BBX gene family of zinc-finger proteins in wintersweet (Chimonanthus praecox) have yet to be elucidated. In this study, a group IV subfamily BBX gene, CpBBX19, was identified and isolated from wintersweet. Quantitative real-time PCR (qRT-PCR) analyses revealed that CpBBX19 was expressed in all tissues and that expression was highest in cotyledons and inner petals. CpBBX19 was also expressed in all flower development stages, with the highest expression detected in early initiating bloom, followed by late initiating bloom and bloom. In addition, the expression of CpBBX19 was induced by different abiotic stress  and hormone (ABA and MeJA) treatments. Heterologous expression of CpBBX19 in Arabidopsis thaliana (Arabidopsis) enhanced the tolerance of this plant to salt and drought stress as electrolyte leakage and malondialdehyde (MDA) concentrations in transgenic Arabidopsis after stress treatments were significantly lower than those in wild-type (WT) plants. In conclusion, this research demonstrated that CpBBX19 plays a role in the abiotic stress tolerance of wintersweet. These findings lay a foundation for future studies on the BBX gene family of wintersweet and enrich understanding of the molecular mechanism of stress resistance in wintersweet.Zinc-finger proteins are important transcription factors in plants, responding to adversity and regulating the growth and development of plants. However, the roles of the  Chimonanthus praecox), belonging to the Calycanthaceae family, is native to China, has a distinct flowering period (from November to March), strong aroma, and yellow flowers [CpSIZ1 in Arabidopsis enhanced the cold resistance of transgenic Arabidopsis  [CpLEA5 increased the ability of transgenic Arabidopsis to resist low temperature and tolerate salt and alkali [CpFATB in tobacco enhanced the drought tolerance of transgenic tobacco (Nicotiana tobaccum) [CpTAF10 increased the germination rate of transgenic Arabidopsis seeds, and under salt stress, the taproot and lateral roots of transgenic plants showed certain growth advantages compared with WT plants [Wintersweet  , while od alkali . In addiobaccum) . Furtherobaccum) , while oBBX gene family, encoding zinc-finger proteins, is an important family of transcription factors in plants, with functions including response to environmental changes, and plant growth and development [COP1 [Arabidopsis, the BBX protein family is divided into five structural types according to the number of B-box domains and CCT domains: the first type (BBX1-6) has two tandem B-box domains, one VP domain, and one CCT domain; the second type (BBX7-13) has two B-box domains and one CCT domain, but lacks a VP domain; the third type (BBX14-17) contains one B-box domain and one CCT domain; the fourth type (BBX18-25) only contains two tandem B-box domains, and the fifth type (BBX26-32) has only one B-box domain [In plants, transcription factors, as key elements of the expression network, participate in the regulation of expression of numerous genes and are instrumental in regulating plant growth and development and the response to the external environment, including biotic and abiotic stresses ,11,12. Telopment ,14. BBX elopment . In planelopment ,17. The elopment , which ielopment . For exaelopment ,20,21,22nt [COP1 ,23. In A type BBX-25 only BBX gene family members were strongly induced [Arabidopsis, the thermostability of AtBBX18-RNAi plants was improved, while the thermostability of AtBBX18 overexpression plants was decreased. In AtBBX18 overexpression plants, heat stress response genes DGD1, Hsfa2, and Hsp101 were downregulated, suggesting that the gene BBX18 plays a negative regulatory role in the heat-stress response of Arabidopsis [MdBBX genes were upregulated in roots and leaves of apple trees  [MdBBX10 enhanced the resistance of transgenic Arabidopsis to drought, salt, and other abiotic stresses, which was closely related to the enhancement of active oxygen-scavenging ability [SsBBX24 gene expression and protein accumulation in Solanum sogarandinum [BBX genes [CmBBX24 was influenced by GA4/7, and suppressing the expression of CmBBX24 reduced cold and drought tolerance of chrysanthemum [OsBBX2, OsBBX7, OsBBX17, OsBBX19, and OsBBX24 were upregulated at different time points [Arabidopsis leaves with ABA resulted in significant upregulation of BBX11, BBX13, and BBX22 gene expression levels, while BBX2, BBX3, BBX16, BBX18, and BBX19 genes were downregulated by cyclic adenosine diphosphate ribose (cADPR), which is involved in ABA early signal transduction [Under abiotic stresses, such as drought, high and low temperatures, and high salt, multiple  induced ,24,25. Ibidopsis . Under dmestica) . Overexp ability . High sarandinum . ExogenoBX genes ,31,32. Tanthemum . Under tsduction .Under adversity stress, the permeability of plant cell membranes changed. The degree of plant cell membrane damage could be judged by measuring the extravasation of cell electrolytes . MalondiBBX gene, CpBBX19, might be different under abiotic stress. In this study, we identified and isolated a BBX gene, CpBBX19, in wintersweet, and explored the expression patterns and function of this gene. CpBBX19 was classified into the IV subfamily of BBX proteins, and significantly improved the tolerance of transgenic Arabidopsis to salt and drought.The BBX protein family was widely reported to be involved in the process of abiotic stress in plants ,11,12. TCpBBX19 in wintersweet, the roots, stems, cotyledons, and young and old leaves were collected from six-leaf wintersweet, and flowers during the full bloom period were collected from five-year-old plants. Using the wintersweet floral development stages classification method [To analyze the expression pattern of the gene n method , flowersArabidopsis (ecotype Columbia−0) was used for plant transformation. After sterilizing in hypochlorite solution, Arabidopsis seeds were sown on solid Murashige and Skoog (MS) medium, vernalized at 4 °C for 3 days, and then grown in an environment of 22 ± 1 °C with a 16 h light/8 h dark photoperiod. After 2 weeks, the seedlings were transferred into a matrix of vermiculite and peat (1:1 ratio) and cultivated from MS medium under the same conditions as described above.Wild-type 2O [CpBBX19-F/R  according to the manufacturer’s instructions. The resulting fragments were cloned into the pMD19-T vector  and sequenced by TsingKe Company .Total RNA was extracted from the wintersweet tissues according to the manufacturer’s instructions of the RNAprep pure plant total RNA extraction kit . To ensure RNA quality and quantity, RNA extracts were visualized by 1% agarose gel electrophoresis, then a Nano-Drop ND-1000 spectrophotometer  was used for quantification at optical densities of 260 and 280 nm. The cDNA samples were synthesized using a PrimeScript RT reagent kit with gDNA Eraser . Each 10 µL contained 2 µL 5 × gDNA Eraser Buffer, 1 µL gDNA Eraser, and 1μg total RNA. Then, PCR procedure was performed PCR at 42 °C for 2 min; first-strand cDNA was synthesized by reverse transcriptase in 20 μL reaction system containing 10 µL reaction product from the previous step, 1 µL PrimeScript RT Enzyme Mix I, 4 µL 5 × PrimeScript Buffer 2, and 4 µL RNase Free dHBX19-F/R  were desBBX19 upstream promoter sequence was cloned using genomic walking method via Universal Genome WalkerTM 2.0 User Manual kit  by manufacturer’s description. Specific primers (SP1 and SP2)   were desCpBBX19 was determined using the BLAST-Protein program in the National Center for Biotechnology Information (NCBI) database, available online https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 December 2019). Multiple sequence alignments were performed by BioEdit software. The online analysis website MEME (The MEME Suite 5.3.3) was used for protein motif analysis; motif setting value was six; available online https://meme-suite.org/meme/info/status?service=MEME&id=appMEME_5.4.11632023872135256190210 (accessed on 8 July 2021). To understand the distribution of Cis-acting elements upstream of the CpBBX19 promoter sequence, the 2000-bp sequence upstream of CpBBX19 [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 19 March 2021).The sequence of  CpBBX19  was analCpBBX19 were determined under different abiotic and hormonal treatments using the six-leaf plant as the materials [Expression profiles of the gene aterials . Droughtaterials  and 300 aterials  irrigatiaterials , respectaterials , respectCpBBX19 expression patterns were analyzed in different tissues and flowers development stages of wintersweet. RNA was extracted from the wintersweet samples according to the method described in 2.2. The qRT-PCR analyses were performed by Bio-RAD CFX96 (Bio-RAD CFX Manager Software Version 1.6). Specific primers (RT-CpBBX19-F/R) were used for PCR amplification, and the process of qRT-PCR was performed according to the method described by Liu et al. [2, EvaGreen dye, and stabilizers. Each 10 μL reaction mixture contained 5 μL Ssofast EvaGreen Supermix , 0.5 μL each gene-specific primer, and 3.5 μL nuclease-free water. The PCR program comprised preheating at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 5 s, and extension at 72 °C for 5 s. There were three biological replicates for each experiment and three technical replicates for each sample. CpActin genes were employed as the internal reference gene of wintersweet [CpBBX19 was 150 bp, and the primers (RT-CpBBX19-F/R) are shown in u et al. . Accorditersweet . SpecifiCpBBX19 was fused in-frame into the pGWB551 vector with the CaMV35S promoter by Gateway recombination reactions [CpBBX19 construct. Specific primers (pGWB551-CpBBX19-F/R) used in the process are shown in pGWB551 vector is shown in CpBBX19 plasmid was confirmed by sequencing and then transformed into Agrobacterium tumefaciens (strain GV3101 competent cells) via electroporation to further explore the function of CpBBX19.eactions  to generArabidopsis transgenic lines (OE lines) were obtained by transferring the 35S::CpBBX19 recombinant plasmid into wild-type Arabidopsis via the floral dip method [CpBBX19 specific primers (CpBBX19-F1/R1)  were selected for phenotypic investigation; wild-type Arabidopsis (WT) was used as the control.p method . T-0 seep method  was used9-F1/R1) . The PCRtin gene  used as Arabidopsis were irrigated every 2 days with 300 mM NaCl to ensure that the concentration of NaCl solution in the tray remained uniform. To test the drought tolerance of the transgenic lines, WT and transgenic Arabidopsis were irrigated with 30% PEG-6000. MDA content and electrolyte leakage of transgenic Arabidopsis were detected as indicators of stress tolerance. MDA content was determined as described by Heath et al. [To determine the salt tolerance of the transgenic lines, WT and transgenic h et al. , while th et al. .p-value < 0.05 was recognized as statistically significant, and a p-value < 0.01 was classed as extremely significant.IBM SPSS Statistics 23 software package was used for statistical analysis, and Duncan’s multiple range tests were employed to analyze the significance of differences. A CpBBX19 (accession number MZ740468) was obtained. The 5′ untranslated region (5′-UTR) and a 3′ untranslated region (3′-UTR) of CpBBX19 were 475 bp and 428 bp, respectively, and the open reading frame (ORF) of CpBBX19 was 633 bp, encoding 210 amino acids. The predicted molecular weight (MW) of the resulting protein was 23.47 kDa, and the deduced isoelectric point (PI) and instability index were 5.91 and 56.50, respectively. Subcellular localization prediction analysis of the CpBBX19 gene-encoded protein of wintersweet showed that the protein was predominantly distributed in the nucleus.Based on the wintersweet transcriptome sequence in the database , a 1536-Arabidopsis, Gossypium hirsutum, Hibiscus syriacus, Ziziphus jujuba, and Hevea brasiliensis , one element involved in salicylic acid responsiveness (TAC-element), one MYB binding site (MBS) involved in drought-inducibility, three elements related to abscisic acid responsiveness (ABRE), and four elements involved in MeJA responsiveness (TGACG-motif and CGTCA-motif), indicating that CpBBX19 might be related to the stress response in plants.Multiple sequence alignment revealed that the CpBBX19 protein sequence was highly homologous