Upon vascular injury, vascular smooth muscle cells (VSMCs) change from a contractile phenotype to a synthetic phenotype, thereby leading to atherogenesis and arterial restenosis. Myocardin (MYOCD) is essential for maintaining the contractile phenotype of VSMCs. Deletion of MYOCD in VSMCs triggers autophagy. However, the molecular mechanism underlying the effect of MYOCD on autophagy is not clear. In this study, knockdown of MYOCD in human aortic VSMCs (HA-VSMCs) triggered autophagy and diminished the expression of SMC contractile proteins. Inhibition of autophagy in MYOCD-knockdown cells restored the expression of contractile proteins. MYOCD activated the transcription of miR-30a by binding to the CArG box present in its promoter, as confirmed by luciferase reporter and chromatin immune coprecipitation assays, while miR-30a decreased the expression of autophagy protein-6  by targeting its 3′UTR. Restoring the expression of miR-30a in MYOCD-knockdown cells upregulated the levels of contractile proteins. Treatment of VSMCs with platelet-derived growth factor type BB (PDGF-BB) resulted in the transformation of VSMCs to a proliferative phenotype. A low level of miR-30a was observed in PDGF-BB-treated HA-VSMCs, and re-expression of miR-30a led to a decrease in proliferative marker expression. Furthermore, using a wire injury mouse model, we found that miR-30a expression was significantly downregulated in the arterial tissues of mice and that restoration of miR-30a expression at the injured site abolished neointimal formation. Herein, MYOCD could inhibit autophagy by activating the transcription of miR-30a and that miR-30a-mediated autophagy defects could inhibit intimal hyperplasia in a carotid arterial injury model. The switching of vascular smooth muscle cells (VSMCs) from a contractile, quiescent or differentiation phenotype to an adverse, synthetic, proliferative or dedifferentiation phenotype plays an important role in the pathogenesis of various vascular proliferative diseases, including atherosclerosis, restenosis, and aneurysm. Inappropriate VSMC activation and proliferation are critical causes of neointima formation, stenosis and obstructions of vessels. The proliferative phenotype of VSMCs is characterized by proliferation and migration, as well as decreased levels of contractile marker proteins such as alpha smooth muscle actin , smooth muscle 22 alpha (SM22α) and smooth muscle myosin heavy chain (SMMHC). Understanding the potential mechanisms of VSMC phenotypic switching provides a new therapeutic strategy for the treatment of vascular proliferative diseases.SMMHC-CreERT2/MyocdF/F conditional mutant mice, Jianhe Huang et al. confirmed that loss of MYOCD markedly decreased the expression of SMC contractile proteins and induced cell autonomous autophagy [Myocardin (MYOCD) is expressed specifically in cardiac myocytes and VSMCs and plays a critical role in cardiovascular development . It is autophagy . This phIncreasing evidence has confirmed the critical role of autophagy in the smooth muscle cell phenotype switch. Autophagy is one of the key degradation systems devoted to degrading and recycling misfolded proteins and damaged or aged organelles. Autophagy activation can be observed in VSMCs in vascular diseases, including atherosclerosis , hyperteMicroRNAs (miRNAs) are a class of endogenous, 22–24 nucleotide noncoding RNAs that function as posttranscriptional regulators of gene expression. MiRNAs have been found to participate in a variety of biological processes ranging from critical physiological functions to the pathophysiology of many diseases. Recent studies have confirmed the important role of miRNAs in the posttranslational orchestration of autophagy-related genes. MiRNA-30a has been reported to inhibit autophagy by directly targeting beclin 1 in various types of cells –16. HoweIn this study, we revealed a new mechanism by which MYOCD regulated the expression of smooth muscle contractile genes. We found that MYOCD could maintain the expression of SMC contractile proteins by upregulating miRNA-30a-5p and inhibiting VSMC autophagy. We have also demonstrated for the first time that miRNA-30a-5p plays an important role in regulating the phenotypic switch of VSMCs.To test whether MYOCD can regulate the autophagy response, MYOCD was knocked down in HA-VSMCs by transfection with an shRNA MYOCD plasmid. Knockdown of MYOCD was confirmed by western blotting Fig. , and a sNext, we wanted to determine whether the expression of contractile proteins could be restored by 3-methyladenine . HA-VSMCs were transfected with sh-MYOCD plasmid for 4 h and then treated with 3-MA for another 48 h. As shown in Fig. MiRNA-30a-5p has been known to mediate beclin1 suppression by targeting the 3′UTR of beclin1 . Here, wBioinformatics analysis revealed that the promoter regions of miR-30a-5p contain a CArG box motif (CCTTAATGGG) Fig. . To confSince knockdown of MYOCD can inhibit the level of miR-30a-5p, we wanted to know whether recovery of miR-30a-5p expression in MYOCD-knockdown cells could restore the expression of contractile proteins. As shown in Fig. To confirm that beclin1 is a target of miR-30a-5p in HA-VSMCs, cells were transfected with miR-30a-5p mimics, and then the expression of beclin1 was detected by western blotting. The results showed that miR-30a-5p significantly reduced the expression of beclin1 in HA-VSMCs Fig. . To confThen, we hypothesized that miR-30a-5p knockdown could activate autophagy and cause a decrease in the level of contractile proteins. As shown in Fig. PDGF-BB plays a major role in inducing the switch of VSMCs from the contractile phenotype to the proliferative phenotype. Here, PDGF-BB stimulation upregulated the expression of the proliferative markers OPN and PCNA, and downregulated the expression of ACTA2 and SM22α in HA-VSMCs Fig. . The PDGWe next investigated the expression of miR-30a-5p in cells stimulated by PDGF-BB. As shown in Fig. To further confirm the role of miR-30a-5p in VSMC phenotype switching, we performed a wire injury procedure on mouse carotid arteries, followed by local perivascular delivery of miR-30a-5p agomiRs or scramble agomiRs. Three days or 21 days later, injured segments of femoral arteries were harvested and subjected to qRT-PCR and histochemistry staining. Three days after wire injury and local delivery of AgomiRs, the level of miR-30a-5p was detected in carotid arteries by qRT-PCR. Perivascular delivery of AgomiR-30a-5p significantly increased the levels of miR-30a-5p in the injured vessels Fig. . Then, wBy performing MYOCD loss-of-function studies, we demonstrated that MYOCD knockdown triggered autophagy in HA-VSMCs, as indicated by an increased LC3-II/LC3-I ratio, LC3 puncta formation and beclin1 expression but decreased p62 accumulation. This is consistent with the recent literature showing that SMC-restricted MYOCD mutant mice exhibit activation of autophagy and deformation of the arterial structure . MYOCD dPDGF-BB , TNF-α , nicotinmiR-30a-5p was found to suppress the expression of beclin1 in hepatic stellate cells , cardiomTo further clarify whether the effect of MYOCD in regulating autophagy depends on miR-30a-5p, miR-30a-5p mimics were transfected into MYOCD-knockdown cells. The data showed that miR-30a-5p mimics effectively restrained excessive autophagy induced by MYOCD knockdown and restored the expression of contractile proteins. It has been reported that miR-30a-5p could inhibit the activation of nuclear factor κB (NF-κB) in many types of cells –25. NF-κPDGF-BB is a potent mitogen for VSMCs, acting on the PDGF-beta receptor in VSMCs and promoting the migration and proliferation of VSMCs. MYOCD is inhibited by PDGF-BB signaling via transcription factors such as Kruppel-like factor 4 , 31 and Accumulating evidence show that autophagy activation in VSMCs is an essential regulator of the transition from a contractile to a synthetic phenotype, which contributes to neointima formation. High autophagy is observed not only in PDGF-stimulated VSMCs in vitro but also in the VSMCs of injured arteries in vivo using a mouse partial left carotid artery (PLCA) ligation-injury model . Treatme+/Atg5flox/flox mice suggested that loss of basal autophagy in VSMCs increased the susceptibility of VSMCs to death, enhanced ER stress activation, and promoted VSMC inflammation [However, basal autophagy levels are critically required for VSMC survival and vascular integrity during the development and progression of AAs. A recent study using