Background and objectives: The human respiratory syncytial virus (hRSV) is among the important respiratory pathogens affecting children. Genotype-specific attachment (G) gene sequencing is usually used to determine the virus genotype. The reliability of the fusion (F) gene vs. G gene genotype-specific sequencing was screened. Materials and Methods: Archival RNA from Saudi children who tested positive for hRSV-A were used. Samples were subjected to a conventional one-step RT-PCR for both F and G genes and direct gene sequencing of the amplicons using the same primer sets. Phylogeny and mutational analysis of the obtained sequences were conducted. Results: The generic primer set succeeded to amplify target gene sequences. The phylogenetic tree based on partial F gene sequencing resulted in an efficient genotyping of hRSV-A strains equivalent to the partial G gene genotyping method. NA1, ON1, and GA5 genotypes were detected in the clinical samples. The latter was detected for the first time in Saudi Arabia. Different mutations in both conserved and escape-mutant domains were detected in both F and G. Conclusion: It was concluded that a partial F gene sequence can be used efficiently for hRSV-A genotyping. Pneumoviridae, genus Orthopneumovirus, and possesses a single-stranded negative-sense RNA genome that encodes eleven proteins [Human respiratory syncytial virus (hRSV) causes considerable respiratory distress with variable severity in infancy and early childhood ,2. It isproteins , includiproteins , while fproteins .F0 precursor that is cleaved twice, resulting in disulfide-linked F1 (aa 137–574) and F2 (aa 1–109) subunits along with a short peptide, pep27 (aa 110–136). F2 possesses heptad repeat C (HRC), while F1 possesses, at its N terminal, a hydrophobic fusion peptide (FP) followed by two heptad repeats, A and B (HRA and HRB). HRA, HRB, and HRC are essential for envelope fusion to the host cell membrane [F2 possesses five N-glycosylation sites, while HRB, part of F1, possesses a single N-glycosylation site; however, F2 contains more conserved sequence than F1, which is characterized by a high homology among different hRSV genotypes [F2 qualifies it for being a potential target for diagnostic assays.The G glycoprotein is a type II surface protein which is highly glycosylated and possesses a considerable degree of nucleotide variability . It possmembrane ,13. F2 penotypes . The higThe hRSV is classified into two major groups: hRSV A and hRSV B . To dateF2 and the first part of F1 (FP and HRA) and compare it with genotype-specific partial G gene sequencing. Genotyping and sequence analysis of the Saudi hRSV-A strains is another objective of the current study.Typing of hRSV is mainly based on direct G sequencing of sequences flanking HVR2; however, genotype-specific amplification is needed. The F gene could constitute an alternative method for hRSV genotyping; it was found to successfully genotype RSV strains in comparison to the G gene and resulted in the same phylogenetic clusters based on a full genome sequence of the F gene . AccordiEthical release was obtained from both by the College of Medicine, Taif University , and also from the hospital of Paediatrics ethical committee based on the human subjects protection guidelines. The first part of the study was previously published . The guan = 56) samples were found to be weak, with a range of 1 × 102 to 1 × 104.5 copies/mL [A total of 59 archived RNA/DNA samples that showed positive real-time RT-PCR results for hRSV-A, determined in our previous study , were usopies/mL .® 1-Step RT-PCR  that was based on GoScript™ Reverse Transcriptase. Briefly, the program was adjusted for reverse transcription at 45 °C for 45 min, followed by an initial denaturation step at 95 °C for 5 min and 35 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. This was followed by a final 10 min elongation step at 72 °C. The RT-PCR amplicons were subjected to 1.5% agarose gel electrophoresis.An F gene-specific oligonucleotide primer set: Fus-For (1–29) 5′-ATGGAGTTGCCAATCCTCAAAGCAAATGC-3′ and Fus-Rev (622–643) 5′-ATATGCTGCAGCTTTGCTTGTT-3′, that flanks F2 and the first part of F1 (FP and HRA), was designed based on hRSV-A-GZ08-0 (KP218910). G gene genotype-specific primers for ON1/NA1 and GA5 that flank HVR2 in the C-terminal region of the G gene were also designed and used for amplification of the partial G gene based on the result of F gene sequencing. G gene-specific primer sets were as follows: ON1/NA1 (365-384)-For 5′-CTGAGTCAACCCCACAATCC-3′, ON1/NA1-Rev (24–43 G–F intergene UTR) 5′-ATTTGGTCATGGCTTTTTGC-3′ based on based on hRSV-A-GZ08-0 (KP218910) and GA5-For (374–395) 5′-TCCTGCAATCTACAACAGTCAA -3′ and GA5-Rev-(867–889) 5′-CTGTTATGTTGGATGGAGATG-GA-3′ based on RSVA/Homo sapiens/ITA/120/2009 (KF826832) were designed. Reverse transcription was conducted using GoTaqAmplicons with the expected size were excised from the gel and then purified using a gel-extraction kit . The purified amplicons were used as templates for direct sequencing using the same primers as those used for the F and G genes’ amplification. Sequencing was performed commercially using the BigDye v.3.1 Applied Biosystems  kit according to the manufacturer’s protocol. Raw sequences were visualized and analyzed using MEGA 5.2. Homology BLASTn searches from different strains were performed using highly similar sequences (megablast) against published hRSV sequences in the GenBank databases using default algorithm parameters. The gene sequences of both F and G genes were deposited in GenBank under the accession numbers: F (KU924011-KU924023) and G (MK182708-MK182720). CLUSTAL W multisequence analysis was performed and the phylogenetic tree was constructed using the maximum likelihood statistical method with 1000 bootstrap replicates and Tamura–Nei model substitution model. Nucleotide variability among different strains was calculated using the CLUSTALW available in GenomeNet Database .Deduced amino acid sequences of both F and G amino acid sequences were compared using MEGA 5.2. Amino acid substitutions at different functional and structural protein fragments were analysed. The polyphen (polymorphism phenotyping) prediction webtool  was used3.5 copies/mL). The F gene domains of the hRSV-A isolates possess about 10% nucleotide variability. The signal peptide, however, showed higher nucleotide variability. Partial F gene sequencing revealed that the Saudi strains in the current study belong to the NA1 genotype , ON1 , and GA5 genotype   a. Partia 15.38%) a,b. The Five N-glycosylation sites, N27, N70, N116, N120, and N126, were found to be conserved among all the thirteen Saudi strains, except for strain Taif-103, that has lost one of the N-glycosylations at N120 . The fusA duplication of amino acids (QEETLHSTTSEGYLSPSQVYTTS) was found in two regions within the G protein which reflects an 72 nt insert characteristic to the ON1 genotype . L298P, Both of the designed generic primers were successful in amplification of the three different genotypes of hRSV-A, ON1, NA1, and GA5, that were detected among Saudi archival RNA samples. It is also assumed that the generic F primer set could detect all hRSV-A strains, since it showed complete identities to most of the published hRSV-A strains except for a single mismatch detected occasionally. Genotyping based on partial gene sequencing of both F and G genes showed similar results, which denotes that the designed F gene could be used efficiently in genotyping of hRSV-A strains.The Saudi strains in the current study are classified as NA1, ON1, and GA5 genotypes. Both NA1 and GA5 showed higher virulence to the children in comparison to ON1 and other hRSV-A strains ,25,26. TIt is known that the F protein is focused on the signal peptide, p27, transmembrane domain, and ø antigenic site . SimilarThe deduced amino acid sequence of the G gene revealed the presence of L298P, V303A, and Y304H amino acid substitution in all ON1 Saudi strains. Y273N was detected in Taif 23 and L274P was detected in the Taif-24 and Taif-103 strains. Such substitutions are considered noteworthy as they are present next to aa 265–273 (antigenic site) . The P31Direct partial F gene sequencing represents an accurate method for hRSV-A genotyping that matched partial G gene sequencing results. The current study provides evidence of the circulation of GA5, NA1, and ON1 genotypes in Saudi Arabia. Although mutations in conserved or escape-mutant domains were detected in both F and G proteins, most of them do not affect the virus’ virulence.