to the BBX19 protein sequence of iliensis a, indicay MEGE 6 b and shoanalyzed . The CpBCpBBX19 and the IV subfamily of Arabidopsis, identified by MEME software, comprised between 8 and 47 amino acids. Sequence analysis revealed that motif 1 contained the complete structure C-X2-C-X8-C-X7-C-X2-C-X4-H-X8-H of B-Box1. Motif 2 contained the C-X2-C-X8-C part of B-box2, while motifs 3 and 4 contained the last histidine (H) of B-box2 and a more conserved amino acid sequence. Motifs 2, 3, and 4 constituted a relatively complete conserved domain of B-Box2.As shown in CpBBX19 was expressed in the root, stem, leaf, and flower tissues of wintersweet, but the expression levels varied with location. Expression levels of CpBBX19 were relatively high in cotyledons and inner petals of wintersweet, and in other floral organs  and hormone treatments (ABA and MeJA). These treatments were selected based on the cis-acting elements in the CpBBX19 promoter region  at 24 h. . Using the leaves DNA of the T2 lines as templates, the CpBBX19 overexpression lines were identified by PCR , and most of the transcription factor binding sites (TFBSs) are in the region from −1000 bp to +200 bp with respect to the TSS [CpBBX19 revealed the presence of a TATA-box, which is a core promoter element usually located 25–35 bp upstream of the TSS [CpBBX19 also contained a CAAT-box, which also commonly exists in promoters and is usually located 75 bp upstream of TSS [cis-acting elements of abiotic stresses and hormone responses, such as ABRE, ARE, MBS, MYB, MYC, TC-rich, TCA-element, and TGACG-motif, were also present. These observations indicated that CpBBX19 may be involved in abiotic stress and hormone responses. Expression of the BBX gene family members SlBBX7, SlBBX9, and SlBBX20 was induced by cold stress in tomato [GhBBX genes after ABA treatment in Gossypium hirsutum [PavBBX6 and PavBBX9 showed strong induction under ABA, GA, and BR treatment in sweet cherry fruit [OsBBX1, OsBBX2, OsBBX8, OsBBX19, and OsBBX24 [OsBBX2, OsBBX7, OsBBX17, OsBBX19, and OsBBX24 was upregulated by GA, SA, and MeJA hormones at different time points [CpBBX19, with the expression of CpBBX19 being induced by abiotic stress  and exogenous hormones (ABA and MeJA)   in the creatment , and the at 24 h . These dCpBBX19, Arabidopsis plants overexpressing CpBBX19 were obtained. Growth of the transgenic lines was better than that of WT lines under drought stress, and the electrolyte leakage and MDA content of these transgenic lines were much lower than those of WT lines. Under salt stress, all plants (WT and OE lines) were obviously withering, but the electrolyte leakage and MDA content of OE lines were significantly lower than those of WT. This indicated that the transgenic Arabidopsis plants had enhanced salt and drought resistance compared with the WT plants. Some functions of BBX genes have been verified. For example, CmBBX24 transgenic Arabidopsis was more tolerant to low temperature and drought stress than the WT lines were [CmBBX22 in Arabidopsis improved the drought resistance of Arabidopsis [MdBBX10 enhanced the tolerance of transgenic Arabidopsis to salt and drought stress [AtBBX24 could improve the tolerance of Arabidopsis to salt stress [CmBBX19, the drought tolerance of CmBBX19-overexpressing (CmBBX19-OX) lines was lower than that of WT [Electrolyte leakage and MDA content, as an important index of plant stress resistance, can reflect the degree of damage of the plant cell membrane system under adversity . The incnes were ; transgebidopsis ; heterolt stress , and comt stress . Howeverat of WT . In planat of WT ,69. In gBBX gene family in wintersweet. In this study, CpBBX19 from wintersweet was isolated and its expression pattern and functional characterization were investigated. Expression of CpBBX19 was induced by multiple abiotic stresses , as well as by hormone treatments (ABA and MeJA). Heterologous expression of CpBBX19 in Arabidopsis enhanced tolerance to drought and salt stresses. These findings indicate that CpBBX19 plays a role in drought and salt stress responses. Moreover, this study also lays a strong foundation for further investigations into BBX genes of wintersweet and enriches understanding of the molecular mechanism of stress resistance in wintersweet.To date, there are no reports on the


Further, we summarize current therapeutic strategies targeting SLC7A11 as well as novel avenues for treatment.SLC7A11/xCT is an antiporter that mediates the uptake of extracellular cystine in exchange for glutamate. Cystine is reduced to cysteine, which is a rate-limiting precursor in glutathione synthesis; a process that protects cells from oxidative stress and is, therefore, critical to cell growth, proliferation, and metabolism. SLC7A11 is expressed in different tissues and plays diverse functional roles in the pathophysiology of various diseases, including cancer, by regulating the processes of redox homeostasis, metabolic flexibility/nutrient dependency, immune system function, and ferroptosis. SLC7A11 expression is associated with poor prognosis and drug resistance in cancer and, therefore, represents an important therapeutic target. In this review, we discuss the molecular functions of SLC7A11 in normal  Solu surface , 10. The surface , 12. Thelutamate .de novo via a trans-sulfuration pathway in some tissues , however, SLC7A11 remains an important transporter for cancer cells that are largely dependent on extracellular cystine for survival , a tripeptide, through a two-step process  Figure 1survival , 16. In survival , 18. Slclifespan , 20. SLClifespan –23. It iSLC7A11 which allow the cells to adapt and respond to stress  through transcription factors, epigenetic regulators, protein stability, interaction with other proteins, post transcriptional regulation, post-translational regulation, and transporter activity Figure 1o stress . For exao stress . Under to stress , 30. Reco stress .via post-transcriptional mechanisms  cycle (via conversion to α-ketoglutarate)  , 51. Intutarate) . It has utarate) . This isutarate) .Many studies indicate that several SLC7A11-high cancers are sensitive to glucose deprivation, however the exact mechanism here remains an active area of research. The loss of SLC7A11 and SLC3A2 has been shown to promote cancer cell survival under glucose starvation conditions and SLC7A11 overexpression promotes glucose starvation-induced cell death and vice versa , 51. Add-), and hydrogen peroxide (H2O2) formed by reduction-oxidation (redox) reactions. ROS and their products can serve as signaling intermediates that contribute to molecular responses involved in normal biological processes, for example, in stem cell renewal, immune responses, and insulin synthesis  are free oxygen radicals such as superoxide ynthesis . Howeverll death , 61. Thill death . This imll death , 63–65. e import , 9. GSH e import , 66. Cane import . Oxidatisurvival , 51.in vivo . Mutant KRAS has recently been shown to promote SLC7A11 transcription via its cooperation with NRF2 and/or ATF4  tumor growth is significantly impaired  blockade therapy and CD8+ T cells increase ROS levels by releasing interferon gamma (IFNγ) which promotes the binding of transcription factor STAT1 to the SLC7A11 transcriptional start site, downregulating expression. Similarly, interferon regulatory factor 1 upregulation by IFNγ reduces SLC7A11 transcription through Janus kinase (JAK) . Post-imhibition . Suppreshibition . TherefoV600E mutant melanoma, geldanamycin resistance in lung cancer, cisplatin resistance in gastric cancer, temozolomide resistance in glioma and gemcitabine resistance in pancreatic cancer  and gastrointestinal (GI) tract cancers . In prosal cells . SLC7A11al cells , 100. Oval cells . SLC7A11al cells  and an ial cells . SLC7A11al cells , 104. Sunt group . Treatment group . Likewisnt group . Adminisnt group . SLC7A11nt group . It was nt group .Evaluation of 127 esophageal squamous cell carcinoma patients who received radical chemoradiotherapy revealed SLC7A11 and NRF2 overexpression in the tumor tissue. Overexpression of SLC7A11 was associated with lymph node metastasis and shorter overall survival in these patients .via infiltration of tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs). Either depletion of TAMs or MDSCs, treatment with a CSF1 inhibitor, an anti-PD-L1 antibody or Anakinra (an IL1β inhibitor), or SLC7A11 knockdown, all result in a reduction in HCC metastasis (in vitro and in-vivo via reduction in GSH levels and an increase in ROS by inhibiting the CD44v9-SLC7A11 interaction (Higher levels of SLC7A11 are associated with poor differentiation and advanced disease stage in hepatocellular carcinoma (HCC) , 111. Antastasis . xCT inheraction .in vitro and in vivo by elevating the levels of miR-489-3p and suppressing SLC7A11 , miRNA (miR-375), long non-coding RNA and siRNA has been shown to inhibit tumor progression through induction of ferroptosis –118. Tre SLC7A11 . Decreasgulation . SLC7A11in vitro .in vitro and in vivo by inducing ROS accumulation and is associated with downregulation of SLC7A11 protein levels via ubiquitination and degradation  . Elescloradation . CRC ste to CRCs , 124. Ust P70S6K .via SLC7A11. Culturing PDAC cell lines in cystine deficient conditions induces cell death in more than 80% of cells, which is rescued by the addition of antioxidant Trolox. Addition of system in vitro and in vivo when injected subcutaneously in nude mice  is characterized by a poor 5-year survival rate (~10%) due to inherent chemoresistance, barriers to drug delivery, and a lack of early diagnostics. SLC7A11 expression was found to be upregulated in many pancreatic cancer cell lines . Recentl of PDAC . Daher eude mice . Gene onude mice . Bioinfoude mice . One of r growth .via SLC7A11 and fall under the category of class I ferroptosis inducers. However, each one has its pros and cons. For example, due to unfavorable pharmacological properties, sulfasalazine treatment does not result in better outcomes in PhaseI/II clinical trials. However, when delivered as a zinc oxide-sulfasalazine nanoparticle derivative, it has better tumor retention with improved cytotoxic effects and no evident damage to healthy cells  is needed  following inhibition in a variety of cancers. Identifying novel targets  that can be co-targeted with SLC7A11 inhibition in cancer may significantly improve the anti-cancer effects. For example, because of higher dependency of SLC7A11-high cancers on glucose and glutamine, combination targeting strategies including drugs/inhibitors that target/block glucose or glutamine metabolism would increase therapeutic efficacy. Slc7a11 knockout and transgenic mouse models are ideal to study the role of SLC7A11 in tumor progression and maintenance. All currently available SLC7A11 inhibitors, including sulfasalazine and erastin, have either off-target effects or bioavailability issues. Utilizing biocompatible nanoparticles to package and deliver these drugs would enhance their efficacy, increase their bioavailability, and potentially eliminate associated side effects. Therefore, more selective SLC7A11 inhibitors and genetic targeting strategies, including small interfering RNAs (siRNA) and small guide RNAs (sgRNA), would be ideal for clinical purposes.The redox status of cancer cells reflects many aspects of carcinogenesis including cell growth, proliferation, migration, metabolism, invasion, and metastases. Cancer progression and tissue injury are often associated with a state of redox imbalance. To survive under conditions of increased oxidative stress, cancer cells adopt various strategies to produce antioxidants, for example, upregulation of SLC7A11. SLC7A11 functions in a variety of roles in cancer including metabolic reprogramming, nutrient dependency, growth, proliferation, invasion, and drug resistance Figure 1NJ conducted the research and wrote/edited the manuscript. KT researched the manuscript. KD edited the manuscript, supervised, and funded the research. All authors contributed to the article and approved the submitted version.The DelGiorno laboratory is supported by the Vanderbilt-Ingram Cancer Center Support Grant (NIH/NCI 5P30 CA068485), the Vanderbilt-Ingram Cancer Center SPORE in Gastrointestinal Cancer (NIH/NCI 5P50 CA236733), a Vanderbilt Digestive Disease Research Center Pilot and Feasibility Grant (NIH/NIDDK 5P30 DK058404), an American Gastroenterological Association Research Scholar Award (AGA2021-13-02), NIH/NIGMS 1R35 GM142709, and the Sky Foundation, Inc. (AWD00000079).