Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.Bacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.Herein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae.Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within The online version of this article (10.1186/s12864-019-5439-1) contains supplementary material, which is available to authorized users. The CRISPR array features direct repeats (DRs), which vary in size from 21 to 37-bp and can occur in 1 to over 100 times depending on the species and the strain. These repeats are separated by non-repetitive DNA of similar size called spacers [. Cas proteins are encoded by the cas gene cluster in an operon, which is usually located upstream from a CRISPR array. These Cas proteins are required for expression of cas genes, new spacer acquisition and target recognition and degradation [Bacteriophages (phages) are viruses that infect bacteria, by injecting their viral DNA or RNA into bacterial host cells. This foreig'n DNA can then circularize and replicate or integrate into the bacterial host chromosome to form a prophage by site specific recombination mediated by an integrase. Phages are abundant in many ecosystems and are estimated to outnumber bacteria by ten-fold . Some ph) system –13. This) system . CRISPR-) system . A CRISP spacers . The spa spacers , 19. Casradation , 20–24.CRISPR-Cas systems are classified based on Cas protein content and arrangements in CRISPR-Cas loci with two main classes (1 and 2) and at least six types  that have been defined and updated , 25, 26.cas genes, gene arrangement and direct repeats. Signature Cas proteins are the effectors that cleave target DNA/RNA and are used to differentiate between these types. These hallmark proteins are Cas3 for type I, Cas9 for type II, Cas10 for type III, Csf1 for type IV, Cas12 (Cpf1) for type V, and Cas13 for type VI [, in which case the system is dependent on adaptation modules from other systems [There are currently six types of CRISPR systems characterized, which are differentiated based on their signature  type VI , 25, 26. type VI , 26. The type VI , 26. Com type VI . However systems , 16, 23. systems .Spacers are sequences derived from protospacers from foreign invading mobile genetic elements (MGEs) that are incorporated into a CRISPR array between two repeat sequences generally adjacent to the CRISPR leader sequence. The protospacer adjacent motif (PAM) is a sequence consisting of two or three nucleotides immediately before the protospacer sequence and it is necessary to distinguish CRISPR targeted protospacer sequences (non-self) from the system’s own genome (self) .Vibrionaceae have 8 genera that have whole genome sequences available, Aliivibrio, Enterovibrio, Grimontia, Listonella, Vibrio, Photobacterium, Photococcus, Salinivibrio) that are ubiquitous in the marine environment where phages are abundant. There have been limited studies describing CRISPR-Cas systems in this bacterial family and most studies were confined to V. cholerae, a significant pathogen of humans that causes cholera [V. cholerae biotype classical strains that caused the earlier pandemics of cholera [V. cholerae are all present within MGEs.The family  cholera , 32–37.  cholera . A CRISP cholera . The typ cholera , 39. GI- cholera . Recentl cholera . Thus toVibrionaceae through comparative genomics, bioinformatics, and phylogenetic analyses of available genome sequences in the NCBI database. In addition, we examined the genomic context of each system to determine whether it was acquired as a single module or within a MGE. Several variant type I-F CRISPR-Cas systems were identified in V. cholerae and in 41 additional species. The canonical type I-F system and a variant type I-Fv in V. cholerae were present within the genomic island VPI-6. A mini type I-F system (tniQcas5cas7cas6f) was within Tn7-like transposons in V. cholerae, V. parahaemolyticus and over 40 additional species. In V. parahaemolyticus, within the Tn7-associated CRISPR-Cas system was the pathogenicity island containing the type three secretion system 2 (T3SS-2). A putative hybrid type III-B/I-F system, which contained a type III-B cas gene cluster, a cas6f gene and a type I-F CRISPR array was identified in several V. cholerae and V. metoecus strains. The hybrid system was present within a prophage at the same genome location in both species. Multiple CRISPR-Cas system types including type I-C, I-E, I-F, II-B, III-A, III-B, III-D and the rare type IV system were uncovered. Interestingly, the majority of these CRISPR-Cas systems were identified within MGEs that included genomic islands, plasmids and transposon-like elements. A number of novel cas gene arrangements and cas gene contents were found among the systems identified. The data suggest that many variations of Cas protein content exist within the different types, and the acquisition of CRISPR-Cas systems on MGEs is a common feature in this group. Phylogenetic analysis of Cas proteins and their sporadic occurrence within a species also suggested that the CRISPR-Cas systems were acquired by horizontal gene transfer. Overall these data show that CRISPR-Cas systems are phylogenetically widespread but not the predominant defense mechanism of this group.Here, we determined the prevalence, diversity and phylogenetic distribution of CRISPR-Cas systems present within Vibrionaceae available in the NCBI genome database for the presence of CRISPR-Cas systems. We identified eight different system types: type I-C, I-E, I-F, II-B, III-A, III-B, III-D, and IV as well as variants of these types and hybrid systems among 70 species  followed by a type I-F CRISPR array  was present in 13 strains, which encodes variant Cas7fv and Cas5fv proteins  and left end (L) att sites [V. cholerae strains, the Tn7-like region consists of four genes, tnsABC homologs in an operon, and tniQ in an operon with cas5cas7cas6f genes. TniQ shows homology to TnsD, which targets a sequence specific site, attTn7, for insertion. No tnsE homolog was identified, which is usually responsible for directing the element into other MGEs [Our analysis identified five  species . The cantt sites , 46. In her MGEs , 48. TheV. cholerae strains identified the presence of three divergent mini type I-F systems. These mini type I-F systems were associated with at least three divergent Tn7-like transposons , a novel Tn7 transposon attachment sites that was described previously by Peters and colleagues [tnsA is called the right (R) end and the distal end is named the left (L) end  also annotated as guanosine 5′-monophosphate oxidoreductase (guaC)  that contains this metabolic cluster. Overall it appears that these Tn7-like elements have captured not only CRISPR-Cas defense systems but also restriction modification systems.In aC) Fig. b (Table aC) Fig. . The Tn7aC) Fig. b. This iVibrionaceae that contained a copy of this protein and an associated Cas6f. Interestingly, a large number of V. parahaemolyticus strains (> 800 sequenced genomes) in the NCBI genome database contained a Tn7-like associated mini type I-F system. In this species, four divergent mini type I-F systems were identified that were carried within five different Tn7-like transposons, which had three different attachment sites; SRP-RNA on chromosome 1, and IMPDH and YciA (acyl-CoA thioester hydrolase) on chromosome 2  on chromosome 2, the Tn7-like CRISPR-Cas region was directly upstream of the T3SS-2α gene cluster at the yciA R end attachment site , Cas6, and TnsA were co-located on the same contig Fig. . For thed E Fig. . These dies Fig. . For exaV. cholerae strains that contained a putative type III-B/I-F hybrid system that consisted of type III-B operon cas7cas10cas5cas7cmr5cas7 followed by a resolvase and cas6f genes  were aligned using ClustalW and a phylogenetic tree was constructed by the neighbor-joining method [V. cholerae strains and 1 V. parillis (formerly V. cholerae) strain, recovered between 1978 and 2009 from five continents and was associated with the variant type I-F (type I-FV2) systems containing five cas genes. Clade II contained Cas6f from 10 strains with the type III-B/I-F hybrid system, which were isolated mainly in the USA and Haiti with single strains from both the Ukraine and the UK. Clade III contained six strains, containing the canonical type I-F system from three genera, Aliivibrio, Photobacterium and Vibrio. Clade IV contained 17 V. cholerae strains that were recovered between 1981 and 2012 from four continents and was associated with the canonical type I-F system. Within this clade and closely related to V. cholerae were Cas6f from V. metoecus, V. fluvialis, V. navarrensis and five strains of Salinivibrio.  within which were highly variant Cas6f proteins, with some species present on multiple distantly related branches indicating in some species the system was acquired multiple times from diverse sources  , however no protospacer matches were identified. We identified a canonical type I-E system in V. harveyi ATCC 43516 with 37 spacers. In S. sharmensis DSM 18182, the type I-E systems had 79 spacers with protospacer targets in Salinivibrio phage SMHB1  of the excised GI-24 and an empty GI-24 chromosomal insertion site (attB) after excision  was present at the 5′ end of the island at a SRP-RNA insertion site as described in V. cholerae and V. parahaemolyticus. We identified attTn7 sites that encompassed a RM system, the mini type I-F system and the complete CRISPR-Cas type I-C system  domain in 14 of the 18 Cas1 proteins identified. In type III-A system of V. gazogenes CECT 5068 and DSM 212464, the Cas1 protein is fused with RT and Cas6 domains. In 11 strains with either a type III-A, III-B or III-D system, only RT and Cas1 domains are fused. In P. aphoticum JCM 19237, the RT encoding gene is adjacent to the cas1. These RT containing Cas1 proteins have been shown previously to be primarily found in proximity to type III systems, are not specific to any subtype, and function autonomously [In 18 of the strains with a type III system characterized in this study, nomously .V. gazogenes strains with a cas1 gene with cas6 and retron domains and are distantly related to cas1 genes from Photobacterium and Vibrio species. In clade II, the Cas1 from the four strains containing a type III-B cluster together from 4 V. gazogenes strains  such as phages and plasmids. Most of the studies of these systems have been conducted in examined , 66, 67.V. cholerae, canonical and variants of type I-F were identified within genomic islands and mini type I-F systems were present within Tn7-like transposons. The mini type I-F system was also identified in a large number of V. parahaemolyticus strains as well as many additional species. A previous study has suggested that the Tn7-like transposons have coopted the mini type I-F CRISPR-Cas systems to identify and target its insertion site [cas3) and spacer acquisition (cas1 and cas2). Peters and co-workers hypothesize that the CRISPR containing transposon function together to form a functional element that allows for target integration into MGE and/or movement within a genome [V. parahaemolyticus, a significant pathogen of humans, the main virulence mechanism, a T3SS-2 system, is present within a CRISPR-Cas-carrying Tn7-like element. T3SS-2 systems are large genomic regions of > 80-kb and are only present in human pathogenic strains and are absent from environmental isolates [V. parahaemolyticus are all part of CRISPR-carrying Tn7-like transposons and indicate a mechanism by which these virulence factors can be mobilized, which needs to be investigated further.In ion site . The Tn7a genome . In V. pisolates , 52, 53.cas6f followed by a type I-F array, was present in eight V. cholerae strains and two V. metoecus strains. The strains were isolated between 1974 and 2012 from three continents and each strain contained a unique array with between 12 and 50 spacers. Although further experimental evidence is required, the type III-B/I-F could be a true hybrid system. It has been proposed that extensive recombination events within the CRISPR-Cas region may lead to the formation of a hybrid system [A putative hybrid system consisting of type III-B genes and d system , 68. TheVibrionaceae, members of which are marine inhabitants that encounter huge amounts of viruses and thus would be good candidates for