TaglnCreammation . They alammation . TherefoIn the present study, we found that miR-30a-5p was significantly downregulated in PDGF-BB-treated HA-VSMCs in vitro and wire injury-induced mouse carotid arteries in vivo. Liu et al. observed that the expression of miR-30 family members, including miR-30a-e, was significantly reduced in balloon-injured rat carotid arteries compared with uninjured rat carotid arteries . LentiviIn summary, we showed that MYOCD regulated the expression of miR-30a-5p, a potent inhibitor of autophagy, by downregulating beclin1 expression. Our study provided the first evidence that MYOCD knockdown induced the degradation of VSMC contractile proteins by suppressing miR-30a-5p and activating autophagy. Restoring the expression of miR-30a-5p prevented PDGF-BB-induced VSMC phenotype modulation in vitro and inhibited VSMC proliferation in the arterial walls and neointimal hyperplasia in vivo following carotid injury. These studies provide a new potential therapeutic strategy to reduce the progression of intima hyperplasia-related vascular diseases.2 and 95% air.Human aortic VSMCs (HA-VSMCs) were obtained from the American Type Culture Collection  and cultured in Dulbecco’s modified high glucose medium (DMEM-HG)  containing 10% fetal bovine serum  at 37 °C in 5% CO6 cells/mL. The cells were starved in serum-free media for another 24 h and then subsequently treated with 40 ng/mL PDGF-BB  for 48 h.HA-VSMCs were seeded onto 6-well plates at 1 × 10A MYOCD-targeting shRNA plasmid (shMYOCD), a scrambled control shRNA plasmid (shControl), a pcDNA3.1-MYOCD expression plasmid (MYOCD) and an empty vector pcDNA3.1 (pcDNA3.1) have been described previously , 41. The®800CW anti-rabbit secondary antibody and IRDye®680 anti-mouse secondary antibody, were purchased from Li-COR . Signals were detected by an Odyssey™ Infrared Imaging System , and the bands were quantified by Image-Pro Plus 5.1 software .Whole-cell proteins were extracted and subjected to standard western blot analysis as described previously . AntibodReal-time PCR for mRNA and miRNA was performed as described in our previous studies in an Applied Biosystems real-time PCR system . BrieflyFor quantification of GFP-LC3 dots, HA-VSMCs were cotransfected with shMYOCD or shControl together with GFP-LC3 plasmid for 48 h. The numbers of GFP-LC3 dots per cell were quantified from three independent experiments (≥100 cells per experiment).For gene promoter activity assays, the promoter regions of miR-30a-5p were amplified by PCR and then cloned into the pGL3 luciferase reporter vector (WT-pmiR30a-5p-luc). The primers used were as follows: 5′-AGAGCTAGCGAGTAGTATAGGTCCCACTTGGAT-3′ (sense) and 5′-CTTCTCGAGATACCTTCTTTAGCCTTCTGTTGGG-3′ (antisense). Luciferase reporter constructs containing the CArG box-mutated hsa-miR-30a-5p promoter (MUT-pmiR30a-5p-luc) were generated using a QuikChange site-directed mutagenesis kit . The primers used were as follows: 5′-GACTCAAAGATTAACTTTTTTAAAAGGCATGGGATAGAGTTACCCTTTGCTTT-3′ (sense); and 5′-AAAGCAAAGGGTAACTCTATCCCATGCCTTTTAAAAAAGTTAATCTTTGAGTC-3′ (antisense). The vector pGL3 was used as a control. VSMCs were cotransfected with gene promoter plasmids (WT-pmiR30a-5p-luc or MUT-pmiR30a-5p-luc) and the expression plasmids as indicated in the figure legends using X-tremeGENE HP DNA transfection reagent . Luciferase assays were performed using a luciferase assay kit  according to the manufacturer’s protocol. For beclin1 3′UTR reporter activity assays, luciferase-UTR reporter constructs (WT-BECN1-3′UTR) were generated by inserting the beclin1 3′-UTR. The primers used for PCR were as follows: 5′-GTTTAAACGAGCTCGCTAGCCTTTTTTCCTTAGGGGGAGGTTTG-3′ (sense) and 5′-CTCTAGACTCGAGGCTAGCGGCAGTTTTCAGACTGCAGCAAAT-3′ (antisense). The beclin1 3′UTR-MUT luciferase reporter plasmid (MUT-BECN1-3′UTR) was constructed using the following primers: 5′-ATTCGGGTAATATTAAACAGTACATAGGGCAAATACCAAAAAAGAAAAAATC-3′ (sense) and 5′-GATTTTTTCTTTTTTGGTATTTGCCCTATGTACTGTTTAATATTACCCGAAT-3′ (antisense). HA-VSMCs were cotransfected with reporter plasmids (WT-BECN1-3′UTR or MUT-BECN1-3′UTR) and control or miR-30a-5p mimics using X-tremeGENE HP DNA transfection reagent. After 24 h, luciferase activity was measured with a Synergy™ 4 . Transfection efficiencies were normalized by the total protein concentrations of each sample.ChIP analysis was performed as described previously . The croTM EdU DNA Cell Proliferation Kit . Briefly, after being treated with 40 ng/mL PDGF-BB for 48 h, the cells were incubated in 100 μL of 50 μM EdU for 2 h, fixed with 4% paraformaldehyde, and stained with Hoechst 33342. The cells were observed under a laser confocal microscope , and the rate of EdU-positive cells/total cells was counted.Cell proliferation was determined by the incorporation of 5-ethynyl-20-deoxyuridine (EdU) with a Cell-LightMale C57BL/6J (8 weeks old) mice were purchased from the PLA Military Academy of Medical Sciences Laboratory Animal Center  and were randomly divided into three groups. All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and approved by the Animal Ethics Committee of Tianjin University of Science and Technology. Wire-induced carotid artery injury was performed as described previously . Brieflyt test or one-way ANOVA. P < 0.05 was considered statistically significant and expressed as *P < 0.05, **P < 0.01, ***P < 0.001.All of the data were presented as the means ± S.E.M. The differences between two experimental groups were analyzed by using a two-tailed Student’s Original data of western blot for Figure 1Original data of western blot for Figure 2Original data of western blot for Figure 3Original data of western blot for Figure 4Original data of western blot for Figure 5Original data of western blot for Figure 6Reproducibility checklist


Meanwhile, the organic substance may combine with metal cations  through functional groups, such as hydroxide, carbonyl etc., which increases its stability in the industrial waste salt. These findings provide comprehensive information for the resource utilization of industrial waste salt from chemical industry etc.Industrial waste salt is classified as hazardous waste to the environment. The organic impurity and its occurrence in industrial waste salt affect the salt resource utilization. In this paper, composition quantitative analysis, XRD, TG-DSC, SEM/FIB-SEM coupled with EDS, FTIR, XPS and GC-Ms were chosen to investigate the organic impurity and its occurrence in industrial waste salt. The organic impurities owe small proportion (1.77%) in the specimen and exhibit weak thermal stability within the temperature of 600°C. A clear definition of organic impurity, including 11 kinds of organic compounds, including aldehyde, benzene and its derivatives etc., were detected in the industrial waste salt. These organic impurities, owing (C-O/C-O-C, C-OH/C = O, C–C/CH These impurities hold the following properties: the containing of complex functional group structure, such as benzene ring structure, ester group, sulfonamide etc.; owing halogen elements, such as F, Cl. These characteristics make organic impurities have a certain affinity for heavy metals and make the organic impurities owe the thermal stability and non-volatile property in the industrial waste salt.2+, Mg2+, Cu2+ etc. cationic and F-, NO3- etc anionic. While the organic impurities are represented in the form of TOC in the industrial waste salt. The industrial waste salt exhibits two weightlessness processes in the TG-DSC curve, i.e. 45–600°C and 600–1000°C. The organic impurity pyrolysis mainly happen within the temperature of 600°C.(1) The impurities in the industrial waste salt are classified into inorganic and organic impurities. The former include Nix/C = C etc.; while the inorganic impurities are mainly embedded into the bulk crystal. Meanwhile, the organic substance may combine with metal cations through functional groups, such as hydroxide, carbonyl etc., which increases its stability in the industrial waste salt.(2) The organic impurities distribute both on the surface and inside of the solid salt particles, owing complex function group, such as C-O/C-O-C, C-OH/C = O, C–C/CH(3) GC-MS analysis has clearly proved that the main organic impurities include 11 kinds of organic compounds in the industrial waste salt, which contains aldehyde, benzene and its derivatives etc. These organic compounds owe the thermal stability and non-volatile property, increasing the difficulty of resource utilization of the industrial waste salt.


Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV), is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide. An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010. S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV. In this study, the construction, expression and purification of Pseudomonas aeruginosa exotoxin A (PE) without domain III (PEΔIII) as a vector was performed for the delivery of PEDV S-A or S-B. PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice. The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses, but also stimulate PEDV-specific mucosal immune responses in mice. PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection. Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV) is a highly contagious and devastating swine disease that leads to high mortality in piglets as it causes severe diarrhea, vomiting and dehydration  lacking domain III (PE(ΔIII)) is a nontoxic bioadjuvant that enhances cellular and humoral immune responses to a fused target gene  gene PE(ΔIII) PEDV S-A or PE(ΔIII) PEDV S-B. These two genes were synthesized by Sangon Biotech and cloned into the pET28a vector by restriction enzymes NheI and XhoI. The recombinant plasmids pET28-PE(ΔIII) PEDV S-A and pET28-PE(ΔIII) PEDVS-B were confirmed through restriction enzyme digestion and DNA sequencing.In the present study, PE(ΔIII) was constructed, expressed and purified as a vector to deliver PEDV S-A (aa 25–229) or S-B (aa 499–789+ aa 1370–1378) gene segments of the epidemic PEDV isolates of genogroup GII-a. In Fig. E. coli BL21 (DE3) pLys cells. Expression of the recombinant strains was induced by 1 mM IPTG and expressed as an inclusion body. After the inclusion bodies were denatured and renatured, the recombinant proteins were purified through Ni2+ affinity chromatography. The purified proteins were detected through SDS-PAGE  PEDV S-A and pET28-PE(ΔIII) PEDVS-B were transformed into P > 0.05). In Fig. To evaluate the specific humoral immunity of mice immunized with the recombinant proteins, indirect ELISA and neutralizing antibody tests were performed. Serum samples collected at 0, 14, 28, and 42 dpi (days post immunization) were examined by indirect ELISA. As showen in Fig. in vitro. Th1-type (IFN-γ and IL-2) and Th2-type (IL-4 and IL-10) cytokines were examined using a commercially available double-antibody sandwich ELISA kit. As showen in Fig. P < 0.001), and they were significantly higher than those of PEDV S-A group. In Fig. P > 0.05). Cytokines play an important regulatory role in various immune reactions. These results demonstrated that PEDV S-B group could induce Th1-dominant cellular immune responses. Our results were consistent with previous studies, which showed that PE(ΔIII) delivering antigen proteins improved cellular immune responses in vivo  were cultured in Dulbecco’s minimal essential medium  with 10% fetal bovine serum  at 37 °C in 5% COPEDV S-A fused with PE(ΔIII) (GenBank: K01397.1) or PEDV S-B from the variant PEDV CH2019 strain was codon optimized, synthesized by Sangon Biotech, and cloned into pET28a vector for recombinant protein expression. Two recombinant plasmids, namely, pET28-PE(ΔIII) PEDV S-A and pET28-PE(ΔIII) PEDV S-B, were successfully constructed and confirmed through DNA sequencing by Sangon Biotech.E. coli BL21 (DE3) pLys cells, and the recombinant proteins were purified by Ni2+ affinity chromatography.The recombinant plasmids pET28-PE(ΔIII) PEDV S-A and pET28-PE(ΔIII) PEDV S-B were transformed into After purification was performed, the proteins (PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B) were separated through 12% SDS-PAGE to detect the purity of target proteins. The purified target proteins were also confirmed by Western blot analysis. Protein bands were detected using an ECL chemiluminescence system and visualized with Image Lab V. 4.0.1.n = 20). Group A (negative control group) was intranasally (i.n.) immunized with 50 μL PBS; Group B was i.n. immunized with PEDV S-A, which was composed of 30 μg purified PEΔIII PEDV S-A; Group C was i.n. immunized with PEDV S-B, which was composed of 30 μg purified PE(ΔIII) PEDV S-B. After 2 weeks, all mice were boosted with the same vaccine. Serum samples were collected at 0, 14, 28, and 42 dpi and assayed for PEDV-specific IgG or IgA and PEDV-specific neutralizing antibodies. The small intestines of mice immunized with PEDV S-A and PEDV S-B groups were collected at 42 dpi. The extracted liquid was harvested by grinding and centrifuging, and it was assayed for PEDV-specific IgA.Animal experiments were performed in accordance with protocols approved by the animal ethical and welfare committee of Huazhong Agricultural University. Six-week-old female BALB/c mice were purchased from the Hubei Center of Disease Control, China. The mice were randomly divided into three groups (3 with a sucrose discontinuous gradient and incubated at 4 °C overnight. The incubated plates were blocked with 2% BSA-PBST and washed three times with PBST. Samples were diluted (1: 200) with 2% BSA-PBST, added to the plates for 1 h at 37 °C, washed three times, and incubated with HRP-conjugated goat anti-mouse IgG  or goat anti-mouse IgA  for 1 h at 37 °C to detect antibody titers of PEDV-specific IgG or IgA. The optical density (OD) was measured at 450 nm by using a microplate reader.Ninety-six well microtitration plates were coated with 0.5 μg purified PEDV diluted in 0.05 M NaHCOSerum neutralization test was performed to detect PEDV antibodies as previously described , and stimulated with 5 μg purified PEDV, 10 μg/mL concanavalin A, or 100 μL RPMI 1640. Proliferative responses were detected with an MTS assay. OD was measured at 450 nm by using a microtiter plate reader. Stimulation index (SI) was calculated with the following formula: SI = (OD of immunized groups – OD of blank control)/(OD of negative control group – OD of blank control).Splenic lymphocytes were separated from the spleen of immunized mice by using a mouse lymphocyte isolation reagent . The isolated lymphocytes were adjusted to 4×106 cells/mL with RPMI-1640 containing 10% FBS, seeded into 24-well plates, and stimulated with 5 μg/mL purified PEDV. After 24 h of incubation, culture supernatant was centrifuged at 1000 rpm for 10 min. Cytokines  in the culture supernatant were analyzed with a commercial sandwich ELISA kit. OD was measured at 450 nm by using a microtiter plate reader.Splenic lymphocytes were isolated, resuspended at 4×10P < 0.05.Statistical analyses were performed with GraphPad Prism V. 5. Data were represented as the mean ± SEM. One-way ANOVA was conducted to statistically analyze differences between multiple groups. Differences were considered significant when