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Histological analyses revealed that NPA induced an increase in cell size and numerous cell divisions in the cortex and pith, respectively, compared to untreated stem. In the area of NPA application, vascular tissues had significantly wider cambial zones consisting of 5–6 cell layers, whereas phloem and xylem seemed not to be affected. This indicates that stem swelling in the area of NPA application is caused by stimulation of cell division and cell enlargement mainly in the cambial zone, cortex, and pith. Comprehensive analyses of plant hormones revealed that NPA substantially increased endogenous levels of indole-3-acetic acid (IAA) in the swelling area. NPA also increased endogenous levels of cytokinins, jasmonic acid, and its precursor, 12-oxo-phytodienoic acid, but did not increase abscisic acid and gibberellin levels. It was shown, using radiolabeled 14C-IAA, that NPA applied to the middle of internode segments had little effect on polar auxin transport, while 2,3,5-triiodobenzoic acid substantially inhibited it. These results strongly suggest that NPA induces changes in endogenous levels of plant hormones, such as IAA, cytokinins, and jasmonic acid, and their hormonal crosstalk results in a conspicuous local stem swelling. The possible different mode of action of NPA from other polar auxin transport inhibitors in succulent plants is extensively discussed.The mode of action of  In this process, auxin is transported basipetally between cells toward the root tip through the stele in the aboveground parts, and then redirected symmetrically in specific root tip cells, and transported back towards the root elongation zone ,5,6,7. CBryophyllum calycinum, B. daigremontianum, Kalanchoe tubiflora, and K. blossfeldiana, while TIBA and morphactins (IT 3456 and HFCA) completely inhibited it in these species dihydrozeatin, [2H5]trans-zeatin riboside, [2H5]indole-3-acetic acid, [2H4]salicylic acid, [2H2]gibberellin A1, [2H2]gibberellin A4, [2H2]gibberellin A5, [2H2]gibberellin A6, [2H6]cis, trans-abscisic acid, [2H5]benzoic acid , [2H5]jasmonic acid , and [2H5]dinor-12-oxo-phytodienoic acid  were used. The technical details are given in the cited references.Analyses of plant hormones were performed according to the methods reported previously ,47,48,49eviously ,46. PlanB. daigremontianum stems.Similar analyses of plant hormones in relation to conspicuous local stem swelling induced by 0.4% NPA were carried out in 14C]-IAA with a specific activity of 55 mCi/mmol, 0.1 mCi/mL,  at the bottom of 2-mL Eppendorf tubes (for 25 mm stem segments) and 6-mL plastic vials (for 40-mm stem segments), respectively.Following the method reported previously ,10, withw/w) were applied in lanolin as a 2-mm strip to the middle part around the internode segments. Internode segments treated only with lanolin served as the control segments. In another experiment, internode segments (40 mm in length) prepared from the growing internode of intact plants pretreated with NPA or TIBA for 24 h were used to determine polar auxin transport.Internode segments (25 mm in length) excised from growing internodes were used, mainly from the second or third internode from the top of growing plants with active elongation. NPA and TIBA . Therefore, TM-ES, MP-Biomedicals, CA, USA), and then, its radioactivity was determined using a liquid scintillation counter . The experiments were performed in triplicate with 6 segments. The results are expressed as percentages of the mean control values with standard errors (n = 3).After incubation at 23.5 °C for 18 h, a 3-mm piece was cut on the opposite side of the segment. The piece was directly put into a vial containing liquid scintillation cocktails  was conducted using STATISTICA software . When ANOVA indicated significant effects, the means were separated using Duncan’s multiple range test or Student’s B. calycinum and some other succulent plants as well as rooting of shoot cuttings. NPA showed almost no inhibition of polar auxin transport in B. calycinum plants. NPA also induced a significant increase in the levels of IAA, cytokinins, and JA and its precursor, 12-oxo-PDA, at its application site in succulent plants. The crosstalk of these plant hormones might regulate cell division and enlargement, resulting in conspicuous local stem swelling. Further studies on the mechanism of NPA induced local accumulation of these plant hormones will be required in the near future.Unlike TIBA and HFCA, NPA shows unusual physiological effects that differed from other polar auxin transport inhibitors in succulent plants; NPA induced conspicuous local stem swelling in intact plants of


Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic hereditary renal disease, promoting end-stage renal disease (ESRD). Klinefelter syndrome (KS) is a consequence of an extra copy of the X chromosome in males. Main symptoms in KS include hypogonadism, tall stature, azoospermia, and a risk of cardiovascular diseases, among others. Gitelman syndrome (GS) is an autosomal recessive disorder caused by SLC12A3 variants, and is associated with hypokalemia, hypomagnesemia, hypocalciuria, normal or low blood pressure, and salt loss. The three disorders have distinct and well-delineated clinical, biochemical, and genetic findings. We here report a male patient with ADPKD who developed early chronic renal failure leading to ESRD, presenting with an intracranial aneurysm and infertility. NGS identified two de novo PKD1 variants, one known (likely pathogenic), and a previously unreported variant of uncertain significance, together with two SLC12A3 pathogenic variants. In addition, cytogenetic analysis showed a 47, XXY karyotype. We investigated the putative impact of this rare association by analyzing possible clinical, biochemical, and/or genetic interactions and by comparing the evolution of renal size and function in the proband with three age-matched ADPKD (by variants in PKD1) cohorts. We hypothesize that the coexistence of these three genetic disorders may act as modifiers with possible synergistic actions that could lead, in our patient, to a rapid ADPKD progression. PKD1 (OMIM#: 601313) and PKD2 (OMIM#: 173910) [PKD1 variants versus PKD2 variants), and the type of the variant (frameshift/nonsense versus non-truncating variants) [+/Cl− cotransporter. GS is clinically characterized by hypomagnesemia, hypochloremic metabolic alkalosis, hypokalemia, hypereninemia, and hyperaldosteronism with normal or low blood pressure and salt loss [Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in two main genes,  173910) . It is wariants) . However1 OMIM#: 01313 andariants) ,12, inclariants) . The renariants) . Indeed,ariants) ,15. Klinariants) ,17. A noariants) ,19,20,21ariants) , a 20-foariants) . Howeverariants) ,24,25,26alt loss . Most ofalt loss ,29,30,31−1.2623). Mayo clinic imaging classification and the prediction of future eGFR based on the classification were determined in the proband [We compared the clinical evolution of ADPKD in the proband with our three control PKD1 defined cohorts by kidney magnetic resonance imaging (MRI)  and esti proband .ADPKD gross deletion analysis was made by means of multiplex ligation-dependent probe amplification (MLPA) using the SALSA MLPA KIT P351/P352 for PKD1/PKD2 , following the manufacturer’s instructions. Data analysis was done with Coffalyser v6 MLPA Analysis Software (MRC-Holland).Massive parallel sequencing analysis was performed with a custom-targeted next-generation sequencing (NGS) gene panel (Nefroseq v1.2) designed for the study of the full spectrum of genetic nephropathies (which includes 380 genes related to kidney diseases) . The seqPKD1 mutation screening for coding sequences and intron/exon boundaries for exons 27 and 29 were confirmed by Long Range-PCR and direct sequencing. SLC12A3 mutation screening for coding sequences and intron/exon boundaries for exons 16 and 25 was performed by PCR and direct sequencing. PCR conditions and primers (designed with the help of Primer3 plus v04.0 Software) are available upon request. PCR products were sequenced using BrightDye Terminator cycle kit  and run on an ABI3730XL Sequencer .Our pipeline routine for azoospermia studies includes microdeletions of the Y-chromosome, karyotyping, and cystic fibrosis analysis. The Y-chromosome microdeletions analysis was performed by standard fragment analysis, by multiplex PCR of STS markers and direct sequencing (primers are available upon request). The molecular diagnosis of Y-chromosome microdeletions was performed following the recommendations of the EAA/EMQN best practice guidelines . BrieflyThe proband, a 36 year-old male German patient presented in our outpatient clinic for renal evaluation in April 2016. He was the fourth child of unrelated and healthy parents A.2. His father was alive at 77 years old without a history of ADPKD or any other renal disease. His mother died at the age of 51 . His three brothers were asymptomatic based on medical history. There was no history of intracranial aneurysms, ADPKD, or other renal diseases in other family members. Our first physical examination  established a well-developed male , 22.1 Kg/m2), presenting a decreased testicular volume. Blood pressure was 115/70 mmHg without any treatment. His serum creatinine level was 2.9 mg/dL and eGFR of 27 mL/min/1.73 m2. ECG showed a complete blockage of the right branch of the bundle of His, and abdominal MRI documented bilateral, enlarged, cystic kidneys. A TKV, 726 mL  and htTKV, 370 mL/m were measured by manual segmentation using MRI [2  showed bilateral, enlarged, cystic, and echogenic kidneys being diagnosed with ADPKD. Surgical intervention of bilateral inguinal hernias was undertaken in Germany. At this time, the patient was also receiving intermittent treatment with Ramipril (5 mg/daily), which was finally discontinued. His serum creatinine levels were 2 mg/dL with an eGFR of 44 mL/min/1.73 msing MRI . We clasg MRI [2 . SeveralPKD1 in heterozygosis, we classified the patient as PROPKD 4 score (low risk of progression) [PKD1 cohorts used to monitor our proband , but not smaller than PKD1 without hypertension . Moreover, Since genetic studies showed a likely pathogenic missense mutation in ression)  and Mayortension . In factInterestingly, the proband had no clinical features related to GS, at all. He showed that the serum magnesium (1.6–2.0 mg/dL) and potassium levels (3.9–4.7 mmol/L), and venous (pH 7.31–7.41) were within normal limits, although HCO3- levels in blood were inappropriately higher (24–31 mmol/L) for his grade of CRF . It is rCFTR gene were negative.Cytogenetic analysis of peripheral blood showed a 47, XY,+X karyotype D. Y-chroPKD1; NM_001009944.3:c.9499A>T(p.Ile3167Phe) (http://pkdb.mayo.edu/ (accessed on 14 December 2021); ADPKD mutation database), and as a disease (Uniprot), which reflect conflicting interpretations of pathogenicity (ClinVar), as well as VUS (LOVD), VUS (HGMD), and, following ACMG/AMP 2 criteria [PKD1 missense variant NM_001009944.3:c.9756G>C(p.Glu3252Asp) . Both variants were validated by Sanger sequencing in the proband. In addition, NGS analysis also established two SLC12A3 variants, NM_000339.3:c.1928C>T(p.Pro643Leu)  or related to cyst progression. The analysis of CNV by means of MLPA for PKD1, and PKD2, and the rest of the panel  was negative. Genetic findings are shown in NGS studies detected a previously described missense variant in 3167Phe) E, left icriteria , it is l3252Asp) E, right o643Leu) F, left io643Leu) F, right PKD2 loci may accelerate the progression to ESRD approximately 20 years earlier than patients with only PKD1 sequence variants [In ADPKD, the uncontrolled growth of renal cysts increases renal volume and the destruction of the parenchyma progressively, resulting in renal failure in most patients . In addivariants ,7.Cyst growth in ADPKD is associated with increases in epithelial cell proliferation, dedifferentiation, and fluid secretion. Then, the enlargement of cysts affects surrounding