the presence of diverse CRISPR-Cas systems. In addition to type I-F systems, our analysis uncovered type I-E, I-C, III-A, III-B, III-D systems and system variants, as well as type II-B and type IV systems. Thus, there is a diversity of systems present among the different species, but their occurrence is infrequent suggesting that these systems are not a major contributor to bacterial fitness and survival. The multiple variations in the cas gene arrangements and content identified here suggests that gene rearrangements occur frequently and the systems are constantly in flux. In addition, our analysis demonstrates that the majority of systems identified in Vibrionaceae are associated with MGEs and that many of these elements carry additional cell defense systems mainly restriction modification systems and toxin-antitoxin systems. In these MGEs, CRISPR-Cas may be playing more of a role in protecting the MGE they are carried on rather than the strain they are present in, similar to the role of the type I-F system present in phages isolated from cholera stool samples [Vibrionaceae. In addition, the presence of these systems on MGEs carrying additional cargo genes such as virulence genes could suggest additional roles within the cell.This study is the first, large scale genome analysis to characterize the CRISPR-Cas systems in the family  samples , 37. PhyVibrionaceae, these systems are highly diverse with multiple sub-types and sporadic in distribution. The identification of protospacer targets suggests that these systems were active within this family. Importantly, the association of these systems with genomic islands, plasmids, phages, and transposons suggest a vector for their transfer amongst species. Furthermore, the mobility of these systems can lead to novel variant subtypes, which we have identified several examples of, including a type III-B/I-F hybrid system. This work opens the door for future studies in determining how CRISPR-Cas systems affect the survival of these bacteria and potentially novel functions of these systems.The CRISPR-Cas repertoire is an ever-expanding defense system in bacteria. In V. cholerae as well as signature Cas proteins identified in other species as seeds. A complete list of the bacterial strains within Vibrionaceae that contained a CRISPR-Cas system with a CRISPR array (repeat and spacer) and the accession number of the sequence contigs are shown in , transposases (tnp) and site specific attachment (att) sites. In addition the %GC content of each region was determined and compared to the overall %GC content of the chromosome. To identify regions of difference between strain with and without a CRISPR-Cas system, the sequence/contig with the CRISPR-Cas system was compared with related strains or species lacking the system, and figures were generated using Easyfig [The genomic region surrounding the CRISPR-Cas systems were analyzed for the presence of markers of MGEs such genomic islands, phages, and transposons markers included integrases  following the manufacturer’s instructions. In order to detect the excision, a nested two-stage PCR was performed to amplify the empty chromosomal site attB or the circular intermediate attP. The first round of PCR was performed in 20 μl reactions using 10 ng of isolated DNA as template using primer pairs: attB_R1L: agtttggtgcgggtatcaag and attB_R1R: gccactgcgtgactctgtta and for attP the following primers were used: attP_R1R: gctccctccttcaagtaccgctc and attP_R1L: gcgaaactgccaacgcacg. Following the first round of PCR, 1 μl of the reaction product was used as a template for a second round of PCR using attB primers_attBR2L aaagtgggcgagtagggtt and_attBR2R tctggacaccatcatgcaat and attP primers_attPR2R ccgatagcgacaatgacactgc and attPR2L gagacccttgcacccaatccatc. The PCR products were analyzed by gel electrophoresis on a 1% agarose gel stained with ethidium bromide. Experiments were performed with two biological replicates and three technical replicates.PCR assays to detect both the empty site attB following excision of GI-24 as well as the circular intermediate attP of excised GI-24 were performed as previously described . DNA was isolated from Additional file 1:Figure S1. Distribution of CRISPR-Cas system types identified among Vibrionaceae. (A) Percentage break down of all the systems identified by type. Different colors are used to indicate different CRISPR-Cas systems. (B) Percentage of systems identified that are associated with mobile genetic elements. (C) Diversity of each system identified within the Vibrionaceae. Figure S2. Phylogenetic analysis of cas1 and intV from V. cholerae strains containing type I-F system. The cas1 nucleotide sequences were examined for 21 type I-F systems characterized in V. cholerae. For the same 21 strains intV nucleotide sequences from VPI-6 were collected and analyzed. Aligned sequences were used to construct a neighbor-joining phylogenetic tree with bootstrap of 1000. Figure S3. Phylogenetic analysis of cmr1, cas6f and phage associated integrase. The nucleotide sequences for cmr1 and cas6f from V. cholerae and V. metoecus strains containing the putative type III-B/I-F system were examined to construct a phylogenetic tree. Similarly, the nucleotide sequences of the integrase from the prophage associated with the putative type III-B/I-F system were used to construct a phylogenetic tree. Aligned sequences were used to construct a neighbor-joining phylogenetic tree with bootstrap of 1000. Figure S4. Protospacer targets from V. cholerae I-F systems to Vibrio phages. Protospacer targets identified within the I-F arrays of Vibrio cholerae were mapped to the genomes of Vibrio phages X29/phi2, phage Kappa, phage fs2, fs-1, and KSF1-phi. Genomes were constructed using EasyFig. Figure S5. CRISPR-Cas type I-F systems in Vibrionaceae are within MGEs. Comparative analysis of the genomic regions containing type I-F systems demonstrated their presence on genomic islands. (A) The CRISPR-Cas island in V. metoecus YB5B04 is absent from V. metoecus strain OYP8G12. (B) The type I-F system present within an island in V. parahaemolyticus A4EZ703 between VPA0712 and VPA0713 relative to RIMD2210633 that lacks the island. (C) CRISPR-Cas island in V. vulnificus 93 U204 is inserted relative to VV1_0636 and a tRNA in CMCP6 that lacks the island. (D) Comparative analysis of the genomic regions containing type I-F systems demonstrated their presence on genomic islands. Type I-F systems were identified in an island region in four V. fluvialis strains, which was absent in strain S1110. The genome comparisons and gene diagrams were drawn using EasyFig and the grayscale bars represent nucleotide homology. The core genes are colored in black, transposases are colored in pink and integrases are red. Red brackets indicate the genomic location of CRISPR. Figure S6. Phylogenetic analysis of cas8e and int from V. cholerae strains containing type I-E system within GI-24. The cas8e and associated int nucleotide sequences from V. cholerae were collected and analyzed using MEGA. Aligned sequences were used to construct a neighbor-joining phylogenetic tree with bootstrap of 1000. Figure S7. Phylogenetic analysis of type I-E Cas8e from Vibrionaceae. The Cas8e protein sequences were examined for all type I-E systems characterized in this study. Aligned sequences were used to construct a neighbor-joining tree with bootstrap of 1000. Salinivibrio are indicated by green squares and Photobacterium by light blue triangles. Figure S8. Genomic loci analysis of type I-C systems in Vibrionaceae. The genomic region containing type I-C systems identified in Vibrionaceae were analyzed. Gray scale bars represent nucleotide homology. The core genes are colored in black, transposases are colored in pink, resolvase in yellow and integrases in red. Red brackets indicate the genomic location of CRISPR. Figure S9. Type III systems found within V. gazogenes. (A) A type III-B system found in strains ATCC 43941 and ATCC 43942. (B) A type III-B system in strains DSM 21264 and CECT 5068. (C) The type III-A system present in DSM 21264 and CECT 5068 present on chromosome II. Genome comparisons were constructed in Easyfig and gray scale bars represent nucleotide homology. The core genes are colored in black, transposases are colored in pink and integrases in red. Red brackets indicate the genomic location of CRISPR. Figure S10. Phylogenetic analysis of Cas1 domain and Cas10. (A) Cas1 domain sequences and (B) Cas10 sequences from strains containing a type III system and a Cas1 protein were aligned in MEGA7 with clustalW to construct a neighbor-joining phylogenetic tree with bootstrap of 1000. Figure S11. Phylogenetic analysis of Cas10 from the family Vibrionaceae. Cas10 sequences were collected from 31 strains belonging to various species from the family Vibrionaceae containing the type III system. ClustalW was used to align the collected sequences and a neighbor-joining tree was built with bootstrap of 1000 using MEGA7. The protein sequences separated into four distinct clusters. The Salinivibrio are indicated by green squares and the Photobacterium are indicated by light blue triangles. (PPTX 2880 kb)Additional file 2:Table S1. Type I-F systems in V. cholerae. Table S2. Type I-F systems in Vibrionaceae. Table S3. Type I-E systems in V. cholerae. Table S4. Type I-E systems in Vibrionaceae. Table S5. Type I-C systems in Vibrionaceae. Table S6. Type II-B systems in Vibrionaceae.Table S7. Type III CRISPR-Cas systems identified in Vibrionaceae. Table S8. CRISPR-Cas contig accession numbers for species in this study. (XLSX 55 kb)


O-acetyl-renieramycin T isolated from the blue sponge Xestospongia sp. mediated lung cancer cell death via the induction of p53-dependent apoptosis. Importantly, 5-O-acetyl-renieramycin T induced the death of CSCs as represented by the CSC markers CD44 and CD133, while the stem cell transcription factor Nanog was also found to be dramatically decreased in 5-O-acetyl-renieramycin T-treated cells. We also found that such a CSC suppression was due to the ability of the compound to deplete the protein kinase B (AKT) signal. Furthermore, 5-O-acetyl-renieramycin T was able to significantly sensitize cisplatin-mediated apoptosis in the lung cancer cells. Together, the present research findings indicate that this promising compound from the marine sponge is a potential candidate for anti-cancer approaches.Lung cancer is one of the most significant cancers as it accounts for almost 1 in 5 cancer deaths worldwide, with an increasing incident rate. Management of the cancer has been shown to frequently fail due to the ability of the cancer cells to resist therapy as well as metastasis. Recent evidence has suggested that the poor response to the current treatment drugs and the ability to undergo metastasis are driven by cancer stem cells (CSCs) within the tumor. The discovery of novel compounds able to suppress CSCs and sensitize the chemotherapeutic response could be beneficial to the improvement of clinical outcomes. Herein, we report for the first time that 5- Due to the enormously diversified environments found in the oceans, marine organisms are indeed founts of novel candidates for drug discovery . Approxiotherapy . A receng cancer . Accumulg cancer . It has g cancer .Xestospongia sp., which is a marine blue sponge found in the seas around Thailand and the Philippines [O-acetyl renieramycin T (O-acetyl RT) (®) [O-acetyl RT and other renieramycins, such as renieramycin M and renieramycin T, have a strong cytotoxicity, with inhibitory concentration (IC50) values in the range of nanomolar concentrations against many human cancer cell lines, including colon cancer HCT116 and DLD1 cells, breast cancer MDA-MB-435 cells, pancreatic cancer AsPC1 cells, lung carcinoma QC56 cells, and non-small-cell lung cancer cells [O-acetyl RT and its mechanism on human NSCLC has not yet been elucidated. Herein, we report that O-acetyl RT synthesized from renieramycin T induced apoptosis, suppressed CSCs, and sensitized NSCLC H292 cells to cisplatin. These findings might benefit the development and further investigation of this compound as a sensitizer of NSCLC to cisplatin.Renieramycins A−Y are a series of tetrahydroisoquinoline marine alkaloids isolated from lippines ,17,18,19lippines , which elippines . Among tlippines ,22. The etyl RT) , having  RT) (®) . It has er cells ,25. HoweO-acetyl RT on cisplatin-mediated cancer cell death was determined, we investigated the cytotoxic profile of O-acetyl RT compared with doxorubicin and cisplatin, the standard chemotherapeutic drugs for lung cancer therapy, using the MTT cell viability assay. Human lung cancer H292 cells were incubated with various concentrations (0–25 µM) of compounds for 24 h. The results showed that O-acetyl RT had a greater cytotoxic effect on H292 cells than that of doxorubicin and cisplatin  for 24 h and then quantified the apoptotic cells using the Hoechst 33342/propidium iodide (PI) double staining assay. We found that O-acetyl RT was able to induce apoptosis of H292 cells in a dose-dependent manner  . Moreover, necrosis cell death was not detected under all treatments. To confirm the apoptosis-inducing effect of O-acetyl RT, apoptotic cells were determined using the Annexin V/PI apoptosis assay. O-acetyl RT caused an increase in the number of early-apoptotic cells dependent on the treated concentrations . The key proteins controlling apoptosis were determined by Western blot analysis. As shown in O-acetyl RT significantly induced the activation of caspase-3 compared to the non-treated cells . In agreement with such results, the expression of the active form of caspase-9 was found to be significantly upregulated in H292 cells treated with O-acetyl RT at 1 and 5 µM .Having shown the potential of apoptosis induction, we then confirmed the apoptosis effect of F5,12 = 9.021, p = 0.0093) and p53 , and a decrease in the expression of BCL-2  in O-acetyl RT-treated H292 cells compared to non-treated cells.  