Through the transfection of constructs encoding either a wild-type human nucleolin or a mutant nucleolin with the same C-terminal sequence predicted for the autism proband, and by using co-localization with the nucleophosmin  protein, we have shown that the nucleolin mutation leads to mislocalization of the NCL protein from the nucleolus to the nucleoplasm. Moreover, a construct with a nonsense mutation at the same residue, p.Gly664*, shows a very similar effect on the location of the NCL protein, thus confirming the presence of a predicted nucleolar location signal in this region of the NCL protein. Real-time fluorescence recovery experiments show significant changes in the kinetics and mobility of mutant NCL protein in the nucleoplasm of HEK293Tcells. Several other studies also report de novoNCL mutations in ASD or neurodevelopmental disorders. The altered mislocalization and dynamics of mutant NCL (p.G664Glufs*70/p.G664*) may have relevance to the etiopathlogy of NCL-related ASD and other neurodevelopmental phenotypes.Nucleolin  is a major nucleolar protein that plays a critical role in multiple processes, including ribosome assembly and maturation, chromatin decondensation, and pre-rRNA transcription. Due to its diverse functions, nucleolin has frequently been implicated in pathological processes, including cancer and viral infection. We recently identified a  Nucleolin  is one of the most abundant proteins in the nucleolus, accounting for almost 10% of the total protein content ,8.The nucleolin protein consists of three domains: a disordered N-terminal domain containing several acidic regions and phosphorylation sites; four RNA recognition motifs (RRM) or RNA binding domains (RBD) within the central region; and a C-terminal Gly/Arg-rich (GAR) or Arg-Gly-Gly (RGG) domain with several glycine/arginine-rich motifs B 9]. Th. Th9]. Tde novo in an individual presenting with autism spectrum disorder (ASD), which replaced the last 47 C-terminal GAR residues of nucleolin protein with 69 non-GAR residues  and the Special Education Organization (SEO), Iran. The IAU66 proband/father/mother trio was ascertained by Dr. Sasanfar by the Children’s Health and Evaluation project (CHEP), sponsored by the Special Education Organization (SEO), as described previously . The diawww.tcag.ca (accessed on September 2021)). For details on the DNA library preparation and sequencing, as well as the sequence alignment and analysis pipeline, see [de novo variants was performed as described in Harripaul et al. (MedRxiv and Ph.D. thesis) [Whole-exome sequencing of the male ASD individual and both parents was conducted using Illumina HiSeq 2500 . The mutation was confirmed by Sanger sequencing  ,17.TM cloning system . Site-directed mutagenesis was used to generate a p.Gly664Glufs mutation NCL construct, using the forward NCL-G664Fs-F GGCCGCGGCGGCTTTGAGGACGAGGTGGTG GTA and reverse NCL-G664Fs-R TACCACCACCTCGTCCTCAAAGCCGCCGCGGCC, and a p.Gly664* mutation construct, using forward NCL-G664del-F GGCAGAGGCGGCTTTTGAGGACGAGGTGGTG and reverseHEK293T human embryonic kidney cells (ATCC CRL-3216TM) were used for the various localization experiments. Full-length NCL (NM_005381.3) was cloned into the mammalian expression vector pDEST47, C-terminally GFP-tagged using the Gateway®, PolyPlus, Illkirch, France), following the manufacturers’ instructions.NCL-G664del-R CACCACCTCGTCCTCAAAAGCCGCCTCTGCC. All mutations were introduced through PCR-based site-directed mutagenesis, according to the manufacturer’s instructions . To transfect WT and Mut fusion proteins in HEK293T cells, either PolyFect  or polyethylenimine based transfection reagents were used . Transfected HEK293T cells were permeabilized with PBS  and blocked in 10% goat serum for 2 h. Immunofluorescence staining, using monoclonal anti-B23 (Nucleophosmin/NPM) , was conducted overnight, followed by a Cy3-conjugated goat anti-mouse IgG antibody . The cells were then counterstained with DAPI and visualized under a confocal fluorescence microscope.Z-stack images acquired with a frame size of 512 × 512 pixels were used to study NCL (C23) and B23 co-localization. A confocal microscope, Olympus FV1200, using 100X/1.3 NA oil objective, was used to capture DAPI (heterochromatin) at 405 nm, 488 nm for GFP (recombinant NCL-GFP fusion protein), and 588nm for mCherry (B23-Cy3 conjugate).2 (5%)-controlled incubation chambers in the HEK293T cells in 10 independent FRAP assays. In all time-lapse imaging, a series of confocal time-lapse images of frames (512 × 512 pixels) was captured at a 488 nm laser excitation with 0.05 transmissions for GFP-tagged protein, post-bleach recovery. All the FRAP assay images were recorded with a minimum of 10 pre-bleach frames, 250 µs bleach time, with a 405 nm laser line at 100% transmission, and 150 post-bleach frames were recorded at equal time intervals.Exogenous NCL was expressed in HEK293T cells in chambered cover glass culture plates . To capture the recovery and diffusion dynamics, FRAP assays were used. All FRAP experiments were performed in temperature (37 °C)- and CONCL, encoding the nucleolin protein; however, despite causing a frameshift, this does not result in a premature stop codon, and thus, the nonsense-mediated mRNA decay is not predicted . The p.Gly664Glufs*70 mutation replaces the last 47 C-terminal residues of nucleolin protein.The mutation  was identified by the Whole-Exome Sequencing (WES) of a set of ASD trios, as described elsewhere ,17, and https://web.expasy.org/protparam/ accessed on October 2021) [(GGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDHKPQGKKTKFE) with 69 non-GAR residues (EDEVVVEEAEEDLVAEAGEALEGEEASEEAEEEEVTTSHKERRRSLNSFCPSAFPFPFERKDSGVFTVT) D. The coer 2021)  indicateTo study the effect of p.Gly664Glufs*70 on the localization of the NCL protein, N-terminally GFP-tagged WT full-length and mutant truncated NCL proteins were transiently expressed in HEK293T cells. A slightly larger protein showing the insertion of few residues caused by this mutation was shown by anti-GFP western blots of protein extracted from the cell lysate expression recombinant NCL proteins C. The ceAs the RGG domain of NCL protein is believed to be involved in various steps of the ribosome biogenesis, including RNA polymerase (Pol) I transcription and the processing of pre-rRNA, and the assembly and nucleocytoplasmic transport of ribosome particles , we analde novo mutation in the NCL gene. Interestingly, instead of truncating the C-terminal of the NCL protein, this mutation replaces the last 47 C-terminus with 69 non-GAR residues. These substitutions are predicted to have a significant effect on the important chemical properties of the C-terminal region of the NCL protein, such as molecular weight, theoretical pI, Arg and Gly composition, instability index, aliphatic index and GRAVY. The WT residue composition clearly indicates a neutral-positive residue motif (Gly-Arg) (GGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDHKPQGKKTKFE), whereas the Mut C-terminus mostly consists of neutral-negative residues  (EDEVVVEEAEEDLVAEAGEALEGEEASEEAEEEEVTTSHKERRRSLNSFCPSAFPFPFERKDSGVFTVT).Gene-disrupting de novo mutations have become a common theme in the genetics of autism spectrum disorder . In thisThe presence of positively charged residues in the experimentally validated nucleolar localization sequences (NoLSs) dataset of different nucleolar protein highlights the importance of the arginine residues at the C-terminus of NCL . Our nucIn summary, by using co-localization and FRAP analyses, we have shown that the NCL p.G664Efs*70 variant causes disruption of the nucleolar localization signal of the NCL protein. The localization of the NCL protein to the nucleolus is critical, as its known functions are restricted to involvement with the nucleolar machinery; therefore, mislocalization is likely to equate with a loss of function.The FRAP data show altered binding and mobility dynamics of the recombinant NCL p.G664Efs*70 protein. The slow recovery and lower mobile fraction of the wild-type protein indicates the normal mobility and binding of the wild-type NCL protein in the nucleolus, whereas faster mobility and more protein in the mobile fraction indicates more unbound non-functional protein, i.e., more of the protein failed to reach the nucleolus to perform its function C,D.NCL, is not predicted to lead to nonsense-mediated mRNA decay (NMD), according to mutationtaster.org. However, since it was not possible to transport viable mRNA or cells from Iran, it was not possible to validate this prediction in vitro.It should also be noted that the mutation, which is in the penultimate exon of de novo nonsense mutations in NCL. One mutation, Chr2:232321342G>A; NM_005381.3: c.1705C>T; p.