nephrons interrupts kidney function significantly ,41,44. I2) or 42 years (eGFR 12 mL/min/1.73 m2). Furthermore, his eGFR was significantly lower than expected when it was compared with three different age-matched ADPKD cohorts, one with similar htTKV , however, the eGFR predicted established the development of ESRD at 48 years (rapid progression). In fact, his real situation of ESRD had been achieved at age of 40  and c.2891G>A(p.Arg964Gln). However, we have not found electrolyte alterations characteristic of GS in our proband. Thus, the phenotypic effect of such missense variants is difficult to evaluate, and some pathogenic variants may not be sufficient to cause phenotypic changes related to GS [GS caused by SLC12A3 sequence variations is characterized by hypochloremic metabolic alkalosis, hypomagnesemia, RAAS activation with normal or low blood pressure, salt loss, and hypokalemia . Identifed to GS ,30,31, aPKD1 gene associated with other genetic variants (two SLC12A3 variants and an extra X-chromosome). In addition, we cannot rule out that the PKD1 variant c.9756G>C may have a role as a hypomorphic allele [We describe the first report of ADPKD combined with KS and GS, which initially were unnoticed, indicating the need to consider the concurrent existence of additional disorders in cases of atypical ADPKD manifestations. This patient carries clinically significant variants in two different genes and a huge genomic rearrangement (one extra X-chromosome) responsible for additional pathology. Therefore, all these entities in one individual are not common, but it depends on the prevalence of each disease. Taking into account that their prevalences are around one in 500–1000 (ADPKD) ,4 and onc allele ,12, or mc allele . The latPKD1 and PKD2 [PKD1 variants and likely biallelic disease [TSC2-PKD1 [PKD2 and COL4A1 [PKD1, KS, and a compound heterozygous GS-associated variant showed a severe phenotype of ADPKD, characterized by the development of early CRF (at 27 years old) and rapid evolution to ESRD (at 40 years old) (PKD1 (average age of 58 years) [The clinical progression to ESRD and disease prognosis in this patient with ADPKD, KS, and GS (isolated KS or GS not producing CRF) could be potentially synergistic and comparable to individuals with variants in both and PKD2 ,7, hypom disease , the conSC2-PKD1 , co-occuSC2-PKD1 , or duald COL4A1 . In factars old) , associaars old) C. He dev8 years) ,37,38. I8 years) ,65. Ther8 years) ,71,72,73On the other hand, in both ADPKD and KS there is an elevated frequency of intracranial aneurysms and, in the case of associated variants in PKD1 protein and KS, it is expected to have at least additive intracranial aneurysms in disease presentation ,49,50,55SLC12A3 may increase the pathogenic action of PKD1 variants. This is done, we presume, by increasing apoptosis, fibrosis, and producing predominant tubule–interstitial and vascular renal injury, instead of cystic growth, as it has been suggested in murine models. The use of NGS in the genetic diagnosis of different diseases is currently demonstrating the coexistence of several genetic variants in the same patient, which may contribute to the high variability and heterogeneity observed in numerous individuals. This case illustrates the importance of performing genetic testing to establish a complete diagnosis in ADPKD patients with atypical presentations and/or with fertility abnormalities.We present, for the first time a young ADPKD patient with an early, fast progression to ESRD, as well as growth of an intracranial aneurysm, accompanied with masqueraded KS and GS. We think that the presence of a 47, XXY karyotype and compound heterozygous variants in


Wnt1-inducible transgenic mouse model targetingCol1a1-expressing alveolar osteoblasts,odontoblasts, and cementoblasts, we demonstrate that the WNT ligandWNT1 is a strong promoter of cementum and alveolar bone formation invivo. We induced Wnt1 expression for 1, 3, or 9 wk inWnt1Tg mice and analyzed them at the age of 6 wk and 12 wk.Micro–computed tomography (CT) analyses of the mandibles revealed a1.8-fold increased bone volume after 1 and 3 wk ofWnt1 expression and a 3-fold increased bonevolume after 9 wk of Wnt1 expression compared tocontrols. In addition, the alveolar ridges were higher in Wnt1Tg miceas compared to controls. Nondecalcified histology demonstratedincreased acellular cementum thickness and cellular cementum volumeafter 3 and 9 wk of Wnt1 expression. However, 9 wk ofWnt1 expression was also associated withperiodontal breakdown and ectopic mineralization of the pulp. Thecomposition of this ectopic matrix was comparable to those of cellularcementum as demonstrated by quantitative backscattered electronimaging and immunohistochemistry for noncollagenous proteins. Ouranalyses of 52-wk-old mice after 9 wk of Wnt1expression revealed that Wnt1 expression affectsmandibular bone and growing incisors but not molar teeth, indicatingthat Wnt1 influences only growing tissues. To furtherinvestigate the effect of Wnt1 on cementoblasts, westably transfected the cementoblast cell line (OCCM-30) with a vectorexpressing Wnt1-HA and performed proliferation aswell as differentiation experiments. These experiments demonstratedthat Wnt1 promotes proliferation but notdifferentiation of cementoblasts. Taken together, our findingsidentify, for the first time, Wnt1 as a criticalregulator of alveolar bone and cementum formation, as well as provideimportant insights for harnessing the WNT signal pathway inregenerative dentistry.The WNT/β-catenin signaling pathway plays a central role in the biologyof the periodontium, yet the function of specific extracellular WNTligands remains poorly understood. By using a The WNT β-catenin signaling pathway is central for differentiation andproliferation of dental stem cells. The canonical WNT signaling is mediatedvia the binding of a WNT ligand to the 7-pass transmembrane receptorFrizzled (FZD) and the single-pass low-density lipoprotein receptor–relatedprotein 5 or 6 (LRP5/6), leading to dephosphorylation-mediated stabilizationand nuclear translocation of β-catenin .TransgeWNT10A and WNT10B havebeen shown to cause syndromic tooth agenesis  anda nonsyndromic type of tooth agenesis in humans . All other genotype litters wereexcluded from the study. We analyzed male mice at the age of 6 and 12wk and female mice at the age of 52 wk. For each experiment includingstatistical analysis, a minimum of biological triplicates was used.Mice were allocated randomly to DOX-ON, DOX-OFF groups (coin flip),and experiments (micro–computed tomography [CT] and staining) wereperformed in a blinded fashion.To generate Wnt1Tg mice, we crossed tTA mice with ptent1 mice  on a mixAppendix).All mice were maintained in the animal facility of the University MedicalCenter Hamburg-Eppendorf in agreement with our animal ethics committeeand comply with the ARRIVE Checklist  (see morAppendix). For morphological analysis, incisors andmandibular molars were imaged with a high-resolution invertedmicroscope (Olympus DSX500i) and a 10× objective lens .Skulls were analyzed by contact radiography with a Faxitron X-ray cabinet(Faxitron X-ray Corp.), followed by micro-CT scanning  with a voxel size of 15 μm, 55 kV, 145 µA, and 200 ms aspreviously described  andanalAppendix.Histology was performed on undecalcified specimens for histomorphometryand decalcified material for hematoxylin and eosin staining, asdescribed in the Appendix.Immunohistochemistry was performed on 5-µm paraffin sections, asdescribed in the Staining was performed, using the Molecular Instruments HCR v.3.0protocol, for “generic sample on slide.” Fluorescence in situhybridization probes were designed and purchased from MolecularInstruments.Appendix).The degree of tissue mineralization was measured by means of thebackscattered electron intensity, as previously described  , which targets alveolar osteoblasts and odontoblasts. In thismodel, doxycycline (DOX)–dependent Wnt1 transgeneexpression is governed by the Col1a1 promoter-driventetracycline-controlled transcriptional activator(Col1a1-tTA), resulting in conditionalWnt1 expression in cells expressingCol1a1 (under the 2.3-kb fragment promoter) in molars and incisors of 6-wk-old Wnt1Tg mice and controls 1and 3 wk after DOX withdrawal using in situ hybridization . Whereas the expression ofWnt1 was not detectable in teeth sections ofcontrol mice , Wnt1 mRNAexpression was increased upon DOX withdrawal in osteoblasts,osteocytes, odontoblasts, cementoblasts, and cementocytes of Wnt1Tgmice .To analyze short- and long-term effects of romoter) upon DOXor 12 wk . ToconfAppendix , C. WherWnt1 expression , whereas the crowns of themandibular molars were normally formed. However, the visible crownheight appeared shorter in Wnt1Tg mice after 9 wk ofWnt1 expression as compared to those ofcontrols  of Wnt1Tg mice, in which doxycycline has been removed for 1,3, or 9 wk. The 3-dimensional (3D) segmentation images revealed aclear increase of mandibular bone mass in Wnt1Tg mice after 1, 3, and9 wk of c effect  . However, wall thicknessanalysis of the pulp together with von Kossa/van Gieson staining ofnondecalcified sections revealed a decrease in pulp volume and thepresence of mineralized matrix within the pulp cavities of Wnt1Tg miceafter 3 and 9 wk of Wnt1 expression . Interestingly, we observed inthe pulp a significant increase in vascularization upon Wnt1expression . Importantly, after 3 wk and 9 wkof Wnt1 expression, the incisors were filled withectopic calcifying matrix. A high proportion of this matrix consistedof nonmineralized organic tissue (45% after 3 wk ofWnt1 expression and 40% after 9 wk ofWnt1 expression) .We next investigated the mandibular teeth in more detail using micro-CTimaging as well as undecalcified histology . We obsepression .InteresAppendix Fig. 4). The calcein double layersindicated that dentin formation was still evident after 1 and 3 wk ofWnt1 expression . This observation suggests thatWnt1 expression in odontoblasts does notdirectly affect its function. In addition, the pattern of calceinlabels in the pulp indicates that the ectopic formation of thecalcified tissue begins at the root apices and moves coronally towardthe pulp horns.To further analyze the observed increase in calcified tissue in the pulpand the effect on dentin formation, we performed fluorescent imagingof calcein dye, which had been injected 10 and 3 d before sacrifice. Interestingly, this increaseof nonmineralized matrix was not evident in bone . Quantification of fluorescentcalcein imaging further demonstrated high cementum formation after 3wk of Wnt1 expression, whereas no double calceinlabels could be detected after 9 wk of Wnt1expression . However, an importanthistological finding was also the severe periodontal breakdown betweenthe molar roots after 9 wk of Wnt1 expression . Taken together, these resultssuggest that Wnt1 is a strong promoter of cementumformation. However, long-term exposure of Wnt1 mayinduce periodontal destruction and ectopic calcification.We next studied the root cementum in Wnt1Tg mice . Micro-C1Tg mice . The acepression . In factrol mice .In addipression ,confirmTo further characterize the ectopic calcification, we used immunostainingfor noncollagenous matrix proteins of Wnt1Tg and control mice .OsteopoWe next analyzed the calcium content using quantitative backscatteredelectron imaging and compared it with those of bone, dentin, andcementum . The bacWnt1 affects the periodontium ofaged mice. To investigate this, we analyzed 52-wk-old mice after 9 wkof Wnt1 expression . Here we observed thatWnt1 stimulates proliferation  but not differentiation of OCCM-30cells .Given these results, we finally determined the effect ofWnt1 on cementoblasts remain subject to furtherresearch.Taken together, our results demonstrate that Wnt1 promotes cementum andalveolar bone growth. However, the exact molecular mechanisms ofWNT10A andWNT10B, which appeared to cause tooth agenesis) have beeWnt1 for cementum formation and periodontalhomeostasis. More specifically, our histological analyses together withmicro-CT imaging revealed that Wnt1 is a strong promoter ofalveolar bone and cementum formation. This is in line with a previous studyshowing that constitutive activation of β-catenin in the