and CD44 , respectively. In addition, this CSC-suppressing activity of the compound was supported by the depletion of CD133-positive (CD133+) cells  in the O-acetyl RT-treated cells, as determined by flow cytometry  in H292 cells  of O-acetyl RT for 24 h, followed by treatment with 50 µM cisplatin for 24 h, and cell viability was measured using MTT assay. O-acetyl RT . The cell viability of H292 cells treated with cisplatin was reduced by 56%, whereas a combination of 0.01 or 0.05 µM O-acetyl RT with cisplatin was more effective than cisplatin alone and reduced cell viability by 62% and 81%, respectively. This effect was further confirmed using a colony formation assay . O-acetyl RT and 50 µM cisplatin significantly induced apoptosis compared to the group treated with cisplatin alone . Western blot analysis showed that the pretreatment of 0.05 µM O-acetyl RT significantly increased the expression of the active form of caspase-3  and caspase-9   and pro-apoptotic protein BAX was significantly upregulated  in the O-acetyl RT pretreatment. Pretreatment with 0.05 µM O-acetyl RT could increase the expression of p53  but the level of p53 expression was not altered in the O-acetyl RT pretreatment group when compared to the group treated with cisplatin alone (p > 0.9999). It has been reported that cisplatin can activate the ERK pathway leading to the induction of p53-dependent apoptosis in lung cancer cells [O-acetyl RT clearly increased the p-ERK level in H292 cells .To test whether cetyl RT A  D,E. Figuer cells . In lineO-acetyl RT sensitization. The spheroid number and area of the group pretreated with O-acetyl RT, following by treatment with 50 µM cisplatin, was also lower than those of the group treated with cisplatin alone  and CD44  but the results were not significantly different from those of the group pretreated with O-acetyl RT and the group treated with cisplatin alone , a critical gene related to survival and stemness, whereas total AKT was not changed in comparison to that of cisplatin used alone. Previous results have suggested that cisplatin increased CD133+ cells subpopulation in NSCLC cells, which is a responsible for drug resistance [O-acetyl RT on the number of CD133+ cells subpopulation in NSCLC using flow cytometry. As shown in + cells subpopulation was increased in H292 cells treated with cisplatin alone, while pretreatment with O-acetyl RT was able to reduce the number of CD133+ cells subpopulation . These results indicate that pretreatment with O-acetyl RT significantly reverses cisplatin-induced CD133+ cells subpopulation in H292 cells.We further observed the inhibition of cisplatin-induced stemness with in alone A–C. We ain alone D,E. In aO-acetyl-renieramycin T is a derivative of renieramycin T, which was isolated from the blue sponge Xestospongia sp. found in the seas around Thailand [50 at 96 h ranges from 0.01 to 0.12 µM [O-acetyl RT on NSCLC cells, H292, A549, and H23 cells. H292 cells were more sensitive to O-acetyl RT than A549 and H23 cells. Our data indicated that O-acetyl-RT has a strong cytotoxic effect on human H292 cell, with an IC50 at 24 h of 0.66 µM. Moreover, O-acetyl RT can also induce p53-dependent apoptosis in H292 cells (O-acetyl RT (0–5 µM) for 24 h caused a dose-dependent increase in p53 protein level. O-acetyl RT at concentrations ≥ 0.1 µM was sufficient to activate the induction of p53 and apoptosis in H292 cells. Treatment with O-acetyl RT at 0.05 µM for 24 h increased p53 protein levels but did not induce apoptosis in H292 cells  medium, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine, phosphate-buffered saline (PBS) and trypsin-EDTA were obtained from Gibco . 3--2,5-diphenyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), Hoechst33342, propidium iodide (PI), cisplatin, doxorubicin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, Co. . The following primary antibodies, caspase-9 (#9502), caspase-3 (#9662), p53 (#9282), BCL-2 (#4223), BAX (#5023), AKT (#9272), phosphorylated AKT (#4060), ERK (#4695), phosphorylated ERK (#4370), Nanog (#4903), CD44 (#9502), GADPH (#5174) and β-actin (#4970), were obtained from Cell Signaling Technology . CD133 (#CA1217) was obtained from Cell Applications . The respective secondary antibodies, anti-rabbit IgG (#7074) and anti-mouse (#7076), were obtained from Cell Signaling Technology .Xestospongia sp. was collected by scuba diving at a depth of 3–5 m in the vicinity of Sichang Island, Chonburi Province, Thailand, in October 2016 with the assistance of the Aquatic Resources Research Institute, Chulalongkorn University. The fresh sponge was identified based on the light bluish-gray color, bulbous surface lobes, numerous and moderate size of oscules and easily crumble texture [The Thai blue sponge The collected blue sponge was homogenized and suspended in the phosphate buffer solution (pH 7). Then, a solution of 10% potassium cyanide (KCN) in the phosphate buffer solution (pH 7) was added dropwise to the suspension until the overall KCN concentration reached 10 mM . ThereafO-acetyl RT by esterification. Renieramycin T  was dissolved in dry pyridine (1.0 mL). Acetic anhydride  was added to the reaction mixture, which was then stirred at room temperature (25 °C) under an argon atmosphere for 3 h . The organic layers were combined, washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by flash chromatography using silica gel and a mixture of hexanes:EtOAc (4:1) solution as stationary phase and mobile phase, respectively to furnish O-acetyl RT  as a yellow amorphous solid. The spectroscopic data of O-acetyl RT was matched with the previous report [The naturally produced renieramycin T was employed as a synthetic precursor for chemical modification to yield  for 3 h . Then, ts report .O-acetyl RT was prepared in DMSO and was further diluted in a complete medium to the desired concentrations. The final concentration of DMSO in each treatment was less than 0.1% which showed no signs of cytotoxicity. Cisplatin was dissolved in 0.9% sodium chloride (NaCl) and was diluted in complete medium to provide the indicated working concentrations. The solution was aliquoted and stored at −20 °C until further use.2 in a humidified incubator.Human non-small cell lung cancer (NSCLC) cell lines, H292, A549 and H23 cells were obtained from the American Type Culture Collection . H292 and H23 cells were cultured in RPMI 1640 medium. A549 cells were cultured in DMEM medium. The medium was supplemented with 10% FBS, 2 mM L-glutamine and 100 units/ml of each penicillin and streptomycin at 37 °C with 5% COO-acetyl RT on NSCLC cells was determined by MTT assay. In brief, cells were seeded into 96-well plate at 1 × 104 cells/well, allowed to adhere overnight and then treated with different concentration of O-acetyl RT  for 24 h. After that, 100 μL of MTT solution (400 µg/ml) was added, incubated for another 4 h at 37 °C. The formazan crystal product was dissolved in 100 μL of DMSO. The optical density was measured at 570 nm using a microplate reader . For O-acetyl RT sensitization in H292 cells, the cells were exposed to O-acetyl RT for 24 h, and then after washout of O-acetyl RT, cells were incubated in medium with or without cisplatin (50 µM) for an additional 24 h.Cell viability inhibition effect of O-acetyl RT at non-toxic concentration  for 24 h were seeded in 24 well plates at a density of 100 cells/well and incubated for 7 days. The colony was fixed with methanol at 4 °C for 15 min and stained with 0.1% crystal violet for 15 min. Colony formation was assessed using a phase-contrast microscope  equipped with a DP70 digital camera system .H292 cells pre-treated with O-acetyl RT, followed by incubation in medium with or without 50 µM cisplatin, were cultured in serum-free medium in 24-well ultralow attachment plates at a cell density of 2500 cells/well. After 7 days, the spheroids were determined using phase-contrast microscopy. The relative spheroid number and area were calculated compared with those of the non-treated cells.H292 cells pretreated with 0.01 or 0.05 µM Apoptotic cell death was determined by nuclear staining with a Hoechst 33342 and PI. After being treated with the indicated concentration, H292 cells were stained with 10 μg/mL Hoechst 33342 for 15 min at 37 °C and then stained with 5 μg/mL PI. Cells were imaged randomly using the fluorescent microscope . Apoptotic cells with nuclear condensation and DNA fragmentation were analyzed and expressed as a percentage of apoptotic cells.O-acetyl RT, H292 cells were collected and washed in cold PBS, pH 7.4. The cells were then dispersed in binding buffer containing annexin V-FITC and PI as recommended in the manufacturer’s instructions . Live, apoptotic and necrotic cells were determined using Guava easyCyte flow cytometer .After treatment with O-acetyl RT treatment, H292 cells were incubated with RIPA lysis buffer supplemented with protease inhibitor cocktail  for 1 h on ice. The extracted proteins (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further transferred to 0.45 μm nitrocellulose membranes . The membranes were blocked in Tris-buffer saline containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h at room temperature and incubated with the appropriate primary antibodies at 4 °C overnight. The membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The membranes were detected using an enhancement chemiluminescent detection system  and subsequently exposed to X-ray film. The protein band was analyzed using ImageJ software .After O-acetyl RT were trypsinized and incubated in PBS supplemented with 3% BSA for 30 min on ice. After that, cells were incubated with an anti-CD133 antibody for 1 h at 4 °C. The cells were then incubated with an Alexa488-conjugated secondary antibody  for 30 min at 4 °C in the dark and washed again before analysis using Guava easyCyte flow cytometer .To determine CD133 expression by flow cytometry, H292 cells treated with p < 0.05 significance level.All data from at least three independent experiment are expressed as mean ± standard deviation (SD). Statistical differences between multiple groups were analyzed using analysis of variance (ANOVA), followed by Turkey’s post-hoc test for individual comparisons at the O-acetyl RT is able to induce apoptosis and suppress the expression of CSC markers in H292 cells. We also found that O-acetyl RT had the ability to increase cisplatin-induced apoptosis and to decrease the number of cisplatin-induced CD133+ cells. These results provide novel and significant data indicating that O-acetyl RT might be a promising candidate as a sensitizer in cancer chemotherapy for reducing resistance and suppressing tumor progression.Our results provide novel and significant data indicating that