(Gln569*), was reported for a female subject described as having global developmental delay, a thyroglossal cyst, obesity, short stature, attention deficit hyperactivity disorder, localized hirsutism, deep set eyes, low-set ears, large fleshy ears, dental crowding, bilateral single transverse palmar creases, short tapering fingers, short, broad feet, and small nails. The second mutation, Chr2:232322432G>A, NM_005381.3:c.1369C>T, p.(Arg457*), was reported for an individual described as having ‘abnormality of head or neck, abnormality of musculoskeletal system, abnormality of the nervous system, and growth abnormality. It should be noted that, in addition to the predicted loss of the NCL C-terminus, these mutations result in the loss of one or more of the RRMs. Additionally, these mutations would be predicted to lead to nonsense-mediated mRNA decay (using www.mutationtaster.org (accessed on October 2021)), and hence, possibly more severe consequences than for the mutation we report here, as both the mRNA and protein would be affected . Two individuals diagnosed with autism spectrum disorder were reported as having de novo loss-of-function mutations in NCL [Several additional de novo mutations in nucleolin have been identified and reported through large genome or exome sequencing studies. In the Deciphering Developmental Disorders Study , two pat−6) at the intron 5/exon 6 junction. If this were to result in removal of exon 6, the resulting transcript would remain in-frame; (2) A splice donor variant (chr2:232321995_232322004del) that only affects a non-canonical exon ; (3) A stop gain just 20 codons ahead of the stop codon in the terminal exon, p.Arg691* , which would remove the C-terminal 20 amino acids, but leaving the majority of the GAR or RGG domain intact, and, as with the mutation identified in the proband, is not predicted to trigger NMD. The gnomAD v3.1.2 dataset also lists 12 frameshifting indels; however, upon close inspection, these are in a region listed as a “low complexity region, variant annotation or quality dubious”, and they all pair-off with each other in the individual reads to give non-frameshifting variants.In population databases for whole-exome and genome sequence data such as gnomAD and ExAC, loss of function (LoF) variants are significantly underrepresented ). In the most recent gnomAD release (as of November 2021), v3.1.2 non-neuro controls , there are just three potential LoF variants identified: (1) A splice acceptor variant  protein 1 [PIDD1 mutations segregating in autosomal recessive non-syndromic intellectual disability [MECP2 have also been shown to disrupt nucleolin-rRNA-mTOR-P70S6K signaling [NCL has been reported as a susceptibility gene associated with bipolar disorder [NCL loss-of-function variants and ASD, and we note shared biological pathways with a number of other proteins that have been associated with ASD and other neurodevelopmental disorders.In contrast, constraint measures for missense and nonsynonymous rotein 1 . Recentlsability ,25,26,27sability . In addisability . Mutatioignaling . In addidisorder . Our res


Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class Gammaproteobacteria. Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates.There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of IMPORTANCE Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens. Acinetobacter baumannii, Pseudomonas aeruginosa, and members of the family Enterobacteriaceae. Horizontal gene transfer on small genetic elements, such as plasmids, is common among Gram-negative bacteria, facilitating the rapid spread of newly evolved resistance mechanisms. Furthermore, Gram-negative bacteria possess an outer membrane (OM), which vastly restricts the accessibility of targets for drugs with intracellular modes of action. A dried-up antibiotic development pipeline is aggravating this situation and drastically illustrates that new, resistance-breaking antibiotics active against these highly threatening pathogens need to be discovered and developed now  Gram-negative pathogens is the recently discovered natural product darobactin A (DAR A). It is produced by several tabolite . AdditioS-adenosylmethionine (SAM) (RaS) enzyme DarE . Its biosynthesis is encoded by a small ∼6.5 kb) biosynthetic gene cluster (BGC) that codes for the precursor DarA, three transport-related proteins , and the radical yme DarE , 5. This kb biosydatabase .in silico translate into manufacturable products with the given biosynthetic machinery and, if so, how their activity patterns compare to that of DAR A, the goal was to make these predicted compounds accessible. In this study, we present the mutasynthetic production and antimicrobial characterization of the analogs DAR B to F identified in silico.To test whether the sequences observed Yersinia, Vibrio, and Pseudoalteromonas of the gammaproteobacteria clade (1-N2-W3-S4-K5-S6-F7) is overall dominant. However, a few genes were identified that encode precursor peptides with one or two amino acid substitutions within the heptapeptide core. Among those, the sequence predicted to encode DAR B (W1-N2-W3-T4-K5-R6-F7) particularly caught our attention, since it was a precursor peptide gene that was present in addition to a DAR A-encoding gene within multiple Photorhabdus strains. Precursor-encoding genes that should result in DAR C (W1-S2-W3-S4-R5-S6-F7), DAR D (W1-N2-W3-S4-R5-S6-F7), and DAR E (W1-S2-W3-S4-K5-S6-F7) instead of the Dar A precursor were identified in Yersinia strains. In addition, the sequence W1-K2-W3-S4-K5-N6-L7, which we termed DAR F, was discovered in Photorhabdus thracensis (formerly temperata) strain DSM15199. In this case, a DAR A precursor was also present, and alignment of the precursors from various Photorhabdus strains indicated the DAR F sequence to be related to the DAR B precursor  or very similar (DAR C and D) positions  analysis. The identity of the molecules was corroborated by tandem mass spectrometry (MS/MS) fragmentation analysis. In general, the OH-b1* and OH-b2* fragments, indicative of hydroxylated tryptophan , as well as the C-terminal fragments y1 and y2, could be observed . DAR C alone could not be fragmented, due to a poor production titer. The retention times were similar to that of DAR A, with DAR B and F eluting slightly earlier. However, the intensities of the ions corresponding to the desired molecules showed a high degree of variation   were only detected in traces, with DAR C showing the lowest intensity of all  . DAR F ay of all . On the After corroboration of the identity of the molecules by MS/MS fragmentation analysis, the BGCs were recloned into the optimized heterologous expression system  to reach1H and 13C NMR spectra of DAR B in D2O showed seven Cα methine signals for the peptide backbone . The amino acid sequence could be determined by a thorough 2-D NMR analysis to be Trp1-Asn-Trp2-Thr-Lys-Arg-Phe. Heteronuclear multiple-bond coherence (HMBC) correlations further confirmed ring closure between Cβ of Trp2 (δH = 6.24 ppm) and the aromatic ring of Trp1 (δC = 149.59 ppm) via a bridging oxygen atom, as well as the C-C bond between Cβ of lysine (δC = 52.40 ppm) and the aromatic ring of Trp2 (δH = 6.99 ppm). Characteristic 1H signals in this respect are the doublet of doublets at 6.99 ppm for Trp 23 and the doublet at 6.24 ppm for Trp 17. In addition, the 13C shift of Lys 32 at 52.40 ppm is quite typical for the DAR derivatives. Due to the proximity of the observed heteronuclear single quantum coherence (HSQC) correlations, assignment of the proton signals for the lysine residue proved difficult. On the other hand, the 13C signal of Lys 33 was identified by its HMBC coupling with the