More active C3S and β-C2S promote the alkali activation of CSS, whereas the less active γ-C2S hinders the depolymerization of SSS. The incorporation of BFS does not change the hydrate, whose seed effect is helpful for accelerating the depolymerization and polycondensation of CSS and SSS, especially for SSS, and makes the hydrate increase significantly. Owing to the high SiO2 and Al2O3 contents of SSS, the C-(A)-S-H chain length is increased, thus facilitating the polycondensation effect. In this study, the optimal NH/NS of CSS and SSS is NH/NS= 1:2, and the optimal liquid/solid ratio is 0.29. Compared to CSS–BFS, the C-(A)-S-H gel produced by SSS–BFS has lower Ca/Si and Al/Si ratios. Unlike CSS, pure SSS is inappropriate as an alkali-activated precursor and needs to be co-activated with BFS.In order to compare the properties of alkali-activated carbon steel slag (CSS) and stainless steel slag (SSS), the effects of sodium hydroxide/sodium silicate solution mass ratio (NH/NS), liquid/solid ratio and blast furnace slag (BFS) dosage on the compressive strength, hydration products and hydration degree of CSS and SSS were studied. Furthermore, a combination of X-ray diffraction (XRD), thermo-gravimetric analysis coupled with differential thermal analysis (TGA-DTA), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope-energy dispersive spectrometer (SEM-EDS) were used to characterize the morphology and structure of alkali-activated CSS-BFS and SSS-BFS cementitious materials. As the results revealed, the primary hydrate of alkali-activated CSS and SSS is C-(A)-S-H with Q With the continuous development of global steel industry in recent years, bulk solid wastes like steel slag and blast furnace slag (BFS) have tended to grow. Taking the two major types of steel slag, carbon steel slag (CSS) and stainless steel slag (SSS) ,2,3, as The methods of steel slag activation mainly include thermal activation ,17,18, m2. Gonzalez et al.’s 4− to accelerate the polycondensation reaction, thereby leading to increased strength. The excessive NaOH (NH/NS ≥ 1:1.5) leads to a hyper fast reaction, where the limited hydrates accumulate rapidly on the surface of partial precursor particles to hinder the continuation of the reaction. As a result, there is a limited increase in strength.In Non-evaporable water is also known as bound water. The higher content of bound water implies greater total production of alkaline aluminosilicate hydrate . From Fi3S, the C-S-H and C-A-S-H peaks already appear for the pure CSS specimen at an age of 3 d, which is accompanied by the disappearance of C3S peak. β-C2S and γ-C2S react slowly and continuously, whose peaks decay gradually with increasing age but still exist on 90 d. The BFS incorporation promotes the alkali activation, which is beneficial to the formation of well-crystallized C-S-H and C-A-S-H. This is manifested by markedly weakened β-C2S and γ-C2S peaks, as well as sharp and heightened peaks of C-S-H and C-A-S-H. No obvious changes are observed in the peaks of RO phase (Mg1−xFexO), wustite (FeO) or magnetite (Fe3O4), suggesting that these phases do not participate in the alkali activation reaction.2S and bredigite (Ca7Mg (SiO4)4) decrease slightly with age  tetrahedra’s Si-O bond  tetrahedrons and [AlO4] tetrahedrons  in γ-C2S unlike the two different coordination states of [CaO6] and [CaO8] in β-C2S, thus forming a more stable crystal structure. The inferior hydration activity is also attributed to the absence of active oxygen atoms with high charge density in γ-C2S [The leading component of steel slag studied herein is C is tiny . The hydin γ-C2S . 2, slightly higher Al2O3 and lower CaO contents than CSS, and the oxide composition between SSS and BFS is closer, but pure SSS exhibits weaker alkali activation properties because of its higher crystallization degree -S-H [−1 towards a low wave number direction and the formation of a new absorption peak near 670 cm−1 -S-H gel in a Q-(A)-S-H ,53,54, w670 cm−1 . The C--S-H is formed by depolymerization and recombination of BFS to enhance the strength. On the other hand, porous C-(A)-S-H with large specific surface area acts as a “seed” [−1 in SSS shifts significantly towards the low wave number -S-H, and Si-rich raw materials are conducive to the production of more C-(A)-S-H hydrate. Unlike CSS, the Si-O bond of alkali-activated pure SSS shows unobvious depolymerization due to the high γ-Ca “seed” ,56, adsoe number . Once de3S, β-C2S in CSS and γ-C2S, bredigite in SSS. However, the inert phases, such as the RO phase (Mg1−xFexO), wustite (FeO), magnetite (Fe3O4) in CSS and cuspidine (Ca4Si2O7F2), magnesiochromite (Mg-Cr2O4), fluorite (CaF2) in SSS, do not participate in the reaction (SEM image)  . To someIn this paper, the hydration properties of alkali-activated CSS/SSS–BFS cementitious materials are studied. The BFS content, NH/NS and liquid/solid ratio all affect the strength and hydration progression of the studied materials. The following conclusions are drawn:(1) The quantity of C-(A)-S-H, the primary hydrate of alkali-activated steel slag cementitious materials, determines the materials’ hydration properties, such as the strength and non-evaporable water content. C-(A)-S-H increases with the increasing BFS content. For CSS and SSS, the optimal alkali dosage is NH/NS = 1:2, which can balance the depolymerization and polycondensation, and the optimal liquid/solid ratio is 0.29, which ensures the formation of the gel skeleton and the compactness of paste.2S, γ-C2S and chemical components, including CaO, SiO2 and Al2O3, determine their alkali activation performance as a precursor. SSS is not easily alkali-activated due to the stable crystal structure of γ-C2S, which is reflected in the delay or even termination of depolymerization. Nevertheless, its high Al2O3 and SiO2 contents elevate its potential as a precursor. Without doubt, depolymerization is important for steel slag, especially for SSS.(2) The contents of steel slag’s mineral components β-C2, slightly higher Al2O3, and lower CaO, resulting in SSS-BFS gel with lower Ca/Si and Al/Si ratios than CSS-BFS gel.(3) BFS’s beneficial seed effect on the alkali activation of steel slag is reflected in two aspects: The production of C-(A)-S-H through the alkali activation and the promotion of steel slag depolymerization. The increase in the BFS content is helpful for producing C-(A)-S-H gel with high Ca/Si and low Al/Si ratios, thus improving the gel density. Compared with CSS, SSS has higher SiO(4) The inert phases in the steel slag like RO and magnesiochromite do not participate in the alkali activation reaction. In the gel system, they are encapsulated by C-(A)-S-H to form a weak link. This proves, from another perspective, that the alkali-activated steel slag possesses sealing properties against harmful metals like Cr.(5) Considering the effect and cost of alkali activation, pure CSS can be used as a precursor, while SSS needs to be activated jointly with BFS.


NOTCH3 was determined by bioinformatic data mining. We verified the IA-related indicators of NOTCH3. Association was found between IA and the NOTCH3 SNPs rs779314594, rs200504060 and rs2285981. Levels of NOTCH3 mRNA were lower in IA tissue than in control tissue, but higher in peripheral blood neutrophils from IA patients than in neutrophils from controls. Levels of NOTCH3 protein were lower in IA tissue than in cerebral artery tissue. NOTCH3 also decreased the expression of angiogenesis factors in human umbilical vein endothelial cells. Variation in NOTCH3 and alteration of its expression in cerebral artery or neutrophils may contribute to IA. Our findings also describe a bioinformatic-experimental approach that may prove useful for probing the pathophysiology of other complex diseases.Intracranial aneurysm (IA) incidence is about 1~2%. However, the specific mechanisms of IA onset and development need further study. Our objective was to discover novel IA-related genes to determine possible etiologies further. We performed next-generation sequencing on nineteen Chinese patients with familial IA and one patient with sporadic IA. We obtained mRNA expression data of 129 samples from Gene Expression Omnibus (GEO) and made statistical computing to discover differentially expressed genes (DEGs). The screened IA-related gene  Intracranial aneurysm (IA) occurs in 1%–2% of the general population , 4. SmokThere are some new findings of IA. Hemodynamic factors, such as irregular shear stress and oscillatory shear index, may contribute to the development and rupture of IA , 8. Singin silico analyses, followed by verification using immunohistochemistry of IA and cerebral artery tissue.IA is associated with many hereditary diseases, and many genes have been associated with IA . Build oWe preliminarily screened IA-related genes from high throughput genomic and transcriptome data. In order to screen IA-related mutant genes, we performed high-throughput genome sequencing for 20 Chinese subjects  with IA as a discovery cohort  and 1B. -3), immune system process , anatomical structure development , and nervous system development  (There were 369 genes shared between the mutant genes identified in our next-generation sequencing and the DEGs identified from GEO transcriptome sequencing data  and 2C . These s49×10-2) . From th49×10-2) . However49×10-2) .NOTCH3 SNPs related to IA in our discovery cohort: allele T of rs779314594, allele A of rs200504060, and allele T of rs2285981. Bioinformatic software tools predicted that allele T of rs779314594, allele A of rs200504060, and allele T of rs2285981 lead to