IL21R promoter region in B cells was assessed with ChIP-qPCR. We demonstrate an increase in IL-21R expression in total and memory B cells from RA subjects, which correlated with responsiveness to IL-21 stimulation. Stimulation of naïve RA B cells with IL-21 and CD40L resulted in an increase in differentiation into plasmablasts and an increase in IL-6 production in comparison to healthy controls, which was dose dependent on IL-21 stimulation. IL-21R expression on memory B cells in RA synovial fluid was comparable to peripheral blood making our study pertinent to understanding B cell responses in the joint and site of inflammation. We identified an increase in SP1 protein and mRNA in RA B cells and demonstrate an increase in binding of SP1 to the IL21R promoter region, which suggests a mechanism by which IL-21R expression is enhanced on B cells in RA. Taken together, our results indicate a mechanism by which IL-21 enhances B cell development and function in RA through an SP1 mediated increase in IL-21R expression on B cells.B cells are implicated in rheumatoid arthritis (RA) based on the presence of autoantibodies and the therapeutic response to B cell depletion. IL-21 has a significant role in B cell development and function. Here we assess B cell responses to IL-21 and the mechanisms responsible for altered IL-21R expression in RA. Flow cytometry of PBMC and cultured B cells was used to quantify protein and mRNA levels of IL-21R, IL-21 signaling through pSTAT3, specificity protein 1 (SP1) and to determine cytokine production (IL-6) and maturation status of B cells in RA and healthy control subjects. SP1 binding to the  Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by the presence of rheumatoid factor and anti-cyclic citrullinated peptide autoantibodies (ACPA) , 2. AlthPRDM1, a critical transcription factor for plasma cell development . Diluted samples were treated with hyaluronidase (VWR) and benzonase (Sigma) for 30 minutes at 37°C, centrifuged and resuspended in 2 mL hemolytic buffer. Samples were quenched with 30 mL PBS, centrifuged, resuspended in 10% RPMI, filtered through a 100 μm cell strainer and washed with 10% RPMI media.C (TUGh4), STAT3 (M59-50), from BD, SP1 (D4C3) from CST and IL-6 (MQ2-13AS) from eBioscience. IL-6 and IgM levels were determined after brefeldinA (Biolegend)/monensin (Biolegend) stimulation for 4 h, fixed with cytofix (BD), permeabilized with cytoperm (BD) followed by intracellular staining. Transcription factor staining was conducted according to the manufacturers protocol (BD). Where the mean fluorescent intensity (MFI) is analyzed we utilized the geometric mean fluorescent intensity. All flow cytometry experiments were acquired on a BD FACSCanto II (BD) and data were analyzed with FlowJo software (Tree Star).PBMC were rested in XVIVO 15 (Lonza), stained with a viability dye (eBioscience) and blocked with Human TruStain FcX (Biolegend). PBMCs were incubated with CD19 (HIB19), CD20 (2H7), CD24 (ML5), CD10 (HI10a), IgM (MHM-88), CD3 (UCHT1), CD8 (RPA-T8), CD45RO (UCHL1), CD45RA (HI100), CD138 (MI15), IL-21R (17A12), from Biolegend; CD38 (HIT2), CD27 (L128), CD4 (SK3), CD27 (L128), Blimp-1 (5E7), γRPL13A RNA probe was used as a positive control and a no probe negative control was included. The following Type I RNA probes were obtained from eBioscience: IL21R, SP1, PRDM1, SOCS1, SOCS3, and miR155.Intracellular RNA flow cytometry was conducted using the manufacturer's protocol . PBMCs were rested in XVIVO15 (Lonza). Cells were stimulated with media or IL-21 (50 ng/mL) (Miltenyi) for 45 min. PBMC was fixed with methanol and labeled with surface antibodies, intracellular CD20 antibody , and pSTAT3 (pY705) . Cells were stimulated with IL-10 (10 ng/mL) (BD) for 45 min or IL-4 (50 ng/mL) (R&D Systems) for 15 min. Cells stimulated with IL-10 were analyzed for pSTAT3 fold change levels and those stimulated with IL-4 were analyzed for pSTAT6 (pY641) . PSTAT3 fold change was calculated by the geometric mean of the stimulated well divided by the geometric mean of the unstimulated well.Example gating for B cell populations and IL-21R gating of a bimodal population is visualized in Figure For 24 or 48 h cultures, PBMC was thawed and stimulated with IL-21 (50 ng/mL) (Miltenyi) with and without CD40L (5 μg/mL) (Peprotech). Four hours prior to harvest, brefeldinA/monensin were added to the cultures. Cells were harvested and stained for flow cytometry.For plasmablast cultures, naïve B cells were isolated using the human naïve B cell isolation kit II (Miltenyi) and purity was assessed following 12-day cultures. Samples with purity >90% were plated at two million cells per well and cultured with IL-21 (15 pg/mL or 50 ng/mL) (Miltenyi) and CD40L (5 μg/mL) (Peprotech).An aliquot of PBMC was removed to determine levels of SP1 and IL-21R prior to stimulation to obtain baseline levels. PBMC were plated with 0.1 μM Okadaic acid or DMSO control for 24 h.Serum from RA subjects was assessed for anti-cyclic citrullinated peptide 3 IgG (Inova Diagnostics), anti-rheumatoid factor IgM (Abcam), IL-6 (eBioscience), and IL-10 (eBioscience) levels according to the manufacturer's instructions.B cells were isolated by negative selection using the human B cell isolation kit II (Miltenyi). Cells were fixed with 4% paraformaldehyde for 10 minutes at 37°C. The cell pellet was frozen at −80°C until sample preparation. Magnetic beads were blocked (PBS/0.5% BSA) and incubated with either 10 μg SP1 antibody  or control IgG overnight at 4°C. Nuclei were isolated the following day and sheared using a truChIP Chromatin Shearing Reagent Kit (Covaris), an AFA microTUBE (Covaris) and a M220 focused ultrasonicator (Covaris). Conditions for sonication: 20% duty cycle, 30 min. Sonicated samples were immunoprecipitated with magnetically labeled SP1 antibody or control IgG overnight at 4°C. The next day samples were reverse cross-linked. The Qiagen QIAquick PCR purification kit was used to purify the DNA.IL21R gene are: site 1: forward (5′-GTCCCACAGGTCCCTAAGGT−3′) and reverse (5′-CCAGCAACTTCCCTGAGATG−3′); site 2: forward (5′-AAGGTAAGGCTGGGCTCAAT−3′) and reverse (5′-GCAATCTCAGGTCACTGCAA−3′).Gene expression analysis was performed using SYBR green real-time quantitative PCR on an ABI7900 HT analyzer (Applied Biosystems). Primers used to detect SP1 binding in the IL-21R promoter region were: forward (5′-GGTCCCTAAGAGGGAAGTGTCA−3′) and reverse (5′-GCACCCACTGTCACCAAAGG−3′) as previously published by Wu et al . Primershigh compared to controls or IL-21Rlow, was assessed with a two-sided Mann Whitney U or Student's t test of which a P value of <0.05 was considered significantly different. A Mann Whitney U or Student's t test was selected after determining the normality of the groups with a D'Agostino-Pearson omnibus normality test. Correlations were assessed by calculating the Pearson's correlation coefficient, r.Data was analyzed using GraphPad Prism. Statistical significance between controls and RA; or between RA-IL-21Rhigh cohort was defined as subjects for whom the gMFI of IL-21R is two standard deviations (SD) above the mean of the control group. The RA-IL-21Rlow cohort was defined by the IL-21R gMFI within two SD of the mean gMFI of the control group , memory (p = 0.0087), and naïve (p < 0.0001) B cells  suggesting that enhanced pSTAT3 response was not due to alterations in signaling via γC or a target downstream of it   and RA low IL-21R (RA-IL-21Rlow) groups. We defined RA-IL-21Rhigh as subjects for whom the gMFI of IL-21R is two standard deviations (SD) above the mean of the control group. RA-IL-21Rlow were RA subjects with IL-21R gMFI within two SD of the mean gMFI of the control group  has been reported to regulate IL-21R expression in T cells . We asses Figure . In addis Figure . SP1 mRN Figures  and thiss Figure .IL21R promoter in B cells. As before, we analyzed B cells from controls, RA-IL-21Rhigh and IL-21Rlow subjects and performed ChIP-qPCR on purified B cells; we analyzed the SP1 binding site that were identified in the IL21R promoter in T cells  which has been shown to control the dephosphorylation of SP1 . Dephospl Figure . These fWe find that RA subjects with elevated IL-21R expression demonstrate hyper-responsiveness to IL-21, increased plasmablast differentiation and IL-6 production. These features could participate in early RA by promoting the development of autoantibodies and the persistent inflammation in late disease via the production of IL-6. These findings are consistent with the observation that circulating plasmablasts are increased in the setting of active RA disease and supports a role for the IL-21/IL-21R pathway in autoantibody formation in RA . A role hit-bet+ B cells as described in human SLE  in RA subjects compared to controls in the naïve B cell population (in vivo, increased IL-6 production in response to IL-21, by both memory B cells and plasmablasts, may be a driving factor in the expansion of T follicular helper and Th17 cells in RA and ultimately promote B cell maturation and autoantibody production (Multiple factors may influence the enhanced plasmablast development and IL-6 production by B cells in RA patients. Our group previously observed an increase of approximately 2% in unconventional memory cells (CD19pulation . Unconvepulation . The freoduction , 43, 44.+ B cells compared to controls while MS subjects did not. This finding was modest, and not observed in memory or naïve B cell subsets. Notably, there are several papers in the literature that have assessed IL-21R expression in SLE subjects. These papers provide opposing views to whether SLE subjects express increased, decreased or no difference in expression compared to controls (IL21R SNP rs3093301 in SLE (SLE subjects demonstrated enhanced IL-21R expression on total CD19controls –47. Le Ccontrols . SLE is 1 in SLE , 49, whi1 in SLE . This su1 in SLE  and our In this study we determined the mechanism that leads to enhanced IL-21R in RA, show that IL-21R mRNA levels are increased in RA B cells and investigated the role of SP1 in the regulation of IL-21R expression. Augmented transcriptional activity of SP1 has been described in fibroblast-like synovial cells and in bone marrow mononuclear cells in RA –53. T ceSOCS3 in B cells in RA. As SOCS3 is a negative regulator of IL-21 signaling, it may contribute to the enhanced response to IL-21 seen in RA B cells. Prior studies have found SOCS1 and SOCS3 transcripts in PBMC to be increased in RA (SOCS1 and SOCS3 expression in B cells would be reflected in this analysis. No differences in miR155 expression between RA and controls in our cohort were observed which is in contrast to a report from Alivernini's group that miR155 levels are elevated in B cells in RA (SOCS3 expression in RA could contribute to enhanced IL-21/IL-21R signaling.Other mechanisms may contribute to increased IL-21R expression or IL-21 responsiveness in the setting of RA. We observed a significant decrease in ed in RA . Due to ls in RA . These dDefining the role of IL-21/IL-21R signaling in RA is important because of the interest in blocking this pathway therapeutically by targeting IL-21, IL-21R  and the PRDM1 expression, plasmablast differentiation and IL-6 production. Mechanistically we showed that the increased IL-21R was due to enhanced binding of the transcription factor, SP1. Together, these studies highlight the importance of IL-21 signaling in B cells and provide insight into how to target therapies to the IL-21 signaling pathway in RA.We have shown that IL-21R expression is increased on total and memory B cells in peripheral blood and synovial fluid from RA subjects. Increased IL-21R in peripheral blood correlates with enhanced response to IL-21 with respect to All subjects gave written informed consent in accordance with the Declaration of Helsinki and according to the Institutional Review Board-approved protocols at both the Benaroya Research Institute, and the VA Puget Sound Health Care System.ED designed the research studies, conducted experiments, acquired data, analyzed data, and wrote the manuscript. AM conducted experiments, acquired data, and analyzed data. AH wrote the manuscript. JC and BN recruited subjects. JB recruited subjects, designed the research studies and wrote the manuscript.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.