proton of Lys 31, while the 13C signal of Lys 35 was mainly assigned based on its chemical shift  , which is probably derived from the purification procedure and might form a salt with the alkaline amino acid residues present in the molecule. The key nuclear Overhauser effect spectroscopy (NOESY) and ROESY correlations are in accordance with those published for DAR A (The ulation) . DistincYersinia genus, had 8- to 16-fold-higher MIC values than DAR A. DAR B displayed comparable or even better activities index mean values were 1.09 for both strains, indicating an additive effect -HSQC NMR spectra of the transmembrane β-barrel domain of BamA bound to either DAR A or DAR B superimposed essentially perfectly, indicating that the two ligands bind in identical fashion and cause the same structural rearrangements of BamA . The. TheE. c of BamA . Notablyaumannii , which iDAR A has good exposure in mouse models of infection, with blood levels maintained above the MIC for 8 h and a half-life of 1 h . To gainPhotorhabdus, which are known symbionts of entomopathogenic helminths that feed on insect larvae , and are released by the nematode. The latter mechanism will also release potential competitors, which are then inhibited by DAR. Even though the natural product was first identified from Photorhabdus strains, the corresponding BGC is more widespread among gammaproteobacteria. All of these  are reported to be associated with higher organisms , despite being the only precursor in the respective BGC. The predicted derivatives were not directly accessible by fermentation approaches. However, heterologous expression opens up the possibility to biosynthesize specialized metabolites, even though the BGC is silent under laboratory conditions. Previously reported DAR A expression constructs were successfully modified to generate the desired analogs. However, DAR B alone was isolated in a scale of several mg, while heterologous expression of DAR D and E resulted in ∼1 mg of pure compound from ∼100 liters of fermentation broth, and DAR C was not isolated at all. The identity of the molecules could be confirmed by MS/MS fragmentation analysis and was verified by NMR experiments in the case of DAR B. However, this synthetic biology approach does not clarify whether these analogs are real natural products or if these sequences only represent snap shots in evolution that will be deselected, since they are not easy to biosynthesize. It also might be that the modifying DarE is not adapted to the altered sequence of the core heptapeptide yet, so that much of the precursor is not converted into the final bicyclic compound. It can be assumed that it will depend on the bioactivity of the analog whether it is worthwhile from an evolutionary point of view to keep the sequence and to optimize biosynthesis of the metabolite. For DAR C and E, the K-to-R shift at position 5, an amino acid that participates in the aromatic-aliphatic covalent C-C bond, resulted in severely impeded production compared to the production of DAR A. Further investigation of DarE’s flexibility will be part of future research.Interestingly, some of the E. coli strain NRZ14408), tetracycline , gentamicin (E. coli strains NRZ14408 and MMGI1) and last-resort carbapenems  showed MICs similar to those of wild-type E. coli. According to the 2019 CDC report on antimicrobial resistance, carbapenem-resistant Enterobacteriaceae (CRE), and carbapenem-resistant Acinetobacter in particular, are “urgent threats” to the modern health care system (Klebsiella pneumoniae in an intensive care unit in Paris (March 2021) could not be prevented, although strict additional infection control procedures were applied.Concerning their mode of action, the DARs bind to the promising target BamA, which is located in the OM of Gram-negative bacteria and not yet employed for any antibiotics in application. The MICs observed for DAR A and B proved the compounds to possess activity against MDR pathogens. Clinical isolates resistant to rifampicin . Lipid A biosynthesis is essential for most Gram-negative bacteria, but certain colistin-resistant strains of A. baumannii, including AYE, are capable of survival without lipid A , Yersinia pseudotuberculosis ATCC 6904, Yersinia aldovae ATCC 35236, and Yersinia bercovieri ATCC 43970 for DAR analogs A, C, D, and E, respectively, were grown in 10-ml fermentations for 10 days at 28°C and 220 rpm. Aliquots were taken at time points of days 3, 7, and 10. Twenty-fold-concentrated supernatants were dried by rotary evaporator and resuspended in MilliQ water. Five-microliter amounts of 1× supernatant and 20× concentrated supernatant were pipetted onto Mueller-Hinton II agar lawns with E. coli strain MG1655 and observed for zones of inhibition.Strains including E. coli strains for cloning and expression were grown in LB broth or on agar medium supplemented with appropriate antibiotics or supplements at 37°C or 30°C using standard working concentrations. Plasmid DNA was isolated using the innuPREP plasmid minikit 2.0  according to the manufacturer’s protocol. Genomic DNA was extracted using the innuPREP bacteria DNA kit . PCR amplification for cloning purposes was performed using Q5 DNA polymerase  according to the manufacturer’s instructions. Restriction digestion was performed using standard techniques and employing NEB enzymes . DNA fragments were analyzed on and excised from 1% or 2% Tris-acetate-EDTA (TAE)-agarose with GeneRuler 1 kb plus  as the marker. DNA for cloning purposes was purified using the Zymoclean large-fragment DNA recovery kit according to the manufacturer’s instructions. DNA concentrations were determined photometrically with an Eppendorf BioSpectrometer , using a 1-mm-light-path UV cuvette. DNA fragments to be fused by isothermal assembly were gel purified and fused using self-made isothermal assembly master mix  using 5′-GCTCTAAGGAGGTTATAAAAATGCATAATACCTTAAATGAAACCGTTAAA-3′/5′-TAGGTTTATTGCTTAATTCGTTTAGTGCTT-3′, amplifying the lacZ spacer from pCRISPOMYCES-2 using 5′-CGAATTAAGCAATAAACCTAAAGTCTTCTCAGCCGCTACA-3′/5′-ACCTGATGGGATAAGCTTTAATGTCTTCACCGGTGGAAAG-3′, amplifying the rest of the P. khanii DSM3369 BGC using 5′-TAAAGCTTATCCCATCAGGTTATTT-3′/5′-CGAAAACTCACGTTAAGGGATTACGCCGCGATGGTTTGTTTTATT-3′, and subsequent isothermal assembly of the fragments to yield the final plasmid. After transformation and selection on LB with kanamycin, apramycin, IPTG , and X-Gal  , blue colonies were picked and the correct assembly of the plasmid was corroborated by test restriction. Amino acid modifications were designed in silico and ordered as complementary oligonucleotides (Table S1) with 4-nucleotide overlaps of the pNBDaroMod backbone. Oligonucleotides were annealed and assembled into pNBDaroMod using the protocol described in Cobb et al.  with a 2-fold excess of DAR B in a sitting-drop vapor diffusion experimental setup. Crystals in space group I2 were grown in 0.2 M calcium chloride dihydrate, 0.1 M HEPES, pH 7.5, 53% (vol/vol) polyethylene glycol 400 (PEG 400) and directly flash frozen in liquid nitrogen. Data were collected at the SLS beamline X06DA  at 100 K, indexed and integrated with XDS (7NRF (7P1C.Samples of the BamA β-barrel (BamA-β) for crystallization were produced as described previously . BamA-β with XDS , and scawith XDS . The strDS  and analyzed using NMR Sparky ([eviously , 33. TheR Sparky .Plasma protein binding  was dete7P1C.The atomic coordinates have been deposited in the RCSB Protein Data Bank and are available under accession code