loss-of-function. Allele T of rs779314594 and allele A of rs200504060, both in the conserved domain of NOTCH3, were predicted to alter an amino acid residue and therefore potentially the protein structure (http://gnomad.broadinstitute.org/)   27]. Wh. WhNOTCHte.org/) .We tested NOTCH3 expression in the IA and cerebral artery by immunohistochemistry. Staining showed significantly lower NOTCH3 expression in the IA  and 5C. https://www.ncbi.nlm.nih.gov/geo, GSE106520) [Due to the role of the immune response in IA rupture, we retrieved data on NOTCH3 expression in peripheral blood neutrophils from individuals with IA and healthy controls from the GEO database (E106520) . NOTCH3 IA has been associated with IL-1β, IL-6, TNF-α, MMP9, MMP2, NF-κB, MCP-1, and VCAM1 , 18–20. NOTCH3 as a key IA-related gene through a series of bioinformatic analyses followed by validation with IA patients’ samples and cell culture. First, we analyzed next-generation sequence data and discovered 6649 deleterious SNPs in 4537 genes in IA patients. Second, we collected RNA-sequencing data from GEO datasets and identified 1403 DEGs associated with IA. Third, we focused on 369 genes shared between the genes that were mutated in our next-generation sequencing data and the DEGs from the GEO data. Finally, we took the intersection of four highly relevant IA-related GO-term genes and decided to focus on NOTCH3 because of its previously defined role in the cerebral artery. We then performed immunohistochemical studies on IA and cerebral artery tissue and showed NOTCH3 down-regulation in IA. Further, we found that NOTCH3 knockdown down-regulated angiogenesis molecules in HUVECs. However, the mRNA expression of NOTCH3 increases in peripheral blood neutrophil.In this study, we identified NOTCH3 remain familial accumulations. They locate in the exons regions of NOTCH3 gene, and may affect the function of protein, because the variations alter the amino acid residue of original protein so that the structure of the NOTCH3 protein was changed [In this study, we found that IA may be associated with allele T of rs779314594 (P = 0.001), allele A of rs200504060 (P = 0.004), and allele T of rs2285981 (P = 0.004) in discovery cohort . However changed , SIFT [4 changed , CADD [4 changed , PolyPhe changed , and Mut changed   contribute to IA, perhaps directly as well as indirectly by affecting the levels of downstream IA-related factors   and whole exome sequencing (n=10)  . Variatihttps://www.ncbi.nlm.nih.gov/geo, GSE106520).We obtained high-throughput RNA data from 129 samples across eight series of data for IA and cerebral artery tissue from the Gene Expression Omnibus (GEO) . We alsolimma package (version 3.8) with batch normalization to integrate different data platforms into one matrix [Differentially expressed genes (DEG) between IA and healthy cerebral artery tissue were identified using R and Bioconductor , 54. Fire matrix . Then ale matrix . StatistTo identify likely IA-related genes, we combined the next-generation sequencing data and high-throughput RNA data and analyzed them using BiNGO , a Gene Ontology (GO) program that assesses the overrepresentation of GO categories in a biological network . DEGs weIn order to ascertain the association between identified SNPs of NOTCH3 and sporadic IA patients, we performed KASP (Kompetitive Allele Specific PCR) for SNP genotype  Co., Ltd). The primers and probes were designed by the Laboratory of the Government Chemist (LGC) . The DNAIA specimens were donated by three patients who underwent clipping at Fuzhou Second Hospital Affiliated to Xiamen University. All three subjects signed informed consent forms. The sample acquisition was approved by IRB (SQ2018-003). After the aneurysm was securely clipped, a small piece of tissue was removed from the IA crest and considered as IA wall tissue. Cerebral artery samples from the control group were taken from autopsies performed in the Department of Pathology at Fuzhou Second Hospital Affiliated to Xiamen University. The two sets of specimens were immediately stored at −80°C  and processed for experimentation within one month. Tissues were fixed in 4% paraformaldehyde for 24 hours, paraffin-embedded, and sliced to a thickness of 4 μm. Specimens underwent antigen retrieval in 10 mM sodium citrate (pH 6.0) containing 0.05% Tween 20 for 3 min at maximum strength in a pressure cooker, then allowed to cool to room temperature. This was followed by blocking with 5% goat serum for 30 min at 37 °C to prevent nonspecific staining. The samples were subsequently incubated overnight at 4 °C with anti-NOTCH3 primary antibody . After 16 hours, the samples were rinsed with TBST and then incubated for 1 h at room temperature with secondary antibodies. DAB chromogen was added for 4 min to a final concentration of 0.05%, and slices were sealed with neutral resin. Finally, the sections were observed and images captured with an inverted microscope .Before fixation, a small piece of tissue was taken for genotyping rs779314594, rs200504060, and rs2285981 of NOTCH3 by Sanger sequencing  [2. Subculture was performed when the cells reached 90–95% confluency. Cells within five passages were used for invitro studies.HUVECs  were maintained in Endothelial Cell Medium  supplemented with 1% endothelial growth supplement and 5% fetal bovine serum (FBS) at 37 °C in an incubator with 95% humidified air and 5% CORecombinant lentivirus was transfected into HUVECs using the Lentivirus transfection system  according to the manufacturer’s instructions. For lentivirus construction, short hairpin (sh)RNA clones were inserted into pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO puromycin lentiviral vectors . Cells were infected with the virus in the presence of Polybrene . At 48h later, HUVECs were cultured in medium containing puromycin for the selection of stable clones. Clones in which NOTCH3 was stably knocked down were selected and verified by Western blotting and RT-qPCR. The following previously published shRNA sequences were used :NOTCH3 top strand,GATCCGGGGGACCTGCCGTGGCTATATTCAAGAGATATAGCCACGGCAGGTCCCCCTTTTTTG;NOTCH3 bottom strand,AATTCAAAAAAGGGGGACCTGCCGTGGCTATATCTCTTGAATATAGCCACAGGTCCCCCG;negative control sequence top strand,GATCCGGGGGACCTGCCGTGGCTATATTCAAGAGATATAGCCACGGCAGGTCCCCCTTTTTTG;negative control bottom strand,AATTCAAAAAAGGGGGACCTGCCGTGGCTATATCTCTTGAATATAGCCACGCAGGTCCCCCG.https://pga.mgh.harvard.edu/primerbank/) [Total RNA was extracted from cells using Trizol reagent  as described by the manufacturer’s instructions. Reverse transcription was performed using a Reverse Transcription Kit . All reactions for real-time PCR were carried out in triplicate in a Bio-Rad Cycler system  using the SYBR Premix  and analyzed using the 2ΔΔ cycle threshold method. Levels of mRNA were presented relative to those for GAPDH. All primers were sourced from PrimerBank (erbank/) . Primer t-test. Statistical analyses of allele T of rs779314594, allele A of rs200504060, and allele T of rs2285981 was performed by Fisher’s test. A value of P<0.05 was regarded as significant. All analyses were performed using SPSS 22.0 .Statistical comparisons of two groups were analyzed using Student’s Subjects (or their parents or guardians) have given their written informed consent.The research institute’s committee has approved the study protocol of population genetics on human research (20170035). Immunohistochemistry of IA and healthy cerebral artery specimens was also approved (SQ2018-003).Supplementary FiguresSupplementary TablesSupplementary Table 1Supplementary Table 3


Chemokines are essential guidance cues orchestrating cell migration in health and disease. Cognate chemokine receptors sense chemokine gradients over short distances to coordinate directional cell locomotion. The chemokines CCL19 and CCL21 are essential for recruiting CCR7-expressing dendritic cells bearing pathogen-derived antigens and lymphocytes to lymph nodes, where the two cell types meet to launch an adaptive immune response against the invading pathogen. CCR7-expressing cancer cells are also recruited by CCL19 and CCL21 to metastasize in lymphoid organs. In contrast, atypical chemokine receptors (ACKRs) do not transmit signals required for cell locomotion but scavenge chemokines. ACKR4 is crucial for internalizing and degrading CCL19 and CCL21 to establish local gradients, which are sensed by CCR7-expressing cells. Here, we describe the production of fluorescently tagged chemokines by fusing CCL19 and CCL21 to monomeric red fluorescent protein (mRFP). We show that purified CCL19-mRFP and CCL21-mRFP are versatile and powerful tools to study CCR7 and ACKR4 functions, such as receptor trafficking and chemokine scavenging, in a spatiotemporal fashion. We demonstrate that fluorescently tagged CCL19 and CCL21 permit the visualization and quantification of chemokine gradients in real time, while CCR7-expressing leukocytes and cancer cells sense the guidance cues and migrate along the chemokine gradients. Typical chemokine receptors are heptahelical class A G-protein coupled receptors (GPCRs) that signal through Gi-proteins for guiding cell migration [Chemokines are best known for their central role in orchestrating cell migration. Chemokines and their cognate receptors not only regulate leukocyte trafficking during homeostasis and inflammation; they also guide and position cells in organogenesis and control the growth and spread of numerous tumors ,2,3. Cheigration . Ligand igration . In addiigration . The actigration .The homeostatic CC chemokines CCL19 and CCL21 are constitutively expressed primarily