Human placentation is a highly invasive process. Deficiency in the invasiveness of trophoblasts is associated with a spectrum of gestational diseases, such as preeclampsia (PE). The oncogene B-cell lymphoma 6 (BCL6) is involved in the migration and invasion of various malignant cells. Intriguingly, its expression is deregulated in preeclamptic placentas. We have reported that BCL6 is required for the proliferation, survival, fusion, and syncytialization of trophoblasts. In the present work, we show that the inhibition of BCL6, either by its gene silencing or by using specific small molecule inhibitors, impairs the migration and invasion of trophoblastic cells, by reducing cell adhesion and compromising the dynamics of the actin cytoskeleton. Moreover, the suppression of BCL6 weakens the signals of the phosphorylated focal adhesion kinase, Akt/protein kinase B, and extracellular regulated kinase 1/2, accompanied by more stationary, but less migratory, cells. Interestingly, transcriptomic analyses reveal that a small interfering RNA-induced reduction of BCL6 decreases the levels of numerous genes, such as p21 activated kinase 1, myosin light chain kinase, and gamma actin related to cell adhesion, actin dynamics, and cell migration. These data suggest BCL6 as a crucial player in the migration and invasion of trophoblasts in the early stages of placental development through the regulation of various genes associated with the migratory machinery. The oncogene B-cell lymphoma 6 (BCL6), which is a crucial regulator of B-lymphocyte development, is a transcriptional modulator and often deregulated in lymphomas ,2. AlongInterestingly, the gene expression of BCL6 has been reported to be increased in preeclamptic placentas ,10,11,12A healthy placenta is the key to a successful pregnancy and the life-long health of the offspring  and humaPE, which is the most common disease in pregnancy, is associated with substantial maternal and fetal morbidity and mortality worldwide ,25. It aWe hypothesized about the role of BCL6 in the motility of trophoblasts in early placental development. In the present study, we show that BCL6 is involved in the regulation of migration and invasion on multiple levels of trophoblastic cells derived from first trimester placental tissue. Moreover, a comparison to previous transcriptome data of BCL6 in epidermoid carcinoma cells reveals that BCL6 targets the same genes in trophoblastic cells and malignant cells, likely to modulate cell invasion.To examine whether BCL6 is expressed in early EVTs, placental tissue sections from first trimester placentas were immunohistochemically stained for BCL6. As shown in To underline the observation, individual cell tracking using time-lapse microscopy, which is a well-used assay employed to document the motility of single cells ,34,35, wMoreover, HTR and HIPEC cells knocked down of BCL6 H,I were The interplay of cell adhesion and actin dynamics is crucial for migration . To addrDynamic focal adhesion (FA) is crucial for the regulation of cell attachment . To lookThe actin cytoskeleton is the major source of internally generated force that regulates FA dynamics, cell shape, and migration . To addrBCL6, which is a transcriptional repressor, affects a great body of genes related to various signaling pathways ,43. To ep-FAK) and F-actin . Microscopic examination revealed a clear difference between the two opposite fronts formed by HTR cells treated with sicon and by HTR cells treated with siBCL6#1 , suggesting that the depletion of BCL6 renders them non-migratory or rather stationary. To underline this observation, HTR cells depleted of BCL6  (p-FAK (Y397) (p-paxillin (p-FAK (Integrin clustering causes FAK autophosphorylation and affects cell motility . Paxillin (S258) A, in addK (Y397) B, for mipaxillin C,D, FAK n  and below 3 (red color code), respectively, are depicted in a heatmap (PAK1), actin gamma (ACTG1), myosin light chain kinase (MYLK), caveolin 1 (CAV1), and matrix metalloproteinase-2 (MMP2), in HTR cells depleted of BCL6 , which is a player in actin dynamics that reactivates cofilin [The interaction of adhesion receptors such as integrins with the ECM transduces environmental signals into the cellular apparatuses, such as the actin cytoskeleton, which is important for maintaining the cell shape, contractility, tension, and motility. The dynamic actin cytoskeleton provides both the protrusive and contractile forces required for cell migration via a combination of actin polymerization and depolymerization, actin filament crosslinking, and the interaction of myosin-based motors with actin filaments . Intrigupaxillin , stathmipaxillin , vimentipaxillin , and filpaxillin . In factigration ; MYLK, wilaments ; TMSB4X,rization ; and pro cofilin . These dp-FAK, p-Akt, and p-Erk1/2 and is associated with a decreased migratory and enhanced stationary cell type. In support of this notion, reduced BCL6 leads to altered gene levels of important regulators for MAPK/Erk and PI3K/Akt, such as CAV1 [RPS6KA) [DUSP16) [MMP2. The inhibition of these multiple stated upstream kinases reduces cell adhesion and deregulates actin dynamics, hampering the recruitment and activation of FA components such as paxillin and FAK. This deregulation contributes to decreased cell migration and invasion in cancer and trophoblastic cells.Furthermore, integrin adhesion activates FAK, MAPK/Erk, and PI3K/Akt signaling . Actuall as CAV1 , ribosom[RPS6KA) , and dua[DUSP16) . Multipl[DUSP16) ,71. Addi[DUSP16) . Here, wTM (Thermo Fisher Scientific), as previously reported [BeWo , HTR-8/SVneo (HTR) , HIPEC-6reported ,15,74. F® M2 ; rabbit polyclonal antibody against FAK ; rabbit monoclonal antibody against p-FAK (Y397) ; rabbit polyclonal antibody against AKT ; rabbit polyclonal antibody against p-AKT (S473) ; rabbit polyclonal antibody against ERK1/2 ; rabbit monoclonal antibody against p-ERK1/2 (T202/Y204) ; rabbit monoclonal antibody against STAT3 ; and rabbit monoclonal antibody against p-STAT3 (Y705) .Western blot analysis was performed as previously described ,75. The p-FAK (Y397) ; monoclonal mouse antibody against paxillin ; and rabbit monoclonal antibody against p-paxillin (Y118) (#2541S) and mouse monoclonal antibody against α-tubulin . The F-actin cytoskeleton was stained using phalloidin . FITC-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch (Cambridgeshire). DNA was stained using 4′,6-diamidino-2-phenylindole-dihydrochlorid (DAPI) (Roche). Imaging of the immunofluorescence slides was performed with an Axio Imager 7.1 microscope  or by confocal laser scanning microscopy , as described previously [Indirect immunofluorescence staining was performed as previously described ,76,77. Teviously ,76,77.TM FLEX peroxidase blocking reagent  was used to stain formalin-fixed, paraffin-embedded first trimester placental tissue sections from healthy individuals, as previously described [A standard staining procedure with DAKO EnVisionescribed . A mousep-paxillin, and F-actin  were quantified by using the LAS AF Lite software from Leica. The mean signal size, mean gray intensity of paxillin, and active integrin area were analyzed with NIH ImageJ, as previously reported [The image acquisition was performed with the same wave intensity and exposure time. Images shown in figures are maximum intensity projections of confocal Z-sections. The mean fluorescence intensities of multiple proteins , and incubated at 37 °C for 20 and 60 min. Non-adherent cells were removed by careful washes with PBS and adherent cells were counted. Adherent cells were fixed with paraformaldehyde containing Triton X-100 and stained. Quantification of the cell area from slides released at 20 and 60 min post plating was analyzed using ImageJ.For cell spreading/adhesion assays, four-chamber slides were coated with 2 µg/mL fibronectin for 15 min , as previously described . Cells wTo evaluate the dynamics of the actin cytoskeleton, cells were starved in serum-free medium for 12 h. Cells were treated with latrunculin B  for 90 min to depolymerize actin fibers. The drug was washed out with PBS and the cells were released into serum-containing medium for 30 and 60 min. The cells were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 for 15 min at room temperature before processing for immunofluorescence staining.4) and HIPEC (5.5 × 104) and BeWo cells (5.5 × 104) were seeded in each well of the culture-inserts. Culture-inserts were gently removed after at least 8 h. The cells were acquired and imaged at indicated time points with bright-field images. Four pictures of each insert were taken and the experiments were performed in triplicate and with at least three independent times. The open area was measured using the AxioVision SE64 Re. 4.9 software (Zeiss).Cell migration assays were performed, as previously reported , with cuFor the invasion assay, HTR cells depleted of BCL6 or control HTR cells were used as previously described . IndividThe motility assay was performed with an AxioObserver.Z1 microscope (Zeiss), imaged with an AxioCam MRc camera (Zeiss) equipped with an environmental chamber to maintain proper environmental conditions . Time-lapse movies of 13 h were analyzed and quantified by using ImageJ 1.49i software  with the manual tracking plugin, and Chemotaxis and Migration Tool , as described previously .GAPDH (Hs_02786624), BCL6 (Hs00153368_m1), PAK1 (Hs00945621_m1), ACTG1 (Hs03044422_g1), MYLK (Hs00364926_m1), CAV1 (Hs00971716_m1), MMP2 (Hs01548727_m1), and CDH1 (Hs01023895_m1) were obtained from Applied Biosystems (Darmstadt). Real-time PCR was performed with a StepOnePlus Real-time PCR System and the data were analyzed via StepOne Software v2.3 (Applied Biosystems). The final results are presented as relative quantification (RQ), as previously described [Total RNAs were extracted with the EXTRACTME Total RNA Kit without DNase digestion, according to manual instructions . Reverse transcription was performed using Go Script Reverse Transcription Mix, as instructed . The primers and probes for escribed ,82.® UltraTM RNA Library Prep Kit for Illumina® , following the manufacturer’s recommendations. Differential expression analysis between two conditions/groups  was performed using the DESeq2 R–package and 60,450 genes were analyzed. The genes found by DESeq2 were further depicted in different pathways by GO enrichment analysis and KEGG pathway enrichment analysis.For transcriptomic analysis, the HTR cell line was treated with siRNA targeting BCL6#1 and #2 or control siRNA for 48 h. The experiment was repeated three independent times and total RNAs were extracted with the EXTRACTME Total RNA Kit, according to manual instructions  and without DNase digestion. A total amount of 1 μg RNA per sample was sent to Novogene Co., Ltd.  and used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNextt-test (two-tailed and paired or homoscedastic) was used. An unpaired Mann–Whitney U test (two-tailed) was used to analyze the statistical significance of the single cell tracking and fluorescence intensity quantification for the proteins ). Differences were considered statistically significant when p < 0.05. For transcriptomic analysis, a differential expression analysis of two conditions was performed using the DEGseq R package. The p values were adjusted using the Benjamini andTo evaluate the significance of the cell viability and invasion assay, Student’s (Fold Change)| of 1 were set as the threshold for significantly differential expression. Student’s t-test (two-tailed and paired or homoscedastic) was used to evaluate the significant difference between diverse groups with real-time PCR.Hochberg methods. A corrected p-value of 0.005 and |log2Taken together, in combination with our previous results ,14,15, wACTG1, CAV1, MYLK, and PAK were also found to be deregulated in a transcriptomic analysis of epidermoid carcinoma cells A431 [Given that BCL6 is an important oncogene involved in diverse malignant entities, studies on its presence in trophoblasts could provide novel insights into common signaling pathways shared by malignant progression and placental development, as suggested by multiple deregulated signaling pathways related to cell motility and migration . Indeed,lls A431 , indicat


Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses  and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.Best known as licorice,  Glycyrrhiza is a genus of medicinal perennial herbs within the legume family (Fabaceae), native to Asia and the Mediterranean region and currently distributed in temperate and subtropical regions of the world [Glycyrrhiza uralensis F., G. inflata B., and Glycyrrhiza glabra L.  is the most common general lab technique to accurately determine gene expression levels due to its high throughput, accuracy, and sensitivity ,11. StabGlycyrrhiza.Biosynthesis and accumulation of trace-amount bioactive metabolites in medicinal plants are regulated by multiple intrinsic and extrinsic factors through corresponding genes and gene networks. Glycyrrizin, the major active compound of licorice, only happens in roots and rhizomes . MoreoveGlycyrrhiza mainly distributes in dry or semi-dry areas, including Eurasia, northern Africa, and western Asia. Drought and salt stresses, the most common abiotic factors in licorice habitats, have been reported to induce the accumulation of glycyrrhizin [G. glabra [Glycyrrhiza species is still lacking in general, especially for two other congeneric species with pharmaceutical potentials, G. uralensis and G. inflata. yrrhizin . Plant hyrrhizin . Abscisiyrrhizin , and secyrrhizin . Althoug. glabra  have beeGlycyrrhiza species under different abiotic stresses or hormonal treatments. To examine this hypothesis, we (1) evaluated the stability of 14 candidate reference genes, (2) selected optimal reference genes under abiotic stresses and hormonal treatments, and (3) compared the suitable reference genes in three congeneric Glycyrrhiza species under the osmotic stress. In addition, we surveyed and summarized the reference genes previously used within Leguminosae plants.In the present research, we hypothesized that there are overlapping reference genes recommended for congeneric including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2), were selected and evaluated. The expression levels of the candidate reference genes were determined as quantification cycle (Cq) values. As shown in DREB possesses the lowest gene expression variation in G. uralensis, with a narrow range of Cq values from 24.26 to 28.96, revealing that DREB might have a stable expression level under different treatments. Conversely, TUB has Cq values that range from 22.74 to 34.74, so TUB is probably not a good choice for a reference gene in G. uralensis  and G. inflata (20.17), indicating the highest expression level of TIF1 in the two species, while EF1 (27.31) or CAC (25.62) was expressed at low levels in G. uralensis or G. inflata, respectively  condition, the most stable reference genes were stKeeper . In G. ihe roots .DNAJ, CAC, and UBC2 recommended by geNorm in G. uralensis leaves; ABCC2, CYP, and TIF1 recommended by NormFinder; DREB, RAN, and CS recommended by the BestKeeper; and CYP, ABCC2, and DNAJ recommended by Delta CT. In the roots of G. uralensis under salt stress, the top three most stable candidate reference genes were DREB, UBC2, and DNAJ identified by geNorm; DNAJ, TUB, and DREB identified by NormFinder and Delta CT; and DREB, COPS3, and UBC2 identified by BestKeeper , the most stable reference genes were stKeeper . In G. ihe roots .UBC2, CS, and CAC recommended by geNorm under ABA treatment in G. uralensis leaves; CS, CAC, and ABCC2 recommended by NormFinder and Delta CT; and TUB, R3HDM2, and CS recommended by BestKeeper. In the roots of G. uralensis under ABA treatment, the top three most stable candidate reference genes were CAC, RAN, and UBC2 identified by geNorm, NormFinder, and Delta CT amd COPS3, R3HDM2, and TUB identified by BestKeeper , the most stable reference genes were stKeeper . In G. ihe roots .DREB, UBC2, and DNAJ recommended by geNorm in G. uralensis leaves; ABCC2, CAC, and CYP recommended by NormFinder; R3HDM2, ABCC2, and EF1 recommended by BestKeeper; and CAC, ABCC2, and UBC2 recommended by Delta CT. In the roots of G. uralensis under MeJA treatment, the top three most stable candidate reference genes were ABCC2, CS, and EF1 identified by geNorm; CAC, TUB, and UBC2 identified by NormFinder and Delta CT; and EF1, COPS3, and R3HDM2 identified by BestKeeper , the most stable reference genes were stKeeper . In G. ihe roots .G. uralensis, the most stable reference gene were CS, DNAJ, and DREB in the leaves and DNAJ, UBC2, and CAC in the roots  is not necessary. For all the experimental conditions in G. uralensis and G. inflata, the first V-value less than 0.15 occurred at V2/3  values (cutoff = 0.15). When pairwise variations Vn/ at V2/3 , suggestGlycyrrhiza species are different under various experimental conditions and that no one of the top three candidates was shared among all experimental conditions tested  is a key gene in glycyrrhizin biosynthesis and mainly expressed in root or rhizome in licorice [β-AS was also upregulated by drought stress, which is in close correlation to glycyrrhizin accumulation [β-AS to validate the reliability of the selected reference genes under different conditions.licorice ,28. Prevlicorice , and expmulation . We thusG. uralensis and G. inflata, β-AS expression profiles under all the experimental conditions were different when normalized to the most suitable and the least suitable reference genes, and the differences fall into three types: (1) β-AS expression was changed dramatically between control and stress conditions when using the most suitable candidate reference genes for normalization, but there was no obvious differences in the expression levels of β-AS when the least suitable reference gene were used . (2) β-AS expression had no obvious difference between control and stress conditions (p > 0.05) when using the most suitable candidate reference genes for normalization, while the difference became significant when the most unstable reference was used (p < 0.05) . (3) The expression of β-AS presented significantly difference between control and stress conditions when normalized with the most and least reference genes, but the significance level of the differences was changed   . All theβ-AS expression under same experimental conditions were different when normalized to the most, the two most, and the three most suitable candidate reference genes. Under ABA treatment in G. uralensis, when the most suitable reference gene (CAC) was used for normalization, the expression of β-AS under stress conditions showed no obvious differences (p < 0.05), but it changed dramatically in the expression levels of β-AS between control and stress conditions when using the two (CAC and RAN) or three most  suitable candidate reference genes for normalization   . The samreatment . TherefoAmmopiptanthus mongolicus, Arachis hypogaea L., Cajanus cajan (Linn.) Millsp., Caragana intermedia, Caragana korshinskii Kom, Cassia obtusifolia L., Cicer arietinum L., Cyamopsis tetragonoloba L.Taub, Glycine max (L.) Merr., Glycyrrhiza glabra L., Oxytropis ochrocephala Bunge, Robinia pseudoacacia L., Trifolium repens L., Vigna angularis (Willd.) Ohwi, Vigna mungo L., Vigna unguiculata L., Medicago sativa L., and Pisum sativum L.) under different experimental conditions, including abiotic stresses and hormonal treatments, by searching “NCBI” or “Web of Science” with the key words “Leguminosae” and “reference gene”. In this study, we added G. uralensis and G. inflata to this list.Reference gene selection has been reported in 18 Leguminosae species , ubiquitin , and tubulin  were the top three choices, and the frequency of recommendation was 12.64%, 11.49%, and 10.34%, respectively. In this study, our results further confirm this observation. When research involves the drought stress, UBC2 can be used as the internal control for gene quantification studies among the three licorice species, G. uralensis, G. inflata, and G. glabra , 15.15% , and 20% , respectively  selected form the RNA-Seq data of Glycyrrhiza species. In total, CAC, UBC2, and ABCC2 were the three most stably expressed genes under different experimental conditions tested in the two Glycyrrhiza species. In the previous study, CAC was identified as the most stable reference gene in chickpea under drought stress condition [CAC gene was the least stable under ABA treatment, osmotic stress, and cold stress [CAC gene was most stable gene of G. uralensis under ABA and MeJA treatment, whereas CAC was the most stable gene of G. inflata under salt stress and ABA treatment . Results showed that the top three most suitable reference gene in G. uralensis and G. inflata were R3HDM2, CAC, and TUB and COPS3, R3HDM2, and DREB, respectively. In the present study, we evaluated the optimal internal reference genes of licorice under different treatment conditions, and the results showed that the optimal internal reference genes were different in root and shoot for the same conditions  the most and least suited, (2) the two most and least suited, (3) the three most and least suited genes, and (4) all the candidate reference genes. Therefore, reliable reference genes are essential for the accurate normalization of gene expression levels.This point is strongly supported when we analyzed the expression pattern of  inflata . TherefoGlycyrrhiza species; UBC2 was the only optimal reference gene shared among G. glabra, G. uralensis, and G. inflata under the osmotic stress  under abiotic stresses or hormonal treatments . We found ACT and TIF1 were the most unstable reference genes in G. uralensis and G. inflata, and it has been proven that it will cause false results using the unstable reference gene for expression normalization  used in this study were provided by Gansu Jin You Kang Pharmaceutical Technology Co., Ltd., Lanzhou, China. Glycyrrhiza seeds were soaked in H2SO4 (18 mol/L) for 30 min and then washed several times with water to remove the hard shell [Seeds  medium plates with or without 100 mM mannitol (osmotic stress) or 100 mM NaCl  and grown for four weeks. Leaves and roots of every 20 seedlings were harvested with liquid nitrogen as one sample and stored at −80 °C . The seeTM RT reagent Kit with gDNA Eraser  following the manufacturer’s instructions. Quantitative PCR were carried out in 384-well blocks using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus)  on LightCycler 480  according to manufacturers’ instructions. Total RNA was isolated using the HiPure Total RNA Mini Kit  according to the kit instructions. The RNA quality and concentration were measured with agarose gel electrophoresis and a spectrophotometer . Removal of genomic DNA contamination and first strand cDNA synthesis were performed using the PrimeScriptACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, and