dental mesenchymeleads to differentiation of cementoblasts and induces excessive cementumformation in mice . It, therefore, remains to be determinedwhether the pOBCol2.3 promoter used in this study to regulateWnt1 (Here we present, for the first time in vivo, a novel role of in mice . HoweverateWnt1  is also Wnt1 not only serves as a stimulantfor cementoblasts but also regulates alveolar and mandibular bone formation.This confirms our previously published data on the bone-anabolic effect ofWnt1 (Wnt1 in young mice resulted in periodontal bone loss. Weinterpret this as a sign of overstimulation as cementum formation needs tobe orchestrated with the formation of periodontal fibers to achieve properperiodontal anchorage. It, therefore, seems likely that long-termWnt1 expression causes an increase of dysfunctionalcementum, in which PDL fibers cannot properly anchor anymore (Wnt1induction. Although we did not investigate the expression pattern ofWnt1 in aged mice, our analysis suggests that thedetrimental effects of Wnt1 might therefore be morerelevant for growing teeth. In this regard, it might also be interesting tostudy vascularization processes during bone formation, which have not beenanalyzed in this mouse model so far, although WNT signaling is crucial forangiogenesis (Wnt1 induction. Therefore, it seems thatWnt1-expressing odontoblasts induce angiogenesis byattracting endothelial cells (Furthermore, we show that  ofWnt1 . Indeed, anymore . This isogenesis . We obseal cells . This,hWnt1 can increase bone formationand therefore prevent alveolar bone loss while having a supporting effect oncementum formation. Nevertheless, long-term exposure will potentially leadto unwanted calcified pulp tissue as well as periodontal bone loss at leastin growing teeth. This seems also important to consider when osteoanabolicmedication harnessing the WNT pathway is given to young patients.Altogether, our findings open up a new paradigm in dental regeneration asβ-catenin activating drugs such as BC21  can haveWnt1 for tooth development that can be employed forfuture regenerative strategies in dentistry.Taken together, our results provide new insights into the role ofC. Nottmeier, J. Petersen, T. Koehne, contributed to conception, design, dataacquisition, analysis, and interpretation, drafted and critically revisedthe manuscript; N. Liao, contributed to conception, design, dataacquisition, analysis, and interpretation, critically revised themanuscript; A. Simon, M.G. Decker, M. Kaucka, contributed to dataacquisition, analysis, and interpretation, critically revised themanuscript; J. Luther, M. Schweizer, contributed to data acquisition andanalysis, critically revised the manuscript; T. Yorgan, contributed to dataacquisition and interpretation, critically revised the manuscript; E.Bockamp, contributed to data acquisition, critically revised the manuscript;B. Kahl-Nieke, M. Amling, T. Schinke, contributed to data interpretation,critically revised the manuscript. All authors gave final approval and agreeto be accountable for all aspects of the work.Click here for additional data file.Supplemental material, sj-pdf-1-jdr-10.1177_00220345211012386 for Wnt1Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Mannerby C. Nottmeier, N. Liao, A. Simon, M.G. Decker, J. Luther, M.Schweizer, T. Yorgan, M. Kaucka, E. Bockamp, B. Kahl-Nieke, M. Amling,T. Schinke, J. Petersen and T. Koehne in Journal of DentalResearch


However, whether differential BoLA-DRB3 affects BLV infectivity remains unknown. In a three-year follow-up investigation using a luminescence syncytium induction assay for evaluating BLV infectivity, we visualized and evaluated the kinetics of BLV infectivity in cattle with susceptible, resistant and neutral BoLA-DRB3 alleles which were selected from 179 cattle. Susceptible cattle showed stronger BLV infectivity than both resistant and neutral cattle. The order of intensity of BLV infectivity was as follows: susceptible cattle > neutral cattle > resistant cattle. BLV infectivity showed strong positive correlation with PVL at each testing point. BLV-infected susceptible cattle were found to be at higher risk of horizontal transmission, as they had strong infectivity and high PVL, whereas BLV-infected resistant cattle were low risk of BLV transmission owing to weak BLV infection and low PVL. Thus, this is the first study to demonstrate that the BoLA-DRB3 polymorphism is associated with BLV infection.Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. Polymorphism in bovine lymphocyte antigen  Deltaretrovirus of family Retroviridae, is the etiological agent of enzootic bovine leukosis (EBL), the most common neoplastic disease in cattle [+ B-cell expansion, while only a small percentage develop EBL after a long latency period. Currently, vaccines or therapeutic procedures for preventing BLV transmission are lacking, because of which, the virus has spread worldwide. EBL was also added to the International Organization of Epizootics (OIE)-listed diseases, infections, and infestations in 2020 [Bovine leukemia virus (BLV), an oncogenic member of the genus n cattle ,2. Most  in 2020 . Current in 2020 , 94.2% o in 2020 , 36.7% o in 2020 , approxi in 2020 . BLV inf in 2020  as it af in 2020 ,13. Repo in 2020 . In addi in 2020 .5 cells in blood, cattle secrete BLV in nasal secretions, saliva [The free virus is unstable, and herd infection occurs mainly via BLV-infected cells. Horizontal transmission is recognized as the major route of BLV infection . Either , saliva , and mil, saliva . Further, saliva . Therefo, saliva .(BoLA)-DRB3 [https://www.ebi.ac.uk/ipd/mhc/group/BoLA/, accessed on 2 September 2021). The BoLA-DRB3*015:01 and DRB3*012:01 alleles are known susceptibility-associated markers related to high PVL. In contrast, the BoLA-DRB3*009:02, DRB3*014:01:01 [DRB3*002:01 alleles [BoLA-DRB3 alleles were not significantly associated with PVL in vivo [BoLA-DRB3 alleles are believed to determine cattle–specific differences resistant to BLV disease progression. However, whether polymorphisms in BoLA-DRB3 alleles are related to BLV infectivity is not known.PVL is strongly associated with the highly polymorphic bovine leukocyte antigen LA)-DRB3 ,31,32. I14:01:01 ,31,33,34 in vivo . Thus, BBoLA-DRB3 alleles were associated with BLV infectivity using white blood cells (WBCs) from BLV-infected cattle with different BoLA-DRB3 alleles and PVL.In present follow-up study spanning three years, we assessed whether different BoLA-DRB3*015:01 and DRB3*012:01 alleles are associated with high PVL of BLV. The BoLA-DRB3*009:02, DRB3*014:01:01 and DRB3*002:01 alleles were associated with high PVL resistance [BoLA-DRB3 alleles were not significantly associated with PVL in vivo [n = 179) from Farm A in Tochigi prefecture, Japan, and analyzed the BoLA-DRB3 alleles and BLV infection. DNAs were extracted from blood samples to be typed BoLA-DRB3 allele using sequence-based typing (SBT) method. Twenty alleles of BoLA-DRB3 in the IPD-MHC database were identified in our samples. As shown in BoLA-DRB3 allele is known to be lesser than that of other alleles [BoLA-DRB3 allele data, 179 cattle were grouped into three cattle groups: resistant cattle, susceptible cattle, and neutral cattle, which were defined to be carrying at least one susceptible, resistant or neutral allele in their genomes, respectively  and neutral cattle (p = 0.00669)  ,36,37,3805 cells B. Furthe0.00669) B. In con0.00669) .5 cells in blood samples, respectively [5 cells, as described previously [5 cells. The 35 susceptible cattle (56.5%) had high PVL, with mean PVL of 32,218 copies/105 cells, ranging from 10,154 to 65,564 copies per 105 cells, while the 23 susceptible cattle (37.1%) had low PVL, with mean PVL of 2746 copies per 105 cells, ranging from 9 to 9761 copies per 105 cells. In contrast, only six resistant cattle had high PVL and the mean PVL was 28,688 copies/105 cells, ranging from 12,984 to 39,614 copies per 105 cells, and the remaining 27 resistant cattle (55.1%) had low PVL and the mean PVL was 1275 copies/105 cells, ranging from 14 to 8122 copies per 105 cells (5 cells).In particular, the BLV provirus can be detected in milk, nasal mucus, and saliva samples of dairy cattle with PVL of more than 10,000, 14,000, and 18,000 copies/10ectively ,28. Thereviously . Based o05 cells . In addiTaken together, the high infection rate in susceptible cattle with high PVL was compared to that in neutral and resistant cattle. Our results revealed the presence of BLV-infected resistant and susceptible cattle with different levels of PVLs, which were sufficient for the three-year follow-up study.BoLA-DRB3*015:01 or DRB3*012:01 allele, eight cattle carrying a resistant BoLA-DRB3*009:02 or DRB3*014:01:01 allele, and one cattle carrying a neutral allele. The tested cattle with different levels of PVL were assessed using CoCoMo-qPCR-2. All susceptible cattle (S1-15) had high PVLs that exceeded 10,000 copies/105 cells, one neutral cattle N1 also carried 12,423 copies/105 cells, and all resistant cattle (R1-7) had low PVLs of less than 10,000 copies/105 cells. The WBCs obtained from the tested blood samples were co-cultured with the BLV reporter cell line CC81-GREMG, which can respond to Tax expression to express green fluorescence [5 cells), showed obviously weaker BLV infectivity than all susceptible cattle and one neutral cattle, as shown in PVL is a risk factor of BLV infection that correlates with BLV infectivity and is evaluated based on the syncytium formation assay ,26. Nextrescence . Nuclei In this three-year follow-up investigation, we would not constrain any decisions of the farmer as depositary breeding, selling, slaughtering, and eliminating et al. At the beginning, we decided to select 13 out of 24 cattle for BLV infectivity syncytium formation assay, but some selected cattle were sold out or deposited with other vendors’ deposit breeding services, while at the time of our sampling. Therefore, as shown in BoLA-DRB3 alleles are associated with PVL in vivo. In addition, our three-year follow-up study clearly showed that BLV infectivity was weak in resistant cattle, strong in susceptible cattle, and medium in neutral cattle N1. However, the association of the BoLA-DRB3 allele with BLV syncytium formation ability is unknown. Therefore, to clarify the relationship between BoLA-DRB3 allele and BLV infectious ability, we increased the number of tested cattle to 13, which the selected cattle in the depositary breeding service returned to the farm  ranged from 0.7611 to 0.9350 (R = 0.8537 ± 0.0716)  B. This iBoLA complex polymorphisms are one of the most important host factors strongly involved in controlling the subclinical progression of BLV infection by regulating PVL in vivo [BoLA-DRB3 is a highly polymorphic gene that affects susceptibility to BLV-induced B cell lymphoma [BoLA-DRB3*015:01 or DRB3*012:01), three resistant cattle (BoLA-DRB3*009:02 or DRB3 *014:01:01), and one neutral cattle, and showed for the first time that BoLA-DRB3 alleles are associated with BLV infectivity.Among the many risk factors associated with BLV infection,  in vivo ,42,43,44lymphoma ,46, and lymphoma . BLV infBoLA-DRB3*016:01 and DRB3*015:01 have been reported to be associated with high PVL in Japanese Black and Holstein cattle, respectively [BoLA-DRB3*009:02 is known to play an important immunological role in suppressing viral replication, resulting in resistance to disease progression in both Japanese Black and Holstein cattle [BoLA-DRB3*009:02 allele, and they had weak BLV infectivity and low PVL in the long-infected phase. In addition, no BLV transmission was observed over 30 months of contact when cattle carrying resistant BoLA-DRB3*009:02 allele with low PVL were incorporated into a BLV-negative dairy herd. The BoLA-DRB3*0902 cattle with low PVL disrupted the BLV transmission chain [ectively ,45. All n cattle . Cattle on chain . Our reson chain ,48. ThusBoLA-DRB3*014:01:01 has also been reported to suppress both BLV PVL and lymphoma [BoLA-DRB3*014:01:01 allele. Although BoLA-DRB3*014:01:01 suppressed PVL to some extent in vivo, infection suppression was possibly not completely achieved. The PVL of R1 increased and exceeded the threshold value of 10,000 copies/105 cells. Besides, the BoLA-DRB3*002:01 allele [DRB3*014:01:01 