by stroma cells in lymphoid organs ,8,9. In CCR7-guided cell migration is well studied for DCs. Upon pathogen encountering in peripheral tissues, they capture antigens and induce the expression of CCR7. DCs then migrate in a CCR7-dependent and haptotactic manner along an immobilized CCL21 gradient towards lymphatic vessels from where they are flushed into the sinus of the draining lymph node ,16,17. Ai-proteins for signal transduction and hence fail to induce cell migration. However, ACKRs scavenge chemokines and deliver them for lysosomal degradation [The generation and maintenance of local chemokine gradients is critical for efficient leukocyte trafficking. A family of atypical chemokine receptors (ACKRs) was recently identified to internalize and degrade chemokines, thereby ACKRs regulate the local availabilities of chemokines and shape chemokine gradients ,24. ACKRradation . ACKR4  quantification was often restricted to fixed specimen for staining purposes of the chemokines using antibodies, (ii) chemokines had to be printed as gradient to a substrate using highly sophisticated methods, or (iii) chemokine gradients were simply estimated by concurrently applying dyes, e.g., FITC-dextran, with the chemokine. Moreover, the scavenging activity of chemokine receptors was routinely determined by measuring liberated radation . Here, w6-SUMO double tag to the mature form of the chemokines entails three advantages: (i) the chemokine fusion protein is secreted as properly folded protein upon overexpression in mammalian cells. (ii) the His6 tag enables easy affinity purification of the fusion protein over a Ni2+-column. (iii) the SUMO3 tag renders the recombinant protein soluble and can be simply and precisely cleaved off with an impeccable specificity and efficiency using the SUMO protease 2 (SUMOstar™), liberating the native chemokine-mRFP with its correct mature N-terminus [The availability of fluorescently labeled chemokines is highly desired but very limited. Chemokines can be expressed and produced in inclusion bodies of bacteria as recombinant proteins, but the denatured purified chemokine must be properly refolded and contaminating LPS derived from host cells needs to be carefully removed ,27,28. Mterminus ,28.6-SUMO-CCL19-mRFP or pHis6-SUMO-CCL21-mRFP and collected the supernatants after three and five days of culture, as described in 2+-column. The His6-SUMO tag was cleaved off the chemokine and removed by a subsequent affinity purification step using the same Ni2+-column as before, yielding about 25‒50 µg pure, native and mature human CCL19-mRFP or CCL21-mRFP per T175 cell culture flask  where mouse mature bone-marrow-derived DCs are imbedded into a 3D collagen matrix within a narrow observation area connected to two larger reservoirs to one of which we applied 100 nM of chemokine to establish a gradient that is stable for at least 48 h based on the manufacturer’s information. Migrating DCs were recorded by time-lapse video microscopy, tracked and analyzed using the ‘chemotaxis and migration tool’ provided by the manufacturer. i-protein signaling by incubating H1299 cells expressing CCR7-eGFP with PTx abolished directional migration towards CCL19 , monoclonal anti-polyhistidine antibodies conjugated to HRP , rabbit-anti-mRFP , anti-phospho-p44/42 MAPK , anti-p44/42 MAPK (ERK1/2) Antibody , peroxidase conjugated AffiniPure goat-anti-rabbit IgG , Fluo-3 AM , Pertussis Toxin .6-SUMO3 followed by the human mature codon optimized cDNA of CCL19 (amino acids 22‒98) and sub-cloned in pcDNA3-mRFP with a (GGGGS)3 flexible linker revealing pHis6-SUMO-CCL19-mRFP plasmid. To clone pHis6-SUMO-CCL21-mRFP, a second BamHI restriction site was introduced by site directed mutagenesis into pHis6-SUMO-CCL19-mRFP using the primers 5′-GCAGATCGGCGGATCCACCAACGACGCCGAGGATTG and 5′-CAATCCTCGGCGTCGTTGGTGGATCCGCCGATCTGC. The cDNA fragment of CCL19 was replaced by a PCR fragment coding for human mature CCL21 (amino acids 24–134) using the primers 5′-GACCCAAGGATCCGATGGAGGGGCTCAGGACTG and 5′-GTCGACGGATCCGAATTCTGGCCCTTTAGGGGTCTG.The signal peptide of IgGκ (coding for the amino acids: METDTLLLWVLLLWVPGSTG) was ligated to the N terminus of the cDNA encoding His6-SUMO-CCL19-mRFP or pHis6-SUMO-CCL21-mRFP using FuGENE® 6 transfection reagent . The next day medium was changed to DMEM supplemented with only 5% FCS and cultured for another three days. The supernatants containing the secreted chemokines were collected and cells were grown for an additional two days in fresh medium before harvesting it again. Pooled supernatants were centrifuged to remove cell debris and supplemented with 20 mM imidazole before applying on 1 mL Ni2+ agarose matrix packed in a gravity column previously equilibrated with binding buffer . Ni2+ columns were washed with 10 column volumes of washing buffer  and the total captured protein was collected by two sequential elutions with an elution buffer . The column was re-equilibrated with washing buffer for the second purification step. The eluted His6-SUMO-chemokine-mRFP proteins were applied on Vivaspin 20 cross-flow spin columns  with a cutoff of 10 kDa for chemokine concentration and to exchange for the washing buffer. His6-SUMO-chemokine-mRFP proteins were digested using SUMOstar™ protease  for up to 48 h at 8 °C. The digested material was applied onto the re-equilibrated Ni2+ column to retain the His6-SUMO tag and the impurities of the first purification step. The flow-through containing the pure CCL19-mRFP or CCL21-mRFP, respectively, was collected. The chemokine-mRFP containing fractions were concentrated and the buffer was exchanged to PBS on Vivaspin 20 cross-flow spin columns. The last flow-through fraction was collected as negative control buffer solution.HEK293 cells were grown in DMEM medium supplemented with 10% FCS (Lonza) and 1% penicillin/streptomycin  in T175 flasks. Cells were transfected with either pHis3 linker introduced using the primers 5′-GGACTCGAGAGCGGAGGTGGCGGTTCTGGTGGTGGCGGTTCCGGCGGTGGCGGTAGCGTGAGCAAGGGCGAGGAGCTG and 5′-GCCCTCTAGACTACTTGTACAGCTCGTCC (Primer-B). Both PCR products were digested with XhoI, ligated and PCR amplified with Primer-A and Primer-B, digested with HindIII and XbaI, and inserted into pcDNA3.Human ACKR4 cDNA was amplified by PCR using the primers 5′-GAACAAGCTTCATTACGGCCGCTTTGGA and 5′-CTACCTCGAGCCCCAATAGAGAAGGTAGAAGT using a synthetic cDNA  as template. The PCR product was inserted into the HindIII and XhoI restriction sites of pcDNA3. Site-directed mutagenesis was performed to re-introduce a methionine at position 1 using the primers 5′-CCCAAGCTTCATTACGATGGCTTTGGAACAAAATC and 5′-GATTTTGTTCCAAAGCCATCGTAATGAAGCTTGGGTC. Next, ACKR4 was amplified using 5′-GGAGACCCAAGCTTCATTACGATGGC (Primer-A) and 5′-GAGCTCGAGTCCACCAATAGAGAAGGTAGAAGTAGGTTCAGTTGGACC. In addition, eGFP was amplified from CCR7-eGFP  and a GGCCR7-eGPF and ACKR4-eGFP were transiently expressed in HEK293, HeLa, or stably expressed in H1299 cells. HEK293 and H1299 cells were grown in a DMEM medium supplemented with 10% FCS and 1% penicillin/streptomycin, whereas HeLa cells were cultured in RPMI1640 medium with 10% FCS and 1% penicillin/streptomycin.Human monocyte-derived DCs were generated and matured with a cocktail of inflammatory cytokines for 48 h as described previously . Mouse-b® Permeable support, 3421, Washington, DC, USA) with 5 μm pore-sized polycarbonate membranes. Mature MoDC from different donors (105 per well) in medium were allowed to migrate to the lower well containing graded concentrations of chemokines for 180 min at 37 °C in a 5% CO2 atmosphere. Migrated cells were harvested and enumerated by flow cytometry . The number of cells that migrated spontaneously to the lower compartment in the absence of chemokines was subtracted and the percentage of migrated cells was calculated relative to the input of cells.Two-dimensional chemotaxis assays were performed in duplicate in 24-well Transwell™ chemotaxis chambers  were premixed, added to 90 μL cell suspension, and mixed carefully. The mixture was applied to µ-slide chemotaxis chambers and mounted as described in the manufacturer’s instructions. Chemokines were applied to the right reservoir, whereas control buffer from the chemokine purification was added to the left reservoir. Cell migration was monitored by time-lapse video microscopy on a Zeiss Axiovert 200M  equipped with a Tokai Hit INU (Shizuoka Japan) incubation system for at least 3 h for DCs or overnight for H1299 cells. Cell migration was analyzed and quantified using the ‘chemotaxis and migration tool’ software provided by Ibidi. At least 24 cells per condition for every experiment were analyzed. The fluorescent mRFP gradient was stable and recorded before and after the migration assay.Three-dimensional migration through collagen was performed in µ-slide chemotaxis chambers purchased from Ibidi  according to the manufacturer’s protocol. Briefly, mature bone-marrow-derived DCs or H1299 cells were collected and re-suspended in RPMI1640 or DMEM supplemented with 10% FCS and 1% penicillin/streptomycin, respectively, to 10Changes in concentrations of intracellular free calcium was determined in 300-19 pre-B cells expressing CCR7 loaded with Fluo-3-AM and recorded as previously described . As a neBRET was determined essentially as described previously . BrieflyLive-cell confocal imaging was performed on a TCS SP5 II microscope  equipped with a temperature-controlled chamber. The videos were routinely recorded with a 63× objective. Live cell imaging was conducted in 35 mm Mattek No. 1.5 dishes in a medium supplemented with 20 mM HEPES buffer pH 7.5.