The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Å resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display. During maturation, Gag is cleaved into three major structural proteins—matrix (MA), capsid (CA) and nucleocapsid (NC)—and undergoes a dramatic morphological rearrangement  × 100.TZM-b1 cells  overnight at 4 °C. In the subsequent experiments, we characterized CA N21C/A22C before and after assembly and compared with WT CA. WT CA presented as a monomer in SDS-PAGE both before and after dialysis, with no particles in negative-staining EM images A. As expNext, we analyzed the assembly of CA N21C/A22C by HPSEC and AUC. In the HPSEC analysis, we found that the elution volume was approximately 7.85 mL and 5.40 mL for CA N21C/A22C before and after assembly, respectively D. Howevehttps://www2.mrc-lmb.cam.ac.uk/relion/index.php)  , we recodex.php) . The strdex.php) B. The dedex.php) C. Each adex.php) C. Obvioudex.php) D. Navigadex.php) E; these We fitted the crystal structures of the HIV-1 CA pentamer (PDB no. 3P05) and hexamer (PDB no. 3H47) into the T = 4 cryoEM density map, and generated an atomic model for the CA-N21C/A22C particle. We identified two unique clustering interactions involving three capsomers located around a 3-fold axis and a local pseudo 3-fold axis that surrounded a 5-fold axis A. For thWe next compared the intertwined angles of the representative hexamer–hexamer and pentamer–hexamer interactions  for the T = 4 icosahedral association with that of the entire fullerene capsid structure generated by molecular dynamics; the structure was based on cryo-ET images of the native capsid, where 12 pentamers (labelled a–l) were introduced to the hexamer-netting conical entity  Figure . Among tTo investigate the potential to use CA N21C/A22C as a scaffold protein, the Env V3 loop was grafted, replacing residues A88 to R97 in the cyclophilin-binding loop of CA. The chimeric protein—CA N21C/A22C-V3—was expressed and purified as described for the parental CA N21C/A22C. After self-assembly, CA N21C/A22C-V3 presented in hexameric form in non-reducing SDS-PAGE and dissociated into monomers under reducing conditions, and showed reactivity with the CA-specific mAb (A10F9) in WB A. The ch50) with the CA N21C/A22C-V3 and CA N21C/A22C than with CA . As shown in The HIV-1 capsid protein not only plays a crucial role in HIV-1 maturation but also in HIV-1 infection ,43. DuriIn this study, we analyzed a previously engineered a disulfide cross-linked capsid and revealed a novel T = 4 capsid structure that is different to that deduced by Pornillos et al. . The groCardone et al.  reportedThe HIV-1 Gag protein has been investigated in vaccine research , and desIn conclusion, we obtained the T = 4 CA N21C/A22C structure at a lower resolution due to the high heterogenicity of the CA particle, and compared its dihedral angles to the conical capsid; our results suggest some similarity in the curvature between these two types of capsid assemblies. Furthermore, we found that the HIV-1 V3 loop displayed by CA N21C/A22C could induce cross-clade neutralizing antibody responses in BALB/c mice. These findings will provide important information for future antiviral designs targeting capsid assembly and for vaccine development based on the capsid scaffold.


While reperfusion reestablishes oxygen supply, it generates a high concentration of radicals, resulting in tissue dysfunction and damage. Here, we aimed to challenge and achieve the potential of a delivery system based on astaxanthin, a natural antioxidant, in attenuating the muscle damage in an animal model of femoral hind-limb ischemia and reperfusion. (2) Methods: The antioxidant capacity and non-toxicity of astaxanthin was validated before and after loading into a polysaccharide scaffold. The capacity of astaxanthin to compensate stress damages was also studied after ischemia induced by femoral artery clamping and followed by varied periods of reperfusion. (3) Results: Histological evaluation showed a positive labeling for CD68 and CD163 macrophage markers, indicating a remodeling process. In addition, higher levels of Nrf2 and NQO1 expression in the sham group compared to the antioxidant group could reflect a reduction of the oxidative damage after 15 days of reperfusion. Furthermore, non-significant differences were observed in non-heme iron deposition in both groups, reflecting a cell population susceptible to free radical damage. (4) Conclusions: Our results suggest that the in situ release of an antioxidant molecule could be effective in improving the antioxidant defenses of ischemia/reperfusion (I/R)-damaged muscles. An ischemic condition results in an imbalance in the oxygen production and consumption in the cells and tissues, generating a constant influx of pro-inflammatory reactions that render tissues vulnerable to microvascular dysfunction  and to aNatural carotenoids have shown particular antioxidant capabilities to scavenge ROS and enhance the cell’s ability to prevent oxidative stress . AstaxanEnvironmental scanning electron microscopy (ESEM) images reflected a heterogeneous microporous structure of the pullulan/dextran (P/D)-based scaffold A,B, spreP/D/CD-A release product was quantified regarding its concentration and antioxidant capacity. First, a calibration curve using different concentrations of CD-A (0–12.5 µM astaxanthin concentration) versus absorbance at 472 nm was plotted , then thP/D-based scaffolds were autoclaved by UV light and evaluated to determine any sign of in vitro and in vivo toxicity. The toxicity of CD-A and P/D/CD-A release product (12.5 µM) was evaluated after contact with the 3T3 cells for 24 h. This cell line allows the evaluation of toxicity of samples thanks to its high sensitivity. In vivo toxicity was evaluated by monitoring the response of vital organs distant from the implant. No noticeable systemic reaction was triggered by the presence of either the P/D scaffold or the P/D/CD-A antioxidant in the kidney and liver  of the aDuring the in vivo implantation study , animalsHematoxylin eosin staining  of the cp < 0.05) was found in the total CD68 monocyte/macrophage positive population at 7 days of reperfusion between the P/D and the antioxidant-loaded scaffold P/D/CD-A (p < 0.05) was observed between control, P/D, and PD/CD-A samples despite the lower values registered for all three samples. Compared to the sham group, there were no significant changes in the number of cells with positive staining for the CD163 marker after 7 days of reperfusion in the P/D/CD-A group. However, a significant increase of M2 repair macrophages (p < 0.05) was observed in the P/D/CD-A group after 15 days. Monocytes/macrophages are phagocytic cells consisting of two cellular subtypes: (i) M1 related to the inflammatory process and positively labeled with the anti-CD68 antibody; (ii) M2 related to the tissue regeneration process and showing positive labeling with anti-CD68 and anti-CD163 antibodies. Evaluation after 60 min of reperfusion demonstrated no significant inflammatory response given by positive CD68 or CD163 markers in any of the I/R groups. An example of histological images is presented in P/D/CD-A , being aP/D/CD-A . After 6The effect of CD-A local treatment on nuclear factor-erythroid 2-related factor 2 (Nrf2) translocation in the gracilis muscle was studied under I/R conditions. Nrf2 is a crucial transcription factor, which regulates antioxidant defense comprising antioxidant enzymes heme oxygenase-1 (HO1) and NAD (P) H: quinone oxidoreductase 1 (NQO1) . RegardiA positive tissue staining with Perls/DAB  was differentiated from red blood cells, heme or non-heme iron fixation. After 60 min and 7 days of reperfusion, the P/D/CD-A group showed a decrease in the Perls/DAB positive staining compared to the sham group. However, after 15 days of reperfusion, a slight increase in the non-heme iron fixation in the muscle area next to the implanted scaffold in either the sham or the antioxidant group suggested a retention by macrophages. Positive heme iron fixations of the red blood cells were noticed in all the groups C and a nAs the main results of this study, a polymeric system composed of a mixture of two polysaccharides, pullulan and dextran, might allow the delivery of an antioxidant molecule such as astaxanthin in an easy and controlled manner. This system could be used as an in situ antioxidant treatment modality to enhance the muscle damage induced by I/R injury. In this study, the I/R protocol consisting of the femoral artery clamping in the section between the popliteal and the deep femoral artery during 45 min was chosen. This model was described as the most suitable model of chronic mild ischemia and the most representative of the degree of ischemia seen in patients . Under lPrevious studies have reported the capacity of antioxidants to attenuate oxidative stress after hind-limb I/R injury ,31,32,33Further, the biocompatibility of P/D scaffolds has been widely evaluated in vitro and in vivo. No negative side effects have been reported in a wide range of applications in this regard ,20,21,37One of the most important cytoprotective systems against oxidative stress is the Keap1-Nrf2 complex . Nrf2 is2+) such as hemoglobin, myoglobin, and cytochromes, and as a metal cofactor  present in several enzymes including Fe-S cluster proteins in mitochondria. Iron homeostasis has been associated with oxidative stress and ROS generation [3+ and release of Fe2+ ions [2O2 to generate highly reactive hydroxyl radicals and result in cellular injury [Iron is a crucial component in living organisms. It plays a vital role as a structural component of heme-containing proteins , Hydroxypropyl-β-cyclodextrin (DS = 0.67), potassium ferrocyanide , 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), trisodium trimetaphosphate , sodium chloride , and isopropanol  were purchased from Sigma-Aldrich Co. LLC . Pullulan (MW 200000) was purchased from Hayashibara Inc., Okayama, Japan. Dextran (MW 500000), with a degree of branching of 5%, was obtained from Leuconostoc mesenteroids . Acetone, methanol, hydrochloric acid, hydrogen peroxide, and chloroform were all purchased form Carlo Erba Reagents S.A.S . EnVision Dako kit (ref K4063 and ref K4010), Dako REAL peroxidase-blocking solution, and Negative Control mouse IgG1 solution were provided by Dako . Mouse anti-rat CD68 and CD163 primary antibodies were obtained from Bio-Rad . Anti-phospho-Nrf2 (S40) antibody was purchased from Abcam . The water used was double distilled and deionized. Mouse fibroblast cells CCL 163  were purchased from ATCC-LGC Standards S.a.r.l. . Minimum essential medium-L-glutamine (MEM), fetal calf serum (FBS), penicillin–streptomycin- amphotericin (PSA), and trypsin/EDTA solution (TE) were purchased from GIBCO .w/v). Then, 10 g of the polysaccharide solution was withdrawn, to which 1 mL of NaOH 10 M was added, and incubated at 50 °C for 20 min. Next, 300 mg of sodium trimetaphosphate (STMP) (30% w/v) in 1 mL of water was added as a chemical crosslinking agent. Finally, the mixture was poured into a glass mold of 10 cm × 5 cm × 1 mm. The resultant hydrogel was washed extensively in PBS pH 7.4, freeze-dried , sterilized under UV light, and stored at room temperature until use. Polysaccharide-based scaffolds were prepared according to the protocol proposed by Autissier et al. . Brieflyv/v 1:1) solution was mixed with hydroxypropyl-β-cyclodextrin  dissolved in 12.5 mL of 95% methanol and put in an atmosphere of nitrogen. The mixture was sonicated for 5 min at 35 °C (ultrasonic bath bandelin sonorex rx-100-h) and stirred overnight under light protection. The solution was dried under vacuum, recovered with double distilled water, freeze-dried, and stored at −4 °C until use. To facilitate natural astaxanthin loading into the scaffolds, hydroxypropyl-β-cyclodextrin was previously mixed with astaxanthin according to the following preparation method: Natural astaxanthin  in acetone/chloroform  or 2.5 mg CD-A reconstituted in 10 µL saline solution (12.5 µM) for the antioxidant group (P/D/CD-A). Five minutes were required for the solutions to be completely absorbed by the scaffolds. P/D and P/D/CD-A scaffolds were characterized by environmental scanning electron microscopy (ESEM) using a Philips XL 30 ESEM-FEG  at an accelerating voltage of 15 keV and at a pressure of 3.5 Torr. To determine the release of CD-A complex from the hydrogel, a calibration curve by plotting the CD-A absorbance (OD) at 472 nm against concentration of the complex (0–15 µM) was established . The CD-A release from the scaffold complex was determined as follows: P/D/CD-A scaffolds were immersed in PBS 0.1 M, pH 7.4 at 37 °C, to allow the release of CD-A into the solution. Then, 50 μL of the release solutions were collected at 5, 10, 20, 30, 40, and 60 min. The absorbance of P/D/CD-A release products was recorded at 472 nm, and OD values were reported to the percentage of CD-A concentration based on the calibration curve.The antioxidant scavenging capacity of CD-A before and after release from the P/D scaffolds was evaluated using the ORAC  method . BrieflySolutions of fluorescein (4 nM), 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) , and Trolox  were prepared in PBS. Fluorescein solution (150 μL) was added to each well (96-well microplate), and then 25 μL of the P/D/CD-A scaffold release product, blank (PBS), or Trolox were distributed in the wells, prior to the addition of AAPH (25 μL). Then fluorescence decay was monitored (485/ 528 nm Ex/Em) at 37 °C for 60 min, and data were taken every minute. The area under the curve (AUC) of relative fluorescence was calculated. The AUC values for CD-A release products and Trolox were reported to the AUC value calculated for AAPH (stressor), to indicate the AUC net values. ORAC values were expressed as Trolox equivalent in μM  and calculated as the slope ratio from curves, AUCnet, versus the concentration of antioxidant and Trolox, respectively (AUCnet = AUCsample − AUCblank).v/v) FBS and 1% PSA. Then, the cells were seeded in a 96-well microplate at a cellular density of 1 × 104 cells/well and cultured overnight. After MEM removal, CD-A (1.25 to 5 μM) or P/D/CD-A scaffold release product, MEM , or 10% DMSO  were added to the wells. After 24 h incubation in cell culture conditions, the solutions were discarded and the MTT assay was performed. In this regard, the MTT solution  was added to the cells and incubated for 3 h at 37 °C. Then, 200 μL of isopropanol was added for 20 min. Next, the absorbance was recorded at 570 nm . The samples were considered non-toxic if cellular viability was higher than 70% compared to NC . Cell viability was assessed using the MTT assay. To this end, mouse fibroblast cells (3T3) were grown in MEM, supplemented with 10% (n = 12), sham (n = 12), and antioxidant (n = 12) . Hind-limb I/R surgeries were performed on male rats weighted 250–300 g  using an I/R model adapted from Luyt et al. . Animals(n = 12) . Control(n = 12)  were eva2O2 incubation [Animals were sacrificed 1 h, 7 days or 15 days post-surgery. Gracilis muscles were collected and immersed in 4% formaldehyde during 24 h, dehydrated, and embedded in paraffin according to the standard protocols. Sections at 4 μm thickness  were stained with hematoxylin and eosin. Total phagocytes and type-2 macrophages were identified by anti-CD68 and anti-CD163 antibodies labeling respectively (1:30 working dilutions). Anti-phospho Nrf2 (1:100 working dilution) immunomarker was used to collect information about the muscle general oxidative stress status after I/R injury. DAKO EnVision+ System and 3,3′-diaminobenzidine was used as a chromogen. Iron accumulation in healthy and I/R muscles was quantified by Perls reaction amplified by 3,3′-diaminobenzidine (DAB). After antigen retrieval, samples were incubated in a solution  containing 1% HCl and 1% potassium ferrocyanide, followed by HSamples images were analyzed by two independent methods: (i) A mathematical quantitative program: A saturation analysis which highlighted the positive stains in the images created by different immunostaining markers using Matlab software . Each image was separated into its components: hue, saturation, and value color bands. Then thresholds were defined to determine the mask for the region of color corresponding to the positively stained cells represented by brown color spots. Total image pixels of each image were used to calculate the percentage of positive staining as a pixel difference.(ii) A qualitative method: The analysis of the proximal and distal sections of the muscle was performed by a physician/pathologist in an independent and random manner. The tissue response was scored  according to a relative scoring system : – = no p-value < 0.05. All experiments were repeated at least three times to ensure the reproducibility of each test. Results were expressed as the mean ± SD and statistical analyses were done using one-way ANOVA followed by Tukey’s HSD (honestly significant difference) post hoc test . Statistical significance was set at Reactive oxygen species (ROS); ischemia/reperfusion (I/R); hydroxypropyl-β-cyclodextrin (CD); hydroxypropyl-β-cyclodextrin-astaxanthin (CD-A); pullulan/dextran-based scaffold (P/D); pullulan/dextran-based scaffold loaded with hydroxypropyl-β-cyclodextrin-astaxanthin (P/D/CD-A scaffold); oxygen radical absorbance capacity (ORAC); area under the curve (AUC); nuclear factor-erythroid 2-related factor 2 (Nrf2); heme oxygenase-1 (HO1); NAD (P) H: quinone oxidoreductase 1 (NQO1).