UBC2) were selected from published literature [G. uralensis genome (http://ngs-data-archive.psc.riken.jp/Gur-genome/index.pl#) and G. inflata RNA-Seq database (SRA accession: PRJNA574093) using TBLASTN program in BioEdit software. Four new generation candidate genes  were selected from the RNA-seq data and expressed consistently in different RNA-Seq samples. qRT-PCR primers were designed using PrimerQuest Tool, INTEGRATED DNA TECHNOLOGIES (IDT) (https://sg.idtdna.com/Primerquest/Home/Index). All primers were synthesized by TSINGKE . The primer information used in this study were listed in A total of 14 candidates were selected, and the stability was evaluated. Ten commonly used candidate housekeeping genes , for investigating the optimal number of reference genes for accurate normalization, was also performed by geNorm software. The threshold was commonly set at 0.15, and additional reference genes are not required when Vn/n+1 is below 0.15. NormFinder identifies suitable reference genes on the basis of stability value (SV); the candidate having the lowest SV is the optimal reference gene. BestKeeper and Delta CT rank the stabilities of candidates on the basis of SD and CV values; the most stable reference gene is the one having the lowest SD value.Expression stability of the 14 candidate reference genes were evaluated by four Microsoft Excel-based computational programs, geNorm , NormFinThe comprehensive ranking of expression stability of candidate reference genes were conducted using RefFinder, which is a system to integrate results obtained by the geNorm, Normfinder, BestKeeper, and Delta Ct method . Based oGlycyrrhiza species were also analyzed. The results were visualized by Venn Diagrams, and it was plotted using the OmicShare tools, a free online platform for data analysis (www.omicshare.com/tools).The top three most suitable reference genes selected by RefFinder under the conditions of osmotic stress, salt stress, ABA treatment, and MeJA treatment were compared; the suitable reference genes under osmotic stress condition among the three congenic Survey of the reference genes used within Leguminosae plants was conducted by searching keywords of “Leguminosae” and “reference” on Web of Science and PubMed. A total of 18 different Leguminosae species under various experimental conditions, including abiotic stresses and hormonal treatments, were included in the analysis. The top three most suitable reference genes for any given Leguminosae species under specific experimental condition  were used to calculate the frequency of recommendation.Glycyrrhiza species under four experimental conditions  were examined for gene expression in order to identify reliable reference genes. Different reference genes were identified in different tissues under different experimental conditions as well as in different species. In addition, we also documented the reference genes that have been used in qRT-PCR analysis among 18 different Leguminosae plants under the same abiotic conditions with current study, i.e., osmotic/salt stress and ABA/MeJA treatment. Among the 132 genes tested, even the routinely used reference genes showed variable expressions under different experimental conditions. Therefore, accurate measurements of gene expression for functional characterization require suitable reference genes to avoid misinterpretation of qRT-PCR results, and a thorough evaluation of reference genes is strongly recommended.In this study, the leaves and roots of two


Natural killer (NK) cells are cytotoxic innate lymphocytes that are well-known for their ability to kill infected or malignant cells. Beyond their roles in tumor surveillance and anti-pathogen defense, more recent studies have highlighted key roles for NK cells in a broad range of biological processes, including metabolic homeostasis, immunomodulation of T cells, contact hypersensitivity, and pregnancy. Consistent with the breadth and diversity of these functions, it is now appreciated that NK cells are a heterogeneous population, comprised of specialized and sometimes tissue-specific subsets with distinct phenotypes and effector functions. Indeed, in addition to the conventional NK cells (cNKs) that are abundant and have been well-studied in the blood and spleen, distinct subsets of tissue-resident NK cells (trNKs) and “helper” Group 1 innate lymphoid cells (ILC1s) have now been described in multiple organs and tissues, including the liver, uterus, thymus, adipose tissue, and skin, among others. The cNK, trNK, and/or helper ILC1 populations that co-exist in these various tissues exhibit both common and distinct developmental requirements, suggesting that a combination of lineage–, subset–, and tissue–specific differentiation processes may contribute to the unique functional properties of these various populations. Here, we provide an overview of the transcriptional regulatory pathways known to instruct the development and differentiation of cNK, trNK, and helper ILC1 populations in specific tissues in mice. Natural killer (NK) cells are cytotoxic innate lymphocytes that were first identified in 1975 based on their capacity to spontaneously kill tumor cell lines without prior immunization . Over thThe balance of evidence suggests that mouse NK cells and helper ILC1s are distinct lineages in mice, arising from separate lineage-committed progenitors under homeostatic conditions . They doWhile most studies on NK cells have focused on population(s) abundant in the blood and spleen, now commonly referred to as conventional NK cells (cNKs), unique tissue-specific and/or tissue-resident NK cell (trNK) populations have recently been described in diverse tissues, including the uterus, thymus, intestine, adipose, skin, peritoneal cavity, and salivary, lacrimal, and mammary glands. As described below, many of these unique trNK populations exhibit distinct tissue-specific phenotypes, functions, and developmental requirements. In particular, recent studies have highlighted notable differences in the transcriptional regulation of trNK, cNK, and helper ILC1 development, suggesting that distinct differentiation processes support the unique functional properties of tissue-specific trNKs. Here, we review current literature on the transcriptional pathways known to control the development of various trNK populations in mice, with a particular focus on regulatory mechanisms that are unique to trNKs as compared to cNKs and helper ILC1s in each tissue.−2B4+CD27+Id2hiIL-7Ra+/− cells in the bone marrow that lacked all mature immune cell lineage markers (Lin−), including classical NK cell markers such as NKp46 (+CD11b− cells being less mature (but more proliferative) and CD27−CD11b+ cells being most mature  and Nuclear factor interleukin-3 regulated (Nfil3), both of which are expressed at or prior to the NKP-ILCP developmental branch point and are important for proper cNK and helper ILC lineage differentiation , 26–34. Nfil3-deficient mice also have severe and early defects in NK cell development, reflected in a near-complete loss of cNKs in the periphery and significantly reduced numbers of NKPs, iNKs, and mNKs in the bone marrow , 31, 33.Rroid) also critically regulate cNK development and are important for maintaining proper Id2 expression in differentiating cNKs  and Signal transducer and activator of transcription 5 (Stat5), the histone H2A deubiquitinase, Myb-like, SWIRM and MPN domains 1 (Mysm1), and the long non-coding RNA (lncRNA), RNA-demarcated regulatory region of Id2 , GATA binding protein 3 (Gata-3), Runt-related transcription factor 3 (Runx3), and Zinc-finger E homeobox-binding 2 (Zeb2). Similar to T-bet, KLF2 restricts abnormal proliferation and supports survival in maturing cNKs, and KLF2-deficiency reduces the number of mature cNKs in the periphery . Gata-3 −CD49b+Eomes+ cNKs, the liver harbors a unique population of CD49a+CD49b−Eomes− ILC1s that are tissue-resident in parabiotic mice (−]. Liver ILC1s reside in the liver sinusoids and have been shown to mediate memory-like immune responses in models of contact hypersensitivity (CHS) and viral infection , CD11b, CD122, and Ly49 receptors such as Ly49A, Ly49D, Ly49G2, and Ly49H , 63, 64.+ NK cells following adoptive transfer into intact (un-irradiated) hosts  hosts , supportd) hosts . Additiod) hosts , 56, 64.Distinct developmental requirements include findings that liver ILC1s require Promyelocytic leukemia zinc finger (PLZF) and the Aryl hydrocarbon receptor (AhR) for development, two transcription factors that are dispensable for cNK development , 69. LosNfil3 has been reported as dispensable for liver ILC1, but essential for liver cNK, development in some studies , 64, 70,Development of liver ILC1s also uniquely depends on Homolog of Blimp-1 (Hobit), a transcription factor that supports T cell tissue-residency, in part by suppressing genes involved in tissue egress . Notably−CD49b+Eomes+ cNKs, tissue-resident CD49a+CD49b−Eomes+ trNKs, and CD49a+CD49b−Eomes− helper ILC1s  harbor fewer tNKs overall, and those present have a CD11bhiKLRG1hiCD27lo phenotype typically associated with mature cNKs  contain several tissue-resident NK and helper ILC1 populations with unique phenotypes and functions –101. Amo−CD49b+ cNK-like cells and CD49a+CD49b− cells are also present  have a relatively intact SG NK compartment , comprised of both Eomes+ NK cells and Eomes− helper ILC1s, which have been implicated in colitis-associated inflammation   , 105. In (RORγt) , 105. Th unclear , 103. In−CD49b+Eomes+ cNKs, and CD49a−CD49b−Eomes+ and CD49a+CD49b−Eomes+ NKs that have been called immature NK cells by some groups  have been shown to contribute to obesity-related metabolic dysfunction, in part by promoting the differentiation of inflammatory M1 macrophages –110. Thee groups , 110. Eoa+CD49b− , 110. Paresident , 110.− trNK populations in the AT have unique surface phenotypes. Most are CD90hiCD69hi and variably express many cNK-associated molecules  but not CD127   . And lasunclear) .−CD49b+Eomes+ cNKs, mouse skin harbors a distinct population of CD49a+CD49b−Eomeslo/− cells that are CD69hiCD127− and are largely tissue-resident in parabiotic mice  contains both CD49aer ILC1s , 64, 113nd CD103 , 113. Innd CD103 . Transcrnd CD103 . In contnd CD103 . Additiond CD103 .+CD49a− cNK-like cells, the kidney contains a minor subset (15–20%) of CD49a+CD49b− cells that are tissue-resident  was specifically associated with tissue damage following ischemic acute kidney injury, suggesting a unique pathogenic role for these cells  populations have also been described in the mammary glands (MG)  . Both su Eomeslo . In addi Eomeslo . Functio Eomeslo .+ NK and Eomes− helper ILCs with still undefined tissue-residency properties  also contains several distinct populations of Eomesoperties . CD49b+Cnfection . Howevernfection . These f+CD3− cells has been shown to localize to the exocrine pancreas in young non-obese diabetic (NOD) mice and to infiltrate the endocrine pancreas in adult NOD mice, possibly reflecting a role in diabetes-related autoimmunity  and chemokine receptors , and low or no expression of CD62L and CCR7  and persist in the liver without recirculation, suggesting they likely represent bona fide liver trNKs/ILC1s (Although this review has focused principally on studies involving tissue-resident NKs and helper ILC1s in mice, it is important to note that similar tissue-associated populations have now been described in diverse human tissues, including the uterus, adipose, tonsils, intestines, liver, kidney, and lung , 119. Mond tools . Neverthand CCR7 . For exaand CD69 . These CKs/ILC1s . NotablyKs/ILC1s . AlthougIt is now appreciated that NK cells are capable of mediating adaptive immune responses in certain settings. Indeed, memory or memory-like responses have been described for tissue-resident NKs and helper ILC1s in the liver, skin, and uterus , 124. WhNV-P and AB wrote the manuscript and designed the figure.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


