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BACKGROUND Pancreaticoduodenectomy combined with revascularization (PDR) is the main surgical procedure for resectable pancreatic ductal adenocarcinoma (PDAC) with venous system invasion, but this procedure is discouraged in elderly patients because of physical complexity. Our aim was to explore the differences of perioperative and survival in patients of different ages who underwent PDR. MATERIAL AND METHODS We reviewed data from PDAC patients undergoing PDR from 2007 to 2018. Patients were subdivided into 3 groups according to age:60 years, 60-70 years, and ≥70 years. Postoperative complications and long-term survival were compared among the 3 groups. RESULTS From 626 patients, 185 had en bloc venous resection who underwent PDR (103, 55, and 27 patients from young to elderly). Increasing age was linked to a higher prevalence of ICU management (P=0.035) and more serious complications (grade ≥III, P=0.043); overall mortality was 8.1% and did not significantly differ among age-matched groups. Further, there was no difference in overall survival (OS) or progression-free survival (PFS) based on age (60, 60-70, ≥70, median OS were 9.7, 8.4 vs 9.1 months, respectively, P=0.787; median PFS were 6.9, 6.1 vs 8.4 months, respectively, P=0.603). However, patients60 years whose tumors invaded the superior mesenteric vascular had better survival outcomes when compared with the other 2 groups (11.5 vs 8.4, 9.1 months, P=0.049). CONCLUSIONS The results show that age should not be considered an absolute contraindication for PDR, as elderly patients can achieve the same surgical efficacy and long-term survival prognosis.

Numerous long noncoding RNAs (lncRNAs) are aberrantly expressed in pancreatic cancer (PC); however, their functions and mechanisms in cancer progression are largely unknown. In this study, we identified a novel PC-associated lncRNA, RUNX1-IT1, that was significantly upregulated in PC patient samples from multiple centers and associated with poor prognosis. In vitro and in vivo, alterations in RUNX1-IT1 expression markedly affected PC proliferation, migration and invasion. RUNX1-IT1 contributed to the progression of PC by interacting with the adjacent gene RUNX1. Rescue experiments showed that RUNX1 reduced the cancer-promoting effect of RUNX1-IT1. RNA-seq analysis after silencing RUNX1-IT1 and RUNX1 highlighted alterations in the common target C-FOS. Mechanistically, we demonstrated that RUNX1-IT1 was a trans-acting factor that participated in the proliferation, migration and invasion of PC by recruiting RUNX1 to the C-FOS gene promoter. Furthermore, RUNX1-IT1 enhanced the transcription of the RUNX1 gene, indicating its potential as a cis-regulatory RNA involved in the upstream regulation of RUNX1. Overall, RUNX1-IT1 is a crucial oncogenic lncRNA that activates C-FOS expression by regulating and recruiting RUNX1 and is a potential prognostic biomarker and therapeutic target for PC.

Background Although gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro. Methods Immunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178. Results We found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan–Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters. Conclusions Our results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