Molecular mechanisms of EBV-driven cell cycle progression and oncogenesis involve diverse genes and signal pathways. Here, we review the molecular mechanisms of EBV-driven cell cycle progression and promoting oncogenesis.The early stage of oncogenesis is linked to the disorder of the cell cycle. Abnormal gene expression often leads to cell cycle disorders, resulting in malignant transformation of human cells. Epstein–Barr virus (EBV) is associated with a diverse range of human neoplasms, such as malignant lymphoma, nasopharyngeal carcinoma and gastric cancer. EBV mainly infects human lymphocytes and oropharyngeal epithelial cells. EBV is latent in lymphocytes for a long period of time, is detached from the cytoplasm by circular DNA, and can integrate into the chromosome of cells. EBV expresses a variety of latent genes during latent infection. The interaction between EBV latent genes and oncogenes leads to host cell cycle disturbances, including the promotion of G G1, after which they enter S phase. There is a precise mechanism of cell cycle regulation in a normal cell. Malfunctions in cell cycle give access to cells to gain uncontrolled growth characteristics, primarily hyperproliferation and a low rate of apoptosis, ultimately leading to oncogenesis , latent membrane proteins LMP-1 and LMP-2 , EBV-encoded small RNAs (EBER1 and EBER2) and microRNAs (miRNAs). During different stages of B cell differentiation 1/S cell cycle progression, such as an inhibitor of differentiation or DNA binding 1 (Id1), the CDK inhibitor p27Kip1, CDK2, and Rb [1/S cell cycle progression, were observed in LMP-1, LMP1Δ204–208, 1–220, and 1–231. LMP-1 regulates telomerase activity through the p16INK4A/Rb/E2F1, PI3K-AKT and JNK signaling pathways to promote cell immortalization [Latent membrane protein 1 (LMP-1) is considered to be an oncogenic protein whose signal region contains three sites: carboxy-terminal activating region 1 (CTAR1) 194–232 a.a.), CTAR2 (351–386 a.a.) and CTAR3 (275–330 a.a.) that directly interact with some cell factors and activate NF-κB, JNK , p38 MAPK, JAK/STAT, and PI3K/Akt signal pathways which involved in cell cycle progression . There i, and Rb . Increas2 a.a., C2/M cell cycle kinase Cdc2 [kip, CDK2, and Rb, thereby, promoting G1/S phase transition, conferring cells proliferation advantages, and anti-apoptosis ability.CTAR2 is known to engage the JNK and NF-κB pathways. Elipoulos et al. reported that induction of LMP-1 directly activated functional JNK . Then Waase Cdc2 . JNK actase Cdc2 . CD40, aase Cdc2 . The conase Cdc2 . Lo et aase Cdc2 . Xiao etase Cdc2 . Comprehin vivo situation. This indicated that EBV involved in the initial steps of lymphomagenesis of GC-derived B cell lymphomas directly [1/S transition and hyperproliferation of B lymphocytes through promoting p27kip1 degradation at the early stage of lymphomagenesis [kip1 [0/G1 phase [kip.Latent membrane protein 2A (LMP-2A) of EB virus is expressed during different latency stages of EBV-infected B cells. Mancao et al. demonstrated that LMP-2A could rescue BCR-GC B cells from apoptosis in an directly . Wasil edirectly . LMP-2A directly . Engels directly . LMP-2A directly . Fukuda directly . LMP-2A directly , 38. Swadirectly , and subdirectly . LMP-2A agenesis . Fish etis [kip1 . Incroccis [kip1 . Wang etG1 phase . As was Epstein–Barr virus nuclear antigen 1 (EBNA-1) is an essential viral protein, expressed in virus latency as well as EBV-associated neoplasm . The EBV1. The cooperation between EBNA-2 and EBNA-LP (leader protein) can induce cyclin D2 expression in resting B cells. LMP-1 and c-Myc are directly activated by EBNA-2, indicating that activation of c-Myc by EBNA-2 is an important step in the process of EBV-induced proliferation and immortalization [Epstein–Barr virus nuclear antigen 2 (EBNA-2) is initially expressed after the infection of EBV, which is absolutely necessary for virus-mediated transformation. Cyclin D2 and CDK4 are both elements of the basic cell cycle machinery and driving cell cycle progression in early Glization . EBNA-2 lization . These s2/M and hypophosphorylated during G1/S phase [1/S transition [1/S transition. Szymula et al. found that EBNA-LP did not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. And they also found that EBNA-LP was essential for the survival of EBV-infected naïve B cells [Epstein–Barr nuclear antigen leader protein (EBNA-LP) is a phosphoprotein . Phospho/S phase . Kato etansition . A stronansition . Co-expr B cells . These f0/G1 phase that was concomitant with the conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein; They also found that p21WAF1/CIP1 expression was elevated following RNAi-mediated knockdown of EBNA-3A in LCLs [INK4A expression that is necessary for the proliferation of EBV-transformed B cells [Epstein–Barr virus nuclear antigen 3A (EBNA-3A) has been shown to play a role in the regulation of cell survival in B cells immortalized by EBV . Tursiel in LCLs . Myc-int in LCLs . Carboxy B cells .in vitro through manipulation of a number of major cellular pathways. Moreover, EBNA-3C can stabilize c-Myc and enhance c-Myc-dependent transcription. EBNA-3C residues 130 to 190 recruit and modulate the activity of retinoblastoma (Rb) and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA-3C in bypassing the cell cycle checkpoints [1/S transition, is well established. E2F1 plays a dual role in controlling cell growth and apoptosis. For example, elevated expression of E2F1 promotes cell cycle progression by driving quiescent cells into S phase [INK4A and p14ARF was essential for LCL growth, was reported by Maruo et al. [1 to S transition. Accordingly, EBNA-3C inhibits p73, p14ARF and p16INK4A to promote G1/S transition. At the same time, the combination of EBNA-3C and E2F6 can effectively block E2F1-mediated apoptosis and promote cell proliferation.Epstein–Barr virus nuclear antigen 3C (EBNA-3C) is a small subset of latent antigens critical for the transformation of human primary B lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) ckpoints . An EBV ckpoints . The inhckpoints , 65. Sahckpoints . The p73ckpoints . The rol S phase . However S phase . Saha et S phase . E2F6 is S phase . EBNA-3Co et al. . These sAdditionally, Rovedo et al.  showed t1/S transition finally [1/S transition by activating the oncogenic PI3K/Akt signaling pathway and inhibiting the tumor suppressor PKR and the cell cycle inhibitors p21 and p27.EBV-encoded small RNAs  are transcribed by RNA polymerase III into non-translated RNAs of 167 and 172 nucleotides, respectively, and form stem-loop structures by intramolecular base-pairing, giving rise to double-stranded RNA like structures. EBERs are considered as reliable markers for in situ hybridization to detect EBV infection in clinical samples of gastric carcinoma, lymphoma, nasopharyngeal carcinoma and etc. –78. Herb finally . In shorEBV has been recently found to encode microRNAs (miRNAs), which expressed in infected B cells and some EBV-associated neoplasms. EBV can encode approximately 23 precursors and 44 mature miRNAs. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts  miRNAs have nearly 30 kinds of BART miRNAs, which overexpressed in EBV-associated malignancies. Lung et al. found that down-modulation of LMP-2A expression by miR-BART22 might permit the escape of EBV-infected cells from host immune surveillance, which may facilitate NPC carcinogenesis . Lei et in vivo . Hooykaain vivo . Lung etin vivo .These findings indicate that EBV-encoded microRNAs play a contributing role in EBV-associated malignancies. EBV-miR-BHRF1-2 interacts with the tumor suppressor gene PRDM1 and plays an important role in cell proliferation. EBV-miR-BARTs can escape immune surveillance by down-regulating LMP-2A, and promote cell proliferation by down-regulating Wnt’s inhibitory gene and tumor suppressor PTEN.kip1, p21WAF1/CIP1, p16INK4A, p73, etc. The time from viral infection to tumorigenesis is usually shorter in EBV-associated neoplasms with specific oncogene activation. EBV-encoded genes rapidly lead to oncogenesis by activating cellular oncogenes or interacting with proteins in host cells. The latent proteins and miRNAs encoded by EB virus in host cells alone or in combination drive the cell cycle through a variety of pathways.As mentioned above, EBV plays a key role in driving cell cycle and oncogenesis of EBV-positive neoplasms. Multiple genes and signal pathways are involved in the occurrence of EBV-related neoplasms, including the interaction of virus gene and host genes Fig. . EpsteinIdentifying the molecular mechanism of EBV-driven cell cycle progression and oncogenesis may help to diagnose and guide clinical medication. Nevertheless, the precise mechanisms still remain unclear, especially those related to the dilemma between virus infection and the host cell. There are many kinds of literature about EBV driving host cell cycle and promoting oncogenesis, but there are few kinds of literature on how the expression changes of host cell genes affected by EBV. EBV-encoded genes, such as LMP-1, EBNA-1, and EBNA-3C, have been shown to interact with E2F1 to affect cell growth; however, its concrete mechanism is not yet clear and needs further study. E2F1 is expected to be a new therapeutic target for EBV-associated malignancies.EBV driving cell cycle and promoting oncogenesis is a very complex process. It is also necessary to further reveal its molecular regulatory network and key nodes to find more precise molecular targets and provide an effective solution for the prevention and treatment of EBV-related neoplasms.