VK has been historically known to be required for the control of blood coagulation, and more recently, bone development and homeostasis. Our understanding of the VK metabolism and the VK-related molecular pathways has been also increased, and the two main VK-related pathways—the pregnane X receptor (PXR) transactivation and the co-factor role on the γ-glutamyl carboxylation of the VK dependent proteins—have been thoroughly investigated during the last decades. Although several studies evidenced how VK may have a broader VK biological function than previously thought, including the reproduction, little is known about the specific molecular pathways. In vertebrates, sex differentiation and gametogenesis are tightly regulated processes through a highly complex molecular, cellular and tissue crosstalk. Here, VK metabolism and related pathways, as well as how gametogenesis might be impacted by VK nutritional status, will be reviewed. Critical knowledge gaps and future perspectives on how the different VK-related pathways come into play on vertebrate’s reproduction will be identified and proposed. The present review will pave the research progress to warrant a successful reproductive status through VK nutritional interventions as well as towards the establishment of reliable biomarkers for determining proper nutritional VK status in vertebrates.Vitamin K (VK) is a fat-soluble vitamin that vertebrates have to acquire from the diet, since they are not able to  Fat-soluble vitamins, including vitamin A, D, E, and K, are essential micronutrients for vertebrate’s development. In general, fat-soluble vitamins have a relatively heterogenic chemical structure, with most of them being terpenes or containing long terpene chains attached to an aromatic moiety. The exception is vitamin D (VD), which has a triene moiety instead of the aromatic one. Additionally, specific amounts and chemical forms of fat-soluble vitamins need to be provided in the diet, since these organic compounds cannot be endogenously synthesized by vertebrates, at least not in adequate amounts. Again, VD could be considered the exception. Although mammals are able to photoconvert 7-dehydrocholesterol into pre-vitamin D3 in the skin and only a minor supply is provided by the diet, no strong evidences of VD photosynthesis have been documented in fish species until now . FurtherIn general, three different VK metabolites (or K vitamers) can be found . All of All of the K vitamers from the diet can be absorbed at the intestine, including those that originated from the intestinal microbiome ,8. Recenpregnane X receptor  [γ-glutamyl carboxylase (ggcx) [As long as the VK was only considered to be involved on the hepatic γ-carboxylation of clotting factors, the liver was considered to be the uniquely significant storage site of VK . Nowaday or sxr) ,18,19,20e (ggcx) ,22. Furte (ggcx) . In thise (ggcx) . Indeed,e (ggcx) . Neverthe (ggcx) ,26. Furte (ggcx) .Because VK stores are very labile and rapidly respond through specific pathways to nutritional status , as well as other conditions, such as pharmacological treatment with VK anticoagulants ,29,30,31In addition to its potential antioxidant function, VK may exert their biological functions by two different molecular pathways: acting as a co-factor of GGCX and transactivating the NR PXR. How VK might be up taken, metabolized, transported, and exert their effects at a transcriptional level or through the carboxylation of VKDPs in vertebrates is summarized in Ciona intestinalis) and invertebrates [GGCX is a transmembrane protein that is localized at the endoplasmic reticulum (ER) and it catalyzes the conversion of specific glutamate (Glu) residues to γ-carboxyglutamate (Gla) residues, a process called γ-carboxylation, where VK acts as a co-factor . GGCX actebrates ,64,65,66tebrates . Indeed,tebrates . Howevertebrates ,67. Of n-/- mice, they exhibited early postnatal lethality due to severe bleeding [vkorc1 and vkorc1l1 genes in vertebrates has been reported [vkorc1 expression is found in liver, consistent with being the main isoform that is responsible for recycling VK epoxide originated from hepatic γ-carboxylation of hemostasis-related VKDPs [vkorc1l1 gene expression in vertebrates was found in brain and ovaries, but also high expression levels were reported in organs, like kidney, eye, and testis [Carboxylation requires the abstraction of a proton from the four-carbon of the glutamate residue by reduced VK, which results in the conversion of VK into VK epoxide. The VK epoxide must be recycled, a reaction that is catalyzed by two VK 2,3-epoxide reductases (VKORs) . VKORs ableeding , which sbleeding , where tbleeding ,57,60,61reported ,56,60,61d testis ,56,58. Rd testis  pointed Ciona intestinalis VDR/PXR [The other main pathway of the VK is the transactivation of the NR PXR . PXR is  VDR/PXR . As a NR VDR/PXR . In cont VDR/PXR  and thus VDR/PXR ,72. PXR  VDR/PXR . Our und VDR/PXR ,71,74. H VDR/PXR ,37.pxr transcripts have also been detected in osteoblastic and neural cells, as well as in the endothelial cells of the blood-brain barrier (BBB) [pxr expression in tissues/organs was reported in different species [pxr transcripts were specifically detected at brain (granular layer), eyes (inner nuclear and nerve fiber layers as well as rod nucleus), cartilage , bone, intestine, liver, gonads, spleen, and kidney [In mammals, PXR is mainly expressed in liver and intestine, where the PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues. However, the control of retinoid metabolism, inflammation, iron, glucose, lipid, cholesterol, bile acid, and bilirubin homeostasis, as well as the regulation of bone mineral density, were also demonstrated ,70,75,76er (BBB) ,18,19,77 species ,37,78,79d kidney ,80, wideReproduction is a tightly regulated biological process that guarantees species perpetuation and increases intra-specific genetic variability, allowing for offspring to increase its fitness and adaptation to the environmental changes and avoiding the extinction. Although vertebrates exhibit a diverse set of modes of sexual reproduction (including hermaphroditism), with oviparous, ovoviviparous, or viviparous offspring production, semelparity or iteroparity , or multiple reproductive cycles along the year), as well as with synchronous, group-synchronous, and asynchronous gonadal development; it requires a myriad of precisely orchestrated events, governed by host signaling pathways that are considerably conserved along its evolution . A great3)), differentiates and leads to the formation of the spermatogonia B, which is characterized by a more rapid mitotic division potential [neuregulin 1 and 3 . Similar to FSH, retinoic acid (RA) also has the capacity to induce the transcription of both nrg1 and 3, through the binding to its own receptors, as demonstrated in Nrg1 Ser−/−mutant mice  maintenance, as well as the one of cadherins, integrins, and laminins to form protein complexes to attach Sertoli cells to elongated spermatids.Gonad development and maturation is a process that is controlled through different signaling pathways at the local level . In maleotential ,88,89,90iewed in ). In parpxr, ggcx, and vkors were specifically found in the pituitary, testis, and ovaries in different vertebrate species, from fish to mammals [cyp3a1 through PXR by VK might be a key step on the control of progesterone, cholesterol, and testosterone synthesis [Pxr-deficient rodents shows poor breeding success [cyp3a4, cyp3a23, cyp3a11, cyp2b6, cyp2b9, cyp2c55, cyp2c8, cyp2c9, cyp2c19 and cyp1a, but also phase II enzymes (eg. UDP-glucuronosyl transferase (ugt), sulfotransferase (sult), and glutathione S-transferase (GST) enzymes), both controlling the synthesis and metabolism of steroids such estrogen (E2), among other compounds  dehydrogenase 4 and retinol dehydrogenase 12 genes that are involved in steroidogenesis [cyp11a—a rate-limiting enzyme in testosterone synthesis—where mRNA levels were positively correlated with the MK-4 concentration in testis [Nowadays, it is clear that both main molecular pathways of VK, PXR transcriptional activation, and γ-carboxylation, are present in main tissues involved in vertebrate’s reproduction, the brain and the gonads. Transcripts of  mammals ,80,92,93 mammals ,94, tranynthesis  and thus success . Furtheriewed in ); being iewed in . In thisogenesis . The undn testis . The effn testis ,55 and fn testis . The impn testis . These rn testis .Solea senegalensis) broodstock fed control and dietary VK1 supplemented diets during six months, Fernández and colleagues [Cyprinus carpio var. Jian) fed increased levels of dietary VK [vkorc1l1 was found to be expressed in the testis of vertebrates, including rodents [It seems like VK has still conserved its role as an important antioxidant molecule from invertebrates. Because spermatozoa have limited antioxidant defense mechanisms and a limited capacity for detection and repair of DNA damage, these cells are among the most vulnerable to oxidative stress. Indeed, oxidative stress has been linked to male infertility, inducing reduced sperm motility, sperm DNA damage, and increased risk of recurrent abortions and genetic diseases, although certain levels of reactive oxygen species (ROS) are required for the maturation of spermatozoa, acrosome reaction, capacitation, hyperactivation, and spermatocyte fusion in humans (reviewed in ). Althoulleagues  found loetary VK , and theetary VK . As prev rodents  and Sene rodents . Further rodents . Thus, V rodents ).In mammals, spermatozoa will mature at epididymis through the interaction with different molecules, proteins, and microRNAs (miRs) (included in exosomes or not), and finally induced to be released by testosterone ,104,105.vkorc1 and vkorc1l1 as well as ggcx in male and female gonads suggest that VK is required and recycled for vertebrate’s reproduction [In females, the gametogenesis process is also quite conserved along evolution, and the detailed review performed by  in mammaoduction ,56,92. N2, in addition to FSH, play an important role. Only the most mature antral follicles (most sensitive to FSH action) go on towards its maturation until the dominant follicle recruitment. LH surge-induced factors are essential for the oocyte meiotic resumption, cumulus expansion, until the ovulation, and luteinization in mammals. In this sense, maturation-promoting factor (MPF) leads to oocyte meiotic resumption, while epidermal growth factor-like factors (EGF-like factors) activate the expression of prostaglandin synthase 2 (ptgs2), hyaluronan synthase 2 (has2), and tumor necrosis factor (TNF) α-induced protein 6 (tnfaip6) in order to the cumulus expansion to take place. For follicle rupture in mammals, the action of matrix metalloproteinases (MMPs), tissue-type plasminogen activator (PLAT), and a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (ADAMTS1) is required [endothelin 2 (edn2), cGMP-dependent