Background C-Myc aberrations confer a more aggressive clinic behavior in diffuse large B-cell lymphoma (DLBCL). Matrine is an alkaloid extracted from Sophora flavescens Ait. It possesses anti-cancer property through inhibiting the cell proliferation and inducing the apoptosis. The present study aimed to explore the underlying mechanisms of matrine in suppressing the cell growth of DLBCL. Methods The influence of matrine on the viability of cultured DLBCL cell lines SU-DHL-16 and OCI-LY3 cells were determined by CCK-8. Apoptosis and cell cycle were measured by flow cytometry after matrine exposure. Western blot was taken to investigate the expression of activated Caspase-3, cleaved PARP, c-Myc, phospho-c-Myc (Ser62), CaMKIIγ, phospho-CaMKIIγ (Thr287), CDK4 and CDK6 after matrine treatment. Cycloheximide chase analysis was used to determine the c-Myc protein half-lives before and after matrine treatment. Growth salvage analysis was taken by ectopic expression of c-Myc. Results In cultured DLBCL cells, matrine suppressed cell viability in a concentration and time dependent fashion. Matrine treated SU-DHL-16 and OCI-LY3 cells for 48 h with IC50 value of 1.76 mM and 4.1 mM, respectively. Matrine induced apoptosis through a caspase-independent pathway and caused G0/G1 cell cycle arrest in a concentration dependent manner in DLBCL cells. The protein expression of c-Myc was inhibited while the transcription of c-Myc was not reduced by matrine. c-Myc protein half-lives were decreased from 30.4, 69.4 min to 16.6, 15.9 min after matrine treatment in SU-DHL-16 and OCI-LY3, respectively. As a critical protein kinase of c-Myc, CaMKIIγ phosphorylation at Thr287 was found to be down-regulated and c-Myc phosphorylation at Ser62 was reduced together after matrine treatment in DLBCL. The growth suppression of SU-DHL-16 cells induced by matrine was rescued by over-expression of c-Myc achieved by recombinant adenovirus infection. The decreased expression of CDK6, not CDK4, induced by matrine was rescued by ectopic expression of c-Myc protein. Conclusions This study has shown for the first time that matrine suppresses cell growth of DLBCL via inhibiting CaMKIIγ/c-Myc/CDK6 signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is severely affecting the health and lives of patients. Clarifying the composition and regulatory factors of tumor immune microenvironment (TIME) is helpful for the treatment of PDAC. We analyzed the unique TIMEs and gene expression patterns between PDAC and adjacent normal tissue (ANT) using Gene Expression Omnibus (GEO) to find new immunotherapy targets. The Cancer Genome Atlas (TCGA) datasets were used to elucidate the possible mechanism of which tumor-associated macrophages (TAMs) changed in PDAC. We found that the composition of TAMs subtypes, including M0, M1, and M2, was different between PDAC and ANT, which was validated in recently published single-cell RNA-seq data. Many immune cells interacted with each other to affect the TIME. There were many DEGs enriched in some pathways that could potentially change the immune cell composition. KRT6A was found to be a DEG between PDAC and ANT that overlapped with DEGs between the M0-high group and the M0-low group in TCGA datasets, and it might alter and regulate TAMs via a collection of genes including COL5A2, COL1A2, MIR3606, SPARC, and COL6A3. TAMs, which could be a target of immunotherapy, might be influenced by genes through KRT6A and indicate an undesirable prognosis in PDAC.

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261C). The structure revealed a well defined electron density for p261C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å2. Comparative structural analysis of p59–p261C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.

Background: Due to the limitations of strategies for its early diagnosis and treatment, pancreatic cancer (PC) remains a substantial human health threat. We previously discovered a methylation-mediated lncRNA, LINC00261, which is downregulated in PC tissues. However, the underlying role of LINC00261 in PC remains largely unknown. Methods: Quantitative real-time PCR and in situ hybridization were performed to evaluate the expression levels of LINC00261 in PC, adjacent nontumor and normal pancreas tissues. The clinical significance of LINC00261 was assessed in multicenter PC samples. The functions of LINC00261 in PC were investigated by gain- and loss-of-function assays in vitro and in vivo. Potential downstream pathways and mechanisms were explored via RNA sequencing and bioinformatic analyses. RNA immunoprecipitation and chromatin immunoprecipitation assays were used to validate the underlying mechanisms. Pyrosequencing and targeted demethylation of the LINC00261 promoter were performed to explore the upstream epigenetic mechanisms and therapeutic potential. Results: LINC00261 was significantly downregulated in PC tissues, and its expression was positively associated with the prognosis of PC patients. Phenotypic studies indicated that LINC00261 overexpression significantly suppressed PC cell proliferation, migration and metastasis in vitro and in vivo. c-Myc was identified as a downstream target of LINC00261. LINC00261 repressed c-Myc transcription by physically interacting and binding with the bromo domain of p300/CBP, preventing the recruitment of p300/CBP to the promoter region of c-Myc and decreasing the H3K27Ac level. Moreover, the methylation level of the LINC00261 promoter was high in PC tissues and was correlated with poor prognosis. Targeted demethylation of the LINC00261 promoter inhibited PC progression both in vitro and in vivo. Conclusions: Our findings indicate that methylation-mediated LINC00261 suppresses PC progression by epigenetically repressing c-Myc expression. These findings expand the therapeutic potential of LINC00261, possibly providing evidence to support the development of epigenetic drugs or therapeutic strategies. This research adds further insights into the etiology of PC and indicates that LINC00261 may be a prognostic and therapeutic target in PC.

