intIn the past half century, N fertilizer is the main contributor to global crop production increases that support two billion more people on Earth. Nevertheless, NUE has become a major constraint on agricultural productivity and environmental sustainability worldwide. Understanding nitrate signaling and regulation and their interaction with hormones is central to meet the global challenges, which demands extensive research under the new paradigms.The author confirms being the sole contributor of this work and approved it for publication.The author declares 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 reviewer EW and handling Editor declared their shared affiliation.


Chemoresistance is a significant factor associated with poor outcomes of osteosarcoma patients. The present study aims to identify Chemoresistance-regulated gene signatures and microRNAs (miRNAs) in Gene Expression Omnibus (GEO) database. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) included positive regulation of transcription, DNA-templated, tryptophan metabolism, and the like. Then differentially expressed genes (DEGs) were uploaded to Search Tool for the Retrieval of Interacting Genes (STRING) to construct protein-protein interaction (PPI) networks, and 9 hub genes were screened, such as fucosyltransferase 3 (Lewis blood group) (FUT3) whose expression in chemoresistant samples was high, but with a better prognosis in osteosarcoma patients. Furthermore, the connection between DEGs and differentially expressed miRNAs (DEMs) was explored. GEO2R was utilized to screen out DEGs and DEMs. A total of 668 DEGs and 5 DEMs were extracted from GSE7437 and GSE30934 differentiating samples of poor and good chemotherapy reaction patients. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform GO and KEGG pathway enrichment analysis to identify potential pathways and functional annotations linked with osteosarcoma chemoresistance. The present study may provide a deeper understanding about regulatory genes of osteosarcoma chemoresistance and identify potential therapeutic targets for osteosarcoma. Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. The worldwide morbidity rates of osteosarcoma are approximately with an average incidence of 3.1 per million for each stage and 4.4 per million for groups <25 years old. Additionally, there is a bimodal age distribution: individuals aged 25–60 years and elderly individuals, respectively. In America the Annual age-standardized incidence of osteosarcoma has reached stabilization from 1976 to 2005 [Many factors are associated with tumor genesis, including high birth weight , pubertaMultidrug resistance protein 1 (MDR1) that is encoded by gene ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1) has been shown to serve as a plausible factor in doxorubicin resistance, which was validated to be linked with poor outcomes in many osteosarcoma studies , 17, buthttp://www.ncbi.nlm.nih.gov/geo/geo2r/). To further understand the function of genes, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Protein-Protein Interaction (PPI) networks, and the connections among DEGs and DEMs were performed in sequence. We selected chemoresistance development related key genes and provided theoretical foundations for modifying and improving osteosarcoma treatment methods.In the present study, we analyzed the data of GSE87437 and GSE30934 submitted by Serra Mand and Kobayashi E. et al., respectively, to get 668 differentially expressed genes (DEGs) and 5 differentially expressed miRNA (DEMs) between samples of poor and good chemotherapy reaction patients in GEO2R  and the latter one was based on GPL10312 platform (3D-Gene Human miRNA Oligo chip v12-1.00).P < 0.05 and |logFC|⩾ 1 were considered as cutoff criterion. All results of DEGs were downloaded in text format, hierarchical clustering analysis being conducted later in Morpheus (https://software.broadinstitute.org/morpheus/).GEO2R, an R-associated web application, was applied to filtrate DEGs between good chemotherapy reaction samples and poor chemotherapy reaction samples. In