protein kinase 2 (prkg2), interleukin 6 (il6), and synaptosome associated protein 25 (snap25) genes through the action of progesterone receptors , and Erequired . In the required ,108, beiiewed in ). BecausN-acetyl cysteine and ascorbic acid, are all able to inhibit apoptosis in antral follicles in the absence of FSH [Danio rerio) embryos under VK-induced deficiency was found [Oxidative stress may also affect ovaries, being ROS involved in the initiation of apoptosis in antral follicles caused, for instance by several chemical and physical agents in mammalian species (reviewed in ). Antrale of FSH . Thus, Vas found . Neverthas found .2+ homeostasis in particular tissues (such as testis) due to the limited carboxylation of these VKDPs, might be an additional relevant pathway on reproduction.In addition to the potential effects of VK on testosterone synthesis and the antioxidant status, VK might also affect gametogenesis through other particular pathways, such as some of the VKDPs and its carboxylation status. In this sense, protein S (PROS1) and GAS6 are also expressed in the testis of Sprague–Dawley rats , and botIn vertebrates, the HPG axis control gametogenesis and reproduction performance in a tightly regulated manner . In femaHypothalamic neurons synthesizing KISS play a key role in the central regulation of the timing of puberty onset and reproduction in mice . Saedi apxr were also specifically found to be expressed in the brain and pituitary in both genders from different vertebrate species [pxr transcript (sspxr1) in Senegalese sole to be expressed in the granular layer [The impact of VK on neuronal development and homeostasis should not be also neglected when considering the tight control of brain over reproductive system through the HPG axis. GAS6 also has a central role in the development and survival of nervous system. As previously commented, GAS6 activates the TAM family of receptor tyrosine kinases, particularly involved in the proliferation and survival of GnRH neurons, allowing their migration from the olfactory bulb to the hypothalamus, as demonstrated by in vitro assays (reviewed in ). Furthe species ,37,78,93ar layer , a densear layer  and it rgnrh expression in female mice [q/11 and Gs , which in turn induced inositol trisphosphate (IP3) and diacylglycerol (DAG) synthesis. The former stimulates Ca2+ release from the smooth ER, whereas the latter leads to protein kinase C (PKC) activation. Ca2+ and PKC-mediated the activation of calcium/calmodulin-dependent kinase II (CAMK II) and (mitogen-activated protein kinase) ERK 1/2, respectively, finally inducing lh gene expression. Conversely, when GnRH is pulsed with low frequency, cyclic adenosine monophosphate (cAMP) production occurs through Gs protein recruitment. cAMP increased intracellular levels, activate protein kinase A (PKA), and consequently cAMP response element binding protein (CREB) phosphorylation that enhance the fsh gene expression.On another hand, GnRH signaling is repressed by gonadotropin inhibitory hormone (GnIH) in rat and sheep females (in ). Recentale mice . Becauseiewed by ). High Glpin3) and glycerophospholipid (cept1b and lpin3) metabolism and/or signaling, in addition to calcium/calmodulin dependent protein kinase II beta (camk2b1) gene [de novo synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from DAG, critical for homeostasis of cellular lipid stores and membranes, but also to induce the expression of LH in the pituitary, as demonstrated by in vitro studies [2+ from the ER controls the activation of calcium/calmodulin kinase I (CAMKI) and II (CAMKII), as well as cell junctions at pituitary, Leydig and Sertoli cells might be an additional pathway where VK may act through these ncRNAs targeting camk2b1.These intracellular communications and signaling pathways might be also altered by VK. VK also induced sphingolipid synthesis, a major class of lipids and a vital modulator of neural cell proliferation, differentiation and survival in mammals (reviewed in ). We havb1) gene . While l studies ,139,140.2+ released from ER. While Ca2+ release activates CAMKI, PKA and PKC induce the expression of several steroidogenic genes through ERK 1/2 signaling pathway, in particular: steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme , steroid 17-alpha-hydroxylase/17,20 lyase , 3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 1 (HSD3B1), and/or testosterone 17-beta-dehydrogenase 3  [2+ release at ER and stimulates PKA and phosphatidylinositol 3-kinase (PI3-K) through cAMP. While Ca2+ release from ER regulates Sertoli–Sertoli cells junction communication, PKA activation stimulates the expression of aromatase , tissue-type plasminogen activator (PLAT), insulin like growth factor 1 (IGF-1), and stem cell factor (SCF) genes that potentiates spermatogonia survival. Furthermore, PI3-K stimulates γ-glutamyl transpeptidase (γ-gtp) and lactate dehydrogenase (ldh) expression, the latter being involved in the lactate production for germ cells development. In addition, FSH also activates phospholipase A2 (PLA2) pathway, leading to prostaglandin E2 (PE2) release [star, cyp11a1, hsd3b1, hsd3b2, and cyp17a1 through PKA signaling and producing androgens and progesterone (P4). Instead, in the Granulosa cells of antral follicles, FSH binds to its receptor, activates PKA and PI3-K signaling, inducing activins, inhibins, and follistatin, as well as supporting androgen aromatization to estrogens, oocyte growth, and granulosa cells proliferation through increasing the expression of cyclins, cyclin-dependent kinases, aromatase, and cyp17a1. In parallel, the expression of lhcgr is also induced [progesterone receptors (pr-a and pr-b).In mammalian testis, LH binding to luteinizing hormone/choriogonadotropin receptor (LHCGR) induces cAMP and cyclic guanosine monophosphate (cGMP) increase in Leydig cells. Consequently, PKA, PKC, and protein kinase G (PKG) activity is stimulated, as well as CaHSD17B3) . In Sert release . In the  induced . At the  induced ,142. In 3), and thyroxine . THs coiewed in ). Besideiewed in . How THsiewed in ). While 2, respectively, suggesting a positive THs control of aromatizable steroids in the ovary. In contrast, some studies evidenced a negative relationship between hypothyroidism and increased serum concentration of P4 and PRL, which cause a luteal phase prolongation, as demonstrated by hyperthyroidism induced premature luteolysis in rats (reviewed in [Oreochromis niloticus), T3 stimulates kiss and gnrh gene expression (reviewed in [androgen receptor (ar), steroid 5α-reductase 1  and 2  aromatase (cyp19a1), as anti-Müllerian hormone (amh) and consequently estradiol levels in all vertebrates, as well as insulin-like growth factor binding protein 1 and 3 (igfbp1a and igfbp3), particularly in zebrafish [In mammals, once released to the blood stream, THs can be transported associated with globulin, transthyretin, and albumin  and thusiewed in ). In maliewed in ), while iewed in ). In teliewed in . THs impebrafish .Stress conditions are also important for reproductive success. Under stress, vertebrates increase the production of CRH at the hypothalamus that will stimulate the synthesis of the adrenocorticotropic hormone (ACTH) in the anterior pituitary. In humans, ACTH induces steroidogenesis , but als2 but increases P4 levels [CORT acts on target tissues through mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Besides increasing blood glucose levels, acting on cardiovascular and immune system with anti-inflammatory properties, CORT is also involved in human and murine reproduction (reviewed in ). Increa4 levels ,155.ggcx, vkorc1 and vkorc1l1 were reported to be expressed in the adrenal gland of male and female mice [Although significant levels of several K vitamers (particularly MK-4) were found in tissues, like mammalian thyroid and adrenal gland ,16, and ale mice , little ale mice , severalale mice . In a veale mice , suggestocn−/− mice are poor breeders that are characterized by low testosterone levels, oligospermia and a reduced reproductive organ weight, a condition that can be reversed by OCN administration [Gprc6a−/− and ocn−/− mice have almost identical metabolic and reproductive phenotypes, including reduced testosterone levels and particularly, Leydig cell-specific deletion of GPRC6A (gprc6aLeydig+/−) leads to these reproductive defects in males [Leyding−/−Creb mice [2+ release [star, cyp11a, cyp17, and 3β-hsd genes that are required for testosterone biosynthesis, and representing a complex pancreas-bone-testis axis communication . Iniewed in . Moreoveiewed in , a low tmiR-146 family members (miR146a-1-2-3 and miR-146a) and a higher of let-7 family members , -7a1, -7a-3, -7a-2, -7a-1, -7e (23 nt)) and piR-675//676//4794//5462 was found when compared to that of males fed the Control diet (with higher sperm DNA fragmentation). Both miR146 and let-7 family members are expressed in spermatogonia and only let-7s in spermatocytes, and oppositely involved in the RA-induced spermatogonia differentiation in mice [With the discovery of the key role of non-coding RNAs (ncRNAs) in cell biology, a diverse set of ncRNAs  and P-element–induced wimpy testis (PIWI)–interacting RNAs (piRs)) involved on vertebrate’s reproduction as well as their corresponding DNA/mRNA target sequences were identified. A key role of piRs in male gametogenesis was clearly evidenced by the male sterility in mice lacking the piR pathway . Similar in mice ,174. A b in mice .Fulfilling the dietary recommended intake (DRI) of each nutrient is basic for a proper development and health, and to complete all of the required biological functions, including reproduction. Furthermore, not covering the requirements for a particular nutrient may have deeper impact on the progeny, as it has been found for VK in different vertebrate species ,175. KnoIn particular, VK seems to have an impact on testosterone synthesis among other steroids, oxidative stress response, and osteocalcin signaling in gonads, as well as GnRH, glutamate, and intracellular calcium levels and signaling at hypothalamus and pituitary tissues. While some classical biomarkers of gametogenesis, such as testosterone, LH, and FSH serum levels, are already available for determining the optimal VK dietary level for vertebrate’s reproduction, a better understanding of the precise signaling pathways active in the organs, and tissues that are involved in the complex regulation of reproduction would allow a deeper and complete prediction of the effects elicited by VK nutritional status. Recent results on the potential cell-to-cell communication through circulating ncRNAs revealed an unexpected regulatory pathway, by which VK may exert a role on reproductive performance. Future research on this issue, as well as on the potential role of VK on thyroid and adrenal gland, might bring new light to understand the broad biological role of VK in vertebrates. Last, but not the least, we have recently shown as the use of fish teleosts is an interesting biological model in vertebrates to identify the effects and describe the conserved VK-related molecular pathways by which VK has a great impact, not only in blood coagulation and bone tissue, but also in reproduction. In this sense, the use of these species might help to unveil whether parental VK nutritional status might determine primordial germ cell population (PGCs), sex gender, gonad development, and/or reproductive performance on their progenies.