Acinetobacter baumannii co-infection in patients with avian influenza A (H7N9) virus infection has been described as one of the most prevalent bacterial co-infections. However, the clinical and laboratory features of this entity of H7N9 and A. baumannii co-infection have not been systematically investigated.Bacterial co-infection of patients suffering from influenza pneumonia is a key element that increases morbidity and mortality. The occurrence of A. baumannii. H7N9 patients without bacterial co-infection and patients with A. baumannii-related pneumonia in the same hospital during the same period were recruited as controls. The antibiotic resistance features and the corresponding genome determinants of A. baumannii and the immune responses of the patients were tested through the respiratory and peripheral blood specimens.We collected clinical and laboratory data from laboratory-confirmed H7N9 cases co-infected by A. baumannii co-infection in H7N9 patients. The co-infection resulted in severe clinical manifestation which was associated with the dysregulation of immune responses including deranged T-cell counts, antigen-specific T-cell responses and plasma cytokines. The emergence of genome variations of extensively drug-resistant A. baumannii associated with acquired polymyxin resistance contributed to the fatal outcome of a co-infected patient.Invasive mechanical ventilation was the most significant risk factor for the nosocomial A. baumannii with H7N9 infection is an important issue which deserves attention. The dysfunctions of immune responses were associated with the co-infection and were correlated with the disease severity. These data provide useful reference for the diagnosis and treatment of H7N9 infection.The co-infection of H7N9 patients by extensively drug-resistant The online version of this article (10.1186/s12879-018-3447-4) contains supplementary material, which is available to authorized users. Although influenza viruses by itself can cause highly fatal primary influenza pneumonia, the excess mortality rates during influenza pandemics is mainly caused by secondary bacterial pneumonia . EpidemiAcinetobacter baumannii is one of the major opportunistic pathogens that have been implicated in various nosocomial infections [A. baumannii infection is ventilator-associated pneumonia [A. baumannii infection has been a matter of continuing debate as many patients with severe influenza had major underlying diseases [A. baumannii infection was associated with an increase in attributable mortality, ranging from 7.8 to 23% [A. baumannii is attracting much attention owing to the increase in antimicrobial resistance and occurrence of strains that are resistant to virtually all available drugs [A. baumannii (MDR-AB) strains resistant to all β-lactams, including carbapenems, illustrated the potential of this organism to respond swiftly to changes in selective environmental pressure [A. baumannii (PDR-AB) that can be fully refractory to the currently available antimicrobial armamentarium.fections . The mosneumonia . Althougdiseases , it has 8 to 23% . A. baumle drugs . The rappressure . Though pressure , which lhttp://www.who.int/influenza). Among the H7N9 patients complicated by bacterial infections, MDR-AB was the most common etiology for secondary bacterial pneumonia revealed by independent descriptive studies. Herein, we studied the occurrence of secondary infection by A. Baumannii in these severe H7N9 patients. We sought to investigate the role of dysfunctional immunity in the pathogenesis of severe pneumonia in H7N9 patients nosocomially co-infected with A. baumannii. Our study may improve the understanding of the high pathogenicity of H7N9 in humans and provide useful recommendations for the clinical diagnosis and treatment.From March 2013, hundreds of human cases infected by avian influenza A (H7N9) virus with a mortality of 40% were reported and the epidemic situation seems more severe currently with over 100 new cases within December 2016  blood culture according to standard microbiological criteria [A. baumannii. Among the 24 H7N9 patients, 9 patients were diagnosed as secondary infection by A. baumannii (H7N9-A. baumannii or H7N9-AB case group). Thirteen H7N9 patients without secondary bacterial infection with complete clinical data were termed as H7N9 controls. Fifteen patients with hospital acquired pneumonia due to A. baumannii, but H7N9 negative in the same period of our hospital, were also recruited as A. baumannii controls. The Declaration of Helsinki was strictly followed. The methods were carried out in accordance with the approved guidelines. We obtained written informed consent from all of the participants or their guardians.From December 2013 to April 2014, 24 patients hospitalized for H7N9 influenza in the Shenzhen Third People’s Hospital, Guangdong, China, which is the designated for H7N9 patients in Shenzhen. The laboratory confirmation of H7N9 virus infection were performed using the protocols as described previously [criteria . The patData collection  started immediately after admission and continued on a daily basis. The clinical data were integrated by two independent inputs and checked and verified by the third party. Antimicrobial resistance was determined according to National Committee for Clinical Laboratory Standards guidelines .www.ncbi.nlm.nih.gov). Eleven publications which described the secondary bacterial infections of H7N9 patients were summarized  following the manufacturers’ instructions. The raw data were analyzed using xPONENT 3.1 software (Merck Millipore). Plasmas from seven healthy individuals were used as controls.We collected the plasma of H7N9-The CTL epitope-specific response was measured by performing IFN-γ ELISPOT assays as described previously . PreviouA. baumannii isolates SMGC-AB1 and SMGC-AB2 were sequenced by Illumina HiSeq sequencing platform according to manufacturer’s instructions. The 150-bp length pair-end raw reads were first filtered to remove low-quality reads using the DynamicTrim and LengthSort Perl scripts within SolexaQA [http://soap.genomics.org.cn) [A. baumannii genomes.The genomes of the SolexaQA  and then.org.cn) . The com.org.cn) . The dra.org.cn) . For SNP.org.cn) . The ant.org.cn)  was usedA. baumannii SMGC-AB1 and subsequently polymyxin resistant, i.e. a pandrug-resistant isolate (PDR) named A. baumannii SMGC-AB2 have been deposited at DDBJ/ENA/GenBank under the accessions NIBI00000000 and NIBH00000000, respectively.The Whole Genome Shotgun projects of patient B4-derived original extensively drug-resistant (XDR) t-test or χ2-test was used to determine differences between two groups. The Pearson correlation coefficient and Spearman rank correlation coefficient were used for linear correlation analysis. We calculated receiver operating characteristic curves for predictive analysis. Logistic regression was used for multivariate analysis of the risk factors. These statistical analyses were performed with SPSS 16.0 for Windows . A P-value < 0.05 was considered statistically significant.The Student’s A. baumannii among the reported H7N9 patients was 19.0% (37/195). For the 11 A. baumannii cases whose outcome were available, 10 patients (90.9%) was reported fatal, much higher than the crude mortality rate of 32.9% (56/170) amongst H7N9 patients. Most of the identified A. baumannii were MDR-AB, and with one case of PDR-AB.We have done the meta analyses of the previous studies on the clinical descriptions of H7N9 patients  during the hospitalization or on retrospective testing . Furthermore, the risk factors for the secondary infection of A. baumannii in the H7N9 patients were analyzed. We found that the H7N9-A. baumannii co-infected patients have a significantly longer duration of invasive mechanical ventilation . No significant differences were found for the clinical manifestations at the time of admission of H7N9-A.baumannii cases compared to the H7N9 controls, including the oxygenation index (PaO2/FiO2)    which reflects the lung injury during the infection indicates a prolonged high SRC in the H7N9-A. baumannii cases  and the length of hospital are also longer than the H7N9 controls without co-infection (30.0 ± 11.2 vs 15.5 ± 7.1). All H7N9 controls without co-infection survived, but four of the nine (44.4%) H7N9-A. baumannii patients died. In the H7N9-A. baumannii cases, the patients who died had persistent abnormal oxygenation index compared to that of a gradual improvement in the survivors  Fig. . Furtherols Fig. , indicatses Fig. . All theA. baumannii presented a decreasing trend during the disease course  of plasma IL-6 and IL-8 levels were 0.927 and 0.850 respectively during the second week of hospitalization among H7N9-infected patients, and were 0.881 and 0.833 during the third week , CD8+ T-cell counts (AUC: 0.812) on admission and the restoring time for the abnormal CD4+ T-cell counts (AUC: 0.876) were all predictive for the H7N9-A. baumannii co-infection. Additionally, the traditional biomarkers of bacterial infection, procalcitonin and C-reactive protein levels in blood fluctuated during the disease process, but the two indicators of fatal cases with H7N9-A. baumannii co-infection remained at a higher level during a longer period compared to survived cases  named SMGC-AB2  II group, including MDR-ZJ06, MDR-TJ, BJAB0868 and BJAB07104 from Mainland China, TYTH-1 and TCDC-AB0715 from China Taiwan and AB1656–2 from Korean  and Lys → Thr (L533 T) substitution, respectively. The 5th to 7th mutations causing Val → Ile (V183I and V292I) are all located in a large repetitive protein harboring type I secretion C-terminal target domain. The 4th mutation resulting in no amino acid substitution appeared in a gene encoding NAD-dependent aldehyde dehydrogenase, and the 8th lies in the non-coding region.When comparing the genome of SMGC-AB1 with its polymyxin resistance descendant, SMGC-AB2, only 8 nucleotide point mutations were identified in SMGC-AB2, 7 of which were located in coding regions and 6 lead to nonsynonymous mutation , beyond the functional sulfatase domain (aa 237–532), may not contribute to the resistance. It is reported that A. baumannii can also develop resistance to polymyxin by mutating genes responsible for LPS production [It is surprising that SMGC-AB1 quickly acquired resistance to polymyxin, an antibiotic that is considered to be the last hope to cope with MDR and or XDR gram-negative bacteria. Polymyxins are cyclic cationic peptides with a long hydrophobic tail that interact with the lipid A moiety of lipopolysaccharide (LPS) to disrupt the integrity of outer membranes of gram-negative bacteria. Three major resistance mechanisms to polymyxin in bacteria have been identified: modification of the bacterial outer membrane lipopolysaccharide, proteolytic cleavage of the drug and activation of broad-spectrum efflux pumps, among which modification of lipid A of LPS regulated by the two-component regulatory system PmrAB has been frequently reported in aumannii . It is raumannii . In thislymyxins . In clinoduction . Though A. baumannii and its impacts on the disease severity in H7N9 patients. The dysfunctional immunity in the H7N9 patients correlated to the A. baumannii co-infection. Furthermore, the genome variations of A. baumannii contributed to the acquired polymyxin resistance in the patient with fatal outcome. We suggest that the enhancement of the anti-nosocomial infection measures for the prevention of A. baumannii in the H7N9 patients with risk factors for secondary infections, and the early administration of appropriate antibiotic regimen when such co-infection is detected by frequent microbiological testing.In conclusion, we described the occurrence of secondary infection of A. baumannii co-infection in in patients with avian influenza A (H7N9) virus infection. The occurrence of H7N9 co-infection with A. baumannii is a key factor for the severity of the patients, with a manifestation of lower oxygenation indexes and chest radiograph scores. Dysregulation of immune responses in the H7N9 patients correlates to the susceptibility of A. baumannii co-infection and severe clinical manifestation. Novel polymyxin resistance-associated genome variations of A. baumannii could quickly emerge during the disease process and may contribute to fatal outcome of the H7N9-A. baumannii co-infected patients. Both the immune and bacterial features in the patients may contribute to and act as biomarkers for the severe pneumonia and fatal outcome of the co-infection. These conclusions shed light on the pathomechanism, diagnosis and treatment of H7N9 and bacterial co-infections.Invasive mechanical ventilation is the most significant risk factor for the nosocomial Additional file 1:Table S1. The meta analyses of A. baumannii infection in H7N9 patients. (DOCX 59 kb)Additional file 2:Table S2. Basic characters of the patients. (DOCX 34 kb)Additional file 3:Table S3. Clinical presentation and main lab-findings on admission. (DOCX 34 kb)Additional file 4:Table S4. The multivariate analysis of the risk factors of H7N9 patients co-infected by A. baumannii (n = 13) compared to H7N9 control patients (n = 9). (DOCX 32 kb)Additional file 5:Table S5. The multivariate analysis of the clinical risk factors and indicators of H7N9 patients co-infected by A. baumannii (n = 13) compared to the group (n = 24) combined of H7N9 control and A. baumannii control patients. (DOCX 32 kb)Additional file 6:Figure S1. The longitudinal variation of oxygenation index (PaO2/FiO2) in the H7N9 patients co-infected by A. baumannii. (PDF 190 kb)Additional file 7:Figure S2. The association of T-cell counts with oxygenation index (PaO2/FiO2) in the H7N9 patients. (PDF 237 kb)Additional file 8:Figure S3. The longitudinal trends of CD3+ T-cell counts, CD4+ T-cell counts and CD8+ T-cell counts among the H7N9 patients. (PDF 210 kb)Additional file 9:Figure S4. ROC curve of the plasma levels of cytokines and T-cell characteristics in H7N9 patients. (PDF 431 kb)Additional file 10:Figure S5. The longitudinal variation of PCT in the H7N9 patients co-infected by A. baumannii. (PDF 185 kb)Additional file 11:Figure S6. The longitudinal variation of CRP in the H7N9 patients co-infected by A. baumannii. (PDF 188 kb)Additional file 12:Table S6. Antibiotic susceptibility profiles for A. baumannii SMGC-AB1 and SMGC-AB2. (DOCX 32 kb)Additional file 13:Figure S7. Phylogenetic position of A. baumannii SMGC-AB1 and its polysaccharide antigen gene clusters. (PDF 624 kb)Additional file 14:Figure S8. Comparison of gene distribution in Subsystem subcategory “Resistance to antibiotics and toxic compounds”. (PDF 337 kb)