Myofibrillogenesis regulator 1 (MR-1) is overexpressed in human cancer cells and plays an essential role in cancer cell growth. However, the significance of MR-1 in human ovarian cancer has not yet been explored. The aim of this study was to examine whether MR-1 is a predictor of ovarian cancer and its value as a therapeutic target in ovarian cancer patients.Reverse-transcription polymerase chain reaction (PCR) and quantitative real-time PCR were used to detect MR-1 mRNA levels in tissue samples from 26 ovarian cancer patients and 25 controls with benign ovarian disease. Anti-MR-1 polyclonal antibodies were prepared, tested by ELISA and western blotting, and then used for immunohistochemical analysis of the tissue samples. Adhesion and invasion of 292T cells was also examined after transfection of a pMX-MR-1 plasmid. Knockdown of MR-1 expression was achieved after stable transfection of SKOV3 cells with a short hairpin DNA pGPU6/GFP/Neo plasmid against the MR-1 gene. In addition, SKOV3 cells were treated with paclitaxel and carboplatin, and a potential role for MR-1 as a therapeutic target was evaluated.MR-1 was overexpressed in ovarian cancer tissues and SKOV3 cells. 293T cells overexpressed MR-1, and cellular spread and invasion were enhanced after transfection of the pMX-MR-1 plasmid, suggesting that MR-1 is critical for ovarian cancer cell growth. Knockdown of MR-1 expression inhibited cell adhesion and invasion, and treatment with anti-cancer drugs decreased its expression in cancer cells. Taken together, these results provide the first evidence of the cellular and molecular mechanisms by which MR-1 might serve as a novel biological marker and potential therapeutic target for ovarian cancer.MR-1 may be a biomarker for diagnosis of ovarian cancer. It may also be useful for monitoring of the effects of anti-cancer therapies. Further studies are needed to clarify whether MR-1 is an early diagnostic marker for ovarian cancer and a possible therapeutic target. Ovarian cancer is the leading cause of cancer-related death in women worldwide. The American Cancer Society revealed that 21,550 women in the US were diagnosed with ovarian cancer and 14,600 women died of the disease in 2009 [Myosin light chain-2 (MLC2) plays an important role in cell migration from solid tumors such as ovarian cancer, and its dephosphorylation can induce apoptosis ,4. A recTaking all the evidence into account, we hypothesized that MR-1 may play a role in the development and progression of ovarian cancer, probably by promoting cell proliferation and invasion. The present study examined MR-1 expression in ovarian cancer tissues and a cancer cell line. Overexpression or knockdown of MR-1 in cancer cells was used to assess its role in cell proliferation, adhesion, and invasion. Finally, the response of MR-1 to treatment with anti-cancer drugs was assessed to identify whether it functions as a novel biological marker and therapeutic target for ovarian cancer.All human samples were collected in compliance with the guidelines of the Ethics Committee at the Fudan University Cancer Hospital. Fresh-frozen surgical ovarian tissue samples were collected from 26 patients with ovarian cancer (aged 20-58 years) and 20 control patients with benign ovarian disease (aged 23-55 years) who were admitted to Fudan University Cancer Hospital  between July 2008 and December 2009. All cases were confirmed by pathology. The samples were defatted, immediately cut into pieces of appropriate size on ice, and stored at -80°C for later use. The ovarian carcinoma cell line, SKOV3, and the non-ovarian cancer cell line, 293T, were generous gifts from Dr. Meiqin Zhang, Laboratory of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai.® Premix Ex Taq™ reagent , 0.5 pmol of each primer, 1 μg of cDNA, and the same sets of primers and cDNAs. The conditions were: an initial denaturation step at 94°C for 10 min, followed by 40 cycles of 15 s at 95°C (denaturation), 30 s at 55°C , and 30 s at 72°C (extension). Delta Ct (CtMR-1-CtGAPDH) was calculated for each set of samples and compared.Total RNA was extracted from the cells using the TRIzol reagent  according to the manufacturer's protocol, and the RNA concentration was determined using a Nanodrop spectrophotometer . RT-PCR was carried out using a SuperScript™ one-step RT-PCR kit (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 2 μg of total RNA at 42°C for 45 min, followed by inactivation of the reverse transcriptase at 85°C for 5 min. The cDNA was then subjected to PCR to amplify the MR-1 and GAPDH (control) genes in 50 μL reaction mixture containing the following primers: MR-1 forward 5'-CCCAGAAAGAGGG GCAAGA-3' (P1), MR-1 reverse 5'-TGAGGATGAAGAG GAGGATACCA-3' (P2), GAPDH forward 5'-GGGAGCCAAAAGGGTCATCATCTC-3' (P1) GAPDH reverse 5'-CCATGCCAGTGAGCTTC CCGTTC-3' (P2). The cycling conditions were: initial denaturation at 95°C for 3 min, followed by 36 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. PCR products were resolved and analyzed on a 2% TAE agarose gel containing ethidium bromide. Quantitative real-time PCR for MR-1  was performed in an MJ PCR system  using a 20 μL reaction volume containing 10 μL of SYBRTaq PCRx® DNA polymerase (Invitrogen) to introduce BamHI and XhoI restriction sites using the primers 5'-CGGGATCCATGGCGGCGGTGGTAGCTGC-3'  and 5'-CCGCTCGAGTCAGGTCTGCACCCCAGAC-3' . The cDNA fragment was then cloned into the PET21a (+) expression vector (Invitrogen) to yield PET21a-MR1, which was then transformed into Escherichia coli BL21 (DE3) cells for expression of the MR-1 recombinant protein [Human MR-1 cDNA was synthesized by reverse transcription from the mRNA . A 429- protein . The MR-New Zealand white rabbits were immunized four times with the MR-1 recombinant protein. Sera were harvested 3 days after the final immunization if the rabbits showed a good anti-MR-1-His titer. The anti-MR-1 antibodies R1 and R2 were then affinity-purified using a protein A column . The antibody titers were measured using an enzyme-linked immunosorbent assay (ELISA), and immunoreactivity was confirmed by western blotting with MR-1 recombinant protein and human ovarian cancer cells (SKOV3).Immunohistochemical staining was performed on 6-μm sections from formalin-fixed, paraffin-embedded human tissues. The slides were deparaffinized and rehydrated in graded ethanol solutions. Antigen retrieval was performed by heating the samples in a microwave oven for 20 min in citrate buffer (pH 6.0). The slides were then incubated with the affinity-purified MR-1 antibody (1:200) followed by incubation with a horseradish peroxidase-labeled polymer conjugated to a goat anti-rabbit immunoglobulin antibody . Color was developed using diaminobenzidine as a chromogen and the slides were counterstained with hematoxylin. An anti-His antibody and polyclonal non-immune rabbit IgG were used as negative controls to check the specificity of the MR-1 affinity-purified antibody. Immunocytochemical staining of SKOV3 cells was performed on cover slips by fixing the cells for 20 minutes in acetone, blocking with 2% bovine serum albumin, and then incubating with affinity-purified anti-MR1 antibody followed by DAB staining and hematoxylin counterstaining. Dark brown cytoplasmic staining was defined as positive and no (or faint) staining was defined as negative. The proportion of tumor cells positive for MR-1 varied considerably: diffuse positive (> 50%), focal or heterogeneous (10%-50%), and trace (< 10%).®  according to the manufacturer's instructions. A pMX-mock vector (pMX-puro(+) containing a random non-MR-1 DNA sequence) was used as the control. Puromycin  was added to the cells 48 h after transfection to select stably transfected clones. Single clones were picked from both pMX-MR-1 and pMX-mock transfected samples, and RNA and protein were extracted to check the expression levels of MR-1 by real-time PCR and western blotting. Positive clones were designated pMX-MR-1-293T, and control clones as pMX-mock-293T. 293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with high glucose, 10% fetal bovine serum , and 1% penicillin/streptomycin at 37°C in 5% CO2.The full-length coding sequence for the MR-1 cDNA was subcloned into the pMX-puro(+) vector (Invitrogen) to yield pMX-MR-1, which was then transfected into 293T cells using Fusion 62. After 16 hours, the cells on the upper side of the well were removed and fixed prior to staining with hematoxylin and eosin. Cells migrating to the underside were counted under a microscope. All experiments were repeated at least three times.For the cell proliferation assay, pMX-MR-1-293T and pMX-mock-293T cells were grown at a density of 1000 cells/ml in 24-well plates with regular medium changes. The cell number was counted every 24 hours for 6 days as follows: cells were detached by brief exposure to 0.025% trypsin containing 2 mM EDTA in PBS, washed in culture medium without FBS, and then resuspended in the same medium for manual cell counting. For the invasion assays, Matrigel was thawed at 4°C overnight, diluted in cold serum-free culture medium, plated onto 24-well plates preloaded with Transwell™ culture inserts  and incubated for 5 hours at 37°C for gelling. The cells were then plated onto the Transwell™ inserts  and cultured at 37°C in 