Among the dimerization intermediates, 2-aminophenoxazinole derivatives have the lowest ionization potential and can reduce O2 to superoxide anion. The rate for O-H homolytic bond dissociation is significantly higher than that for C-H bond in non-enzymatic quinoneimine conjugate. However, the last reaction passes irreversibly, reducing O2 to hydroperoxyl radical. The inorganic ferrous iron and the heme group of Drosophila phenoxazinone synthase significantly reduce the energy cost of 2AP H-atom abstraction by O2. We have also shown experimentally that total antioxidant capacity decreases in Drosophila mutant cardinal with L-3HOK excess relative to the wild type Canton-S, and lipid peroxidation decreases in aged cardinal. Taken together, our data supports the conception of hydroxykynurenines' dual role in neurotoxicity: serving as antioxidants themselves, blocking lipid peroxidation by H-atom donation, they also can easily generate ROS upon dimerization, leading to the oxidative stress development.Kynurenines, the products of tryptophan oxidative degradation, are involved in multiple neuropathologies, such as Huntington's chorea, Parkinson's disease, senile dementia, etc. The major cause for hydroxykynurenines's neurotoxicity is the oxidative stress induced by the reactive oxygen species (ROS), the by-products of L-3-hydroxykynurenine (L-3HOK) and 3-hydroxyanthranilic acid (3HAA) oxidative self-dimerization. 2-aminophenol (2AP), a structural precursor of L-3HOK and 3HAA, undergoes the oxidative conjugation to form 2-aminophenoxazinone. There are several modes of 2AP dimerization, including both enzymatic and non-enzymatic stages. In this study, the free energies for 2AP, L-3HOK and 3HAA dimerization stages have been calculated at B3LYP/6-311G//6-311+(O)+G(d) level, both in the gas phase and in heptane or water solution. For the intermediates, ionization potentials and electron affinities were calculated, as well as free energy and kinetics of molecular oxygen interaction with several non-enzymatically formed dimers. H-atom donating power of the intermediates increases upon the progress of the oxidation, making possible generation of hydroperoxyl radical or hydrogen peroxide from O cardinal with impaired phenoxazinone synthase correlates with decrease in both total antioxidant capacity and lipid peroxidation in an age-dependent manner. Our results support the concept of protective role of proteins converting hydroxykynurenines to their dimeric forms, preventing free radicals overproduction and oxidative stress development.Kynurenines, the products of tryptophan catabolism, are compounds with the multiple neuroactive properties. Hydroxykynurenines are redox modulators with a dual role in the oxidative stress development: being able to block lipid peroxidation, they also produce toxic free radicals during the oxidative self-dimerization. The later can occur both enzymatically and non-enzymatically. Computational study of the dimerization process may reveal the stages and intermediates being the source of the reactive oxygen species. This would help to provide therapeutic strategies aimed at the decrease of kynurenines' dimerization toxic effects. We have modeled oxidative conjugation of kynurenines using quantum-chemical approaches, performing calculations for the energies of the dimerization stages, as well as the redox properties of the intermediates. Phenoxazinone synthase provides a structural basis for the full oxygen reduction to water, prohibiting generation of toxic free radicals. The final non-enzymatic dimerization stages are the most probable source of hydroperoxyl and superoxide anion radical. The accumulation of L-3HOK in Drosophila mutant  The complex nature of dimerization processes makes it hard to study all its separate stages experimentally. This requires the usage of computational and modelling approach. Previously, the ability of hydroxykynurenines to donate H-atom and inhibit phenoxyl or methyl peroxy radicals was computationally investigated . Both L-The enzymatic and non-enzymatic R-2AP dimerization pathways share the common stages of initial H-atom dissociation from R-2AP  leading to quinoneimine (R-2APq) production. Enzymatic pathway includes the following stages: conjugation ; H-atom abstraction ; isomerization/ H-atom migration , proton association ; proton dissociation  . We call2 groups are similar among all the studied compounds, though the side chain conformations may vary  is the same for the both pathways shown in . In the form see . The tauH-atom homolytic bond dissociation stages have the highest ΔG values (BDG): the average BDG is 50.056±5.136 kcal/mol for the gas phase, 51.032±5.365 kcal/mol for heptane solution, and 52.335±5.229 kcal/mol for water solution . H-atom abstraction BDG widely vary for the different stages, being maximal for N22  and minimal for N21  . The onl2+) form. We modelled D3 → D5 and D8 → D10 conversions as consisting of three stages: a. aromatic N-H bond dissociation ; b. quinoneimine NH group protonation ; c. charge migration and amine bridge NH group deprotonation . For b. and c., the summary ΔG is negative, except for 3HAAi-D9(H+).For N-H bond dissociation leading to quinoneimine structures formation, BDG is significantly higher than that for O-H bond dissociation . Partially, this may be caused by the addition of a diffuse orbital to O-atom at level III see . HoweverFor non-enzymatic pathway stages where C-H bond dissociation is accompanied by the second ring aromatization (N16) or intramolecular cyclization (N17), BDG is lower than that for O-H BDG values, except for 3HAAi-D in N17. However, C-H bond dissociation rate may be low due to the high value of the activation barrier. In this case, N16 can proceed in two stages: the exothermic keto-enol tautomerization of D1' second ring (N18) and subsequent enol O-H bond dissociation (N19) with BDG close to those for the similar dissociation step of enzymatic pathway (N5). As mentioned before, N17 is likely to proceed via intramolecular cyclization (N20) and subsequent C-H bond dissociation (N21). N21 has the lowest BDG, making D5' a powerful H-atom donor in reactions with molecular oxygen or some other ROS generators. However, D5' C-H bond dissociation may be kinetically inhibited, as well as that for D1'.O-H BDG slightly differs for the kynurenines' derivatives: typically, it is somewhat higher for L-3HOK-D compared to that for 3HAA-D , except in N19. In this case, low BDG value may be explained by the relaxation of the distorted L-3HOK aromatic ring (see below). Contrary, N-H BDG is higher for 3HAA compared to all the other compounds. For 3HAAi-D, O-H BDG is significantly lower compared to the non-ionized form in the gas phase, the difference becoming less pronounced in water solution. This corresponds to our previous data that negatively charged carboxylic group decreases O-H bond dissociation energy in kynurenines–an effect reduced in media with the high dielectric constant . This isThe other probable factors causing ΔG differences for 2AP and kynurenines' dimerization are:-‥ OH: HB), 3HAA/3HAAi-D , L-3HOK-D , L-3HOK-D –ΔG decreases, as a result.HB formation: 3HAAi-D , 3HAA/3HAAi-D , L-3HOK-D , L-3HOK-D –ΔG rises;HB break: 3HAAi-D –ΔG rises.Repulsion of charged groups: 3HAAi-D –ΔG decreases.Deprotonation of neutral compounds: 3HAAi-D –ΔG rises.Aromatic ring distortion: L-3HOK-D ; 3HAA/3HAAi-D –ΔG rises.Aromatic ring relaxation: L-3HOK-D –ΔG decreases.The effects of polar interactions are decreased in heptane and are nearly extinguished in water solution.2+ affecting their redox properties ); BDSC; #3052. Carries mutation in phenoxazinone synthase gene (FlyBase data); L-3НОК overproduction. The strain has been out-crossed to CS for 9 generations.All strains were purchased from Biocollection of I.P. Pavlov Institute of Physiology of the Russian Academy of Sciences, Koltushi, St. Petersburg, Russia. The strains were maintained on the standard yeast–raisin medium at 22±0.5°C or 25±0.5°C and 12:12 daily illumination cycle.th, 13th, 21th and 29th days of imaginal stage . Drosophila heads were homogenized on ice using plastic pestle homogenizer in 600 ul solution containing 0.3 M sucrose , 1mM EDTA, 0.2 M Tris-HCl, pH 7.4, and centrifuged for 10 minutes at 1000g and 4°C. The supernatant was recentrifuged for 20 minutes at 2000g and 4°C, similar to that in [Total antioxidant capacity (TAC) and lipid peroxidation (LP) were estimated in Drosophila heads at the 5 that in , or at 1Protein content in the samples was measured before every experiment by a standard Eppendorf protocol using Biophotometer plus Eppendorf reader  at the Research Centre for Environmental Safety of St. Petersburg State University. 5 μl of each sample was diluted to the final volume of 1 ml with distilled water, and the optical density was measured according to the standard Eppendorf protocol.CS and cd, and merged into a total sample.TAC was measured in Drosophila heads using oxygen radical absorbance capacity fluorimetric method. For this, 25 μl of sample was incubated with 7.5 nM fluorescein  solution  for 30 minutes at 37°C. Then AAPH  was added up to a final concentration of 30 mM . FluoresIntensity of the LP in Drosophila heads was measured using thiobarbituric acid reactive substances (TBARS) colorimetric assay. For this, 200 μl of sample was incubated with 200 μl of 1% solution of orthophosphoric acid and 400 μl of 0.5% solution of thiobarbituric acid for 20 minutes at 100°C. The optical densities of the samples were measured by spectrophotometric microplate reader SPECTROstar Omega at wavelength 532 nm. The contribution of unspecific extinction by measuring the optical density was estimated at 600 nm . The datPearson correlation and linear regression were calculated using Social Science Statistics online resource . StatistS1 TableX–C, O or N atom. Underlined: statistically significant differences .(PDF)Click here for additional data file.S2 Table2. The name of 2AP, L-3HOK, 3HAA or 3HAAi stands for itself and all its dimeric forms. Underlined–non-significant correlation, italics–the moderate correlation.The values are given for the coefficient of determination R(PDF)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S1 FigN–the number of reactions (according to Tables (TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S1 DatasetAtom names, nucleus charges and coordinates  are given.(TXT)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.