ESR1-CCDC170 (E:C) fusion is the most frequent oncogenic fusion driver of the luminal B subtype. Nevertheless, treatments targeting E:C fusion has not been well established yet. Hence, the aim of this study is to investigate potential therapies targeting E:C fusion based on systematic bioinformatical analysis of the Cancer Genome Atlas (TCGA) data. One thousand related genes were extracted using transcriptome analysis, and major signaling pathways associated with breast cancer were identified with over-representation analysis. Then, we conducted drug-target network analysis based on the OncoKB and CIViC databases, and finally selected potentially applicable drug candidates. Six major cancer-related signaling pathways  were significantly altered in E:C fusion-positive cases of breast cancer. Further investigation revealed that nine genes  in coordination with E:C fusion were found to be common denominators in three or more of these pathways, thereby making them promising gene biomarkers for target therapy. Among the 21 putative actionable drugs inferred by drug-target network analysis, palbociclib, alpelisib, ribociclib, dexamethasone, checkpoint kinase inhibitor AXD 7762, irinotecan, milademetan tosylate, R05045337, cisplatin, prexasertib, and olaparib were considered promising drug candidates targeting genes involved in at least two E:C fusion-related pathways.Among the various types of breast cancer, the luminal B subtype is the most common in young women, and  ESR1-CCDC170 fusion-positive subtype, constituting 6% to 8% of the luminal B class, persists to be the most dominant subtype [Breast cancer, aside from skin cancer, is the most commonly diagnosed cancer in women worldwide . Recent  subtype ,6,7,8,9.ESR1-CCDC170 fusion causing chimeric mRNA is known to be formed by a tandem duplication at the 6q25.1 location on a coiled-coil domain containing 170 (CCDC170) adjacent to the ESR1 gene [CCDC170 gene correlates with breast cancer susceptibility [ESR1-CCDC170 fusion-positive cancers treated with endocrine therapy showed reduced treatment efficiency in mouse models [ESR1-CCDC170 fusion-positive breast cancer has yet to be elucidated [SR1 gene ,10. It htibility ,12. ESR1e models . Althougucidated .ESR1-CCDC170 fusion-positive breast cancer through the data analysis of the Cancer Genome Atlas (TCGA) and identified the activated oncogenic pathways. In addition, putative target genes and actionable drugs were inferred and prioritized by performing network analysis using both transcriptomic signatures and drug-target databases, such as OncoKB and CIViC.Here, we systematically analyzed the molecular pathological features of https://gdac.broadinstitute.org). Related clinical feature data, including information about the samples’ mutation annotation format (MAF) files, molecular subtypes, and tumor-node-metastasis (TNM) stages, were obtained from the website mentioned above.Gene level 3  mRNA expression with normalized read count values of TCGA breast cancer carcinoma (BRCA) was obtained from the Broad GDAC Firehose website , we identified 11 samples of CCDC170 fusion, which were cross-checked with increased CCDC170 expression level. Furthermore, only tumor samples that had the barcode 01A (primary solid tumor) were selectively chosen by disregarding other types of tumor samples, 11A  or 06A (metastasized). Among the remaining samples, 50 samples with the highest expression of CCDC170 were confirmed as upregulated controls for analyzing the network within the noncoding region of the fusion gene. From the upregulated control samples, 2 outlier samples were filtered out using the IQR (interquartile range) method. The same number of controls (n = 48) with the lowest expression of CCDC170 were then selected from the remaining samples.A previous study confirmed 319 fusion genes in TCGA clinical breast cancer tumors . Unlike t-tests. To select genes in coordination with CCDC170 in the RNA expression, t-tests were performed between E:C fusion-positive and fusion-negative cases. Mostly affected 1000 genes were selected .RNA expression data from TCGA were made into a two-dimensional matrix composed of the selected 11 fusion samples and 48 control samples. Each column represents the patient ID, while each row represents the gene name. Based on the RNA expression matrix, variance tests were conducted using independent two-sample CCDC170) from the aforementioned RNA expression data were used to perform over-representation analysis (ORA) via ConsensusPathDB . We inputted a gene list with the option of Entrez Gene using pathway-based sets with a minimum overlap input list (n = 2) and p-value cutoff . A total of 113 biological pathways were merged and curated by CPDB from the following sources, according to data from BioCarta (https://maayanlab.cloud/Harmonizome/dataset/Biocarta+Pathways), INOH [q-value < 0.05), were condensed into 15 cancer-related pathways, and their components were 184 genes.The selected 1000 genes that correlated to the reference gene (s), INOH , KEGG [1s), INOH , PID [19s), INOH . In conshttps://civic.genome.wustl.edu/releases), and the “Actionable Variants” data were accessed and downloaded on 17 October 2017 from the OncoKB website (http://oncokb.org/). A total of 673 CIViC variants (181 genes) with expected therapy efficacy in 148 OncoKB actionable variants (53 genes) were integrated. A total of 113 CCDC170-correlated genes were matched to the CIViC and OncoKB variants.The “Clinical Evidence Summaries” data, released on 1 October 2017, were downloaded from the Clinical Interpretations of Variants in Cancer (CIViC) website  was used to visualize target pathways related to DNA damage response. Cytoscape version 3.5.3 was used to analyze and express the complex network between targetable drugs and therapeutic agents. Our study defined statistical significance with a p-value of < 0.05 and false detection rate (FDR) with a q-value of <0.001.The open software R version 3.4.3 was used to process all statistical analyses for selecting genes correlated to ESR1-CCDC170 fusion-positive and 48 fusion-negative patients among 1095 breast cancer patients in Broad GDAC Firehose  and PR-positive (63.6%), whereas fusion-negative patients displayed significantly lower rates of 20.8% and 4.3%, respectively (p < 0.05). Additionally, HER2 immunohistochemistry (IHC) results showed a significantly higher rate of 3+ for fusion-positive patients than for patients with fusion-negative  and luminal B subtypes (45.5%), while 90.9% of the 48 cases with the lowest PIK3CA, CDH1, TP53, BRCA1, and BRCA2) frequently found in BRCA showed no significant difference between the two groups. Based on these results, CCDC170 fusion-positive BRCA patients have distinct pathological characteristics in terms of tumor subtype and triple positive tendency.On the other hand, no significant differences in age, sex, vital status, and TNM stage were observed between the two groups. In addition, the five gene variants (t-test (q < 2.0 × 10−8) and inputted for performing the over-representation analysis (ORA) of the ConsensusPathDB website to select cancer-related pathways. As a result, a total of six cancer-related pathways  were discerned.One thousand genes were obtained by an independent CCDC170 fusion-positive cases compared with the CCDC170 fusion-negative controls , MDM2, CHEK1(Chk1), and CHEK2(Chk2).Of the six pathways, two concerning p53- and ATR/ATM-related signaling pathways were associated with DNA damage response. Mapping with the KEGG pathway revealed 22 genes that are involved in the p53-related pathway and 17 genes in the ATR/ATM-related pathway. Both pathways are highly relevant to the promotion and maintenance of the cell cycle . Genes wAURKB, HDAC2, PLK1, CENPA, CHEK1, CHEK2, RB1, CCNA2, and MDM2 were included in at least three pathways that are important for tumor proliferation and maintenance specific to ESR1-CCDC170 fusion-positive BRCA patients.There were 39 genes involved in multiple pathways , of whicCCDC170 with the differentially expressed gene (DEG) analysis showed similar patterns as the TCGA data obtained above in fusion-positive samples when compared with the control samples (CCDC170 over-expression.Further investigation of the 48 samples with the highest mRNA levels of  samples . This sun = 673) and OncoKB (n = 262). ESR1, CDK4, RAD50, CHEK1, MDM2, and SMO were mapped as targetable genes. Results indicated the following drug-target relationships: letrozole, palbociclib, fulvestrant, AZD9496, and tamoxifen for ESR1; palbociclib, alpelisib, ribociclib, and dexamethasone for CDK4; checkpoint kinase inhibitor AZD7762 and irinotecan for RAD50; cisplatin, prexasertib, and olaparib for CHEK1; milademetan tosylate and RO5045337 for MDM2; and PSI, vismodegib, patidegib, and arsenic trioxide for SMO , endocrine therapy , ERBB-targeted therapy (trastuzumab and pertuzumab), and combination treatment methods according to the pathological and molecular classification of breast cancer . In the Among the repositioned drugs inferred in our study, CDK4/6 inhibitor , cisplatin, and PARP inhibitor are the drugs used as standard treatments for breast cancer patients with or without metastasis. On the other hand, AZD9496, alpelisib, dexamethasone, checkpoint kinase inhibitor AZD7762, irinotecan, cisplatin, prexasertib, milademetan tosylate, R05045337, PSI, vismodegib, patidegib, and arsenic trioxide are seen as putative actionable drugs that can be used for E:C fusion-positive BRCA proceeding in vitro and in vivo validation.AURKB, HDAC2, PLK1, CENPA, CHEK1, CHEK2, RB1, and MDM2 genes, which were included in at least three pathways, are expected to play an important role in the promotion and maintenance of CCDC170-subtype breast cancer. For instance, PLK1 may act as a tumor suppressor gene that regulates estrogen receptor (ER)-regulated gene transcription in breast cancer [RB1 gene, also a tumor suppressor gene, however, is frequently lost in triple-negative breast cancer [CENPA is a significant prognostic marker for ER-positive patients [HDAC2 and CHEK2 genes have been significantly correlated to CCDC170 fusion subtype and have been reported to be associated with DDR functioning [CCDC170 fusion subtype’s relationship with DDR.t cancer ; RB1 gent cancer ; CENPA ipatients ; and HDActioning ,31, whicIn addition, we investigated whether there is a biological difference between the E:C fusion-positive group and the CCDC170 high-expression group without fusion. We found that there showed no major difference in cancer signaling except in several minor pathways, including the cilium assembly pathway and integrins in the angiogenesis pathway .In summary, this study presents core biomarkers and potentially actionable drugs specific to E:C fusion-positive breast cancer. Via in vitro experimentation, these candidates were confirmed to be strongly associated with this type of cancer, and their roles were verified by discerning their associated signaling pathways. We hope that our findings will be the steppingstone for future investigations, leading to the promotion of a targeted cancer therapy.