5% CO® RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. When the cells reached 70-80% confluence, they were transfected with pGPU6-MR-1 short hairpin DNA (shDNA) targeting MR-1. The sequences of the shDNAs were as follows: shDNA 232: 5'-CACCGCTAACAAGGCTTCTCATAACTTCAAGAGAGT TATGAGAAGCCTTGTTAGCTTTTTG-3'; shDNA 442: 5'-CACCGACCGTGTGA AGCAGATGAAGTTCAAGAGACTTCATCTGCTTCACACGGTCTTTTTTG-3'; shDNA 584: 5'-CACCGACCCTCCTAGGCTATTGACTTTCAAGAGAAGTCAATA GCCTAGGAGGGTCTTTTTTG-3'; mock sequence: 5'-CACCGTTCTCCGAACGT GTCACGTCAAGAGATTACGTGAGACGTTCGGAGAATTTTTTG-3'. The pGPU6-shDNA plasmids were constructed by cloning the respective shDNAs into the pGPU6/GFP/Neo vector (Invitrogen). The cells were exposed to 1500 μg/ml G418 (Invitrogen) for 2 days after transfection to select for stably transfected clones. Cells were then plated at a lower density in 96-well plates containing RPMI 1640 and G418 (750 μg/ml) until single colonies were formed. Colonies from the SKOV3-MR-1-shDNA and SKOV3-mock-shDNA-transfecetd cells were then picked and expanded. Real-time PCR and western blotting were used to test the expression levels of MR-1. GAPDH was used as the internal control.SKOV3 cells were cultured in GibcoSKOV3 cells  were cultured in six-well plates in RPMI 1640 under the conditions described above. After 24 hours, 10 μL of serially diluted paclitaxel  or carboplatin   were added to the medium and the cells were cultured for an additional 48 hours. One set of cells was treated with TUNEL reagent (Roche) and counterstained with DAPI, and the number of apoptotic cells (stained by Annexin V-FITC/PI) was determined by flow cytometry . The other set of cells was harvested for RNA and protein extraction, and the expression levels of MR-1 were determined by real-time RT-PCR and western blotting.P value < 0.05 was considered significant.Statistical analysis was performed using Analysis of Variance (ANOVA) and the post-hoc Student-Newman-Keuls test, with SPSS software version 13.0 . The results were expressed as the mean ± SD. A n = 26) than in control patients (n = 25)  compared with SKOV3-shDNA-mock cells Figure . Both thTo identify whether MR-1 is a potential therapeutic target, SKOV3 cells were treated with different concentrations of the anti-cancer drugs, paclitaxel and carboplatin. Untreated SKOV3 cells grew as usual, with a spear-like shape. After treatment with paclitaxel or carboplatin for 48 hours, the number of cells decreased slightly and showed morphological changes. As the drug concentrations increased, the cells gradually shrank and their nuclei developed apoptosis-like changes, such as the appearance of green TUNEL-positive cores and the aberrant accumulation of granules within the nuclei Figure . MR-1 exCancer progression and metastasis is a highly complex multi-stage process involving alterations in gene expression, increased cell adhesion, and changes in cell motility. Cancer cells move within tissues during invasion and metastasis, and cell invasion involves multiple processes that are regulated by various molecules . OvarianThere are no reports examining the role of MR-1 in ovarian cancer. This is the first study showing that expression of MR-1 is increased in ovarian cancer tissues at both the mRNA and protein levels compared with benign control tissues. The immunohistochemistry results show that MR-1 is abundantly expressed in tumor tissues from ovarian cancer patients, particularly those with serous papillary ovarian cancer Figure , and thaMR-1-stably-transfected 239T cells were established to investigate the potential role of MR-1 in cell invasion and tumor progression Figure  and 4C. To assess whether MR-1 is a potential therapeutic target, SKOV3 cells were treated with the anti-cancer drugs paclitaxel and carboplatin, both of which induce apoptosis and are commonly used to treat ovarian cancer . The numThis study shows that MR-1 is overexpressed in ovarian cancer tissue and cell lines. Knockdown of MR-1 expression inhibits cell adhesion and invasion, and anti-cancer drugs decrease the expression levels of MR-1 in cancer cells. Thus, MR-1 may be a novel biological marker and potential therapeutic target for the treatment of ovarian cancer. It could also be used to monitor the effect of anti-cancer therapies. Further studies are needed to clarify whether MR-1 is an early diagnostic marker for ovarian cancer and to develop its full therapeutic potential.MR-1: myofibrillogenesis regulator 1; PcAb: polyclonal antibody; RT-PCR: reverse-transcription polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; shDNA: short hairpin DNA; MLC2: myosin light chain-2.The authors declare that they have no competing interests.RQL designed the study, detected MR-1 expression levels in the transduced cells, measured cell adhesion and invasion, and drafted the manuscript. MS collected all the tissue samples and analyzed the expression levels of MR-1. JJF produced the MR-1 recombinant protein and anti-MR-1 antibodies and performed immunohistochemical staining. XG identified and characterized apoptosis induced by the anti-cancer drugs and edited the final manuscript. LG conceived of and supervised the project and reviewed the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/270/prepub


The introduction of C-C chemokine receptor type-5 (CCR5) antagonists as antiretroviral therapy has led to the need to study HIV co-receptor tropism in different HIV-1 subtypes and geographical locations. This study was undertaken to evaluate HIV-1 co-receptor tropism in the developing world where non-B subtypes predominate, in order to assess the therapeutic and prophylactic potential of CCR5 antagonists in these regions.+ and CD8+ T cell counts.HIV-1-infected patients were recruited into this prospective, cross-sectional, epidemiologic study from HIV clinics in South Africa, Uganda and India. Patients were infected with subtypes C  or A or D (Uganda). HIV-1 subtype and co-receptor tropism were determined and analyzed with disease characteristics, including viral load and CD4+ count.CCR5-tropic (R5) HIV-1 was detected in 96% of treatment-naïve (TN) and treatment-experienced (TE) patients in India, 71% of TE South African patients, and 86% (subtype A/A1) and 71% (subtype D) of TN and TE Ugandan patients. Dual/mixed-tropic HIV-1 was found in 4% of Indian, 25% of South African and 13% (subtype A/A1) and 29% (subtype D) of Ugandan patients. Prior antiretroviral treatment was associated with decreased R5 tropism; however, this decrease was less in subtype C from India  than in subtypes A  and D . R5 virus infection in all three subtypes correlated with higher CD4+ count correlated with R5 prevalence, while treatment experience was associated with increased non-R5 infection in all subtypes.R5 HIV-1 was predominant in TN individuals with HIV-1 subtypes C, A, and D and TE individuals with subtypes C and A. Higher CD4 Human immunodeficiency virus type-1 (HIV-1) is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have demonstrated that globally, the most prevalent forms of HIV-1 are subtypes (clades) C, B and A . Subtype+ T-cell depletion and disease progression  TE patients). The predominant subtypes in Uganda were A/A1  and D . Other subtypes representing ≥ 1% of the Ugandan cohort were A/D (8%), AE (2.4%), C (2.2%), and Complex (1.5%).Samples from patients with subtype C virus from India and South Africa and subtypes A/A1 and D from Uganda that were initially tested for co-receptor tropism using a single 1 mL aliquot of patient plasma yielded the following reportable results, respectively, when RNA amplification was performed with the standard primers: 240 (86%), 205 (75%), 236 (81%) and 136 (67%)  of both TN and TE patients infected with non-B subtypes . There were difficulties recruiting TE patients in India and Uganda, which may indirectly reflect the availability of ART, estimated by the World Health Organization to reach only 31% of HIV-infected persons in these countries .