This study highlights the role of Nup93-chromatin interactions in driving triple-negative breast cancer propagation through modulation of the actin cytoskeleton, cell migration and proliferation. LIMCH1, a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis.Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing  The nuclear envelope (NE) does not merely represent a protective membrane barrier regulating molecular trafficking between the nucleoplasm and the cytoplasm. Recent findings demonstrate that NE proteins play a crucial role in genome organization and transcription control , cell miNUP93 silencing impaired BCC 3D migration. This effect correlates with dramatic changes occurring in the actin cytoskeleton (AC), including the formation of compelling stress fibers and large focal adhesions. RNAseq demonstrated that NUP93 silencing up-regulated genes associated with ECM organization and down-regulated genes related to cell migration, EMT, and cell proliferation. More intriguingly, the combination of RNAseq data with profiling of Nup93–chromatin interactions revealed that Nup93 directly interacts with and regulates the expression of a subset of genes involved in AC remodeling, stress fiber formation, and ECM contact. Noteworthy, modulating the expression of one of these genes, LIMCH1, partially reverted the phenotype induced by NUP93 silencing. Our data, confirmed by in vivo experiments and by immunofluorescence of human triple-negative, claudin-low breast cancer samples of different stages provide mechanistic evidence of the role of Nup93 in breast cancer progression and highlight potential novel targets for the development of anti-metastatic therapies.A small subset of these nucleoporins  has been associated with tumorigenesis, mainly linked to altered nuclear transport activities . InteresRecent evidence suggests that targeting the AC represents a promising strategy in cancer therapy . For insNUP93 silencing in triple-negative, claudin-low MDA-MB-231 (P < 0.05) (P < 0.001) . We found a partial recovery of the total protein level through Western blot  in cells expressing the RNAi-resistant version of NUP93 upon KD  compared with cells in which Nup93 was depleted  , appeara< 0.001) , and for< 0.001) . These c< 0.001) . In partNPC core  Fig S2)NUP93 silern blot . Similardepleted .NUP93 silencing also increased the level of FAK (P < 0.01). This effect was also observed in NUP155 KD and TPR KD  . Indeed, adhesion assay did not show clear differences between NUP93 silenced and control cells   showed partial functional recovery in the 3D matrix invasion assay compared with wild-type NUP93 silenced cells   (P < 0.01) without compromising adhesion (P = 0.193). However, because Nup93 depletion induced less clear AC changes in H1299 compared with MDA-MB-231, it is possible that additional mechanisms compromising the migratory ability of these cells are involved upon NUP93 KD.Altering the AC can in principle modify the way cells sense the surrounding microenvironment and migrate through it. To test this hypothesis, we suspended MDA-MB-231 within 3D matrices embedded in multi-channel microfluidic devices . Confocal live imaging was used to track the path followed by each individual, viable cell for at least 24 h . Our res < 0.01) , and 2.  = 0.07) . Importa < 0.05) . No stat= 0.305) . We needVideo 1Download videoRepresentative movie showing MDA-MB-231 tracking over 10 h. The cells were embedded in a 3D matrix, incubated for 24 h, and then imaged. Video 2NUP93 KD MDA-MB-231 tracking over 10 h. The cells were embedded in a 3D matrix, incubated for 24 h, and then imaged. Download videoRepresentative movie showing Taken together, collected data suggest a potential role of Nup93 in the AC organization and, as a consequence, may impact the cell migratory ability.P = 0.485) (P = 0.335) (P = 0.792) (P < 0.05) (P < 0.001) (P < 0.001)  . We also= 0.335) , indicat= 0.792) . However < 0.05) , an even < 0.05) . Based o < 0.05) . We foun< 0.001)  and mean< 0.001)  of the Y< 0.001)  were higVideo 3NUP93 KD (right) MDA-MB-231. No clear differences in NPC density were observed. Download videoRepresentative movie showing 3D reconstructions of Nup153 stained nuclei in control (left) and NUP93 silencing can partially affect the assembly of FG Nups within the NPC, we did not observe mislocalization of nuclear proteins, including lamin A/C, which might suggest a defect in nucleocytoplasmic transport. To further strengthen this point, we considered the nuclear localization of FAK. If changes to the Nup93 sub-complex  were responsible for an altered nucleocytoplasmic transport, we would expect a consistent nuclear accumulation of FAK upon depletion of each specific nucleoporin. Conversely, whereas NUP93 KD induced FAK nuclear translocation, the level of nuclear FAK detected in NUP205 KD cells was comparable with control cells , response to wounding  and negative regulation of cell proliferation  , positive regulation of cell motility , mesenchyme development, neural crest development/differentiation , and EMT  . This dataset identified 158 DE genes comparing human triple-negative breast cancer and normal breast tissue. We found that nine genes were commonly down-regulated in Nup93-depleted BCCs and human normal tissue. Gene ontology analysis classified some of these genes under cell cycle  and wound healing . Importantly, STMN1 is inversely correlated to stress fiber formation, whereas PKM2 is a key glycolytic enzyme which can impact cancer cell migration, matrix metalloproteinase expression, and FAK/integrin activation  . In addid WWTR1) . The samilencing . These fer cells , which stivation .Taken together, the data show that Nup93 either directly or indirectly contributes to the expression of key genes involved in ECM remodeling, cell invasion, and proliferation.Our group has recently discovered that Nup93 and Nup153 can influence the cell gene expression by binding genome regulatory structures called SE . ProfiliNUP93 KD in MDA-MB-231 BCCs.Motif enrichment analysis considering all the identified peaks (Cut&Run) upon Table S1 NUP93 KD. This overlap was significant using random genomic regions as background (bootstrap P < 0.001).Previous studies demonstrated that Nup93 can interact with SE regions, which are characterized by high density of H3K27ac marks . This inNUP93 KD. Enrichment analysis showed that up-regulated genes were associated with biological processes including actomyosin structure organization and non-integrin membrane–ECM interactions . Two genes stood out because of their known role in AC organization, LIMCH1 and EDN1   . We alsoer peaks .Overall, our results show that Nup93 directly regulates the expression of a set of genes affecting AC remodeling and cell migration in triple-negative, claudin-low BCCs.NUP93 silencing. In particular, RNAseq data suggested that cells in which NUP93 was silenced might be undergoing a mesenchymal to epithelial transition while also experiencing a reduction in cell proliferation. The reduced proliferation was also confirmed in microfluidic invasion assay where NUP93 silenced BCCs not only migrated significantly less than control cells but also formed smaller micrometastases   (P < 0.001)  . After 4 wk, animals were euthanized and primary tumors analyzed. Cells lacking Nup93 were significantly underrepresented in the tumor compared with control cells   . To testE system . One speE system  was sele< 0.001)  and decr< 0.001) . In addinalysis) . Overall< 0.001) . The samNUP93 silenced cells did not significantly contribute to breast tumor formation and propagation in vivo, we then hypothesized that the level of Nup93 and other proteins directly regulated by Nup93  could be correlated with the progression of the disease in patients. We then collected two samples of stage I and two samples of stage III–IV human triple-negative breast cancer from independent donors. Immunofluorescence analyses revealed that the level of Nup93  was higher in stage III-IV than in stage I samples   . These q samples .Together, these results point toward a potential role of the NPC during tumor propagation and disease progression beyond its function of regulator of nucleocytoplasmic transport. It is important to highlight that Nup93 might not be the only nucleoporin involved in breast tumor propagation because preliminary proteomics data showed higher expression of this and other nucleoporins  in MDA-MB-231 than in the non-tumorigenic breast epithelial cell line MCF10A .Supplemental Data 1.Raw data of the Tandem Mass Tag experiment reported in NUP93 silencing resulted in the formation of a dense network of stress fibers without any indication of NE damage. Most intriguingly, RNAseq suggested that cells in which NUP93 was silenced acquired both a less proliferative and less invasive behavior, possibly linked to the onset of a mesenchymal to epithelial program. Correlation of RNAseq and Cut&Run data highlighted that Nup93 was directly involved in the transcriptional regulation of a subset of these genes. For instance, Nup93 directly regulated the expression of genes associated with AC remodeling and ECM synthesis . As a proof of principle, here we focused on LIMCH1 as a key regulator of stress fiber formation and AC remodeling. Whereas previous studies already demonstrated the active role of this and other genes in stress fiber formation . MCF-10A were cultured in Mammary Epithelial Cell Growth Medium BulletKit (Lonza).NUP93 siRNA (used for NUP93 KD) and luciferase siRNA (used for control cells) (NUP93) 5′-GCGCUAAUUUACUACUGCA-3′((Luciferase) 5′-UAUGCAGUUGCUCUCCAGC-3′Silencing was performed using siLentFect lipid reagent (Bio-Rad) and validated siRNAs. More in detail, the following sequences were used to design l cells) :(NUP93) NUP107, NUP133, NUP155, TPR, and NUP205 were commercially obtained from Thermo Fisher Scientific. Oligos silencing LIMCH1 and EDN1 (SMART Pool containing four different oligos per target) were commercially obtained from Dharmacon.Oligos silencing Before silencing, the cells were washed with PBS and incubated with Opti-MEM (Thermo Fisher Scientific). Cells were used for any further characterization/assay only after 72 h silencing.NUP93 was generated. The NUP93 sequence recognized by the siRNA was modified by introducing the following mutations: GCCCTTATATAGTAGTGGA.For Nup93 rescue, an MDA-MB-231 cell line overexpressing an RNAi-resistant version of hNUP93 (The shERWOOD-predicted RNAi sensor for hNUP93  was insehNUP93 .NUP93 miR-E sequence:TGCTGTTGACAGTGAGCGCCAGGAAAGTGTGGAAGAGAGATAGTGAAGCCACAGATGTATCTCTCTTCCACACTTTCCTGATGCCTACTGCCTCGGATGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTATAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGALuciferase miR-E sequence:2) for 24 h before starting live imaging on a laser scanning confocal microscope  equipped with environmental chamber. The cells were live tracked for 24 h.3D models were developed based on our previous studies . Briefly2) for 2 h and then washed twice with PBS. Next, the cells were fixed and imaged with a laser scanning confocal microscope .Cells were suspended in cancer medium and seeded in eight-well ibidi slides at a density of 3,000 cells/well. The cells were kept in humidified incubators  by vortexing. Next, 1.5 μl 10% NP-40 was added to the resuspension and incubated for 5 min on ice. The suspension was then centrifuged at 4,000g at 4°C for 5 min. The resulting supernatant represented the cytoplasmic fraction while nuclei were in the pellet. The pellet was resuspended in 100 μl buffer  by tapping the tube. Nuclei were then centrifuged at 4,000g at 4°C for 5 min. Finally, the pellet was dissolved in lysis buffer for Western blot  and mass spectrometry. Regarding mass spectrometry, the samples were labeled with Tandem Mass Tag reagents (Thermo Fisher Scientific) and analyzed with a Fusion Orbitrap Tribrid Mass Spectrometer (Thermo Fisher Scientific). Protein and peptide identification were performed with Integrated Proteomics Pipeline–IP2 (Integrated Proteomics