total, 21 samples in GSE87437 and 24 samples in GSE30934 were divided into two groups, respectively, and the concrete grouping schemes were already shown in microarray data. The https://david.ncifcrf.gov/) provided comprehensive information for list of genes by GO and KEGG pathway analyses. In addition, GO enrichment analysis included three different aspects: biological process (BP), molecular function (MF), and cellular component (CC) [P < 0.05, the results of which were downloaded in text format.The online tool, Database for Annotation, Visualization and Integrated Discovery  . KEGG enent (CC) . The scrhttps://string-db.org/), a database covering 9,643,763 proteins from 2,031 organisms, and the result whose minimum interaction score was 0.4 was visualized in Cytoscape [k-core = 2, and max. depth = 100 and hub genes were exported. The functional enrichment analysis of genes in each module was performed in DAVID. Besides, the genes in each module were uploaded to DAVID and KEGG pathway enrichment analysis was conducted with the condition of P < 0.05.To analyze the connection among proteins, DEGs were uploaded to Search Tool for the Retrieval of Interacting Genes , an integrated miRNA target prediction platform including 10 databases , was utilized to explore the correlation between DEMs and DEGs. Besides, different colors were used to indicate the degrees of connections. For example, red color represented strong correlation.DEMs were acquired by the parallel method of DEGs mentioned above. miRWalk1.0 , including positive regulation of transcription and DNA-templated, positive regulation of sequence-specific DNA binding transcription factor activity, nitric oxide mediated signal transduction, positive regulation of transcription from RNA polymerase II promoter, and regulation of phosphatidylinositol 3-kinase signaling. As for molecular function (MF), the DEGs were enriched in estrogen response element binding, Rac guanyl-nucleotide exchange factor activity, calcium ion binding, zinc ion binding, and phosphatidylinositol-4,5-bisphosphate 3-kinase activity. Besides, Cellular Component (CC) analysis showed that the DEGs were enriched in proteinaceous extracellular matrix, cell surface, P granule, integral component of plasma membrane, and endocytic vesicle membrane, as shown in KEGG pathway analysis showed that DEGs were mainly involved in tryptophan metabolism, oxytocin signaling pathway, glyoxylate and dicarboxylate metabolism, cAMP signaling pathway, and dopaminergic synapse .The PPI networks of DEGs were composed of 432 nodes and 428 edges . Then th9 hub genes were screened out, including zinc ribbon domain containing 1 (ZNRD1), myosin heavy chain 7B (MYH7B), G protein-coupled receptor 68 (GPR68), catalase (CAT), fucosyltransferase 3 (Lewis blood group) (FUT3), interphotoreceptor matrix proteoglycan 2 (IMPG2), G protein-coupled receptor 180 (GPR180), alanyl aminopeptidase, membrane (ANPEP), and cyclin dependent kinase 1 (CDK1) . Next, sP < 0.05 and |logFC|⩾ 1.0 , the relationship between miRNAs and DEGs was acquired and different kinds of colors were on behalf of the number of miRNA–DEG pairs in different database which stand for the degrees of connection. For example, red color represented to a strong correlation , upregulated DARPP-32/downregulated ZNRD1, overexpressed miR-508-5p/ZNRD1/ABCB1 activities, respectively –28, but δ) signaling pathway. Besides, hsa-miR-543 was also proved to be linked with tumor staging [Compared with that of good response, 5 DEMs were acquired in GSE30934 in poor chemotherapy response samples . Among t staging , cell pr staging . The stu staging . Besides staging . ConsequIn summary, we identified 668 DEGs and 5 DEMs from GEO2R between good chemotherapy response samples and poor chemoresistance samples in osteosarcoma. And many of them, such as ZNRD1, GPR68, CAT, FUT3, ANPEP, CDK1, and hsa-miR-543, might be key genes related to osteosarcoma chemoresistance. These findings provided a series of promising treatment targets and enlightened us on the further investigations of the molecular mechanisms.