+ cell count and treatment experience, which can be considered a marker of advanced disease. Indian subjects with subtype C virus were almost exclusively infected with R5 regardless of their CD4+ count, HIV-1 disease status, or treatment experience. The prevalence of R5 virus was similarly high in TN South African patients with subtype C infection but, while most common and relatively well-preserved, was somewhat lower in the South African TE cohort of this study (71%). The difference observed between R5 prevalence in India and South Africa may be a result of differences in CD4+ cell count. Compared with the Indian cohort, there was a higher proportion of individuals in the South African cohort with a D/M phenotype (25%) despite similar time since HIV diagnosis and similar exposure to prior therapies. Exclusive use of X4 was rare (4%).Most (83%) study subjects with reportable tropism results had only R5 HIV-1 detectable, including 90% of TN and 72% of TE patients. However, tropism distribution varied by subtype, CD4env variable loops [Our findings are consistent with previous reports of subtype C strains from India and elsewhere, where R5 HIV-1 is maintained even in advanced stages of disease. For example, some similarities can be observed between the findings of the present study and a previous study that assessed co-receptor tropism of HIV-1 strains in China: mainly that R5 tropism was seen exclusively in subtype C infections, and remained stable over time . These dle loops , and conle loops ,21,37-40The distribution of HIV-1 subtypes in India and South Africa was consistent with published epidemiologic data . However+ cell count as a predictor of R5 infection in both TN and TE patients with subtype B and C HIV-1 infections [+ count as a significant predictor of CCR5 tropism for subtypes C, A/A1, and D. In Ugandan patients with subtype A/A1, R5 use was negatively associated with the presence of CDC category B disease, with a negative trend for category C.Multivariate analyses of other cohorts have identified higher CD4fections ,44. In t® assay to provide results in different non-B subtype HIV-1 viral subtypes collected in developing countries. While initial testing using standard amplification primers with just a single 1 mL aliquot of patient plasma yielded a lower reportable rate (67-86%) than that expected from experience with subtype B virus, non-reportable samples that were re-tested with the original Trofile® assay (South Africa and Uganda) or enhanced assay (India) using modified primers optimized for non-B subtype virus, yielded a much higher percentage of reportable results (90-97%), and the distribution of R5 and non-R5 HIV-1 did not change. These improvements in amplification primers - which also retain high performance for subtype B specimens and are currently the primers used regularly for RNA amplification in the Trofile assay - may be advantageous when Trofile testing is used in clinical populations where non-subtype B virus is common [This study provided an opportunity to assess the ability of the Trofiles common .Globally, these data offer guidance on the potential use of CCR5 antagonists and tropism tests in resource-poor clinical settings. Since HIV transmission appears to be mostly associated with R5 HIV-1 regardless of subtype -48 and a+ cell count was highly correlated with CCR5 use regardless of subtype, a further understanding of HIV-1 co-receptor use in individuals and populations infected with non-subtype B HIV-1 is still needed. Given the heterogeneity of HIV-1 co-receptor use in non-subtype B HIV-1, there remains a need for prospective determination of HIV-1 tropism prior to the initiation of CCR5 antagonist therapy in geographic locations where the burden of infection is greatest.This study examined the correlates of HIV-1 co-receptor tropism in virus from individuals with non-B subtype HIV-1 infection from India, Uganda and South Africa. While only R5 virus was detectable in the majority of TN individuals infected with subtypes C, A and D, infection with only R5 virus was maintained, albeit at lower levels, in TE individuals. However, R5 prevalence varied by subtype: patients with subtype C, especially in India, were almost exclusively infected with R5, while subtype D patients were more frequently infected with D/M viruses. The high prevalence of R5 HIV-1 in subtype C infections makes a CCR5 antagonist an attractive option for ART-based prevention strategies in areas where subtype C predominates. While a higher CD4Quazi Ataher and Simon Portsmouth are employees of Pfizer Inc. and own Pfizer stock. Sybil Eng, Anna Greenacre and Randy Tressler were employees at Pfizer Inc. at the time that this study was conceived and conducted. Randy Tressler currently owns Pfizer stock and is eligible for a Pfizer pension. Laura Napolitano is an employee of Monogram Biosciences and its parent company, LabCorp, and owns LabCorp stock. Andrew Kambugu has received honoraria from Abbott. Robin Wood has received advisory fees from Merck, GlaxoSmithKline, Abbot and Tibotec. Sharlaa Badal-Faesen declares that she has no competing interests.QA conducted analyses of clinical samples and led manuscript preparation. LAN led and performed the standard and optimized primer data analysis. SP and SE participated in data interpretation. AG conducted statistical analyses of study data. AK, RW and SBF participated in study design, enrolled study subjects and collected clinical samples for analysis. RT conceived of the study, and participated in its design and in data interpretation. All authors reviewed and edited the manuscript.


Imiquimod, a nucleoside analogue of the imidazoquinoline family, is being used to treat various cutaneous cancers including squamous cell carcinoma (SCC). Imiquimod activates anti-tumor immunity via Toll-like receptor 7 (TLR7) in macrophage and other immune cells. Imiquimod can also affect tumor cells directly, regardless of its impact on immune system. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells (SCC12) and A20 was involved in this process. When A20 was overexpressed, imiquimod-induced apoptosis was markedly inhibited. Conversely, knockdown of A20 potentiated imiquimod-induced apoptosis. Interestingly, A20 counteracted activation of c-Jun N-terminal kinase (JNK), suggesting that A20-regulated JNK activity was possible mechanism underlying imiquimod-induced apoptosis of SCC12 cells. Finally, imiquimod-induced apoptosis of SCC12 cells was taken place in a TLR7-independent manner. Our data provide new insight into the mechanism underlying imiquimod effect in cutaneous cancer treatment. Squamous cell carcinoma (SCC) is one of epithelial cancers, which is originated from the upper layers of skin epidermis. The incidence of SCC is relatively high, ranking as the second most frequent type among the non-melanoma skin cancers Imiquimod (R-837) is an immune response modifier, activating macrophage and other cells via Toll-like receptor 7 (TLR7). Imiquimod provokes Th1 cell-mediated immune response via inducing the secretion of proinflammatory cytokines such as interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) As notified, NF-κB is the important key player in the control of keratinocyte growth and carcinogenesis. The activity of NF-κB is strictly controlled by sophisticated network of negative and positive regulators. We found that A20, one important negative regulator for NF-κB, was highly increased in SCC cells. Since imiquimod affects NF-κB pathway in a TLR-dependent and/or -independent manner in other systems, we investigate whether the effect of imiquimod is related with A20 in SCC cells. Our data provide evidence that imiquimod induces apoptosis of SCC cells via regulation of A20.This study was approved by the Institutional Review Board of Chungnam National University Hospital. All human skin samples were obtained under the written informed consent of donors.Imiquimod was purchased from Santa Cruz Biotechnologies . The following primary antibodies were used in this study: A20 , PARP , caspase-3, ERK, phospho-ERK, JNK, phospho-JNK, p38 MAPK, phospho-p38 MAPK , TLR7 , GFP (Santa Cruz Biotechnologies), actin .2O2 for 10 min at room temperature, blocked in 0.1% Tween-20, 1% bovine serum albumin (BSA) in PBS for 30 min, and followed by reaction with appropriate primary antibodies. Sections were incubated sequentially with peroxidase-conjugated secondary antibodies and visualized with Chemmate envision detection kit .Paraffin sections were dewaxed, rehydrated, then washed three times with phosphate-buffered saline (PBS). After treatment with proteinase K (1 mg/ml) for 5 min at 37°C, sections were treated with H5, treated with imiquimod for 24 h, then MTT assay was performed.SV40-transformed human epidermal keratinocytes (SV-HEK), melanocytes and fibroblasts were cultured according to the methods previously reported Cells were lysed in Proprep solution . Total protein was measured using a BCA Protein Assay Reagent . Samples were run on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and incubated with appropriate antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies, visualized by enhanced chemiluminescence (Intron).Apoptosis was detected using FITC annexin V apoptosis detection kit (BD Biosciences). After treatment of imiquimod, cells were washed twice with cold PBS and stained with FITC annexin V and propidium iodide (PI). Cells were then analyzed by flow cytometry.5′-AGATCTATGGCTGAACAAGTCCTTCC-3′, and reverse 5′-CTCGAGTTAGCCATACATCTGCTTG-3′. The amplified full-length cDNA for A20 was subcloned into the pENT/CMV-GFP vector that had attL sites for site specific recombination with a Gateway destination vector. Replication-incompetent adenoviruses were created using the Virapower adenovirus expression system (Invitrogen). The adenovirus was purified with cesium chloride Total RNA was isolated from human embryonic kidney cells 293A and used for cloning of A20 cDNA fragment. The primer set for A20 is as follows: forward 5′-CCGGCACTGGAAGAAATACACATATCTCGAGATATGTGTATTTCTTCCAGTGTTTTTG-3′; #2, 5′-GTACCGGAGTTGGATGAAGCTAACTTACCTCGAGGTAAGTTAGCTTCATCCAACTTTTTTTG-3′. SCC12 cells were transduced with lentivirus, then stable cells expressing shRNA-A20 were selected by puromycin treatment. In parallel, stable cells expressing shRNA-scrambled (shRNA-Scr) was also established as a negative control.For knockdown of A20 expression, we used lentivirus expressing short hairpin RNA (shRNA). The shRNA plamid DNA stocks (SHCLNG-NM_006290) were purchased from Sigma-Aldrich , and recombinant lentivirus was produced as previously reported 5′-TGCTGTGAAATCGATCTCTACCAGATGTTTTGGCCACTGACTGACATCTGGTAGATCGATTTCA-3′.For microRNA (miR) specific for TLR7, target sequences were designed using Invitrogen’s RNAi Designer. The double-stranded DNA oligonucleotides were synthesized and cloned into the parental vector pcDNA6.2-GW/EmGFP-miR . The expression cassette for miR was moved into pENT/CMV vector, and then adenovirus was made as previously reported NF-κB is a regulator for antiapoptotic and proinflammatory responses, and recognized as an important player in SCC Imiquimod is being successfully used for treatment of SCC. As notified, imiquimod can affect keratinocytes independently of immune system, we investigated direct effect of imiquimod on SCC cells. When SCC12 cells were treated with imiquimod, cell death was occurred in a dose-dependent manner . WesternTo address a question whether A20 exerts antiapoptotic role, we exogenously overexpressed green fluorescent protein-tagged A20 (GFP-A20) using a recombinant adenovirus. Flow cytometry analysis showed that overexpression of A20 inhibited markedly the imiquimod-induced apoptosis . ConsistRecently, it has been demonstrated that A20 suppresses activation of c-Jun N-terminal kinase (JNK) by degrading apoptosis signal-regulated kinase 1 (ASK1), eventually leading to inhibition of apoptosis To further address the role of A20 in imiquimod-induced apoptosis of SCC12 cells, we established the stable cell lines in which A20 expression was knockdowned by shRNA. Western blot showed that endogenous A20 expression was efficiently knockdowned by shRNA . ConsistCollectively, these data strongly suggest that imiquimod-induced apoptosis is mediated through the suppression of A20 and activation of JNK. In line with this, pretreatment of SCC12 cells with JNK inhibitor (SP600125) significantly inhibited imiquimod-induced cell death , potentiSince imiquimod is a specific TLR7 ligand, we wondered if imiquimod-induced apoptosis of SCC12 cells was dependent of TLR7. To this end, we knockdowned TLR7 expression using a recombinant adenovirus expressing miR-TLR7 . When TLImiquimod activates immune system thereby stimulating tumor destruction and/or preventing cancer growth. Besides its potential for inducing anti-tumor immunity, imiquimod can also affect cancer cells directly. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells and antiapoptotic regulator A20 was involved in this process.A20 is a negative regulator in NF-κB signaling pathway. A20 ubiquitinates receptor interacting protein 1 (RIP1), a critical signaling intermediate protein in tumor necrosis factor (TNF)-mediated NF-κB activation, resulting in proteasomal degradation of RIP1 and termination of NF-κB activation In this study, imiquimod induced activation of JNK, which was effectively inhibited by overexpression of A20. Conversely, knockdown of A20 resulted in JNK activation. Thus it can be suggested that JNK activation is a consequence of imiquimod-driven A20 down-regulation. Interestingly, it has been demonstrated that A20 degrades ASK1, an upstream kinase for JNK activation It is thought that imiquimod-induced apoptosis of SCC12 cells is independent of TLR7, a well-established receptor for imiquimod. Interestingly, previous report indicates that imiquimod induces activation of NF-κB and the downstream production of proinflammatory cytokines in the absence of TLR7. TLR-independent effects of imiquimod have been suggested to stem from its interference with adenosine receptor signaling mediated by adenylyl cyclase. In addition, imiquimod exerts direct or indirect adenosine receptor-independent inhibition of adenylyl cyclase activity In summary, we demonstrate that imiquimod induces apoptosis of SCC cells, and that A20 is a critical player in this process. Our results may contribute to a better understanding of action mechanism of imiquimod on cutaneous cancers, and may help to develop new target for SCC.Figure S1Expression of NF-κB target genes in squamous cell carcinoma (SCC) cells. Cellular extracts were prepared and expression of NF-κB target genes was validated using Western blot. As compared to normal human epidermal keratinocytes (NHEK), SCC12 cells show higher expression of several NF-κB target genes.(PDF)Click here for additional data file.Figure S2Expression of A20 in squamous cell carcinoma (SCC) tissues. For simultaneous detection of A20 expression, tissue array analysis was performed. The moderate to high expression of A20 (P 1∼P 11) is observed in about 78% (11/14) patient samples.(PDF)Click here for additional data file.Figure S3Effect of A20 overexpression on imiquimod-induced apoptosis of SCC12 cells. Cells were transduced with adenovirus expressing GFP-tagged A20 (Ad/GFP-A20) or control adenovirus (Ad/GFP), then treated with imiquimod. Caspase activation was determined by Western blot. Cleavage of caspase-9, but not caspase-8, was detected, suggesting that imiquimod induces activation of intrinsic apoptotic pathway.(PDF)Click here for additional data file.Figure S4SCC12 cells were pretreated with JNK inhibitor SP600125 (20 µM), then treated with imiquimod (150 µg/ml). After 24 h incubation, cell viability was determined by MTT assay. Data are expressed as percentage of control. The mean values ± SD are averages of triplicate measurements. (*P<0.01).(PDF)Click here for additional data file.Figure S5SCC12 cells were transduced with adenovirus expressing GFP-tagged A20 (Ad/GFP-A20) or control adenovirus (Ad/GFP), then treated with imiquimod (150 µg/ml) for the indicated time points. Phosphorylation of ASK1 and MAPKs was detected by Western blot. Imiquimod induces phosphorylation of ASK1 and JNK, which is inhibited by overexpression of GFP-A20.(PDF)Click here for additional data file.


Arabidopsis thaliana, the atypical leucine-rich repeat receptor-like kinase STRUBBELIG (SUB) is involved in signal transduction in several developmental processes including the formation of carpels, petals, ovules and root hair patterning. The three STRUBBELIG-LIKE MUTANT (SLM) genes DETORQUEO (DOQ), QUIRKY (QKY) and ZERZAUST (ZET) are considered central elements of SUB-mediated signal transduction pathways as corresponding mutants share most phenotypic aspects with sub mutants.During plant tissue morphogenesis cells have to coordinate their behavior to allow the generation of the size, shape and cellular patterns that distinguish an organ. Despite impressive progress the underlying signaling pathways remain largely unexplored. In DOQ corresponds to the previously identified ANGUSTIFOLIA gene. The genetic analysis revealed that the doq-1 mutant exhibits all additional mutant phenotypes and conversely that other an alleles show the slm phenotypes. We further provide evidence that SUB and AN physically interact and that AN is not required for subcellular localization of SUB.Here we show that AN is involved in SUB signal transduction pathways. In addition, they reveal previously unreported functions of AN in several biological processes, such as ovule development, cell morphogenesis in floral meristems, and root hair patterning. Finally, SUB and AN may directly interact at the plasma membrane to mediate SUB-dependent signaling.Our data suggest that  Tissue morphogenesis and cellular patterning require extensive cellular communication. In plants the coordination of cellular behavior within a tissue is intrinsically linked to cell wall biogenesis and dynamics, as plant cells are connected through semi-rigid cell walls that drastically limit their relative movement. It is a major current challenge in plant biology to understand the mechanistic basis of intercellular communication and its connection to the cell wall during tissue morphogenesis.sub mutants show frequent defects in the initiation and outgrowth of the outer integument. In addition, sub mutants exhibit twisted stems, petals and carpels/siliques. These phenotypes indicate a role for SUB in the control of integument initiation and outgrowth as well as stem and floral organ shape [SUB also plays a role in internode length (and thus stem height), a trait that is potentially important for optimizing yield in crop plants.In Arabidopsis, intercellular signaling mediated by the atypical leucine-rich repeat transmembrane receptor-like (LRR-RLK) STRUBBELIG (SUB) is essential for a number of developmental processes -6. Ovulean shape ,2,6. SUBsub mutants. Therefore, it was postulated that SUB signaling plays a role in orienting cell division planes in initiating integuments and floral meristems and thus influences the morphogenetic behavior of cells in a tissue context [SUB, also known as SCRAMBLED (SCM), is involved in root hair patterning [sub mutations lead to a randomization of root hair patterning such that root hairs develop ectopically or are not formed in the correct files.At the cellular level, frequent misorientations of cell division planes were observed in e.g. L1 and L2 cells of young apical and floral meristems of tterning ,4. In thSUB in coordinating cellular behavior in tissue morphogenesis and cell patterning, SUB acts in a non-cell-autonomous fashion and mediates inter-cell-layer signaling across histogenic cell layers in the ovule, the floral meristem [In accordance with a perceived role of meristem  and the meristem .SUB belongs to the LRRV/STRUBBELIG-RECEPTOR FAMILY (SRF) family ,9 and haSUB suggested that the organ or cell-specific aspects of