Additional file 1: Supplementary Figure 1. Matrine induced the expression of apoptosis-related proteins in NKTCL cells. NK92 cells (5 × 105) were treated with 1.96 mM matrine for 48 h, followed by western blot. NK92 cells treated without matrine were used as control. (A) Representative WB result of Caspase-3 and cleaved Caspase-3. (B) Representative WB result of PARP and cleaved PARP. (C) Representative WB result of Bcl-2. (D) Representative WB result of Bax. (E) WB result of GAPDH, the loading control for A, B, C and D. Supplementary Figure 2. Matrine induced the expression and phosphorylation of STAT3 in NKTCL cells. NK92 cells (5 × 105) were treated with 1.96 mM matrine for 48 h, followed by western blot. NK92 cells treated without matrine were used as control. (A) Representative WB result of phosphorylation of STAT3 at Tyr705. (B) Representative WB result of STAT3. (C) WB result of GAPDH, the loading control for A and B. Supplementary Figure 3. Matrine decreased the expression of c-Myc protein in NKTCL cells. NK92 cells (5 × 105) were treated with 1.96 mM matrine for 48 h, followed by western blot. NK92 cells treated without matrine were used as control. (A) Representative WB result of c-Myc. (B) WB result of GAPDH, the loading control for A. Supplementary Figure 4. Matrine promoted c-Myc protein degradation in NKTCL cells. Cycloheximide chase assay was used for the half-time of c-Myc protein. NK92 cells (1 × 106) were treated with or without 1.96 mM matrine for 12 h. Cells were then treated with cycloheximide (100 μg/mL) for the indicated minutes, and western blotting was performed. NK92 cells treated without matrine were used as control. (A) Representative WB result of c-Myc in matrine treated NK92 cells. (B) WB result of GAPDH, the loading control for A. (C) Representative WB result of c-Myc in the control NK92 cells. (D) WB result of GAPDH, the loading control for C. Supplementary Figure 5. MG132 prevented matrine-induced c-Myc protein degradation in NKTCL cells. NK92 cells (5 × 105) were treated with 1.96 mM matrine, 10 μM MG132 with or without 1.96 mM matrine, respectively, for 6 h, followed by western blot. NK92 cells treated without matrine and MG132 were used as control. (A) Representative WB result of c-Myc. (B) WB result of GAPDH, the loading control for A. Supplementary Figure 6. Matrine inhibited NKTCL cells through CaMKIIγ/c-Myc pathway. NK92 cells (5 × 105) were treated with 1.96 mM matrine for 48 h, followed by western blot. NK92 cells treated without matrine were used as control. (A) Representative WB result of p-c-Myc (Ser62). (B) Representative WB result of c-Myc. (C) Representative WB result of CaMKIIγ. (D) Representative WB result of LMP1. (E) WB result of GAPDH, the loading control for A, B, C and D.

Pancreatic cancer (PC) is a severe disease with the highest mortality rate among various cancers. It is urgent to find an effective and accurate way to predict the survival of PC patients. Gene set variation analysis (GSVA) was used to establish and validate a miRNA set-based pathway prognostic signature for PC (miPPSPC) and a mRNA set-based pathway prognostic signature for PC (mPPSPC) in independent datasets. An optimized miPPSPC was constructed by combining clinical parameters. The miPPSPC, optimized miPPSPC and mPPSPC were established and validated to predict the survival of PC patients and showed excellent predictive ability. Four metabolic pathways and one oxidative stress pathway were identified in the miPPSPC, whereas linoleic acid metabolism and the pentose phosphate pathway were identified in the mPPSPC. Key factors of the pentose phosphate pathway and linoleic acid metabolism, G6PD and CYP2C8/9/18/19, respectively, are related to the survival of PC patients according to our tissue microarray. Thus, the miPPSPC, optimized miPPSPC and mPPSPC can predict the survival of PC patients efficiently and precisely. The metabolic and oxidative stress pathways may participate in PC progression.

The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.