TP53 coding protein sequence are involved in cancer onset and progression. The Hippo pathway is a key regulator of developmental and regenerative physiological processes but if deregulated can induce cell transformation and cancer progression. The p53 and Hippo pathways exert a plethora of fine-tuned functions that can apparently be in contrast with each other. In this review, we propose that the p53 status can affect the Hippo pathway function by switching its outputs from tumor suppressor to oncogenic activities. In detail, we discuss: (a) the oncogenic role of the protein complex mutant p53/YAP; (b) TAZ oncogenic activation mediated by mutant p53; (c) the therapeutic potential of targeting mutant p53 to impair YAP and TAZ oncogenic functions in human cancers.p53 protein is a well-known tumor suppressor factor that regulates cellular homeostasis. As it has several and key functions exerted, p53 is known as “the guardian of the genome” and either loss of function or gain of function mutations in the  TP53 gene exhibits a high mutation rate in human tumors (about 50–70%) which results either in loss or gain of function of oncogenic mutant p53 proteins [The p53 protein is a key factor for the safeguard of different cellular processes such as gene expression, cell cycle regulation, DNA damage repair, ageing, cell migration inhibition, and apoptosis. It was first identified in 1979 as a partner of the T antigen in SV40 viruses ,3,4. Theproteins ,7,8.The Hippo pathway is evolutionarily conserved and tightly controls embryos and adult tissue organ size and growth. It is heavily involved in the control of proliferation, organ size and shape during development, stem cell maintenance, metastasis, tissue regeneration, apoptosis, senescence, and differentiation. It is regulated by many factors such as cell density and polarity, metabolism, senescence, and DNA damage ,10,11,12p53 is a short-lived protein  that shuttles from cytoplasm to nucleus in response to different stresses that can erode the genome integrity  ,22. p53 The most important biochemical activity of p53 is the ability to regulate gene transcription. Through its TA domain, p53 interacts with TBPs and TAFs inducing the recruitment of the RNA Polymerase II and consequently the transcriptional activation of its targets genes harboring a p53 responsive element in the DNA sequence (p53RE) . p53 traWAF1/CIP1P21 transcription that inhibits cyclin dependent kinases/cyclin complexes. This results in the activation of the G1 checkpoint [14-3-3σ transcription, which inhibit Cdk1/cyclin B complexes [The p53 protein exerts an important role in regulating cell cycle checkpoints ,35. In peckpoint ,37,38. Meckpoint . When aceckpoint . In respeckpoint ,43,44,45omplexes . Moreoveomplexes ,48 and iomplexes .trans-activates different genes involved in cell cycle arrest, DNA damage repair or apoptosis [TP53 is deleted or mutated, DNA damage will persist and cells replicate transmitting altered genetic heritage; this mechanism could lead to a neoplastic transformation. Moreover, p53 exerts other functions contributing to the maintenance of genome integrity: it exerts a 3′–5′ exonuclease activity [One of the main functions of p53 is the maintenance of genome integrity. It has been shown that different types of DNA damage, such as ionizing radiation, chemicals, radiomimetic drugs, ultraviolet radiation, and some pharmacological treatments can induce DNA damage . When thpoptosis ,52. If Tactivity ; p53 preactivity . p53 canactivity ; moreoveactivity ,58.INK4P16 and PML, two key factors in senescence, are p53 transcriptional targets [The p53 family members are involved in regulating ageing processes ,59,60. M targets . Normal  targets  that bot targets .BAX [PUMA [FAS-R promoting apoptosis [IGF-BP3 coding Igf-bp3 protein that can bind to the growth factor IGF-1 and inhibits proliferation stimuli transduced by IGF-R receptors; p53 can also interact with WWOX inducing apoptosis synergistically [The most studied p53 function is the induction of apoptosis. The first evidence came from a seminal work of Oren’s lab, in which p53 reintroduction in myeloid leukemia cells-deficient for p53 promoted apoptosis ,68. StudBAX , PUMA [5AX [PUMA  and FAS-poptosis . Moreovestically . Withoutstically . Treatmestically ,77.TIGAR and GLS2 target genes [GLS2 promoter inducing its transcription. This activation leads to increased glutamine metabolism and reduced ROS production [SIRT1, Aromatase, Acad11, Lipin1, MCD, and DHRS3) or also activate proteins that regulate cholesterol efflux (Caveolin 1) [p53 plays a role in the regulation of cell metabolism. It