allele or DRB3*002:01 allele. Thus, it is considered that resistant DRB3*009:02 allele can strongly suppress the development of high PVL than DRB3*014:01:01 allele or DRB3*002:01 allele. It may be that DRB3*009:02 combines with other host factors to thoroughly suppress high PVL.The lymphoma . The tes1 allele  has beenBoLA-DRB3*016:01 was over 30%, DRB3*009:02 was 2.0~5.8%, and DRB3* 014:01:01 was 1.4~2.5%. In Holstein cattle herds, the distribution of allele frequencies for BoLA-DRB3*015:01 was 19.4~ 35.4%, DRB3*009:02 was 5.2~7.1%, DRB3* 014:01:01 was 2.4~7.1%, and DRB3*002:01 was 4.5~8.6% [BoLA-DRB3 allele frequencies in our selected Farm A also was in the above distribution ranges. Thus, Farm A can be a representative of Japanese farms, and it can interpret Japanese farms. Besides, a strong positive correlation was observed between BLV infectivity and PVL. Therefore, our results are considered that can reflect the state of the entire farm.In Japanese Black herds, the distribution of allele frequencies for 4.5~8.6% . The disBoLA-DRB3 allele with BLV infectivity. However, two major hypotheses were considered. One was that the resistant allele was involved in eliminating BLV infections to reduce its original integration; therefore, PVL did not increase. The other was that resistant alleles regulate viral replication, which lowers PVL production, resulting in weaker BLV infectivity than in susceptible cattle. The resistant BoLA-DRB3 allele directly or indirectly affects the BLV infectivity to reduce BLV transmission. In addition, the levels of anti-gp51 antibodies differ significantly in resistant and susceptible cattle [R cattle have lower PVL and weak BLV infectivity than susceptible cattle, indicating that R cattle are low risk of BLV transmission. The amount of PVL increased or changed over time. Resistant and susceptible cattle with the same amount of PVL were not detected in the tested cattle at any time point. Therefore, it was difficult to directly compare the direct association of the e cattle , indicat5 cells in blood samples, respectively [5 cells. The range of PVL in S14 was also 5274–16,532 copies/105 cells. Therefore, we believe that BLV-infected susceptible cattle might produce BLV in their secretions and milk. BLV-infected susceptible cattle were found to have a significantly higher risk of horizontal transmission. Similarly, vertical transmission risk and horizontal transmission appeared to be extremely high for dams and cattle with susceptible alleles compared to those with resistant alleles [BoLA-DRB3 allele can effectively inhibit or prevent BLV infection, as observed in this study. Thus, BLV-infected resistant cattle are low risk of BLV transmission because of weak BLV infection with low PVL. In addition, PVL was maintained at low level in most dams with resistant alleles, thereby reducing the risk of vertical BLV transmission [BoLA-DRB3 allele in cattle, the development of breeding strategies aimed at improving resistance to infectious diseases and designing of broadly effective vaccines against susceptible cattle should be considered in the future.Although some resistant cattle are infected with BLV, they maintain low levels of PVL due to their long infectious period. Furthermore, a considerable proportion of resistant cattle are BLV-negative. In contrast, most susceptible cattle are easily infected with BLV and have high PVLs in the short post-infection period. Previous reports have shown that the BLV provirus may be detected in milk, nasal mucus, and saliva of dairy cattle with PVLs > 10,000, 14,000, and 18,000 copies per 10ectively . In addiectively . Our tes alleles . In contsmission . In contIn Japan, the number of cattle raised per farm is 93.9. We selected an entire herd of 179 cattle containing Holstein cattle, Japanese Black, and F1 hybrids, which is about twice as large as the average scale. Blood samples were collected from 179 cattle of Farm A in Tochigi Prefecture, Japan, and stored in ethylenediaminetetraacetic acid (EDTA). Serum was collected to detect BLV antibodies. This study was approved by the animal ethical committee, and the animal care and use RIKEN animal experiments committee  and the animal care committee of the Institute of Livestock and Grassland Science, NARO .® Genomic DNA Purification Kit  at each time point from the blood samples according to the manufacturer’s instructions.DNAs were extracted using the WizardBLV PVL was measured in all DNA samples from blood using the BLV-CoCoMo-qPCR-2 method  as described previously ,38,50,51BoLA-DRB3 alleles were genotyped using the polymerase chain reaction (PCR)-sequence-based typing (SBT) method as described previously [BoLA-DRB3 exon 2 using PCR. The PCR products were purified to sequence using the Big Dye Terminator v1.1 Cycle Sequencing Kit. The sequence conditions were as follows: 25 cycles at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 2 min. Sequence data were analyzed using ASSIGN 400 ATF software  to identify the BoLA-DRB3 alleles. Susceptible cattle identified as carrying at least one susceptible BoLA-DRB3 allele, resistant cattle carrying at least one resistant BoLA-DRB3 allele, and neutral cattle carrying other BoLA-DRB3 alleles in their genome.eviously . BrieflyAnti-BLV gp51 antibodies in all serum samples were measured using an anti-BLV antibody ELISA kit , according to the manufacturer’s instructions.5 cells/well) were applied to the BLV reporter cell lines, CC81-GREMG (5 × 104 cells/well), [The blood samples were treated with red blood cell lysis buffer  to remove red blood cells, and the pellets were washed with cold phosphate-buffered saline (PBS) and then resuspended in Dulbecco’s modified Eagle’s medium (DMEM)  with 10% fetal bovine serum  . WBCs (1s/well), ,26,39,54s/well), ,56 was up < 0.05, and strongly significant at p < 0.01 and p < 0.001.The correlation coefficient (R) between the syncytium and PVL was calculated using Excel with the Pearson function. Analysis of variance followed by Tukey’s’ post-hoc test was used to determine the significance of the means of PVL for multiple comparisons. Differences were considered to be significant at BoLA-DRB3*015:01 or DRB3*012:01 allele showed strong infectivity and high PVL in their blood. Resistant cattle carrying the BoLA-DRB3*009:02 or DRB3*014:01:01 allele maintained weaker BLV infectivity and lower PVL at each checking point than the susceptible and neutral cattle. Although we did not directly compare BLV infectivity in the same amount of PVL of susceptible cattle and resistant cattle in vitro, the BoLA-DRB3 allele was found to be directly or indirectly associated with BLV infectivity. Consequently, breed selection based on resistant BoLA-DRB3 allele is an effective strategy for reducing and controlling BLV infection.We measured BLV infectivity in WBCs via syncytium formation assay among susceptible, resistant, and neutral cattle at three years of follow-up. The susceptible cattle carrying the


The binding energies for all tested compounds indicated promising binding affinities. A noticeable superiority for the pentacyclic alkaloids particularly, crambescidin 786 (5) and crambescidin 826 (13) has been observed. Compound 5 exhibited very good binding affinities against Mpro , nucleocapsid phosphoprotein , and nsp10 . Compound 13 showed promising binding affinities against Mpro , spike glycoproteins , and nucleocapsid phosphoprotein . Such promising activities might be attributed to the long ω-fatty acid chain, which may play a vital role in binding within the active sites. The correlation of c Log P with free binding energies has been calculated. Furthermore, the SAR of the active compounds has been clarified. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) studies were carried out in silico for the 15 compounds; most examined compounds showed optimal to good range levels of ADMET aqueous solubility, intestinal absorption and being unable to pass blood brain barrier (BBB), non-inhibitors of CYP2D6, non-hepatotoxic, and bind plasma protein with a percentage less than 90%. The toxicity of the tested compounds was screened in silico against five models ). All compounds showed expected low toxicity against the tested models. Molecular dynamic (MD) simulations were also carried out to confirm the stable binding interactions of the most promising compounds, 5 and 13, with their targets. In conclusion, the examined 15 alkaloids specially 5 and 13 showed promising docking, ADMET, toxicity and MD results which open the door for further investigations for them against SARS-CoV-2.The huge global expansion of the COVID-19 pandemic caused by the novel SARS-corona virus-2 is an extraordinary public health emergency. The unavailability of specific treatment against SARS-CoV-2 infection necessitates the focus of all scientists in this direction. The reported antiviral activities of guanidine alkaloids encouraged us to run a comprehensive in silico binding affinity of fifteen guanidine alkaloids against five different proteins of SARS-CoV-2, which we investigated. The investigated proteins are COVID-19 main protease (M COVID-19 is a disease caused by a new strain of the coronavirus. This disease first appeared in Wuhan, China at the end of December 2019. Two months later, the disease became widespread in China [Coronavirus viruses belong to the order Nidovirales in the subfamily Coronavirinae (family Coronaviridae) . They arCoronaviruses mostly cause insignificant respiratory infections, including the common cold. However, more recent emerging coronaviruses can cause more serious diseases, including severe acute respiratory syndrome (SARS-CoV) and the Middle East respiratory syndrome (MERS-CoV) ,7. SARS-Generally, viral proteins can be classified according to their functions into two major groups as structural and non-structural proteins . StructuCryptotethya crypta [Cryptotethya crypta. Both compounds hinder viral DNA polymerase and consequently, DNA synthesis in particular herpes simplex virus type 1 and type 2, vaccinia and varicella zoster viruses [Dysidea avara, and cyanovirin-N, a protein isolated from cultures of the cyanobacterium  Nostoc ellipsosporum [Given the fact that oceans and seas cover almost 70% of the earth, and consequently, contain the largest ecological diversity of biological species, marine natural products (MNPs) attract much interest. This includes metabolite congers from the marine sponge a crypta . MNPs, ma crypta ,33,34,35 viruses . Additiososporum . Meanwhisosporum .Batzella, Crambe, Monanchora, Clathria, Ptilocaulis, and some starfishes, such as Fromia monilis and Celerina heffernani [Crambe crambe and Monanchora unguifera and their synthetic analogues displayed significant inhibitory activity against gp120-CD4 binding, motivate CD4-p56lck dissociation, and prevent HIV-1 cell fusion [Polycyclic guanidine alkaloids (PGAs) represent a major group of marine metabolites common to Poecilosclerida sponges including ffernani ,39,40. Sffernani , these mffernani ,42. PGA ffernani ,48,49,50ffernani ,52, antiffernani ,54, antiffernani ,56,57,58ffernani ,60. Moreffernani ,65,66,67l fusion ,69,70,71Monanchora n. sp. [As part of our research into MNPs together with the global effort to find new robust antiviral drugs capable of combating COVID-19, we report here on the potential interactions between five SARS-CoV-2 proteins and fifteen structurally diverse polycyclic guanidine-containing alkaloids isolated from the Pacific marine sponge a n. sp. .pro) , (ii) spike glycoproteins , (iii) nucleocapsid phosphoprotein , (iv) membrane glycoprotein , and (v) nsp10  were downloaded from Protein Data Bank (http://www.pdb.org). Molecular Operating Environment (MOE) was used for the docking analysis [The crystal structures of the target proteins: (i) COVID-19 main protease  of the compounds were determined using Discovery Studio 4.0. Initially, the Chemistry at Harvard Macromolecular Mechanics (CHARMM) force field was applied, then the compounds were prepared and minimized according to the preparation for small molecules protocol. Then ADMET descriptors protocol was applied to carry out these studies ,78.The toxicity parameters were calculated using Discovery Studio 4.0. Daclatasvir was used as a reference drug. Initially, the CHARMM