The ongoing treatment modalities for breast cancer (BC) primarily rely on the expression status of ER, PR and HER-2 receptors in BC tissues. Our strategy of chemosensitization provides new insights to counter chemoresistance, a major obstacle that limits the benefits of chemotherapy of mammary cancers.By utilizing a murine breast cancer model employing NSG mice bearing orthotopic triple-negative breast cancer (TNBC) xenografts, we have evaluated the ability of phytochemical curcumin in chemosensitizing BC to 5-Fluorouracil (5-FU) chemotherapy and the differential modulations of cellular events in response to this strategy, independent of their receptor status.in vivo and in silico evidence substantiates the superior therapeutic index of this combination.A significant synergistic antitumor potential was observed in the murine model with a sub-optimal dose treatment of 5-FU plus curcumin, as evaluated by a reduction in the tumor-related parameters. We authenticated the pivotal role of thymidylate synthase (TS) in regulating the 5-FU–curcumin synergism using the TNBC pre-clinical model. Our study also confirmed the pharmacological safety of this chemotherapeutic plus phytoactive combination using acute and chronic toxicity studies in Swiss albino mice. Subsequently, the molecular docking analysis of curcumin binding to TS demonstrated the affinity of curcumin towards the cofactor-binding site of TS, rather than the substrate-binding site, where 5-FU binds. Our concomitant This is the first-ever pre-clinical study portraying TS as the critical target of combinatorial therapy for mammary carcinomas and therefore we recommend its clinical validation, especially in TNBC patients, who currently have limited therapeutic options. The highly heterogeneous nature of breast cancer (BC) is a major hurdle in developing single mode of treatment against diverse BC subtypes. Luminal tumors respond moderately well to hormonal interventions while human epidermal growth factor receptor-2 positive (HER-2+) tumors can effectively be managed using a diverse array of anti-HER-2 therapies. Despite this improvement in BC treatment modality, only 20% of triple-negative breast cancer (TNBC) and basal-like breast cancer (BLBC), devoid of estrogen (ER), progesterone (PR) and HER-2 receptors, respond well to standard chemotherapy, while the occurrence of these subtypes of BC is increasing in an alarming rate, worldwide –3.de novo synthesis of 2’-deoxythymidine-5’-monophosphate (dTMP) (5-Fluorouracil (5-FU) is the first rationally designed anti-metabolite administered against a variety of solid tumors including BC, where the drug inhibits thymidylate synthase (TS), the key enzyme for e (dTMP) . Though e (dTMP) , 7, makee (dTMP) . TS state (dTMP) –10, while (dTMP) –13. Apare (dTMP) –17.Curcuma longa is an extensively studied phytochemical owing to its various therapeutic utilities in vitro and in vivo. Numerous reports, including that of ours, have profoundly established the anti-proliferative, anti-angiogenic, anti-metastatic and pro-apoptotic properties of curcumin  mice with 2 drug combinations i.e., 10 µM 5-FU with either 10 µM or 5 µM curcumin. Notably, the combination with 5 µM curcumin was equally effective as the other, though the former combination did not show a synergism in vitro. This prompted us to continue with this particular dosage which also stays approximate to the maximal bioavailable concentration of the curcumin. Hence, we utilized a human TNBC xenograft model in NSG mice, which explicitly demonstrated the superior anti-tumor effect of the 5-FU-curcumin combination, which specifically leads to the down-regulation of TS pathway. Furthermore, our precise studies in the TNBC model corroborated the role of TS to serve as the key regulator of this synergism. Our subsequent molecular docking studies showed the interaction site of curcumin on TS and explained its affinity towards TS alone or in the presence of FdUMP, the active metabolite of 5-FU. Hence, this is the first pre-clinical study to date that portrays TS as a promising clinical target for curcumin-mediated chemosensitization of mammary tumors towards 5-FU chemotherapy, with conclusive in vivo and in silico evidence.The ability of this combination to overcome the undesirable effects of 5-FU chemotherapy by curcumin-mediated chemosensitization of BC The breast cancer cell line MDA-MB-231 and human embryonic kidney cells 293T (HEK293T) were purchased from ATCC and maintained in appropriate medium. Mycoplasma tests were performed on parent cell lines and stable cell lines every 6 months using PCR method.5-FU was purchased from Calbiochem . Curcumin, X-treme GENE™ HP DNA transfection reagent and antibody against vinculin were procured from Sigma-Aldrich . Antibodies against p-ERK1/2, p-p38, p-JNK, p-Akt, p-IKK, c-Myc and caspases were obtained from Cell Signaling Technology . Antibodies against p-p65, MDR-1, VEGF, Bcl-2, cIAP1, XIAP, survivin, TS, PARP, Cox-2 and GAPDH were purchased from Santa Cruz Biotechnology . Antibody against ABCG2 was purchased from Abcam .6 MDA-MB-231 cells in PBS. Tumor growth was monitored by palpitation twice a week. Palpable tumors were identified 15 days post-injection and the animals were randomly assigned to four groups (n=5/group). Treatments were started from the 16th day of injection. Group1 was treated with vehicle alone, Group II received an intraperitoneal injection of liposomal curcumin at 25 mg/kg body weight on alternate days, Group III received an intraperitoneal injection of 5-FU dissolved in PBS at a dose of 20 mg/kg body weight twice weekly and Group IV received both 5-FU and curcumin. Curcumin was encapsulated in unilamellar liposome formulation made of phosphatidylcholine and cholesterol. Animals in group IV received a single intraperitoneal injection of liposomal curcumin at 25 mg/kg body weight as a pre-treatment and after 6 h, these animals again received IP injections of liposomal curcumin at 25 mg/kg (as the maximum retention time of curcumin within the body is 3 h), along with 5-FU dissolved in PBS at a dose of 20 mg/kg body weight. Drug treatment was continued up to 6 weeks, animals were euthanized and the tissue samples were collected for further analyses.Xenograft studies were conducted according to protocol #RD3585, under the approval of the University of Florida Institutional Animal Care and Use Committee in accordance with federal, state and local guidelines. 6-8 weeks old, female, nulliparous mice of the strain NOD/SCID (Nonobese Diabetic/Severe Combined Immunodeficiency) gamma, the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice, commonly known as NSG mice, underwent orthotopic, mammary fat pad injection of 4x10Group I- IP injection of the vehicle (liposome) alone .Group II- IP injection of liposomal curcumin 25 mg/kg body weight .Group III- IP injection of 5-FU 20 mg/kg body weight dissolved in PBS (twice weekly).Group IV- IP injection of liposomal curcumin 25 mg/kg body weight .On the days of treatment with the combination of curcumin and 5-FU, the animals received,a) IP injection of liposomal curcumin 25 mg/kg body weight (pre-treatment).b) 6 h post first curcumin injection, two separate IP injections of liposomal curcumin 25 mg/kg body weight and 5-FU 20 mg/kg body weight dissolved in PBS, respectively (twice weekly).To evaluate the regulatory role of TS in the synergism, MDA-MB-231 cells were infected with lentiviral TS shRNA and injected orthotopically to the mammary fat pad of NSG mice.Lentiviral shRNA vector pLKO.1 (designated #89- obtained from Addgene), lentiviral TS shRNA vector , target sequence: 5′CCGGCTTTGGGAGATGCACATATTTCTCGAGAAATA TGTGCATCTCCCAAAGTTTTTG-3′and lentiviral pSMPUW-GFP vector  were used to produce recombinant lentivirus in HEK293T cells.Below mentioned are the other lentiviral TS shRNA vectors obtained from Sigma and screened for the study:5′CCGGCAGGTGACTTTATACACACTTCTCGAGAAGTG TGTATAAAGTCACCTGTTTTTG-3′ (TRCN0000045664),5′CCGGGCAAAGAGTGATTGACACCATCTCGAGATGGT GTCAATCACTCTTTGCTTTTTG-3′ (TRCN0000045666)5′CCGGGCTGACAACCAAACGTGTGTTCTCGAGAACACA CGTTTGGTTGTCAGCTTTTTG-3′ (TRCN0000045665)5′CCGGCCCTGACGACAGAAGAATCATCTCGAGATGATT CTTCTGTCGTCAGGGTTTTTG-3′ (TRCN0000045663)6 HEK 293T cells plated in to 150 mm dishes were transfected with 10µg of TS shRNA plasmid, pLKO.1 vector or pSMPUW-GFP, 5µg psPAX  and 5µg pMD2.G (VSV-G envelope expressing plasmid) with Fugene transfection Reagent (Qiagen), according to manufacturer’s instructions. 