BACKGROUND: To identify key microRNAs (miRNAs) and their target mRNAs related to gemcitabine-resistant pancreatic cancer (PC) and investigate the association between gemcitabine-resistant-related miRNAs and mRNAs and immune infiltration. METHODS: Expression profiles of miRNAs and mRNAs were obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed miRNAs and mRNAs (referred to as “DEmiRNAs” and “DEmRNAs”, respectively) were distinguished between gemcitabine-resistant PC cells and its parental cells. The DEmRNAs targeted by the DEmiRNAs were retrieved using miRDB, microT, and Targetscan. Furthermore, GO and KEGG pathway enrichment analysis and GSEA were performed. The Kaplan-Meier plotter was used to analyze the prognosis of key DEmiRNAs and DEmRNAs on PC patients. The relationship between the key DEmRNAs and tumor-infiltrating immune cells in PC was investigated using CIBERSORT method using the LM22 signature as reference. Key infiltrating immune cells were further analyzed for the associations with prognosis of TCGA PAAD patients. RESULTS: Four DEmiRNAs, including hsa-miR-3178, hsa-miR-485-3p, hsa-miR-574-5p, and hsa-miR-584-5p, were identified to target seven DEmRNAs, including MSI2, TEAD1, GNPDA1, RND3, PRKACB, TRIM68, and YKT6, individually, in gemcitabine-resistant PC cells versus parental cells. Gemcitabine-resistant PC cells were enriched in proteasome-related, immune-related, and memory CD4(+) T cell-related pathways, indicating a gemcitabine therapeutic effect on PC cells. All four DEmiRNAs and almost all DEmRNAs had an impact on the prognosis of PC patients. All seven DEmRNAs had remarkable effects on CD4(+) memory T cells, which were affected by the gemcitabine therapeutic effect. Effector memory CD4(+) T cells rather than central memory CD4(+) T cells predicted a good prognosis according to the TCGA PAAD dataset. CONCLUSIONS: Gemcitabine resistance can alter the fraction of memory CD4(+) T cells via hsa-miR-3178, hsa-miR-485-3p, hsa-miR-574-5p and hsa-miR-584-5p targeted MSI2, TEAD1, GNPDA1, RND3, PRKACB, TRIM68, and YKT6 network in PC.

Pancreatic ductal adenocarcinoma (PDAC) is a genetic disease and a leading cause of cancer-related mortality. However, the molecular mechanism underlying PDAC progression remains unclear. In this study, we first confirmed that ECM1 is significantly upregulated in PDAC tissues and that its high levels of expression are closely associated with an advanced histologic grade and a poor prognosis using The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. We then found that miR-23a-5p binds directly to the ECM1 3’-untranslated region (3’-UTR), thereby inhibiting ECM1 expression. Functional studies revealed that the induced expression of ECM1 promoted oncogenic abilities and reversed the suppressive effects induced by miR-23a-5p. Collectively, our findings indicate that ECM1 is a proto-oncogene and show that targeting the miR-23a-5p/ECM1 axis may represent a promising therapeutic strategy for PDAC.

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space group P1, with unit-cell parameters a = 86.2, b = 88.9, c = 94.68 Å, α = 93.82, β = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.

In humans and other mammals, the α-chain of interleukin-2 (IL-2) receptor (CD25) is induced and expressed on the cell surface after lymphocyte activation and is released from the membrane of activated cells as a smaller soluble form (sCD25). However, little is known about avian sCD25. In the present study, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) to detect serum sCD25 in ducks, and we used flow cytometry (FCM) to analyze the frequency of CD25(+) cells in the peripheral blood of ducks infected with H9N2 or H5N1 avian influenza virus (AIV) or serotype II Riemerella anatipestifer (RA). Using the AC-ELISA, duck sCD25 molecules were detected in the supernatant and lysates of concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC), and in the serum of ducks infected with H5N1 virus and RA. However, no sCD25 was detected in the serum of H9N2 AIV-infected ducks. FCM analysis revealed that CD25(+) cells were upregulated within the PBMC of RA-infected ducks throughout the experiment until death, while in the PBMC of H9N2- and H5N1 AIV-infected ducks, the frequency of CD25(+) cells increased in the early stage of infection and then returned to a lower level. Our findings confirm that the dynamics of sCD25 and CD25(+) cells are different in the peripheral blood of ducks infected with H9N2 virus, H5N1 virus, and RA.