SUB-mediated signaling are not integrated at the SUB receptor, but involve other components that act together with, or downstream of SUB [SUB we have previously identified three complementation groups sharing the sub-like mutant (slm) phenotypes [SLM-sensitive gene expression. Taken together the results indicated that SLM genes contribute to SUB signal transduction. The corresponding genes are called QUIRKY (QKY), ZERZAUST (ZET), and DETORQUEO (DOQ) [QKY encodes a putative membrane-localized protein with four C2 domains thus potentially connecting SUB to membrane-associated Ca2+- and phospholipid-dependent signaling [Signaling by atypical kinases is poorly understood in plants ,11. In am of SUB . In ordeenotypes . In addiEO (DOQ) . InitialDOQ gene. We show that doq-1 is a mutant allele of the ANGUSTIFOLIA (AN) gene. The doq-1 mutant carries a point mutation in the AN gene and we further demonstrate that doq-1 shares phenotypes with other an alleles and conversely, other an alleles show all slm phenotypes tested. These results rule out the possibility that doq-1 is an atypical allele. In addition, we provide evidence that SUB and AN can physically interact and that AN does not influence subcellular SUB distribution. Together our results reveal that AN is involved in SUB-dependent signaling events.In this work we focused on the DOQ in a 330.6 kb interval at the top of chromosome 1 (see Methods). This interval included AN, a gene previously described to affect trichome branching, leaf morphology, and silique shape [doq-1 trichomes are underbranched. Together with the previously described narrow leaf phenotype of doq-1[DOQ and AN functions are related. We therefore compared the doq-1 mutant leaf and trichome phenotype with three reference an alleles, an-1, an-2, an-EM1 and two an-2 35S::YFP:AN rescued lines. In doq-1 mutants, reductions in trichome branching approached levels seen in the an-1, an-2, an-EM1 alleles -binding domain.We tested whether the slm phenotypes had not been reported for an mutants, the question arose whether doq-1 is an atypical an allele showing new phenotypes or whether all an alleles share the slm phenotypes. We therefore compared slm phenotypes between doq-1, the an-1, an-2, an-EM1 alleles and an-2 35S::YFP:AN rescued lines.As most doq-1, all an alleles showed premature opening of flowers and twisted petals -sensitive recycling -7. As threporter  in rootsA Figure . Expresssub phenotype is sensitive to the genetic background [ERECTA (ER), encoding a LRR-RLK [SUB interact synergistically as sub er double mutants exhibit strongly reduced plant height [ER locus [doq-1 (Ler) plants and asked whether a wild-type copy of ER would affect the doq-1 phenotype. Interestingly, the transgene had a similar effect on plant height but not on other aspects of the doq-1 and sub-1 phenotypes  was slightly reduced as well, despite the presence of wild-type ER, though not to the extent as in doq-1 (Ler). This suggests that AN affects plant height in part independently of ER. Full rescue of plant height in doq-1 ER plants indicates the presence of additional modifiers that influence this trait in Ler.The ckground  and it w LRR-RLK , and SUBt height . To inveER locus ,19, intoAN is involved in the SUB-dependent signaling mechanism. First, our genetic data show that all tested phenotypic aspects of sub are shared by an mutants. Second, DOQ/AN and SUB influence the expression of a common set of target genes. For example, 62 percent of genes misexpressed in sub flowers are also misexpressed in doq-1/an[an and er mutations synergistically affect internode length and plant height, as was observed for sub and er[AN and what for the function of SUB?Multiple lines of evidence indicate that AN was initially identified by its narrow leaf phenotype and trichome phenotype [AN in a regulatory network controlling branching initiation [slm phenotypes of an mutants reported in this study indicate that AN is involved in a SUB dependent signal transduction cascade. This function is separate from the first two functional aspects derived from leaf shape and trichome phenotypes, as sub mutants do not share these phenotypic aspects [AN gene encodes a protein with homology to CtBP/BARS [henotype . The clohenotype ,20. As citiation ,13. In titiation . The newtBP/BARS ,22. CtBPtBP/BARS . CtBPs htBP/BARS ,25. A potBP/BARS . A possitBP/BARS  and membtBP/BARS . In supptBP/BARS .AN fit into the SUB-mediated signaling pathway? AN is unlikely to be a direct or indirect transcriptional target of SUB signaling. For example, SUB expression is only minimally altered in doq-1 flowers at various stages [35S::SUB transgene failed to rescue the phenotype of doq-1 mutants indicating that SUB is not directly regulated at the transcriptional level by AN. At the same time, AN expression was not found to differ between floral tissue of wild type, sub-1 and other slm mutants [AN and SUB regulate each other’s activity at the transcriptional level.How does s stages . In addi mutants   Heynh. lines were used in this study: Columbia (Col-0) and Landsberg (erecta mutant) (Ler) wild-type strains, doq-1[an-1, an-2 [an-EM1[an-2 35S::YFP:AN. an-2 35S::YFP:AN plants were generated by cloning the CDS of the YFP:AN fusion into the pPAMPAT vector containing the 35S promoter (GenBank accession AY027531). Plasmid pKUT196 was described previously [The following ns, doq-1, an-1, a-1, an-2 , an-EM1[2 [an-EM1, an-2 35eviously .GL2:GUS line (Ler) [an mutants by backcrosses. For GUS assays, plants were grown on MS plates for 4 days.Plants were grown on soil at 24°C with 16 hours of light per day. The er/Col mapping population doq-1 was localized to the upper end of chromosome 1 between markers F10O3(481D) and NF21M12 [Using a LThe Matchmaker yeast two-hybrid system (Clontech) was employed and experimental procedures followed the manufacturer’s recommendations. The pACT-AN construct was described previously . For theTo assay possible interactions in yeast pGBKT7 plasmids containing SUBECD, SUBICD and SUBICD-R599C were cotransformed with pACT or pACT-AN into yeast strain AH109. Transformants were selected after 3 days on SC medium lacking Leu and Trp (−LW) at 30°C. To examine yeast two-hybrid interactions, the transformants were grown on solid SC medium lacking Leu and Trp (SC-LW) or Leu, Trp, and His (SC-LWH) at 30°C.AN and SUB cDNA were cloned into gateway entry vector pDONR 201. The following primers were used for gateway cloning: for amplifying AN cDNA: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGCAAGATCCGTTCG and GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCGATCCAACGTGTGATAC; for amplification of the full length SUB cDNA: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGCT TTACAAGATGGGAAGTGT and GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGATCATATGTTGA AGATCTTGG; for the amplification of the ICD version of SUB: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCatgTATAACCGATATTACAGTGGAGC and GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGATCAT ATGTTGAAGATCTTGG. AN cDNA was cloned into pGEX2TMGW  to generate an N-terminal fusion with Glutathion-S-Transferase (GST:AN) and the SUB and SUBICD cDNAs were cloned into pETG-40a  to generate an N-terminal fusion with maltose-binding protein .E.coli. The protein was purified and used to generate antibodies in rabbit . The antibody serum was affinity purified and checked for its specificity by MALDI-TOF analysis.To produce anti-AN antibody, AN was expressed as a GST-fusion protein in Interactions between SUB and AN were studied using purified proteins that were expressed in bacteria. The bacterial cells BL21-CodonPlus (DE3)-RIL containing the IPTG inducible constructs  were grown at 37°C and 220 rpm to an OD 600 of 0.8-0.9 and then the cultures were induced by adding IPTG to final concentration of 1 mM. The induced cells were then grown further for 5 hours (at 37°C for GST-tagged AN constructs and 20°C for MBP-tagged constructs) and cells were harvested by centrifugation. Cells were lysed in Tris-lysis buffer (Tris (pH 7.5) 50 mM, NaCl (100 mM), EDTA (1 mM), EGTA (1 mM), NP-40 (1%), Lysozyme (200 μg/ml), DTT (1 mM), Protease inhibitor cocktail (Sigma)) and sonicated three times for one minute each. The supernatant was collected by centrifugation at 4°C. MBP-tagged proteins were purified by incubation with amylose resin overnight at 4°C. After several washings, part of the resin was boiled with SDS-PAGE gel loading buffer to get purified protein for analysis. The remaining resin was used for incubation with equal amounts of GST:AN lysate for 4 hours at 4°C followed by several washings. Finally, the beads were boiled in SDS-PAGE gel loading buffer at 96°C for 10 min and equal amounts were loaded on a gel followed by western blotting. Detection was done using primary anti-AN antibody and secondary anti-rabbit antibody using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).Histochemical localization of ß-glucuronidase (GUS) activity in whole-mount tissues was performed as described previously . ScanninThe authors declare that they have no competing interests.LF, MH, KS designed the study. YB, LF, PV and HB performed experiments. YB, LF, PV, HB, MH and KS analyzed the data. MH and KS wrote the manuscript. All authors read and approved the final manuscript.


Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors.Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown.Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature.Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated. Kinesin-II motor subunits contain three predicted coiled coil domains in the middle, and always form a heterodimer Drosophila kinesin-II motor subunits, KLP64D (A-subunit) and KLP68D (B-subunit)  , using Creviewed ina-b-c-d-e-f-g’. The ‘a’ and ‘d’ positions are predominantly occupied by hydrophobic amino acids. Whereas charged amino acids dominate in the ‘e’ and ‘g’ positions  and subsequent immunostaining (ii and iii) of the western blots revealed that His-KLP68DS was soluble . As a result, the lower band in i (lane 3) stained more by the Coomassie blue. The affinity purified His-KLP68D/64D-S and His-KLP64D/68D-S heterodimers eluted in similar volume fractions upon gel filtration, . We also found that both the band intensities are comparable for purified heterodimer   analysis, which is the appropriate secondary structure determination tool used for coiled-coil estimation l values . We alsool (TFE) , known t222 which is most sensitive of changes in helical structure  and His-KLP68D/64D-S (mT = 298.8 K) would unfold rapidly between 293 and 310 K  e.g., the kinesin heavy chain (khc) neck coiled coil, was reported to have a dissociation free energy of 57.7 kJ/mol Xenopus, C. elegans and Drosophila are quite diverse (18% to 20% identity), which could account for the difference in the estimated functional characteristics.To resolve this, we estimated the thermodynamic parameters, thermal stabilities of the heterodimers by measuring the [Θ] values between 5 and 90°C (278–363 K). The results were consistent at all wavelengths monitored. Therefore, we decided to analyze the melting profile at [Θ]tructure , and usend 310 K . The ent (Eq. 6)  and the o be low , indicatThe CD analysis, however, provides an estimate of the biophysical properties at thermodynamic equilibrium, which does not necessarily correspond to the mechanical properties of the stalk heterodimer. Indeed, single-molecule analysis showed that kinesin-1 neck coiled coil is mechanically stable, though it is thermodynamically less stable The secondary structure predictions and CD analysis suggested that the heterodimeric KLP64D/68D-S should form a coiled coil. Hence, we modelled its structure using the pig tropomyosin coiled coil as a template (PDB:1C1G) The simulations also revealed that the number of predicted hydrophobic interactions and favourable electrostatic interactions, assessed from the models at ambient temperature , are considerably less in KLP64D/68D-S than those in the CC. While both the hydrophobic and hydrophilic amino acids are evenly distributed in the C-terminal part of the stalk, hydrophobic residues are less in the N-terminal region. Incidentally, the number of probable salt bridges predicted to be present in KLP64D/68D-S were little more than those found to remain in the CC after the 20 ns simulations . These eThe radius of gyration (Rg) for both the proteins did not alter significantly at 300 K , implyinA comparison of the root-mean-square fluctuations (RMSF) of Cα atoms of KLP64DS and KLP68DS residues at 300 K, with their corresponding chains in the CC model, suggested that some of the loops in both the chains, fluctuate significantly at 300 K . Certainvice versa  of both KLP64DS and KLP68DS reduced significantly, as compared to that of the C-terminal regions (SC1 and SC2) , although the later was used as a starting template. Subsequent data analysis provided some new insights such as the flexibility of the N-terminal domain and complementary segmental instability. These possibilities were further tested by copurification studies and CD analysis. For this, we cloned multiple N- and C-terminal stalk fragments and expressed them in E. coli . This shE. coli ; KLP68D-E. coli . SeveralE. coli , indicatE. coli , therefoThe following, combinatorial coexpression and affinity copurification analyses of the stalk fragments revealed that two different C-terminal fragments of KLP64DS (-SC1 and -SC2-T), as well as the KLP64D-SN2, could pull down the full length KLP68DS , whereasDrosophila, Xenopus and C. elegans are highly divergent, but they display grossly similar heterodimerization properties First, this analysis established that the stability of complementary fragments is not sufficient to induce an assembly in the absence of the C-terminal regions. Since the both KLP64D-SN1 and -SN2 failed to associate with the KLP68D-SN1 and –SN2, respectively . Second,222/[Θ]208 ratios are 0.71 and 0.85, respectively, indicating that these are unlikely to form coiled-coils on their own. The structures were also unstable and rapidly unfolded in 5–30°C (278–303 K) range  and His-KLP68D-SN2 (23.4%), at 5°C (278 K) . In addiK) range . AlthougIn summary, the comparative analysis of the KLP64D and KLP68D stalk fragments by a combination of biochemical, spectroscopic and bioinformatics tools showed that they form an unusual heteroduplex with part coiled coil structure at the C-terminal ends, whereas most part of the N-termini remained loosely attached . It is aThe MD simulations provided some intriguing insights. It showed that the N-terminal parts of the KLP64D/68D-S heterodimer could not retain the coiled coil structure, whereas the C-terminal and middle part of the stalk domain retain their coiled coil tertiary structure. Moreover, the 468E–475G region in the N-terminus of KLP64DS chain unfolded during the simulation stalk and behaved as a hinge in the stalk heterodimer, providing more flexibility to the N-terminal half of the stalk. In addition, while some stretches of α-helices retain their secondary structure; other parts unfolded during simulation. Therefore, the model for KLP64D/68D-S after 20 ns of simulation became markedly different from the starting model. This indicated that the simulation method could be used to gather coarse-grain, macroscopic views the of dynamic protein structures.trigger sequence consisting of combination of charged and hydrophobic residues in certain heptad positions trigger sequence, the C-terminal parts of the predicted kinesin-II stalk domains are essential for assembling a parallel coiled coil heteroduplex not only in Drosophila but also in XenopusC. elegansa’ and ‘d’ positions in the heptads could initiate and stabilize the KLP64D/68D-S assembly in a cooperative fashion. A similar process was observed with modified GCN-4 peptide in lipid environment a’ positions, which favoured heterodimerization with MATa1, and their replacement with isoleucine  favoured homodimerization Drosophila kinesin-I showed that such atypical heptads add mechanical stability to a coiled coil by providing a greater refolding barrier The coiled-coil assembly is often initiated from a consensus C. elegans suggested that the N-terminal stalks are held together, but the CD analysis indicated only the C-terminal region  would be coiled coil Forster resonance energy transfer (FRET) analysis of kinesin-II stalk heterodimer in Neurospora kinesin-1 (Nckin-1) contains a soft coiled-coil neck. Its replacement with a comparatively stable coiled coil resulted in reduced speed and run-lengths at increasing loads Processive motor proteins function as dimers, which enable alternate hand-over-hand movement of a single molecule unpublished). Therefore, we propose that the N-terminal segment of the kinesin-II stalk would undergo dynamic transition between the coiled coil and unstructured forms during the motor walk  and purified using affinity chromatography and gel filtration as described in the following section.Recombinant KLP64D and KLP68D stalk fragments were PCR amplified from the cDNA with appropriate sets of primers. They were cloned into either the multiple cloning site-1 (MCS1) of pETDuet™-1 vector (Novagen Inc. USA) in frame with the 6xHistidine (His) tag at the N-terminus, or into the MCS2 of the same vector with a stop codon at the end of desired ORF . The fraE. coli BL21(DE3) cells. E. coli suspension cultures carrying respective DNA constructs were grown at 37°C till OD600 reached 0.6 to 0.8 and then induced with 0.5 mM IPTG for 4 hours at 28°C. The cells were harvested and stored at –80°C. For purification of protein, the cells were resuspended in lysis buffer  10 mL of lysis buffer per gram of cell pellet was used. The cells were sonicated briefly on ice and then centrifuged at 4°C for 45 min at 70000 g. Ni-NTA agarose (Qiagen Inc.) was used to purify the recombinant proteins from the supernatant. The Ni-NTA agarose beads were washed with wash buffer . The purified proteins were assessed by SDS-PAGE, and, the concentration was estimated by Bradford method as well as by spectrophotometric absorption at 280 nm.The recombinant proteins were purified using a protocol described earlier 2, 500 mM NaCl] containing 2.5 mM DTT (TMN-D) at 1 ml/min flow rate, and 1.5 ml fractions of the eluate were collected by using Frac™-920 . The volume fractions were analyzed by SDS-PAGE, and the suitable ones were pooled and concentrated using Centricon® spin concentrators (Millipore (India) Ltd., India). For Circular Dichroism (CD) study, the buffer of the concentrated fractions was further changed to 10 mM sodium phosphate (pH 7.5) containing 0.2 mM DTT by using PD-10 desalting column  as per standard protocols.Affinity purified proteins were analyzed by gel filtration by using HiLoad 16/60 Superdex™-75pg  in TMN buffer   using Yang’s method N) and unfolded (U) states:The temperature dependence of the CD spectra was monitored at 222 nm, with the sample placed in a cuvette of 0.1 cm path length. Temperature was raised from 5 to 90°C with a data pitch of 0.1°C along with simultaneous recording of the spectra at 2°C intervals from 5 to 25°C (278 to 298 K) and at 5°C intervals from 25 to ∼90°C (298 to ∼363 K). The protein unfolding results were analysed using a simplified apparent two state-equilibrium model between the folded (The fractions of the unfolded protein at different temperatures (T) were calculated using the following equation:0Y is the observed mean residue molar ellipticity of the protein at 222 nm, at a given temperature (T); NY and UY represent the intercepts; and; Nm and Um are the slopes of native and unfolded baselines.T was calculated using The apparent equilibrium constant of unfolding R is the gas constant; mT = T. Therefore, at mT, The stability curves were obtained by non-linear least square analysis of The purified recombinant proteins or pull-down products were separated by SDS-PAGE and transferred to a PVDF membrane  according to the suppliers