48 h post-transfection, media containing the lentiviral particles were harvested and centrifuged at 3000 rpm at 4°C for 10 min. The supernatant was collected, mixed with 1/3rd volume of fresh media and 1:1000µl polybrene was added.5x106 MDA-MB-231 cells were plated to 150 mm dishes, incubated overnight and transduced with the lentivirus for 8 h. After incubation, the infection mixture was replaced with fresh media and the lentiviral particles containing media was discarded. This procedure was repeated twice. The efficiency of transfection in HEK 293T and transduction in MDA-MB-231 was assessed using a microscope and photomicrographs were taken. Transduction efficiency was further validated through immunoblotting analysis.5x106 MDA-MB-231TS/MDA-MB-231TS-cells in PBS, respectively. In both groups, tumor growth was monitored by palpitation twice a week. Palpable tumors were identified 15 days-post-injection and the animals in both groups were randomly assigned to four sub-groups (n=5/sub-group). Treatments were started from the 16th day of injection. Sub-group I of group A and B was treated with the vehicle alone, sub-group II of group A and B received an intraperitoneal injection of liposomal curcumin at 25 mg/kg body weight on alternate days, sub-group III of group A and B received an intraperitoneal injection of 5-FU dissolved in PBS at a dose of 20 mg/kg body weight twice weekly and sub-group IV of group A and B received both 5-FU and curcumin. Animals in sub-group IV of groups A and B received a single intraperitoneal injection of liposomal curcumin at 25 mg/kg body weight as a pre-treatment 6 h before administration of combination and these animals again received IP injections of liposomal curcumin at 25 mg/kg along with 5-FU dissolved in PBS at a dose of 20 mg/kg body weight, since the maximum retention time of curcumin within the body is 3 h. Drug treatment was continued up to 6 weeks, animals were euthanized and the tissue samples were collected for further analyses.6-8 weeks old, female, nulliparous NSG mice (n=40) were used for the experiment. The animals were separated into two groups. Group A and B underwent orthotopic, mammary fat pad injection of 4x10The toxicological analysis of the combination was performed in 6-8 weeks old female Swiss albino mice as per protocol (IAEC/230/RUBY) approved by the Institutional Animal Ethics Committee, Rajiv Gandhi Centre for Biotechnology.Acute toxicity: Doses of 0, 25 and 50 mg/kg of curcumin and 20 mg/kg of 5-FU were given to groups of six mice each. Animals were euthanized on day 8. The liver tissue was analyzed by histopathology using H&E staining and the serum was used to perform Liver Function Test .Sub-chronic Toxicity: Doses of 25 and 50 mg/kg of curcumin and 20 mg/kg of 5-FU were given to groups of six mice each. Animals were euthanized after 90 days and toxicity was measured as described .The tumor and liver tissues from mice and rats were fixed and cryosectioned. Immunostaining of specific proteins in the tissue sections was done using Poly Excel HRP/DAB detection system universal kit for mouse and rabbit primary antibodies  as per manufacturer’s protocol. All the immunohistochemistry images were taken in DMi8 Inverted Fluorescence Research Microscope with DMC 2900 Digital Camera.TUNEL assay was performed to detect apoptosis in formalin fixed, paraffin-embedded xenograft tumor tissue sections using DeadEnd Colorimetric TUNEL System (Promega) following the manufacturer’s instructions .Cytoplasmic and nuclear proteins were isolated from tissue samples and were subjected to Western blotting as described earlier . The quaThe computational studies were carried out using the software package of Schrödinger . The crystal structure of TS was retrieved from Protein Data Bank and used as the initial structure for modeling studies (PDB ID: 1JU6). The atomic coordinates of the ligands, curcumin and 5-fluoro-dUMP (FdUMP) were downloaded from PubChem (CID: 969516) and PDB (PDB ID: 1TLS) respectively. The structure correction of 1JU6 was performed with the module Protein Preparation Wizard. The crystallographic water molecules were removed and polar hydrogens were added. Energy minimization was performed up to an RMSD of 0.3 Å. A 36 Å x36 Å x36 Å receptor grid was generated, that encompasses the binding sites for the substrate and cofactor. The ligands were prepared before docking using the module Ligprep. The ligands were docked onto protein binding sites using Extra Precision (XP) mode of flexible docking .All data were expressed as mean ± SD. Data were analyzed using Prism 6.0 (GraphPad Software). One-way ANOVA measured statistical significance between the conditions. ***P-values ≤0.001, **P-values ≤0.01 and *P-values ≤0.05; ns represents non-significance.in vitro findings  and phospho p65 (3 out of 3) in the 5-FU alone group compared to control and curcumin alone. Supporting our in vitro data, curcumin pre-treatment produced a drastic reduction in 5-FU-induced over-expression of TS and activation of NF-κB in vivo, which was authenticated by the minimal expression of IKK phosphorylation in the group treated with the combination  scored -7.70 kcal/mol. Both curcumin and LY231514 made hydrogen bonds with residue A312. Interestingly, curcumin made additional hydrogen bonds with residues R50 and R78. The interactions with A312 and R50 anchor curcumin deep within the folate binding site. The similar binding affinity of curcumin and LY231514 towards the TS cofactor binding site indicates the efficacy of curcumin to function as a cofactor site-binding inhibitor of TS. 5-fluoro-dUMP (FdUMP), the active metabolite of 5FU, forms a covalent adduct with TS in vivo . The catalytic centre of TS possesses substrate and cofactor (folate) binding sites. A receptor grid, which encompasses both sites, was generated and curcumin was docked on to this grid. Curcumin occupied the TS cofactor binding site with Glide score -7.07 kcal/mol whereas the crystallographic antifolate inhibitor LY231514  with comparable binding energy to that of methotrexate , University of Florida Institutional Animal Care and Use Committee #RD3585.Conception and design: RA. Development of methodology: HN, MZ-K, and RA. Acquisition of data : HN, NA, VV, VL, VA, AS, SM, TH, AK, NN, and MG. Molecular docking studies and computational analysis: AA and SC. Histopathology and immunohistochemistry analysis and verification: SS. Analysis and interpretation of data: HN, NA, BV, SB, AA, SC, MZ-K, and RA. Writing, review, and/or revision of the manuscript: HN, NA, VL, SB, MG, MZ-K, and RA. Study supervision: RA and MZ-K. All authors contributed to the article and approved the submitted version.Financial support for this work has been provided by the Council of Scientific & In-dustrial Research (to HN), Government of India and Department of Biotechnology, Govern-ment of India (to RA). This work was supported in part by Public Health Service grant R01 CA-188132 (to MZ-K) from the National Institute of Health and the Gatorade Trust through funds distributed by the University of Florida, Department of Medicine (to MZ-K).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.