The cytokine interleukin (IL)-2 functions as a growth factor and central regulator in the immune system. Using a recombinant goose IL-2 (goIL-2) monomer expressed in prokaryotic cells as an immunogen, we synthesized 5 goIL-2 neutralizing mAbs to identify the functional domains of goIL-2, and used these mAbs to finely map the functional domains of the goIL-2 protein. The mimotopes of the 5 anti-goIL-2 mAbs, including HHDPWDXLP, ESLSRXXMXXLXP, SHHLPTSXL, HPDPWDAPLSS, and HEPWQLXL, were identified using a phage display library and peptide-competitive enzyme-linked immunosorbent assay (ELISA). These mimotopes constitute 1 conformational functional domain in the goIL-2 molecule—T 11 I 14 K 15 D 16 E 18 K 19 L 20 G 21 T 22 S 23 M 24 K 25 L 29 E 30 L 31 Y 32 T 33 P 34 E 36 S 41 W 42 Q 43 T 44 L 45 Q 46 (domain I). The neutralizing mAbs to goIL-2 inhibited the in vitro lymphocyte proliferation stimulated by domain I peptides of goIL-2. A tertiary structural model of goIL-2 showed that domain I is positioned in helix A, long A–B loop, and the N-terminal of helix B. These data provide a clue for defining the interaction between goIL-2 and its receptor.

Interleukin 2 (IL-2) plays an important role in the growth and differentiation of lymphocytes. To identify the functional domains of duck IL-2 (duIL-2), 4 neutralizing monoclonal antibodies (mAbs) to duIL-2 were used to finely map the functional domains of the duIL-2 protein. The mimotopes of 4 anti-duIL-2 mAbs, including LVXGSMPS, KPHKHHXHHSHM, WXXXKAKP, and HVPNERYPLR, were identified by phage display and peptide-competitive ELISA. These mimotopes constitute an important functional domain, Y(32) approximately T(44) (domain I), in the duIL-2 molecule. The bioactivity of the domain I peptide on in vitro lymphocyte proliferation was inhibited by duIL-2Ralpha. A tertiary structure model of duIL-2 showed that domain I is positioned in the long A-B loop and the N-terminal of Helix B. These data provided experimental evidence for elucidating the interaction between duIL-2 and duIL-2Ralpha.

CD132 is the common gamma chain to a number of cytokine receptor complexes including that for IL-2. To identify the functional domain of chicken CD132 (chCD132), the cDNA of chCD132 was cloned, and a neutralizing monoclonal antibody, raised against a recombinant chCD132 protein, was identified by inhibition of IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD132 molecules are expressed on the surface of splenic mononuclear cells. The functional domain of chCD132 that binds to chicken interleukin 2, Q 84 E 94 L 95 Q 96 N 97 L 98 , was found through phage display and peptide-competitive ELISA, and its critical residue Q 96 was further identified. A tertiary structure model shows that the functional domain is positioned at the elbow-like junction of N and C terminal fibronectin-III domains of chCD132. These data provide experimental evidence for elucidating the interaction between chCD132 and chIL-2.

The IL-2Ralpha chain (CD25, Tac) is an essential component of high affinity IL-2Rs, playing critical role for the immune specificity of antigen-activated T-cell clonal expansion. Up to now, no duck cytokine receptor has been described. Here, the cDNA segment of a duck cytokine receptor (duCD25), encoding a 226 aa precursor protein with a 20 aa signal peptide, was isolated. Then a novel mouse monoclonal antibody (mAb) was generated using the prokaryotically expressed duCD25 protein as immunogen. Using this mAb, the endogenous duCD25 molecule was localized on the surface of duck lymphocytes, and the duck IL-2-induced lymphocyte proliferation was further inhibited. Furthermore, flow cytometry analysis showed that duCD25 positive cells were upregulated in ducks infected with avian influenza virus (H9N2). Our findings confirm that duCD25 is a receptor of duck interleukin-2, and duCD25 positive cells play a potential role in H9N2 virus infection.

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5'-triphosphate of RNA and the 3'-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261C). The structure revealed a well defined electron density for p261C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å2. Comparative structural analysis of p59–p261C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme′s conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.

Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tat•P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tat•AFF4•P-TEFb complex containing HIV-1 Tat (residues 1-48), human Cyclin T1 (1-266), human Cdk9 (7-332), and human AFF4 (27-69). Tat binding to AFF4•P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of Cyclin T1 and the α-3 10 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tat•P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.