force field was applied, then the compounds were prepared and minimized according to the preparation for small molecules protocol. Then different parameters were calculated using toxicity prediction (extensible) protocols.1–15 were isolated and identified from the French Polynesian marine sponge, Monanchora n. sp. For detailed isolation and structural characterizations, see El-Demerdash et al. [Compounds h et al. .5 (crambescidins 786) and compound 13 (crambescidins 826) displayed the best binding modes and free energies among the 15 investigated compounds, they were subjected to molecular dynamics (MD) investigation. A 100 ns MD simulation was performed on compound 5 binding against the COVID-19 main protease, nucleocapsid phosphoprotein and nsp10. Furthermore, a 100 ns MD simulation was performed on compound 13 bounded to the COVID-19 main protease, spike glycoproteins, and nucleocapsid phosphoprotein.Since compound MD simulations were performed using GROMACS 2019 software package where the CHARMM36 forcefield was used for protein topology preparation and the official CHARMM General Force Field server (CGenFF) for ligand topology preparation solvated in a dodecahedron box of common simple point charge (SPC) water model applying explicit-solvent periodic boundary conditions. The 6 solvated complexes were neutralized using sodium and chloride ions. Both systems were subjected to minimization using the steepest descent method through 5000 steps to resolve any steric clashes or inappropriate geometry. Then, to ensure a reasonable starting structure, the system was equilibrated under constant number of particles, volume, and temperature (NVT) ensemble for 100 ps using a Berendsen thermostat. The second round of equilibration was performed under constant pressure  ensemble) using the Parrinello–Rahman barostat for an additional 100 ps. Finally, the position restraints were released, and the system was simulated in the production run under an NPT ensemble (Nosé–Hoover thermostat and Parrinello–Rahman barostat) for 100 ns using a time step of 2 fs. Simulation results were analyzed using Visual Molecular Dynamics (VMD) software, ver.1.9.3 .1–15), previously isolated from the French Polynesian Monanchora n. sp. marine sponge (pro) , (ii) the spike glycoproteins , (iii) the nucleocapsid phosphoprotein , (iv) the membrane glycoprotein , and (v) the nonstructural protein (nsp10) . Comprehensive docking studies were performed using MOE 14.0 software. These docking studies predicted the free energy (ΔG) of binding specifically for the molecules shown in In this work, the binding potential of 15 guanidine-containing marine alkaloids  and 826 (13) exhibited the greatest free energy of docking. Crambescidin 786 (5) showed promising binding affinities against COVID-19 main protease , nucleocapsid phosphoprotein , and nsp10 , compared to the co-crystallized ligands PRD_002214 , MES , and SAM , respectively. In addition, crambescidin 826 (13) showed good binding affinities against the COVID-19 main protease , spike glycoproteins , and nucleocapsid phosphoprotein , compared to the co-crystallized ligands PRD_002214 , NAG , and MES , respectively  and the isopropyl moiety occupied the second pocket of (Mpro). Furthermore, the benzyl acetate moiety occupied the third pocket of the receptor. Moreover, the 5-methylisoxazole-3-carboxamide moiety was incorporated in the fourth pocket  against COVID-19 main protease was as follows: the ligand formed four hydrogen bonds and three hydrophobic interactions. In addition, the 2-oxopyrrolidin-3-yl moiety occupied the first pocket of  bonded with COVID-19 nucleocapsid phosphoprotein through the formation of two hydrogen bonds with Asn154 and Asn75 . Further5) exhibited a binding mode similar to that of the co-crystallized ligands against the COVID-19 main protease, nucleocapsid phosphoprotein, and nsp10. The binding mode of compound 5 against the COVID-19 main protease showed four hydrogen bonds with Thr26, Ser46, and Glu166. In addition, it formed two hydrophobic interactions with Lul166 and Pro168. The long ω-fatty acid chain facilitated the occupation of compound 5 with different pockets of the (Mpro)  . For theh Pro151 . Finally protein .13) exhibited a binding mode like that of the co-crystallized ligands against the COVID-19 main protease, spike glycoproteins, and nucleocapsid phosphoprotein. The binding mode of compound 13 against the COVID-19 main protease showed three hydrogen bonds with Gly143, Thr26, and Glu189. Compound 13 occupied the four pockets of the Mpro due to the presence of a long ω-fatty acid chain  indicated that there were moderate correlations between c Log P and the free energy of binding against SARS-CoV-2 spike glycoproteins, nucleocapsid phosphoprotein, and membrane glycoprotein with R2 values of 0.566, 0.5651, and 0.5199, respectively .The calculated partition coefficients (c Log P) of the examined compounds were calculated using Discovery Studio 4.0 and summarized in ectively . For the1, 2, 3, 4, 8, and 15  than the corresponding members incorporating long alky side chain  indicated that the long chain group is more preferred computationally than the shorter chain against the target receptors. In addition, it was found that the side chains with more hydrophilic characters  were more effective than those with less hydrophilic groups . We then investigated the impact of the main nucleus on the binding affinity of the examined compounds. Depending on the binding modes of compounds 5 and 13 (incorporating bulky nuclei), it indicated that increasing of the main nucleus bulkiness leads to an increase in the binding affinity.Firstly, the effect of the length of the side chain on binding affinity has been explored. The decreased binding affinity of compounds 8) and crambescidin 359 (15), which showed high levels of BBB penetration. Accordingly, it might be suggested that such compounds were expected to be safe to the Central Nervous System (CNS). The bicyclic compounds, 1 and 9, together with the pentacyclic compounds, 5 and 6 and 12 and 13, showed optimal range levels of ADMET aqueous solubility. Intestinal absorption is the percentage of a drug that is absorbed across the gut wall [1, 2, 3, and 8, together with the pentacyclic compound 15 were predicted to have good intestinal absorption levels, while compounds 4, 7, 9, 10, and 14 showed moderate absorption levels. The cytochrome P450 2D6 (CYP2D6) model predicts the potential of a compound to inhibit CYP2D6 enzyme using 2D chemical structure as input. CYP2D6 is an essential enzyme involved in the metabolism of a wide range of substrates in the liver. Therefore, CYP2D6 inhibition is needed as part of the regulatory procedures in the drug discovery process [8). Hepatotoxicity prediction of such compounds revealed that all compounds are non-hepatotoxic except compound monalidine A (8). Consequently, the liver dysfunction side effect is not expected upon administration of these compounds. The plasma protein binding model predicts whether a compound is likely to be highly bound (≥90% bound) to carrier proteins in the blood [8 , their distribution, and their excretion. These properties offer valuable information about possible dose, route of administration, and the safety of the examined drugs. Furthermore, these data help to reduce the risk of a compound’s late-stage attrition. ADMET studies were carried out for 15 guanidine alkaloids. Daclatasvir  was used as a reference drug. ADMET studies include many descriptors. (i) blood brain barrier penetration, which predicts blood brain barrier penetration of a molecule; (ii) intestinal absorption, which predicts human intestinal absorption (HIA) after oral administration; (iii) aqueous solubility, which predicts the solubility of each compound in the water at 25 °C; (iv) CYP2D6 binding, which predicts cytochrome P450 2D6 enzyme inhibition; (v) hepatotoxicity, which predicts the potential hepatotoxicity of a given compound; and (vi) plasma protein binding, which predicts the fraction of drug that while be bound by plasma proteins . Discovegut wall . A well-gut wall . Accordi process . All exahe blood . All comblood [8 .50 which predicts the tumorigenic dose rate 50 (TD50) of a chemical in a rodent chronic exposure toxicity test [50 which predicts the rat oral acute median lethal dose (LD50) in the toxicity test of a chemical [50 mouse model, all compounds showed TD50 values higher than that of the reference drug, Daclatasvir. Regarding the rat maximum tolerated dose model, the compounds showed maximum tolerated doses with a range of 0.027–0.350 g/kg body weight, which are all higher than Daclatasvir (0.022 g/kg body weight). For the rat oral LD50 model, compounds 4–15 showed oral LD50 values ranging from 1.829 to 13.415 mg/kg body weight/day. These values are higher than that of Daclatasvir (0.677 mg/kg body weight/day). For the rat chronic LOAEL model, compounds 1–4 and 8–11 showed LOAEL values ranging from 0.0165 to 0.0450 g/kg body weight. These values are similar or higher than that of Daclatasvir (0.0063 g/kg body weight). Compounds 5–7 and 12–15 showed LOAEL values of ranging from 0.0012 to 0.0019 g/kg body weight, which is less than Daclatasvir.A toxicity prediction was carried out for the 15 guanidine alkaloids based on validated models in Discovery Studio software ,87 as foity test ; (iii) rity test ,90; (iv)chemical ; and (v)chemical ,93. As s5 was subjected to MD with the COVID-19 main protease (PDB ID: 6lu7), nucleocapsid phosphoprotein (PDB ID: 6VYO), and nsp10 (PDB ID: 6W4H), and compound 13 was assessed on the COVID-19 main protease (PDB ID: 6lu7), spike glycoproteins (PDB 30 ID: 6VYB) and nucleocapsid phosphoprotein (PDB ID: 6VYO).Six MD simulations were performed to confirm the stability of the ligands binding to their biological targets over a 100 ns time frame. Compound To analyze the stability of the simulated system, the conformational changes of protein-ligand complexes were analyzed over the course of the 100 ns MD simulation using two methods: RMSD with respect to the initial structure and the radius of gyration shown in , respect5 reached a stable conformation after 30 ns on the COVID-19 main protease active site  and 826 (13) displayed the best binding modes and free energies among the 15 investigated compounds. These docking results were confirmed with the very stable molecular dynamics simulation, opening the door to conduct more research (in vitro and in vivo) on compounds 5 and 13 as expected anti COVID-19 candidates. Compounds 5 and 13 exhibited very promising in silico ADMET results as well as showing high safety margins against five toxicity models. The promising ADMET and toxicity results increase the likelihood of compounds 5 and 13 being used as drugs.Fifteen structurally divergent polycyclic guanidine alkaloids were comprehensively investigated for their virtual antiviral potentials against five SARS-Cov-2 (COVID-19) proteins. The pentacyclic guanidinic scaffolds, crambescidins 786 (