has been shown that p53 regulates glucose uptake and glycolysis reducing the expression of glucose transporters , phosphoglycerate mutase (PGM), and fructose-2,6-bisphosphate levels ,79; moreet genes ,80,81 inet genes ,83 and cet genes ,86,87,88oduction , and theoduction . In detaoduction ,91 or treolin 1) ,97,98,99eolin 1) .p53 metabolic regulation appears crucial in the inhibition of cell transformation and tumorigenesis considering that tumor cells change their metabolism to support their rapid growth. In particular, tumor cells exhibit an increased glucose uptake, glycolysis, lactate production, glutamine consumption, nucleic acid metabolism, and lipid and cholesterol synthesis ,101,102.Cell migration is required for the acquisition of migratory and metastatic behavior in cancer cells. p53 protein regulates cellular expansion, cell polarity formation, and cell protrusion formation, which are required for cell motility . The mosp53 also exerts a key role in the negative regulation of angiogenesis activating a potent inhibitor of this process (TSP-1) and inhibiting the collagen α(II)PH protein involved in the growth of primary human endothelial cells ,126.As seen in all the functions previously described, p53 deregulation is primarily involved in cancer progressions instead of early tumorigenesis . Indeed,TP53 coding gene is frequently mutated in human cancers (50–70% of cases) [TP53 denote increased cancer risk (around 90–95%) frequently in early ages [TP53 mutations are dominant negative and in heterozygous we can observe tetramers composed by native and mutant subunits that inactivate native ones abolishing p53 wild-type functions [WAF1/CIP1P21 activation [The f cases) ,134,135.f cases) ,137,138.f cases) ,139 and f cases) ,132. Mosf cases) ,140 and unctions ,141,142.unctions ,145,146.unctions ,148,149.unctions ,154,155.tivation ,151,156.tivation . ASNS, and EEF1A2), in cell metabolism , in cell cycle progression , in oncogenic transformation, cell migration and invasion , in DNA replication and transcription (POLD2 and RNA pol II E) and in anti-apoptotic signaling . All these mutant p53-dependend upregulated genes promote tumor transformation and progression in different cancer cell lines [Mutant p53 proteins show two critical residues in the N-terminal domain (TA), which are necessary for oncogenic function ,159. Difll lines ,164,165.ll lines . It is sll lines ,168.MSP/MST-1 gene [IL-6) binding to and activating the transcription factor C/EBP β [HSP70, EGFR, hsMAD1, PCNA, and NF-κB transcription and activation [CCNA, CCNB1, CDK1, and CDC25C) resulting in increased cell proliferation [MDR1 gene promoting chemoresistance [ID2 and ID4 transcription promoting tumor neo-angiogenesis [BRCA1 and RAD17 genes impairing the DNA repair mechanism and promoting cancer genomic instability [REGγ, which promotes p53 wild-type, p21, and p16 protein degradation [KLF17 promoting cancer progression in invasive breast cancer cells [Beside metabolic regulation that favors tumor transformation, p53 mutants can reinforce anti-apoptotic signaling downregulating the T-1 gene , or prom C/EBP β . Moreovetivation ,176,177.feration . Moreovesistance ; alternaogenesis ,180. Bintability . Mutant radation  and inhier cells . Increaser cells . In pancer cells . It was er cells ,187,188.PDGFRb in pancreatic cancer metastasis [Mutant p53 proteins are abundantly present in tumor specimens and cancer cells due to increased protein stability and nuclear localization ,189. Thetastasis . Similartastasis . Mutant tastasis . It was tastasis ,201,202.tastasis . Interestastasis . The protastasis . Recent tastasis ,207.Drosophila melanogaster. Loss of function mutation in these genes results in overgrowth and reduced cell death [Questions about developmental processes of multicellular organisms led to the discovery of four tumor suppressor genes in ll death ,209,210.ll death .SOXS2 and SNAI2) [CTGF, ANKRD1, CYR61, Birc2/cIAP1, Birc5, AREG, and cMyc [DDIT4 and TRAIL recruiting the NuRD deacetylase histones complex onto the promoters of selected genes [Mst1/2, the adaptor protein Sav1 (or WW45), Lats1/2 AGC family kinases, and MOBKL1A/B (or Mob1) compose the mammalian Hippo kinase cassette ,212. Mstd SNAI2)   ,223. Somand cMyc ,224. YAPed genes  , Lats1/2 exert an oncogenic function reducing tumor immunogenicity. In detail, Lats1/2 deletion induces interferon I response improving tumor vaccine efficacy; this mechanism opens possibilities in targeting Lats1/2 for cancer immunotherapy and suggests an additional function exerted by the Hippo pathway .The NF2-Frmd6/1-WWC1/2 and Lin-7C/MMP5/Patj/Mpdz