The interleukin 2 (IL-2) immunoregulatory cytokine produces its biological effects by binding sequentially to its cell receptor subunits, alpha (CD25), beta (CD122), and common gamma chain (CD132). In this study, we identified the critical amino acid residues of chicken IL-2 (chIL-2) for binding to chicken CD25 (chCD25) by an Ag-capture ELISA, screening of a phage display peptide library, peptide-competitive ELISA and an in vitro T-cell proliferation assay. Specific ligand elution of phage bound to chCD25 and chIL-2 suggested that the P(35)T(36)C(41)T(42)Q(43)L(46)Q(47)C(48)Y(49)L(50)G(51) motif within chIL-2 molecule interacts with the S(99)F(100)C(101)G(102)M(103)P(104)Q(105)T(106)V(107)P(108)S(111)L(112) motif of chCD25 molecule (chCD25(99-112)), whereas the peptide competition ELISA assay showed the residues (27)KIHLELYTPTETQEC(41) within chIL-2 (chIL-2(27-41)) bound to the chCD25 protein. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-2(27-41) to the chCD25 molecule was inhibited by the chCD25(99-112) peptide. Site-specific mutation of the (35)P and (41)C residues in chIL-2(27-41) resulted in the lack of its ability to induce lymphocyte proliferation and binding to the chCD25 molecule. These findings demonstrate that chIL-2(27-41) and chCD25(99-112) are the binding domains between the chIL-2 and chCD25 molecules, and that the residues P(35) and C(41) within chIL-2(27-41) are the critical sites for its bioactivity and interaction with chCD25.

Interleukin 2 (IL-2) is an essential cytokine that plays a pivotal role in the replication, maturation and differentiation of lymphocytes. In this study, the functional domains of chicken IL-2 (chIL-2) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. Nine neutralizing mAbs to chIL-2 were produced using the recombinant chIL-2 monomer expressed in prokaryotic cells as an immunogen and used to finely map the functional domains of the chIL-2 protein. The mimotopes of nine anti-chIL-2 mAbs, including KIELPSL, EHLDXNDSLYL, NHLXGXY, WHLPPSL, EFKASXL, TENPFPE, SGLYL, AHGYWEL and HHGYWEL, were respectively identified by phage display and peptide-competitive ELISA. These mimotopes constitute three conformational functional domains in the chIL-2 molecule, that is, N(26)K(27)I(28)H(29)L(30)E(31)L(32)P(35)Q(43)Q(44)T(45)L(46)Q(47)C(48)Y(49)L(50) (domain I), E(68)E(69)F(70)K(79)K(82)S(83)L(84)T(85)G(86)L(87) (domain II) and N(88)H(89)G(91)K(104)F(105)P(106)D(107)E(111)L(112)Y(118)L(119) (domain III). The neutralizing mAbs to chIL-2 inhibited the in vitro lymphocyte proliferation stimulated by three peptide domains of chIL-2. The predicted tertiary structure of chIL-2 reveals that domain I was positioned in the long A-B loop and the N terminal of Helix B, domain II was mostly situated in Helix C, and domain III was distributed in the C-D loop and Helix D. These data demonstrate the functional domains of chIL-2 and provide a clue for elucidating the interaction between chIL-2 and its receptor.

The common cytokine receptor γ chain (γc) is shared by at least six cytokine receptors and plays a critical role in the regulation of immune responses. In this study, the functional domains of chicken γc (chCD132) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. One functional mAb to chCD132 was produced using recombinant rchCD132 ectodomain protein expressed in prokaryotic cells as an immunogen and used to finely map the functional domain of the chCD132 protein. The mimotope of anti-chCD132 mAb, QEHQNL, was identified by phage display. The corresponding native epitope in the chCD132 molecule, Q84E94L95Q96N97L98, was identified by peptide-competitive ELISA. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-279-96 to chCD132 molecule was inhibited by chCD13282-101. Site-specific mutation indicates that chCD13282-101 changed the residue Q96 lacked the ability binding to chIL-2 molecule. These findings demonstrates that chIL-279-96 and chCD13282-101 are the functional domains of the interaction between chIL-2 and chCD132 molecules, and that the residue Q96 within chCD13282-101 is the critical site for its bioactivity and chIL-2-binding.