Cardiac cancer represents a rare, largely understudied disease. The aim of this study was to elucidate the underlying pathophysiology of cardiac sarcomas by employing specific network-based methods. Focused interactomes comprised of heart- and tumor-associated gene/proteins were constructed. These networks allowed us to unfold the main pathways leading from heart sarcomas to cardiac diseases. The findings of this study could be utilized in the clinical setting for diagnostic and therapy decision-making.Personalized medicine incorporates genetic information into medical practice so as to optimize the management of chronic diseases. In rare diseases, such as heart cancer (incidence 0.0017–0.33%), this may be elusive. Ninety-five percent of the cases are due to secondary involvementwith the neoplasm originating in the lungs, breasts, kidney, blood, or skin. The clinical manifestations of heart tumors  include heart failure, hypertension, and cardiac arrhythmias of varying severity, frequently resulting in blood vessel emboli, including strokes. This study aims to explain the pathophysiology and contribute to a P4 medicine model for use by cardiologists, pathologists, and oncologists. We created six gene/protein heart-related and tumor-related targets high-confidence interactomes, which unfold the main pathways that may lead to cardiac diseases , i.e., the sympathetic nervous system, the renin-angiotensin-aldosterone axis and the endothelin pathway, and excludes others, such as the K oxidase or cytochrome P450 pathways. We concluded that heart cancer patients could be affected by beta-adrenergic blockers, ACE inhibitors, QT-prolonging antiarrhythmic drugs, antibiotics, and antipsychotics. Interactomes may elucidate unknown pathways, adding to patient/survivor wellness during/after chemo- and/or radio-therapy. Since 1999, when Wulff disputed Newton’s mechanistic point of view on ‘disease’, adopting the Aristotelian one , complexity became an ongoing research subject in medicine and epidemiology . NoteworHeart cancer is rarely encountered, as its incidence falls between 0.0017–0.33% , while hThe clinical manifestations of heart tumors  may include no or minor symptoms, such as the so-called medically unexplained symptoms (MUS)—including nausea, weight loss, fatigue, fever, dyspnea at rest, etc.—or serious problems, such as heart failure, hypertension, cardiac arrhythmia, peripheral emboli, or strokes. Cardiac sarcomas are mainly asymptomatic until reaching an advanced stage, when, chest pain, dyspnea, congestive heart failure secondary to blood flow obstruction, and systemic responses may be manifested. The relevant clinical manifestations in cardiac cancer are chest palpitations, chest pain (most common), cardiac tamponade (as the pericardium is often involved), and/or syncope .The heart cancer diagnosis is usually made late, as it often starts after a stroke caused by a detached tumor tissue or thrombus. Echocardiography, CT scan and/or MRI are the main diagnostic tools in the clinician’s quiver. Unfortunately, no satisfactory published series of cases are available for the establishment of prognosis and treatment statistics. While, chemotherapy is generally preferred for heart metastatic tumors, surgery is suggested in heart sarcomas, even though their underlying biology is still under-investigated. Heart failure induced by heart sarcomas is a major complication that warrants special attention, as, frequently, its underlying mechanisms are relatively unknown. Sparse published information challenges diagnosticians and therapists and begs for education and training. The rarity of the disease suggests personalized management and thoughtful treatment. The genetic profiles are unelucidated. More importantly, the rara avis itself and the location raise ethical issues urging for non-interventional research options. Thus, to explain the pathogenesis of cardiac sarcomas and their manifestations would be of value to cardiologists, pathologists, and oncologists, who normally deal with patients suffering from heart neoplasms. We constructed six different interaction networks- one for each type of primary heart sarcomas: (i) angiosarcoma, (ii) undifferentiated pleomorphic sarcoma, (iii) Rhabdomyosarcoma (iv) Leiomyosarcoma, (v) Myxofibrosarcoma, and (vi) Synovial sarcoma. The constructed interactomes , which include nodes of gene/gene products of known and/or predicted interactions, are described in Eighteen novel intermediate nodes , are distributed in the six networks. In total, 18 highly connected nodes , interleukin 6, G protein subunit beta 3,) were revealed.Heart failure, coronary artery disease, and hypertension epidemics are major public health problems of high prevalence, whose pathophysiology has been insufficiently examined, especially when implicated in other diseases . Heart cIn a previous work, we had constructed the heart cancer interactions network (HFCC1) ,15. Thisoncogenesis  ,19,20,21heart failure  ,23,24ischemia  hypertension  ,23,25atrial fibrillation  ,27atherogenesis  ,28inflammation ,oxidative stress  renin angiotensin-aldosterone system  endothelin system (EDN1) α-adrenergic signaling  Our constructed interactomes involve genes with their products that been implicated in various pathological mechanisms:cell survival  homeostasis  hemostasis  hematopoiesis  endocrine  metabolism  autonomic nervous system  ,29Additional physiology mechanisms are also involved, influencing:(i)Angiosarcoma: (ii)Undiferrentiated pleomorphic sarcoma: (iii)Rhabdomyosarcoma: (KRAS)(iv)Leiomyosarcoma: (HRAS mutation)(v)Myxofibrosarcoma: (vi)Synovial sarcoma: (SS18-SSX fusion)More specifically, genes known ,36,37 toThe highly connected nodes (hubs) are cyclin D1, endothelin 1 (endothelin pathway), matrix metallopeptidase 2 (implicated in protein or RNA binding), fibroblast growth factor 2 , interleukin-8, interleukin-6 (inflammation marker), G protein subunit beta 3 , nitric oxide synthase 3 (involved in cardiovascular disorders and hypertension). histone deacetylase 2-implicated in tumorigenesis (HDAC2), chronic obstructive pulmonary disease, lung diseases fibroblast growth factor 2 (FGF2) implicated in cell survival and oncogenesis polycomb group ring finger 5 that is involved in oncogenesis syndecan 2 that has been associated to non-alcoholic fatty liver, hepatic fibrosis, post-traumatic stress disorderdynein light chain LC8-type 1 that is implicated in prostate carcinoma in situ ,40POTE ankyrin domain family member F, that has been related to dilated cardiomyopathy .adducin 1 implicated in the cell volume homeostasis, cell morphogenesis and cellular response to calcium ions adenylosuccinate synthase 2 that is implicated in the de novo pathway and in the salvage pathway of purine nucleotide biosynthesis, it catalyzes the first committed step in the biosynthesis of AMP from IMPcaspase 3, a canonical pro-apoptotic protein cyclin dependent kinase 4—a cell division protein encoded by the CDK4 gene—which is associated to tumorigenesis of variant cancers 2,4-dienoyl-CoA reductase 1 protein encoded by DECR1 gene participating in the beta-oxidation and metabolism of unsaturated fatty enoyl-CoA esters E1A binding protein p300, associated to various syndromes and epithelial cancers high mobility group AT-hook 2 which is a transcription factor related to malignancy and poor prognosisISG15 ubiquitin like modifier—a small ribosomal subunit is a multi-modal unit implicated to immune response and more interestingly to cancer stem cells in a tumor nibrin, a protein involved in DNA damage repair , whilst nerve growth factor receptor—a neurotrophic factor involved in the regulation and survival of certain neurons and pancreatic beta cells platelet and endothelial cell adhesion molecule 1—a protein that removes aged neutrophils from the body, involving in leucocyte transmigration and angiogenesis .Seventeen novel genes were detected: Four non-mediating genes were recognized, namely: GRAM domain containing 1B (involved in cholesterol metabolism and oncogenesis), KH domain containing 3 like (involved in oncogenesis), subcortical maternal complex member , unc-51 like kinase 4 (associated to bipolar disorder and schizophrenia).The networks include oncogenes , genes , enzymes , ion channels , transcription factors , proteins , cancer cell apoptosis modulator (RHOB). The created interactomes elucidate the main pathways leading to cardiac diseases , such as the renin–angiotensin–aldosterone system (RAAS) and endothelin system, and excludes others, such as K oxidase or cytochrome P450 pathways in these patients. ,53,54,55Angiosarcomas represent 40% of the cardiac sarcomas, are usually found in the right atrioventricular groove  56], an, an56], The constructed interactome  illustrated in Connections coming through CXCL1, MMP1, TIMP1, CXCL8 connect to IL-6 and subsequently drive to SDC2, FGF2, CDKNA2, TP53. TP53 through MYC connects to KRAS. The latter connects to EP300 and KMTD2 and through CDKNA2 to FGF2 and NRAS, KIT, PLCG1, and KDR.We revealed novel not previously reported interactions in CS 1: platelet and endothelial cell adhesion molecule 1 (PECAM1) and nibrin (NBN). Moreover, we identified one case of this extremely rara avis cancer entity (angiosarcoma) in the Onassis Cardiac Surgery Center in Athens, which is presented for the first time : a GreekThe undifferentiated pleomorphic sarcoma is classified with the code number 8830/3 in the WHO classification of tumors of the heart and pericardium of 2015 .The relevant interactome  is presented in Rhabdomyosarcoma classification number in the ICD-O Classification of Diseases for Oncology of 2015 is 8900/3 . It is aLeiomyosarcoma ICD-O code number in the ICD-O Classification of Diseases for Oncology of 2015 is 8890/3 . This tyMyxofibrosarcoma is coded with the number 8811/3 in the ICD-O Classification of Diseases for Oncology of 2015 . It was This network is the only one that involves RHOB- a cancer cell apoptosis modulator and, currently a research target as a cancer therapeutic. As apoptotic factor, it decreases while tumors proliferate, whilst it has been identified in head and neck, lung, and brain cancers and in adenocarcinomas, as a poor prognosis indicator. HSPB1, IFI6, WWTR1, SMS, NGFR, LGALS3, ISG15, SCD, CYB5A, DECR1, ADD1, ADD3, ADSS, ANXA1, ASS1, CASP3 are also expressed exclusively in CS 5. ADD1 and ADD3 are interconnected with CASP3 their only node. CASP3, in its turn, connects directly to IL-6, NGFR, SDC2, HSB1, ACTA1, and CCND1. DECR1 connects directly to CYB5A, EDN1, SDC2. NGFR interacts with FGF2 and RHOB only. ANXA1, in its turn, has three connections: EDN1, SDC2, CXCL8. LGALS3 interacts only with MMP1, MMP2. CYB5A is linked to SCD, except DECR1. ISG15 and IF16 are directly linked together but through PCNA and then through CCND1 they connect indirectly to the common to all sarcomas’ major hubs FGF2 and IL-6. SMS connects to ASS1 which connects to ADSS and then to NKX2-5, which through GATA4 affects EDN1. In keeping with the ICD-O Classification of Diseases for Oncology of 2015, the synovial sarcoma’s code number is 9040/3 . The intIn sum, we observed that all cardiac sarcomas share forty-four common nodestwo common hubs: IL-6 and FGF2, that may be assumed as typical of the overall entity (heart sarcomas)All six interactions networks are presented in 3 basic color-specified units/clusters. Nodes in a given cluster are most closely connected to those nodes of the corresponding cluster, as compared to the other two clusters. Nodes in the same cluster module can be associated with similar/common biological functions. This information could be expanded to more complicated enriched clusters that represent cardiac sarcoma-related biological processes, in the future. Finally, the wild-type gene inclusion is a limitation in this investigation.The protocol followed is the established one in molecular networking ,61 and ihttps://www.aacr.org/, accessed on 31 March 2021, https://www.cancer.org/, accessed on 31 March 2021), was conducted in the period December 2018–January 2021, revealing 85 gene/protein heart sarcomas-related and tumor-related targets. The interactions among gene/gene products were studied through STRING v10 [k-means clustering in the constructed networks were computed with the usage of algorithms implemented in the STRING database. The nodes in the network were partitioned into three clusters, based on the shortest path (distance) between two given nodes. Intra-cluster edges are represented by solid lines, whereas inter-cluster edges are denoted by dashed lines. An extensive search in literature , databases  and cancer official sites (RING v10 . A relatGenes not previously published to be associated to the specific heart sarcoma type, thus, not been included in the initial set of molecules in String platform so as to be processed are described as ‘novel’ or ‘novelties’ in this text.All six interaction networks are presented in three basic color-specified units/clusters. Nodes in a given cluster are most closely connected to those nodes of the corresponding cluster, as compared to the other two clusters. Nodes in the same cluster module can be associated with similar/common biological functions.Cardiac sarcoma patients might profit from administration of beta-adrenergic blockers, ACE inhibitors, QT-prolonging antiarrhythmic drugs, antibiotics, and antipsychotics. Interactomes may elucidate unknown cardiac implications of cardiac malignancies and contribute to patient/survivors’ wellness during and after chemo and/or radio treatment.