Medicago truncatula. In this study, six MtCRK genes were computationally identified, they were classified into five groups with MtCDPKs based on phylogenetic relationships. Six pairs of segmental duplications were observed in MtCDPK and MtCRK genes and the Ka/Ks ratio, an indicator of selection pressure, was below 0.310, indicating that these gene pairs underwent strong purifying selection. Cis-acting elements of morphogenesis, multiple hormone responses, and abiotic stresses were predicted in the promoter region. The spatial expression of MtCDPKs and MtCRKs displays diversity. The expression of MtCDPKs and MtCRKs could be regulated by various stresses. MtCDPK4, 14, 16, 22, and MtCRK6 harbor both N-myristoylation site and palmitoylation site and were anchored on plasma membrane, while MtCDPK7, 9, and 15 contain no or only one N-acylation site and were distributed in cytosol and nucleus, suggesting that the N-terminal acylation sites play a key role in subcellular localization of MtCDPKs and MtCRKs. In summary, comprehensive characterization of MtCDPKs and MtCRKs provide a subset of candidate genes for further functional analysis and genetic improvement against drought, cold, salt and biotic stress.Calcium-dependent protein kinase (CDPK or CPK) and CDPK-related kinase (CRK) play an important role in plant growth, development, and adaptation to environmental stresses. However, their gene families had been yet inadequately investigated in  Ca2+ is bound with calcium binding proteins (CBP), or Ca2+ sensors, which decode the signals and lead to cellular alterations in biochemical and physiological processes [2+ sensors in plants, including calmodulin (CaM), calmodulin-like protein (CML), calcineurin B-like protein (CBL), calcium/calmodulin-dependent protein kinase (CCaMK) [2+ signal into phosphorylation cascades, which makes CDPKs have a dual function: Ca2+ sensors and responders [Calcium is a ubiquitous second messenger in plant cells involving in growth, development, and responses to environmental stresses . The extrocesses ,4. There (CCaMK) , and cal (CCaMK) . Comparesponders .2+ [CDPKs exist in higher plants, green algae, oomycetes, and protists, but absent in animals and fungi , while C2+ . There i2+ . The mem2+ .Arabidopsis [Arabidopsis [OsAPX2/OsAPX8 and NADPH oxidase OsrbohI expression is regulated by OsCPK12 in rice [+ channel TPK1 is phosphorylated by AtCPK3 to regulate the cytosolic K+/Na+ balance in responses to salt stress [Botrytis cinereal infection via inducing ethylene production through modulating the transcriptional level of ethylene biosynthesis enzyme ACC synthase ACS2 and ACS6 [2O2, which is crucial for accelerating rapid signal propagation to defense response activation [Pst DC3000 and Sclerotinia sclerotiorum resistance in tomato [M. truncatula [M. truncatula, a legume model plant, had been yet inadequately investigated. Although 25 MtCDPK genes were identified and characterized [cis-acting elements, and subcellular localization. In addition, MtCRK genes have not been investigated.CDPKs and CRKs participate in regulation of multiple physiological processes. They are involved in pollen tube elongation , seedlinbidopsis ,23. CDPKbidopsis , while a in rice . Moreovet stress . OsCPK17t stress . GmCDPKSt stress . AtCRK1 t stress ,29. AtCRt stress . CDPKs at stress . AtCPK5,and ACS6 . AtCPK5 tivation . SlCRK6 n tomato . MtCDPK8uncatula ,36,37. Cuncatula ,39. Howecterized , they wecis-acting regulatory elements of MtCDPK and MtCRK gene families in M. truncatula and to analyze their spatial expression profiles and response to abiotic and biotic stresses. In addition, the subcellular localization of selected MtCDPKs and MtCRKs were analyzed to explore the role of N-terminal acylation in protein localization.The objectives of this study were to investigate the gene structure, conserved motifs, phylogenetic relationship, chromosome distribution, gene duplication, and MtCRK gene families was performed based on M. truncatula genome sequences using HMMER3.0, SMART, and CDD software. A total of six MtCRKs were perceived from M. truncatula genome. They and previously identified MtCDPKs were denominated as MtCDPK1 to MtCDPK24 and MtCRK1 to MtCRK6, respectively, depending on their chromosomal positions  to 6710 bp (MtCRK1), encoding protein length varies from 581 (MtCRK6) to 596 (MtCRK5) amino acids (aa). The molecular weight (MW) varies from 65.15 (MtCRK6) to 66.80 kDa (MtCRK4), while the isoelectric point (pI) varies from 8.49 (MtCRK3) to 9.07 (MtCRK2), indicating that MtCRKs are alkaline. The grand average of hydropathy (GRAVY) ranges from −0.415 (MtCRK5) to −0.305 (MtCRK4), indicating that MtCRKs are hydrophilic. All MtCRKs contain both predicted N-terminal myristoylation and palmitoylation sites. In addition, most MtCDPKs harbor these predicted sites, while MtCDPK7 contains only a predicted palmitoylation site and MtCDPK15 contains a predicted myristoylation site. MtCDPK9, 10 and 23 were predicted to have neither myristoylation nor palmitoylation site  phylogenic tree was constructed using CDPK and CRK sequences from MtCDPKs and MtCRKs was analyzed using TBtools software and gene annotation information. The results showed that MtCDPKs and MtCRKs are distributed on six chromosomes  were found to be paralogous, indicating that they belonged to segmental duplications (Ka) and the number of synonymous substitutions per synonymous site (Ks) substitution rates , dehydration-responsive element (DRE1 and DRE-core), drought-inducibility element (MBS), low-temperature-responsive element (LTR), defense and stress-responsive element (TC-rich repeats), stress-responsive element (STRE) and wound-responsive element (WRE3 and WUN-motif). Multiple growth and development elements were investigated, including endosperm-expression element (AACA-motif and GCN4-motif), meristem-expression element (CAT-box and CCGTCC-motif), circadian element, palisade mesophyll cells-expression element (HD-Zip 1), flavonoid biosynthetic genes regulation element (MBSI), zein metabolism regulation element (O2-site), and seed-specific regulation element (RY-element). Overall, two phytohormone responsive elements (ABRE and ERE) and two stress responsive elements (ARE and STRE) were detected in the promoter regions of most genes. But auxin-responsive elements, gibberellin-responsive elements, dehydration-responsive elements, and most of plant growth and development elements were found in the promoter regions of specific genes. The results indicated that MtCDPKs and MtCRKs are involved in plant growth and development as well as environmental stress responses.To gain insight into the potential function and regulatory mechanisms of the genes during plant development and responses to various stresses, the recognized putative MtCDPK and MtCRK genes was analyzed based on microarray data from the Medicago truncatula Gene Expression Atlas . MtCDPK7, 17, 22, and MtCRK3, 6 transcripts level was lower than other genes. MtCDPK8, 16, 18, 19, 22, 23, and MtCRK1 transcript major observed in roots. MtCDPK17 and MtCRK2 were preferentially expressed in flower, while MtCDPK14, MtCDPK20, and MtCRK4 transcript were low in leaf. MtCRK5, MtCDPK9, and MtCDPK20 transcript were decreased during seed development, while MtCDPK4 and MtCDPK19 were increased  was available from MtGEA.The spatial expression pattern of ncreased . The resMtCDPKs and MtCRKs under abiotic stresses, available RNA-seq data were retrieved from NCBI (GEO database). Fragments per kilobase of exon per million fragments mapped (FPKM) values of MtCDPK1, 5, 6, 10, 11, and 13 were lower than 1, which were considered to be barely expressed and not employed for analysis. MtCDPK4, 8, 15, 16, and 22 transcripts were quickly increased after 2 h of cold treatment. MtCDPK2 transcript was increased continuously under drought stress, MtCDPK4, 8, 12, 15, 16, 21, and 22 transcripts were increased and peaked after 2 h of drought treatment, whereas MtCDPK22 transcript was decreased at 6 h. In contrast, MtCDPK23 and MtCRK6 were downregulated after drought treatment. In response to salt treatment, MtCDPK2, 4, 8, 12, and 16 transcripts were upregulated after salt stress, while MtCRK6, MtCDPK7, and MtCDPK17 transcripts were downregulated  is a well-known protein kinase in Medicago truncatula, involving in nodule organogenesis [MtCDPKs and MtCRKs are not distributed on every chromosome, which is consistent with that in poplar [elopment ,8. Comprbidopsis , 31 CDPK in rice , 29 CDPKn poplar , and 49 ica rapa . In the ogenesis . It belon poplar , soybeann poplar , and meln poplar .Arabidopsis, rice, and M. truncatula, which is consistent with that in other plant species [MtCRKs has a similar gene structure with the members in MtCDPK IV, and cluster with the MtCDPK IV group with 100% bootstrap support. Furthermore, the theoretical pI of the members in the MtCRK and MtCDPK IV group tends to be moderately alkaline, while that of in other group shows slightly acidic. The above data are consistent with those in Arabidopsis, Brassica rapa, and watermelon, indicating that CRKs may have evolved from MtCDPK IV [MtCDPKs and MtCRKs were divided into five groups (CDPK I to IV and CRK) based on the phylogenic tree among those in  species ,41,42,43tCDPK IV ,43,47. TM. truncatula and 29 pairs of duplication events were found between M. truncatula and A. thaliana. Segmental duplication gene pairs were found in CDPK and CRK gene families in rice [Arabidopsis and other species have been reported. There are 22 collinear blocks of CDPK and CRK genes between melon and Arabidopsis [CDPK among species is associated with species-specific WGD events [M. truncatula has experienced a high frequency of local genome rearrangement [MtCDPKs and MtCRKs expansion. Ka/Ks ratio was used to determine selection pressure on the duplicated gene pairs. The Ka/Ks ratio of six paralogous genes was below 0.310. This is consistent with that in tomato [Brassica rapa [Gene duplication is a primary driving force for plant evolution and leads to the expansion of gene family. Gene duplication models include segmental/whole-genome duplication (WGD), tandem duplication, proximal duplication, transposed duplication, and dispersed duplication . In our  in rice  and popl in rice . Inter-gbidopsis . The varD events ,8. M. trangement . Our anan tomato  and Brasica rapa . The rescis-acting elements responsible for phytohormones, stresses, growth and development were observed in the promoter regions of MtCDPKs and MtCRKs, indicating the potential role of MtCDPKs and MtCRKs in regulating multiple responses to phytohormones, environmental stresses, and development. The spatial expression of MtCDPKs and MtCRKs displays diversity, for instance, MtCDPK17 was exclusively expressed in flower, and its homologous genes AtCPK17 and AtCPK34 from Arabidopsis were reported to be involved in pollen tube growth [CDPK and CRK family genes are involved in responding to various stresses, such as drought [MtCDPK22 transcript is specifically and strongly expressed under cold stress, and its homologous gene OsCPK17 is involved in cold stress response [MtCDPK8 was highly induced under cold treatment, while its paralogous gene MtCDPK18 was downregulated expression. Transcripts of MtCDPK4, 8, 16, 18, 21, and 22 were responsive to yeast elicitor treatment. AtCPK28, orthologous to MtCDPK16, was verified to be a negative regulator of PAMP-triggered immunity (PTI) [M. truncatula. MtCDPK2, 18, 19, and CRK1, which contains dehydration-responsive elements (DRE1 or DRE-core), were upregulated under drought stress. In Arabidopsis, some AtCPKs are involved in regulating phytohormone and abiotic stresses signaling when specific cis-acting elements were detected in the promoter regions [Multiple e growth . CDPK an drought ,23, salt drought , cold st drought , and fun drought . MtCDPK2response . MtCDPK8ty (PTI) . The res regions ,54. TherS-palmitoylation [M. truncatula.Myristoylation and palmitoylation are unique among lipid modifications . They anoylation ,65. Prevoylation . ChangesCRK genes were obtained from genome databases of Arabidopsis , rice , and Medicago truncatula (http://www.medicagohapmap.org/) respectively. The amino acid sequences of CRKs of Arabidopsis and rice were used as reference sequences to search predicted homologs in Medicago truncatula using HMMER3.0 Software (http://hmmer.org/). The candidate CRK genes were preliminarily identified by screen the gene using threshold of E-value (full sequence and best 1 domain) less than 1E-100. The composition of identified candidate proteins was further verified in SMART databases (http://smart.embl-heidelberg.de/) [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), while the sequences with errors, shorter length (<100 aa), and containing incomplete Ser/Thr kinase domain were eliminated. The longest one was chosen for further analysis if a gene had alternative splice variants.The sequences of erg.de/)  and NCBIhttp://web.expasy.org/protparam/). The EF-hands motif was predicted using the ScanProsite tool (https://prosite.expasy.org/prosite.html). The N-terminal myristoylation and palmitoylation sites were predicted using GPS-Lipid 1.0 program with default settings and high threshold [Molecular weight (MW), isoelectronic points (pI), and the grand average of hydropathy (GRAVY) of MtCDPKs and MtCRKs were predicted using The ExPASY PROTPARAM tool . The phylogenetic tree was constructed by using MEGA7.0 with the neighbor-joining method, and the 1000 bootstrap replicated by the Jones, Taylor, and Thornton amino acid substitution model (JTT model) and keeping the other parameters as a default to determine the