tumor suppressor complexes are involved in the mammalian apical-basal cell polarity (ABCP) and induce YAP/TAZ phosphorylation and inhibition ,219. AMOOrgan size is finely regulated during development, and the Hippo signaling pathway plays a key role in this process . YAP/TAZThe Hippo network is also a key player in the regulation of cell metabolism, and conversely is regulated by metabolic factors. It was shown in zebrafish models, that YAP upregulates nucleotide biosynthesis through the induction of glutamine synthetase. This promotes tissue growth during either development or tumorigenesis . Mst1 kiDown-regulation of the core kinase cassette components or activators of the Hippo pathway results in uncontrolled tissue overgrowth; moreover, YAP/TAZ overexpression induces cell proliferation, inflammation, acquisition of cancer stem cell features, EMT (forming metastases), inhibition of senescence, suppressed anoikis, reduced apoptosis, and drug resistance ,242,243.MCM7 gene and its hosted miR-25, 93, and 106 cluster [MT1DP inhibiting its expression. Down-regulation of MT1DP enhances FoxA1 activity with consequent induction of the oncogenic factor AFP, a classic liver cancer biomarker [MALAT1 expression at both transcriptional and post-transcriptional level facilitating proliferation and enhancing cell migration [RMRP involved in ribosomal RNA processing [Recently it was demonstrated that YAP could regulate non-coding RNAs biogenesis associated with cancer. In detail, at low cell density, nuclear YAP binds to and sequesters a regulatory protein of the miRNA-processing machinery, p72. This leads to widespread miRNA suppression in cells and tumors with concomitant cMyc post-transcriptional induction . In lung cluster . Moreove cluster . In liveiomarker . It is aigration . Anotherocessing . The Lncocessing . LncARSRIt is known that the Hippo pathway and p53 act as tumor suppressors cooperating to induce senescence and apoptosis. In particular, for the Hippo pathway, this is mediated by the canonical function of inhibiting YAP and TAZ oncogenic activation, as previously described, and by additional non-canonical activities in which YAP functions as a tumor suppressor in response to stress conditions. PUMA and p53AIP1 [PML gene is a target of the transcriptional competent complex YAP/p73; this leads to an auto-regulatory feedback loop that stabilizes YAP and maximizes p73’s pro-apoptotic activity [Bai et al., demonstrate that HepG2 cells treated with diverse chemotherapeutics bear increased YAP nuclear accumulation that contributes to chemosensitivity . In deta p53AIP1 . YAP fun p53AIP1 ,259. In activity ,261,262 activity  , but not wild-type p53 protein, can physically interact with YAP . The proferation   to pro-tumorigenic and metastatic inducers (mutant p53/YAP/TAZ). Potentially, compounds that restore mutant p53 conformation to that of wild-type p53 protein might impair various oncogenic pathways promoted by mutant p53, also the oncogenic activity of the mutant p53/YAP protein complex and/or TAZ induction, and could reactivate the Hippo tumor suppressor function. A growing number of small molecules aimed to restore and stabilize the original DBD conformation of p53 have been identified. These include: p53 reactivation and induction of massive apoptosis (PRIMA-1), maleimide-derived molecule MIRA-1/NSC19630, short interfering mutant p53 peptides (SIMPs) (10–15 residues), reactivation of p53 and induction of tumor cell apoptosis (RITA), CP-31398 and many others ,273,274.All these observations highlighted the need to develop new and more effective compounds and strategies, testing also chronic and acute potential toxicity in in vivo treatments. PRIMA-1MET , a compound very similar to PRIMA-1, but much more active at low dosage, restores the pro-apoptotic function of p53 with consequent activation of downstream target genes ,286,287.Further studies are necessary to elucidate mutant p53/Hippo crosstalk with the aim of designing specific combined therapies that could reactivate their pro-apoptotic function and disable their aberrant oncogenic interactions. In line with this, the elucidation of how p53 mutants and YAP interact each other, which domains are involved in this interaction, and if it is a direct or indirect interaction could be important to foster the identification of compounds aimed to destroy their cooperation. The targeting of the oncogenic mediators SREBPs and WIP proteins or the activation of Lats1/2 tumor suppression activities could be also very attractive. This might allow activating tumor suppressor pathways and impairing those with oncogenic outputs.