Applications).Cells were washed with cold PBS on ice, scraped off from six-well plates and centrifuged at 450RNA was extracted using TRIzol and chloroform following standard protocols. The RNeasy Mini Kit was used to purify RNA (QIAGEN). The samples were incubated with DNAse I to degrade genomic DNA (30 min). RNA was then resuspended in RNAse-free water and its concentration quantified with NanoDrop. The Illumina TruSeq kit was used to prepare cDNA libraries. Quality check was performed using Agilent Tape Station and the amount of cDNA was quantified through Qubit fluorometer. The HiSeq2500 was used to sequence the samples (single-end reading).http://homer.ucsd.edu/homer/). Differential expression analysis was performed using R DESEq2 package . Batch effect was added to the design matrix to account for differences introduced by expressing luciferase vectors in cell lines. Significantly DE genes were identified at cutoff of adjusted P-value < 0.05 and absolute log2 fold-change > 1. Heat map was generated using R package gplots (https://cran.r-project.org/web/packages/gplots/index.html). Gene ontology figures were generated with Metascape software (http://metascape.org).Reads were mapped by STAR  to the hg19 reference genome using default parameters. Reads uniquely aligned to exons of RefSeq genes were then quantified by Homer  to identify peaks specific to each condition , which were then merged to get a full list of peaks found in both conditions. Finally, the number of reads in a given peak was quantified for each individual sample. R package DESeq2  was used to find peaks using an experimental design that was similar to RNA-Seq analysis . DESeq2 normalization size factors were estimated as relative ratios of spike-in normalization factors multiplying relative ratios of total mapped reads. The rationale is to account for differences caused by global shift of peak density between samples (estimated by spike-in ratios) plus sample-specific immunoprecipitation efficiency . Peaks were further annotated to its closest associated promoters.Similar to RNA-Seq analysis, reads were mapped by STAR  to hg19 genome but with splicing mode turned off using parameters: “--alignIntronMax 1 --alignEndsType EndToEnd.” Reads were also mapped to yeast sacCer3 genome to quantify the number of spike-in reads. Normalization factors were calculated as the ratio of spike-in reads over human reads. To maximize peak signals, reads from biological replicates were first pooled together by Homer , PHDH1, Cytoskeleton; 1:100 anti-human Paxillin, mouse monoclonal, AHO-0492; Thermo Fisher Scientific; anti-human Nup153, SA1 mouse ascites from Dr B Burke; 1:200 anti-human FAK, mouse monoclonal, AHO-1272; Thermo Fisher Scientific; 1:100 anti-human lamin A, goat polyclonal, sc-6214; Santa Cruz; 1:200 anti-human Nesprin-1, mouse monoclonal, MA5-18077; Thermo Fisher Scientific; 1:100 anti-human YAP, mouse monoclonal, sc-101199; Santa Cruz; and 1:200 anti-human LIMCH1, rabbit polyclonal, PA5-64136; Thermo Fisher Scientific. 1:200 Alexa Fluor secondary antibodies were used. Nuclei were counterstained with 1:500 Hoechst.NUP93 silenced cells).Samples were imaged with Airy Scan 880 Zeiss microscope setting the same configuration  for each condition  considering z-projected stacks of Nup153-stained samples. Regarding cortical actin, Fiji software was used to manually measure the width of the actin layer at the cell periphery after AC staining. For actin stress fiber quantification, Fiji software was used to draw 20-μm long lines within z-projected stacks of AC-stained samples. Representative images were collected from N ≥ 3 independent measurements for each condition. For focal adhesion quantification, images of paxillin-stained samples were thresholded  and the number of paxillin foci identified through the “particle analysis” command using Fiji software. Regarding quantification of the area covered by FAK, lamin A, Nesprin-1, and YAP, images were processed using Fiji software following established protocols  (region g at 4°C for 5 min and the supernatant stored at −80°C until assayed. The protein content was quantified using BCA protein assay (Thermo Fisher Scientific), loaded on 10% SDS gels (75 μg protein per lane), and electroblotted onto nitrocellulose membranes. 12% SDS gels were used for endothelin 1 detection (EDN1). Membranes were blocked with 5% BSA in TBST at RT for 1 h under stirring. Then, they were incubated with primary antibody diluted in 5% BSA in TBST at 4°C overnight under stirring. Membranes were washed in TBST for three times, 5 min each under stirring. Finally, the membranes were incubated with secondary antibody compatible with Odyssey Imaging System at RT for 1 h under stirring. After washing three times with TBST, 5 min each under stirring membranes were imaged using Odyssey Imaging System. The following primary antibodies were used: 1:1,000 antihuman EDN1, rabbit polyclonal, PA3-067; Thermo Fisher Scientific; 1:1,000 antihuman LIMCH1, rabbit polyclonal, PA5-64136; Thermo Fisher Scientific; 1:5,000 antihuman alpha tubulin, mouse monoclonal, T5168; Sigma-Aldrich; and 1:500 antihuman Nup93, rabbit, lab developed.Cells were lysed in 1X lysis buffer  for 10 min on ice, and then centrifuged at 10,000https://www.indivumed.com/ethical-principles).Human triple-negative breast cancer samples were commercially obtained from Indivumed. All samples were collected by Indivumed under informed consent . The samples were deparaffinized followed by antigen retrieval using standard protocols. Briefly, the samples were incubated in sodium citrate buffer, pH 8.5, at 80°C for 30 min, cooled down, and then washed twice with PBS. The samples were incubated with blocking solution  at RT for 1 h. Then, the samples were incubated with primary antibodies at RT overnight, washed three times in PBS for 5 min each, incubated with secondary antibodies at RT for 1 h, and finally washed three times in PBS for 5 min each. The samples were then mounted with Vectashield (Vector Laboratories) and imaged with a Leica SP8 laser scanning confocal microscope. The following primary antibodies diluted in PBS were used: 1:400 antihuman LIMCH1, rabbit polyclonal, PA5-64136; Thermo Fisher Scientific; 1:100 antihuman Nup93, rabbit, laboratory developed; and 1:100 antihuman mAb414, mouse, laboratory developed. 1:200 Alexa Fluor secondary antibodies were used. Nuclei were counterstained with 1:500 Hoechst.6 MDA-MB-231 cells were orthotopically transplanted into the right fourth mammary gland of 8-wk-old Fox Chase CB17-SCID female mice . The cells were prepared as a mixture of 1 × 106 dox-inducible dsRed control cells and 1 × 106 dox-inducible eGFP NUP93 KD cells in 25 μl of DMEM containing 10% FBS, then mixed 1:1 with GFR Matrigel . Mice were continuously treated with doxycycline  to induce expression of siRNA starting at the time of transplantation (“D0 dox”) or starting when tumors were palpable . 4 wk after dox induction, the tumors were harvested and dissociated as previously described 2 × 10NUP93 KD cells were mixed at 1:1 ratio, and then plated in six-well plates at a density of 40,000 cells per well. The cells were treated with 1.5 μg/ml doxycycline either 6 or 24 h after seeding . For proliferation, the cells were treated with 10 μM Edu for 5 h, collected, then fixed/permeabilized, stained, and analyzed according to the Click-iT Plus Edu Assay . DAPI  was used to assess DNA content. For apoptosis, the cells were collected, washed with cold PBS, resuspended in Annexin V Binding Buffer , and stained with Alexa Fluor 647 Annexin V . DAPI  was used to mark dead cells. Each assay was performed in triplicate for each time point. Analysis was carried out on an LSRII (Becton-Dickinson), and data were analyzed with FlowJo software.Dox-inducible dsRed control cells and dox-inducible eGFP t test or one-way ANOVA) were performed with Prism (GraphPad). Details on statistical tests, exact number of independent measurements, and data normalization are reported in each figure caption. Error bars represent SEM. Details on the statistics applied for RNAseq and Cut&Run data analysis are reported in the “RNAseq: library preparation and analysis” and “Cut&Run: library preparation and analysis”’ sections, respectively.Statistical tests (nonpaired GSE137691. The release of the data was approved by the Salk Institute Institutional Review Board.Data discussed in this publication were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number All the raw data contained in this article are available upon reasonable request.


Despite burgeoning development of nanoplatform made in the past few years, it remains a challenge to produce drug nanocarrier that enables requested on/off drug release. Thus, this study aimed to develop an ideal near-infrared light-triggered smart nanocarrier for targeted imaging-guided treatment of cancer that tactfully integrated photothermal therapy with chemotherapy to accurately control drug release time and dosage.Methods: This delivery system was composed of Ag2S QD coating with dendritic mesoporous silica (DMSN), which acted as nanocarrier of doxorubicin localized inside pores. To provide the nanocarrier with controlled release capability, a polypeptide-engineered that structure was reversible to photothermal effect of Ag2S QD, was covalently grafted to the external surface of drug-loaded DMSN.Results: This nanocarrier with the size of 40~60 nm had satisfactory biocompatibility and photothermal conversion efficiency up to 28.35%. Due to acidity-triggered charge reversal of polypeptide, which significantly extended circulation time and improved targeting ability, fluorescence and photoacoustic signals were still obvious at tumor site post-24 h by tail vein injection and chemo-photothermal synergistic therapy obviously enhanced antitumor efficacy. Mild PTT with multiple short-term exposures not only reduced the side effect of overdose drug but also avoided skin damage caused by long-term irradiation.Conclusion: By adjusting irradiation time and on/off cycle, multiple small amount local drug release reduced the side effect of overdose drug and skin damage. This novel approach provided an ideal near-infrared light-triggered nanocarrier with accurate control of area, time, and especially dosage. Many materials act as efficient photothermal converter, such as metal nanomaterials , modifiable group , or temperature-sensitive reversible dynamic self-assembly structure in vivo. Therefore, it is a better choice to use DNA coding technology to prepare thermosensitive genetically engineered polypeptide for NIR light triggered on-demand drug delivery system.Dendritic mesoporous silica (DMSN) with unique central-radial structure is emerging as a promising nanocarrier for biomedical application owning to large pore size and highly surface area compared with conventional mesoporous silica nanoparticle 2S QD delivery system prepared by one-pot method. Followed, genetically engineering polypeptide cys-P-RGD containing cysteine and RGD sequence was covalently grafted to the external surface of DMSN by crosslinking agent to block drug premature leakage and generated Ag2S@M/D-P-RGD. The distribution of Ag2S@M/D-P-RGD was monitored by fluorescence imaging (FLI) and photoacoustic imaging (PAI) in vivo, and after the targeting probe was preferentially taken up by tumor cell through receptor-mediated endocytosis, NIR irradiation not only caused PTT but also triggered polypeptide unfolding to release drug. Without NIR irradiation, the polypeptide recovered to pentamer to block the pore and stopped drug release. The release amount of drug could be on-demand controlled by adjusting the irradiation time and laser on/off cycle. More importantly, this multifunctional nanocarrier had excellent biocompatibility and tumor targeting ability, and mild PTT with short-term and multiple cycle laser irradiation could significantly improve the efficacy while reducing side effect and skin damage. This robust nanoplatform for tumor targeted imaging and on-command controlled combinational therapy might provide an insight for cancer theranostics.Hence, we designed a novel NIR light-triggered multifunctional nanocarrier with tumor targeted fluorescence-photoacoustic bimodal imaging and chemo-photothermal combined therapy Figure . Firstly2S QD with good crystal structure and prominent lattice  of NaSal/CTAB on formation of dendritic structure of Ag2S@M, systematic studies had been conducted  by graphite furnace atomic absorption method. N2 adsorption/desorption isotherm curve  of Ag2S@M was calculated to be 28.35 %  and thoroughly cleaned to determine fluorescence  and characteristic absorption peak of DOX appeared at 490 nm  at pH 5.9 for 2 h, zeta potential transformed to +0.89±0.40 mV compared with -9.85±1.60 mV under neutral condition  effect Various acidic and basic functional groups on amino acid side chain and termini of protein and polypeptide may be charged, making them biological amphoteric ionization property. Amino acids that make up protein may be positive, negative, neutral, or polar in nature, and together give a protein its overall charge. The isoelectric point (pI) of a peptide is defined as the pH at which the peptide has a net charge of zero. At pH below the pI, peptide presents a net positive charge; above the pI, it presents a net negative charge. The isoelectric point (pI) of polypeptide cys-P-RGD designed by us was calculated to be 6.26, which theoretically would be negatively and positively charged in neutral blood and acidic tumor tissue microenvironment, respectively. Since the pI of BSA is 4.7 n Figure A. In a m2S@M-P-RGD with the abundant plasma proteins in blood, which was one major serum protein often found in the protein corona of nanoparticle. After incubation for 12 and 24 h under neutral condition (pH 7.4), significantly less BSA was found on Ag2S@M-P-RGD compared with Ag2S@M, indicating that negatively charged polypeptide on surface prevented proteins from adsorbing on the surface, whereas this prevention was not happened on Ag2S@M. However, the adsorption of BSA by Ag2S@M-P-RGD increased observably under acidic condition (pH 5.4), which proved that nanoprobe reversed to positive charge were beneficial to anchor negatively charged cell membrane proteins . In addition, the hemolysis of nanoparticle would severely limit its delivery in vivo, then such experiment was also carried out for nanoprobe before and after polypeptide modification, the results showed that Ag2S@M-P-RGD did not trigger hemolysis at concentration up to 1.6 mg/mL . And the high temperature induced by NIR naturally increased the diffusion rate of encapsulated DOX, resulting in high uncontrolled fast release (77.87±0.97 % after 60 h at pH 7.4). However, for Ag2S@M/D-P-RGD , and further improved the therapeutic effect P domain was the coiled-coil region of cartilage oligomeric matrix protein, which could self-assemble into pentamer at room temperature and was sensitive to temperature change e Figure A, indicae Figure B, indica4 Figure . The resD Figure B. The revβ3 were used as positive and negative cells, respectively. After the probe that with or without RGD modification was incubated with both cells for 4 h, NIR fluorescence and photoacoustic images revealed that HeLa cells incubated with Ag2S@M-P-RGD exhibited the strongest signal  were investigated for normal cell (3T3 cells) and cancer cells (HeLa and MCF-7 cells) by a standard assay. After incubation of 3T3 cells with 200 μg/mL of Ag2S@M-P-RGD and Ag2S@M-P, the cells of the two groups still maintained high survival rate  was higher than that of probe with RGD-modification, but there was no difference in cell survival rate for MCF-7 cells incubation with or without the targeting ligand of probe.Biocompatibility of probe should be the first consideration for following bio-application. The toxicities of probes without DOX , illustrating that cys-P-RGD could effectively block DOX in the pore and prevent its leakage. When HeLa cells were treated by Ag2S@M-P-RGD (5 μg/mL), applying 10 min NIR irradiation (2.0 W/cm-2) could cause 66 % cell death, indicating that our probe killed most tumor cells through excellent PTT. To further study the photothermal effect of probe, cells were incubated with drug-free probe and treated with different laser intensities and time , 10 min irradiation would cause about 50 % cell death. With the concentration reached 5 μg/mL, the cell survival rate was only 13.07±0.65 %, therapeutic effect was significantly higher than that of free DOX and PTT alone (p<0.01). As polypeptide could be used as blocking agent to realize on-demand controlled release of drug through thermosensitive characteristic, confocal images showed that DOX was mainly dispersed in cytoplasm and nucleus after drug-loaded probe was incubated with HeLa cells and exposed to laser . Similar to the above results, the effects of two chemo-photothermal synergistic therapy groups were significantly better than those of other groups (p<0.05 or p<0.01) after mice were sacrificed at 24 d and measured tumor masses  with tumor targeted fluorescence-photoacoustic bimodal imaging and chemo-photothermal combined therapy was successfully designed and constructed. Based on the cys-P-RGD thermosensitive valve, DOX was blocked in dendritic mesoporous silica embedded Ag2S QD. The reversible polypeptide was manipulated by switching the laser on/off state by virtue of photothermal characteristic of Ag2S QD, which was capable of controlling the release amount of cargo through polypeptide structure change. Both in vitro and in vivo results demonstrated that Ag2S@M/D-P-RGD had satisfactory biocompatibility and targeting dual mode imaging ability, and mild photothermal therapy with multiple short-term laser irradiation could effectively inhibit tumor growth while reducing side effect of excessive drug release and skin damage. This novel theranostic nanocarrier integrated controllable chemo-photothermal synergistic therapy and real-time imaging might hold promise for efficient tumor therapy.In summary, a novel NIR laser-triggered multifunctional probe  were dissolved in 5 mL water and preheated at 80 ºC for 30 min before adding to the above system. After the mixed system was equilibrated for 1 h, 1.5 mL TEOS was added dropwise and gently stirred for 2 h. The resulting Ag2S@M was purified by repeated centrifugation  to remove the unreacted reagent by water and ethanol, then re-dispersed in 50 mL ethanol containing ammonium nitrate (10 mg/mL) and heated to 75 ºC for 6 h to remove CTAB template. After the same purification methods mentioned above, it was resuspended in 30 mL ethanol and reacted with 150 µL MPTES at 60 ºC for 20 h to modify sulfydryl, then washed as above. Ag+ concentration was measured by graphite furnace atomic absorption method.280 mg CTAB and 6 mg Ag2S@M and 2 mg DOX were dispersed into 5 mL water. After stirring for 24 h, 2 mL cys-P-RGD (3 mg/mL) was added and reacted with BMOE coupling agent for 2 h to cap the pore. The mixture was repeated centrifugation  with PBS  to clean free DOX to obtain DOX-loaded nanoparticle (Ag2S@M/D-P-RGD); cys-P was also used to prepare control probe without targeting RGD (Ag2S@M/D-P). BSA had a free cysteine that could be bonded to mercapto-modified Ag2S@M surface via BMOE and was used as control to an analyze the active role of cys-P-RGD. 10 mg Ag2S@M/D-P-RGD was added in 50 µL hydrofluoric acid to dissolve silica, and pH was adjusted to neutrality to determine DOX amount by analyzing fluorescence intensity at 590 nm, loading content (%) = (DOX weight in probe)/(probe weight)×100 % and encapsulation efficiency (%) = (DOX weight in probe)/×100 %.20 mg Agin vitro, HeLa cells were seeded in glass-bottom Petri dishes and Ag2S@M-P-RGD (100 µg/mL) was added after overnight cultured. After further culture for 4 h, PBS was used to remove the unbound probe. Then each well was added 200 µL PBS and irradiated at different laser intensities (0.5~2.5 W/cm2) and different treatment time. The cells were stained with calcein and propidine iodide (PI) to observe the effect of photothermal therapy.To investigate the photothermal property of the probe 2) for 10 min or without any treatment. Then cells were grown for another 24 h and measured cell viability.The combined antitumor therapy was studied based on MTT assay. After the HeLa cells were seeded in the 96-well plate for overnight culture, various probes with different concentrations  were added for incubation for 4 h. After washing with PBS to remove unbound probe, cells were irradiated with 808 nm laser (2.0 W/cmin vitro was determined by Annexin V-FITC/propidium iodide (PI) double staining apoptosis assay. HeLa cells were seeded in 6-well plate and cultured overnight. The cells were treated with free DOX, Ag2S@M-P-RGD and Ag2S@M/D-P-RGD at the same concentration of DOX (5 µg/mL) for 4 h. After cleaning, they were exposed to laser (2.0 W/cm2) for 10 min and continuously cultured for 24 h. Subsequently, cells were stained with Annexin V-FITC and PI according to the protocol and analyzed by FC500 Flow cytometer .The extent of apoptosis 3 were randomly divided into eight groups (n=5) and intravenously injected with 100 mg/kg of different probes: (I) PBS, (II) PBS + NIR×1,(III) free DOX, (IV) Ag2S@M/D-P-RGD, (V) Ag2S@M-P-RGD + NIR×1, (VI) Ag2S@M/D-P-RGD + NIR×1, (VII) Ag2S@M-P-RGD + NIR×3, (VIII) Ag2S@M/D-P-RGD + NIR×3. NIR×1 referred to irradiation of tumor site with 808 nm laser (2.0 W/cm2) for 6 min at 48-53 ºC after injection of 12 h; NIR×3 meant that the tumor was heated up to 48-53 ºC for 2 min three times by the same laser irradiation after 12 h, 15 h and 18 h after injection. The treated HeLa tumor-bearing nude mouse of each group was sacrificed after laser irradiation and tumors were fixed with 4 % paraformaldehyde, dehydrated, embedded and sectioned for H&E and TUNEL staining. The tumor volume and body weight of mice were measured for 24 d. When the experiments were finished, mice were sacrificed and major organs  were collected and weighed. The contents of ALT, aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) in blood were analyzed, and organs were stained with hematoxylin/eosin (H&E). Visceral index was calculated as follows, organ mass/mice mass×100 %. All animal experiments were approved by Animal Experimental Ethics Committee of Huazhong University of Science and Technology.HeLa tumor-bearing nude mice with an average volume of ~200 mm