reliability of the resulting tree [Multiple alignments of CDPK and CRK protein sequences from ing tree .http://meme-suite.org/tools/meme) [The gene structure and conserved motifs were displayed using TBtools software . The conls/meme) . The maxMtCDPK and MtCRK genes, the Circos diagram was illustrated by annotating genes on their specific chromosomal position in the genome annotation using TBtools software. The syntenic gene relationships between the homologs of A. thaliana and M. truncatula were verified and visualized using the Circos tool implemented in TBtools software.For analysis of chromosomal locations of Ka) and synonymous (Ks) substitution rates of paralogous gene pairs of MtCDPKs and MtCRKs were calculated based on the standard genetic codon table using the Nei–Gojobori method (Jukes–Cantor model) in MEGA 7.0 [The nonsynonymous  to predict the putative cis-acting regulatory elements [A 2.0 kb of promoter sequence upstream from the transcription start site in each elements .MtCDPKs and MtCRKs in roots, stem, vegetative bud, leaf, petiole, flower, pods, and seeds and responses to pathogen were extracted from the MtGEA (https://mtgea.noble.org/v3/) [MtCDPKs and MtCRKs in response to cold, drought, and salt stresses was from NCBI GEO with the dataset accession of GSE136739 [MtCDPK and MtCRK gene. The relative transcript level after treatments was calculated compared with the control before treatment (0 h). The clustered heatmap was portrayed based on the relative expression using the TBtools software.Microarray data on the expression profile of org/v3/) . The proSE136739 . FPKM waAgrobacterium tumefaciens EHA105 harboring the constructed vectors or empty vector in combination with 35S::mCherry vector or 35S::AtAKT1-mCherry vector were used to co-transform the abaxial surfaces of leaves of 4-week-old N. benthamiana. Then the tobaccos were grown under normal condition. Before the observation, the tobacco injected with P.M-marker was immersed in 0.3 g/mL sucrose solution for plasmolysis. Fluorescence was observed using confocal laser scanning microscopy  at 72 h after transformation.The coding sequence without the terminal codon of selected MtCDPKs and MtCRKs was amplified using specific primer pairs and inserted into the expression vector pCAMBIA1305-GFP driven by CaMV 35S promoter utilizing homologous recombination . Agrobac


The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1. The SAM and HD domain-containing protein 1 (SAMHD1) is a ubiquitously expressed deoxynucleotide triphosphohydrolase (dNTPase) of 626 amino acids . In gene2 phase of the cell cycle [0 and G1 phases [SAMHD1 converts dNTPs into deoxynucleosides (dN) and inorganic triphosphates (PPPi) and thereby counteracts the de novo dNTP synthesis, primarily conducted by ribonucleotide reductase (RNR) and cellular deoxynucleoside kinases, which are mainly active during the S phase of the cell cycle ,16,17,18ll cycle ,23. SAMH1 phases . Thus, S1 phases ,26,27,28By degrading cellular dNTPs, SAMHD1 is a major regulator of nucleotide homeostasis. A highly balanced dNTP pool is essential for genomic integrity, including proper DNA replication and efficient repair of DNA breaks. An imbalance can lead to a deregulated cell-cycle progression and therefore induce replication stress, which might eventually result in the accumulation of genomic mutations ,30. DuriSAMHD1 gene, as well as silencing hypermethylation of the SAMHD1 promoter, might play a role in tumorigenesis of various forms of cancer such as colon cancer, chronic lymphocytic leukemia, and lung adenocarcinoma [SAMHD1 has also been described to be important for DNA damage repair by promoting homologous recombination (HR) . SAMHD1 arcinoma ,34,35,36SAMHD1 are associated with Aicardi–Goutières syndrome (AGS)—an autoimmune disorder characterized by a progressive inflammatory encephalopathy, spontaneous type I IFN production in the cerebrospinal fluid, and upregulated IFN-stimulated gene (ISG) expression [SAMHD1 [Mutations in pression . AGS higpression ,38. The  [SAMHD1 ,37,39. D [SAMHD1 ,38,40. I [SAMHD1 ,42,43.Besides its protective role in autoimmunity, SAMHD1 was also shown to suppress the antiviral immune response upon infection . HIV-1 r+ T cells [A hallmark of the retroviral life cycle is the reverse transcription of the viral RNA genome, followed by integration of the cDNA into the host genome in form of proviral DNA. Thus, retroviruses require relatively high intracellular dNTP levels for reverse transcription. In nondividing cells, SAMHD1 limits viral infectivity by downregulating cellular dNTP concentrations required for an efficient reverse transcriptase (RT) activity . Initial T cells ,55,56,57 T cells ,42. Whil T cells ,25. Howe T cells ,27,28. O T cells ,59. Howe T cells ,61.In addition to HIV-1, SAMHD1 has been shown to inhibit other lentiviruses, including HIV-2, several simian immunodeficiency viruses (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and equine infectious anemia virus (EIAV) . SAMHD1 Together, these findings show that the SAMHD1-mediated restriction is not limited to HIV and SIVs, but acts on many different retroviruses upon entry of nondividing cells, with the notable exception of foamy viruses, which enter new target cells with an almost completely reverse-transcribed viral genome . AnotherIn addition to exogenous retroviruses, SAMHD1 has also been shown to downregulate the activity of endogenous retroelements. In 2013, Zhao and colleagues found that SAMHD1 inhibits the retrotransposition of long interspersed nuclear element 1 (LINE-1) GFP-reporter elements and identified AGS-associated SAMHD1 mutants to be defective for LINE-1 restriction . AlthougInterestingly, however, all groups found that SAMHD1 is active against LINE-1 retroelements in cycling cells. In addition, Herrmann et al. found that the capacity of SAMHD1 to block the activity of LINE-1 was regulated by phosphorylation of T592 but did not correlate with its dNTPase activity . TogetheDNA viruses also rely on cellular dNTPs for replication of their genome. Consequently, several DNA viruses were found to be negatively affected by the dNTPase activity of SAMHD1. Hepatitis B virus (HBV) belongs to the hepadnavirus family and replicates its viral DNA genome through an RNA intermediate and a reverse-transcription step. In 2014, Chen et al. found that SAMHD1 potently restricts the viral expression and replication of transfected HBV DNA in liver cell lines . The autSAMHD1 was also found to potently inhibit the infection of vaccinia virus (VACV), a member of the poxvirus family, and the α-herpesvirus herpes simplex virus 1 (HSV-1) by limiting intracellular dNTP levels through its dNTP hydrolase activity in nondividing myeloid cell lines ,72. ReceWhile SAMHD1 experienced positive selection during host–pathogen coevolution, several viruses have also developed evasion strategies to antagonize the restriction by SAMHD1 ,79,80. T+ T cells [trans, when delivered into cells by virus-like particles [A hallmark of lentiviral genomes is the presence of small accessory open reading frames. While both HIV-1 and HIV-2 encode the accessory protein Vpr, only HIV-2 and related SIVs contain an additional open reading frame for Vpx ,82. Vpx  T cells ,55,56,89articles ,90. In tarticles ,55. Cullarticles . In casearticles ,96,97,98Vpx gene is only present in two out of eight SIV lineages, the HIV-2/SIVsmm/SIVmac lineage and the SIV rcm/SIVmnd2  lineage. Lim and colleagues analyzed SAMHD1 degradation upon cotransfection with Vpr and Vpx proteins from different SIVs [The genetic conflict between innate immunity and viruses results in the rapid selection of mutations in proteins involved in direct host–pathogen interaction. The groups of Monsef Benkirane and Michael Emerman described this “arms race” for SAMHD1 and Vpx/Vpr ,99. Whilent SIVs . While Vent SIVs . Schwefeent SIVs ,102.Interestingly, another lentivirus, EIAV, has evolved a different strategy to circumvent SAMHD1 restriction in equine macrophages . While em values of HIV-1 RTs to be very low in general and in the range of the low dNTP concentrations found in nondividing macrophages. In contrast, enzymes encoded by viruses that trigger SAMHD1 degradation, such as HIV-2, displayed significantly higher Km values and required higher dNTP concentrations to efficiently generate cDNA [Nondividing myeloid cells, such as macrophages or microglia, are targeted by many lentiviruses during the course of infection. However, not all lentiviruses encode Vpx, or a protein with a Vpx-like function, to inactivate SAMHD1. Instead, to overcome the SAMHD1-driven shortage of dNTPs in nondividing cells, viruses lacking Vpx harbor very efficient reverse transcriptases that polymerize viral cDNA at low dNTP concentrations ,106,107.ate cDNA . InteresInterestingly, however, pretreatment of nondividing macrophages with Vpx-containing virus-like particles further boosts HIV-1 infectivity in macrophages in vitro, despite optimized RT enzymes ,108. At Human herpesviruses (HHV) encode conserved herpesvirus protein kinases (CHPK). This family of serine/threonine kinases includes UL13 of HSV-1 and -2, ORF47 of varicella-zoster virus (VZV), UL97 of HCMV, U69 of HHV-6 and -7, BGLF4 of EBV, and ORF36 of Kaposi’s sarcoma-associated herpesvirus (KSHV) ,110,111.In 2019, Kim et al. and Businger et al. analyzed the role of SAMHD1 in HCMV infection of myeloid cell lines ,76. TheyThe murine homolog of SAMHD1 occurs in two isoforms. Isoform 2, which is expressed at low levels, is differentially spliced, and therefore lacks the regulatory C-terminal phosphorylation site. Isoform 1, however, highly resembles the human protein with an equally abundant expression pattern ,118,119.In line with these findings, a recent study by Zhang and colleagues found that the EBV kinase BGLF4 phosphorylates human SAMHD1 at the T592 site. The phosphorylation abrogated EBV inhibition by SAMHD1 as well as its dNTPase activity . In thei+ hematopoietic progenitor cells [Aside from the phosphorylation-dependent inactivation of SAMHD1, the downregulation of SAMHD1 expression levels has been observed as a viral countermeasure mediated by several DNA viruses. In case of HCMV, Businger et al. found that the infection of primary MDMs results in a decreased steady state expression of SAMHD1 . This isor cells . Hyeon aor cells . In addior cells . Of noteor cells ,129. Alsor cells . In caseor cells ,69. Alsoor cells .SAMHD1 is a potent restriction factor of various viruses with different replication strategies. The growing spectrum of viruses found to be inhibited by SAMHD1 accentuates its importance for the intrinsic immune defense. During mammalian evolution, SAMHD1 has been under positive selective pressure, and an antiviral activity has been described not only for human or primate SAMHD1, but also for proteins from other species, such as equine, feline, bovine, or murine SAMHD1 ,119,131.+ T cells leads to a dramatic decrease in HIV sensitivity against RT inhibitors, especially those that resemble thymidine. By degrading cellular dNTPs, SAMHD1 leads to an enhanced concentration of exogenous NAs and therefore to a more efficient viral inhibition [Viral antagonists are promising drug targets for the development of novel antiviral therapies. Many current antiviral therapies are based on nucleoside analogues (NA) . NAs highibition ,135. Exihibition ,139,140.hibition ,142. Thu


Correction to: Crit Care (2020) 24:140https://doi.org/10.1186/s13054-020-2815-9Following publication of the original article , the autAnother error was identified by the authors‘response authors which reported the references used in the Authors‘response section were incorrect.bold typeface.The updated Authors‘response and additional references are given below and the changes have been highlighted in Authors’ response2, 3*, Lukas Schuierer1, 2, 3Reinhard Hoffmann1: TUM Graduate School, Technical University of Munich (TUM), Germany2: Institute for Laboratory Medicine and Microbiology, University Hospital Augsburg, Germany3: Faculty of Medicine, Augsburg University, GermanyWe thank Drs. Wang and He for their careful evaluation of our paper. First, we agree that detectable herpes simplex virus (HSV) replication is not a rare event in ventilated intensive care unit (ICU) patients . We, howSincerely,Reinhard HoffmannLukas SchuiererThe original article  has been