protocol, and stained with antibodies as described before www.sbg.bio.ic.ac.uk/phyre). The heterodimeric model of KLP64D and KLP68D stalk domains were built simultaneously using A and B chains  of Sus scrofa tropomyosin homodimer (PDB code: 1C1G) as templates in MODELLER v8.2  www.tripos.com). The details are mentioned in one of the previous article + ions were added to KLP64D/68D-S and CC systems, respectively (swift.cmbi.ru.nl/servers) and COILCHECK (http://caps.ncbs.res.in/coilcheck) servers. Other analyses were performed using scripts included with the GROMACS The secondary structure and coiled-coil probability of the KLP64D (AAF50786) and KLP68D (AAA50008) stalks were predicted/calculated using MultiCoil ectively . For solFigure S1Secondary structure predictions of Drosophila kinesin-II stalks. The amino acid sequences of KLP64D (A) and KLP68D (B) was analyzed using PSIPRED (TIF)Click here for additional data file.Figure S2The coiled coils of kinesin-II stalks. The coiled coil forming regions in KLP64D and KLP68D are predicted using MultiCoil program (TIF)Click here for additional data file.Figure S3222/[Θ]208 ratio in 50% TFE decreased to 0.95 from 1.03 in benign buffer.Measuring the coiled coil propensity of KLP64D/68D-S. Far-UV CD spectroscopic analysis of His-KLP68D/64D-S (P2 form), was performed in phosphate (benign) buffer  and the same buffer containing 50% Trifluoroethanol (TFE), respectively. The helical content of His-KLP68D/64D-S in benign buffer (solid black line) is 43.7% and that in 50% TFE is 45.6% (dotted black line). The secondary structure was estimated using Yang method (TIF)Click here for additional data file.Figure S4Distribution of α–helices and turns along the KLP64D stalk unfold at different temperature. Time evolution of the secondary structural elements of the KLP64D stalk domainin KLP64D:68D-S at 278 K (a), 300 K (b) and 373 K (c), presented at every 2 ns for 20 ns duration. Key: H-α-helix, B- β-sheet, T- turn, L- loop, gaps- undefined.(TIF)Click here for additional data file.Figure S5Distribution of α–helices and turns along the KLP68D stalk unfold at different temperature. Time evolution of the secondary structural elements of the KLP68D stalk domainin KLP64D/68D-S at 278 K (a), 300 K (b) and 373 K (c), presented at every 2 ns for 20 ns duration. Key: H- α-helix, B- β-sheet, T- turn, L- loop, gaps- undefined.(TIF)Click here for additional data file.Figure S6Salt bridge oscillations during MD simulation. Time evolution of the lengths of a few selected inter-chain salt bridges along the length of the stalk heterodimer at 278 K (black), 300 K (red) and 373 K (green).(TIF)Click here for additional data file.Figure S7Gel filtration analysis of the affinity purified stable stalk fragments of the KLP64D and KLP68D. Fraction-wise absorption profiles of the eluates were shown at the left panel. The Y-axis indicates absorptions at 280 nm with a vertical bar denoting 10 mAU (milli absorption units) and the dotted lines denote the base line of absorption. The grey bar denotes the elution volume in which the individual fragments were eluted. The Coomassie stained SDS-PAGE of representative fractions under each peak as marked by the numerals are presented in the right panel. Lane E was loaded with the affinity purified sample used for gel filtration.(TIF)Click here for additional data file.Figure S8Combinatorial pull down of kinesin-II stalk fragments with tags swapped. 6xHis-tag (H) is placed on either KLP64DS fragments or on KLP68DS to study the effect of 6xHis-tag on their association with each other. Coomassie stained SDS-PAGE gels indicate the composition of the affinity co-purification of KLP68DS with His-KLP64D-SC1 (lane 1), KLP64D-SC1 with His-KLP68DS (lane 2), KLP68DS with His-KLP64D-SC2-T (lane 3), and KLP64D-SC2-T with His-KLP68DS (lane 4).(TIF)Click here for additional data file.Table S1# His-KLP68D-NS was cloned in pQE30 vector.Detail list of recombinant fragments cloned. The recombinant DNA fragments were cloned in the bacterial expression vector pETDuet-1 . The cloned position MCS1/2 in pETDuet-1 is indicated within parenthesis. Fragments cloned in MCS1 expressed with N-terminal 6xHis tag,and they are denoted with a ‘His-’ prefix, while those in the MCS2 have no such tag. For co-expression, one of the fragments was cloned in the MCS1 and the other in MCS2. In the text, these co-expression clones are denoted as His-X/Y, where ‘X’ is the fragment that was expressed with 6xHis tag from MCS-1, whereas ‘Y’ was expressed from MCS2 without any tag. *GST-KLP64D-NST was cloned in pGEX4T1 vector and (DOC)Click here for additional data file.Table S2222 divided by the predicted molar ellipticity×100. The predicted molar ellipticity is calculated form [Θ]222 = −40000×(1–4.6/n) for chain length dependence of a helix Secondary structure estimations of kinesin-II stalks from the CD spectra. The secondary structure of the recombinant stalk fragments was estimated form the respective CD spectra obtained in the far-UV region (190–260 nm) at 278 K (5°C) by using three different methods as listed in the table. *-Helical content is calculated from the observed [Θ](DOC)Click here for additional data file.Table S3List of Parameters used for MD simulations.(DOC)Click here for additional data file.Table S4List of Salt-Bridges in KLP64D/68D-S after 20 ns at 300 K.(DOC)Click here for additional data file.


In the Val-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF valL was initially annotated as a validoxylamine A 7′-phosphate(V7P) synthase, whose encoded 497-aa protein shows high similarity with trehalose 6-phosphate(T6P) synthase. Gene inactivation of valL abolished both validoxylamine A and validamycin A productivity, and complementation with a cloned valL recovered 10% production of the wild-type in the mutant, indicating the involvement of ValL in validoxylamine A biosynthesis. Also we determined the structures of ValL and ValL/trehalose complex. The structural data indicates that ValL adopts the typical fold of GT-B protein family, featuring two Rossmann-fold domains and an active site at domain junction. The residues in the active site are arranged in a manner homologous to that of Escherichia coli (E.coli) T6P synthase OtsA. However, a significant discrepancy is found in the active-site loop region. Also noticeable structural variance is found around the active site entrance in the apo ValL structure while the region takes an ordered configuration upon binding of product analog trehalose. Furthermore, the modeling of V7P in the active site of ValL suggests that ValL might have a similar SNi-like mechanism as OtsA.Validamycin A  is an effective antifungal agent widely used in Asian countries as crop protectant. Validoxylamine A, the core structure and intermediate of Val-A, consists of two C Streptomyces genus 7N-aminocyclitols and five-membered ring aminocyclitols 7N-aminocyclitols, including acarbose, pyralomicin, salbostatin, cetoniacytone A and Val-A, have gained increasing attention due to their extensive applications in agriculture and medicine. Val-A is an antifungal agent isolated from S. hygroscopicus var. limoneus and S. hygroscopicus var. jinggangensis 5008. It is used in many Asian countries as a crop protectant against Rhizoctonia solani, which is a pathogenic fungus responsible for various diseases in plants Aminocyclitols are a diverse class of bioactive compounds produced by the valABCGKLMN with valL annotated as V7P synthase gene, rendered the surrogate host with validamycin productivity epi-5-epi-valiolone is proposed to be converted to NDP-valienol and validamine 7-phosphate S. hygroscopicus var. limoneusActinoplanes sp. SE50/110 and Streptomyces glaucescens respectively, are also thought to be responsible for the C-N bond formation in acarbose biosynthesis The structure of Val-A consists of an unsaturated valienol unit connected to a saturated validamine unit through a 1-1 α bond, and a glucose unit attached to the validamine moiety E.coli T6P synthase OtsA, ValL was regarded as T6P synthase with relaxed specificity IV cassette in strain 5008. This was achieved by using a pHZ1358-derived plasmid pJTU685, in which valL had been replaced by aac(3)IV between a 2.98-kb left flanking and a 2.12-kb right flanking sequences of valL . The dynamic scattering experiment indicated that ValL forms a dimer in solution(data not shown). It is unclear why ValL forms a dimer. In other organisms, such as Saccharomyces cerevisae, the T6P synthase and phosphatase form a multiunit complex The overall structure of ValL closely resembles the classic fold of “GT-B” glycosyltransferases . It harbThe overall structure of ValL is analogous to that of OtsA. The secondary structure matching between these two structures resulted in a root mean square deviation(RMSD) of 1.83 Å over 369 residues. A notable difference lies in the loop region ranging from Arg12 to Thr35 . As the To probe the mechanism of reaction catalyzed by ValL, its product analogs, trehalose and validoxylamine A, were used to form complex with ValL. The co-crystallization of ValL/trehalose led to the determination of ValL/trehalose complex structure. The electron density at the junction of N-terminal and C-terminal domains unambiguously indicates the location of trehalose embedded in the active site . The loc2 group on validamine 7-phosphate and O3B on donor phosphate As multiple attempts to crystallize ValL/validoxylamine A complex proved to be fruitless, the V7P molecule was modeled into the active site of ValL to illustrate the binding site of its natural product. The modeling result indicates apo ValL active site could accommodate V7P molecule readily. The superposition of ValL/V7P complex and OtsA/V7P complex structures shows that V7P adopts similar positions in both active sites . A SNi-lThe structure of apo ValL contains two molecules in the asymmetric unit. The SSM alignment shows the backbone of chain A and chain B matches well with an RMSD of 0.606 Å across 461 Cα atoms. However, a significant discrepancy has been observed in the region encompassing residue 88–110 . In chaiAlthough V7P binding residues in OtsA are conserved in ValL, Tyr 76 in OtsA is an exception. In the OtsA/V7P complex structure, Tyr76 is responsible for binding of the acceptor phosphate group and positioning the Arg9 to accommodate the substrate S. hygroscopicus var. jinggangensis 5008 and its derivatives were grown either on solid SFM medium or in modified liquid TSBY medium (with 10.3% sucrose). Fermentation was carried out at 37°C for 5 days. E. coli strains were cultivated at 37°C in Luria-Bertani medium or on Luria-Bertani agar plates. E. coli DH10B (GIBCO-BRL), ET12567 (pUZ8002) E. coli hosts for plasmid construction, E. coli-Streptomyces conjugation, and ReDirect technology, respectively valL was achieved by replacing with a cassette of apramycin-resistant marker aac(3)IV using a PCR-based ReDirect technology valL was cloned to pHZ1358 5′-GCCAACCTGATGTGGGCGGCGAACAACTACGGCTGGGACattccggggatccgtcgacc-3′) and valL-targeting-R (5′-TCAGAGGTCTGCTCGTGTCGAGACTGCCGGTGCGGTGTCtgtaggctggagctgcttc-3′), and pIJ773 homologous sequences were in lowercase. E. coli BW25113/pIJ790 bearing pJTU685 was electroporated with the amplified cassette. Mutated plasmid was firstly introduced into non-methylating E. coli ET12567 pUZ8002) through transformation and then transferred into S. hygroscopicus 5008 by intergeneric conjugation valL in the LL2 mutant was confirmed by PCR amplification with the primers of valL-Det-F (5′-CCGGCGTCCTCAACATC-3′) and valL-Det-R (5′ -TGCTCGTGTCGAGACTGC-3′).The inactivation of gene valL was cloned into pJTU968 valL was cleaved by EcoRI and MunI and cloned into pPM927 digested with EcoRI For the complementation of the mutant, PCR-amplified valL was cloned into pET28a vector between EcoRI and BamHI recognition sites. The pET28a-ValL plasmid was transformed into E. coli BL21(DE3), and a single colony was used to inoculate a 10 mL overnight culture containing 50 µg/mL kanamycin. Then the culture was used to inoculate 3L LB broth, and the culture was grown at 37°C until OD600 reached 0.5. The protein expression was induced with 0.3 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), and the incubation was continued for another 12 hours at 18°C. Cells were collected by centrifugation at 6,000 g, 4°C for 10 minutes. The resulting cell pellet was resuspended in 30 mL buffer containing 20 mM Tris, 0.5 M NaCl, pH 7.9. The cells were lysed with French Press, and the cellular debris was removed by centrifugation at 40,000 g for 20 minutes. The recombinant ValL was purified by chromatography with Ni-NTA resin. The bound protein was washed with wash buffer  and eluted with elution buffer . The protein content in each fraction was resolved on sodium dodecyl sulfate (SDS) gel before appropriate fractions were pooled and further purified with size-exclusion chromatography on a Superdex 75 column  equilibrated with 0.5 M NaCl, 20 mM Tris, PH 7.5. The protein was then concentrated with Amicon® Ultra-4 Centrifugal Filter Units, and the concentration was estimated spectrometerically at OD280. Selenomethionine-labeled ValL was expressed as described in the literature and purified as native protein 4, 0.1 mM CaCl2, and 5 g/L glucose. The culture was grown at 37°C until OD600 reached 0.6. Then lysine, phenylalanine, and threonine were added at 100 mg/L, isoleucine, leucine, and valine were added at 50 mg/L, and L-selenomethionine was added at 60 mg/L. After incubation for 15 min, 0.3 mM IPTG was added to induce the protein expression and the incubation was continued for another 12 hours at 18°C.Gene 2, 100 mM Tris-HCl (pH8.5). The mixture was allowed to equilibrate over a well filled with 150 µL precipitant solution. The crystals appeared after overnight incubation at 20°C and reached their maximum size three days later. Selenomethionine-labeled ValL crystals were grown in the same manner. To obtain ValL/trehalose complex structure, 5 mM trehalose was incubated with the enzyme for 30 min prior to mixing with the precipitant solution. A cryo-protectant solution was made by supplementing precipitant solution with 30% trehalose. The crystals were immersed in the cryoprotectant briefly before frozen in liquid nitrogen.Crystals of native ValL were grown by the hanging-drop vapor-diffusion method. Briefly, 1 µL of protein (10 mg/mL) was mixed with the precipitant solution containing 30% (w/v) polyethylene glycol 4000, 200 mM MgClDiffraction data were collected at 100 k from selenomethionine-labeled ValL crystals at the selenium absorption edge with a Mar 350 CCD detector on beam-line BL17U, Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China. The data set was indexed with XDS package and scaled with Scala www.rcsb.org) with the accession codes of 3T5T and 3T7D respectively.The atomic coordinates and the structure factors of apo ValL and ValL/trehalose complex have been deposited in the Protein Data Bank (


Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized.To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition.A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses. The influenza virus particle is spherical, about 100 nm in diameter, and encapsulated by a lipid membrane derived from the host cell. Two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), encoded by the virus genome are localized to the viral envelope. HA binds specifically to sialic acids, which serve as receptors for virus attachment Influenza virus RdRp composed of three virus-coded subunits, PB1, PB2 and PA, and the RdRp in viral particle catalyzes transcription et al. et al. et al. et al. The nuclear membrane is disassembled during S/G2/M-phase, prior to cell division, and subsequently reassembled after cell separation. Furthermore, the cell shape alters dynamically during the cell cycle In this study, using optical tweezers we show that the influenza virus attaches preferentially to cells in the G1-phase rather than those in the S/G2/M- phase. To our knowledge, this is the first successful application of optical tweezers for virus infection into individual cell.In addition, our results indicate that the sialic acid content of the membrane was higher on cells in G1- than in S/G2/M-phase. In concert the difference in sialic acid content of the membrane, we also succeeded to measure the difference in membrane stiffness between G1- and S/G2/M-phase cells using optical tweezers. The chemical and physical changes in membrane were associated with high-level expression of globotriaosylceramide (Gb3), globotetraosylceramide (Gb4), and glycosylceramide (GlcCer) in S/G2/M- than G1-phase cells. Taken together we propose a molecular basis of the cell membrane influencing influenza virus susceptibility.To determine whether influenza virus binds preferentially to cells at a particular phase of the cell cycle, purified influenza virus particles were labeled with a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). First, the binding of the fluorescence-labeled virus to host cells was confirmed by HA titration using chicken erythrocytes see . The DiIInfluenza virus attached cells were easily detected by the fluorescent probe Dil, while cells in S/G2/M-phase were detected by the GFP expressed from pFucci-S/G2/M Green vector. Among 50 Dil-labaled influenza virus attached cells that the pFucci-S/G2/M Green vector were transfected, 48 cells were not observed GFP expression, but only 2 cells were observed GFP expression. It was examined whether the different fluorescence-labeled influenza virus could attach to the specific cell phase. For this purpose the influenza virus particles were labeled with DiO or Syto21 respectively. DiO intercalates into lipid bilayer, whereas Syto21 binds to RNA. As shown in Previously, we developed a thermo-reversible hydrogel and optical tweezers to infect a cell with DiI-labeled influenza virus in a microchip  using glass beads, and subjected to centrifugation at 700×g for 10 min in a TOMY MX-301 centrifuge. The pelleted fraction was then treated with 100 μl of extraction buffer  and centrifuged at 186,000×g in a Beckman Optima ultracentrifuge . Ten microliters of the supernatant clarified by further centrifugation at 186,000×g was subjected to SDS-PAGE and transferred onto a PVDF membrane. To detect sialic acid, the membrane was probed with digoxigenin-conjugated lectins, which was then detected with an alkaline phosphatase-conjugated anti-digoxigenin antibody .G1- and S/G2/M-phase cells , and fixed with 10% formalin in PBST for 30 min. The binding of virus particles to glycopolymers was detected by incubation with an antibody against influenza virus A/PR/8/34 for 60 min at 37°C.Receptor specificity was determined using a solid-phase binding assay as described previously by Guo et al 6 cells in 20 volumes of 2∶1 chloroform:methanol. The extracts were separated by TLC in chloroform:methanol:water (40∶12∶1 v/v/v) and visualized by spraying with orcinol in 70% sulfuric acid.Cellular lipids were extracted from 3×10Type I collagen was coated onto carboxylate particles (2 μm in diameter)  via the cross-linker 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), using the method of Suzuki Figure S1Observation of binding activity of DiI-labeled virus on the cell treated with sialidase. A. DiI-labeled influenza virus was added to sialidase-treated (a) and –untreated (b) H292 cells respectively, and incubated for 15 min. The unbound viruses were removed and cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature and then observed under a Nikon Ti E confocal microscope fitted with a 100× objective lens. Red colored particles indicated by arrow head in white represent virus particle. B. Chicken erythrocyte aggregation by DiI-labeled influenza virus was assayed. Fifty μl of DiI-labeled influenza virus was added in the 50 μl chicken sialidase-treated and -untreated erythrocyte each. After 1h incubation at room temperature, the aggregation of chicken erythrocyte was observed. wells 1, 2, 3 and 4, influenza virus (+); well 5, influenza virus (−); wells 1 and 2, DiI-unlabeled virus; wells 3 and 4, DiI-labeled virus; wells 1 and 3, sialidase-untreated chicken erythrocyte; wells 2 and 4, sialidase-treated chicken erythrocyte.(EPS)Click here for additional data file.Figure S2Observation of various fluorescence-labeled influenza viruses on cell membrane. The various fluorescence-labeled influenza viruses were added on the H292 cells that were transfected with pFucci-S/G2/M Green vector and incubated for 15 min. The unbound influenza viruses were removed and cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and then microscopic observation was carried out under the ×100 objective lens. A. DiI-labeled influenza virus binding onto H292 cells. B. DiO-labeled influenza virus binding onto H292 cells, C. Syto21-labeled influenza virus binding onto H292 cells. Red colored particles indicated by arrow head in white represent virus particle, green color represents the expressed GFP.(EPS)Click here for additional data file.Figure S3Suction of a single cell using a capillary. Cells in the chamber were washed with 0.04% EDTA in PBS and suctioned with a glass capillary. The cell indicated by the arrowhead was manipulated. Left, before suction; right, after suction; below, manipulated cell.(EPS)Click here for additional data file.Movie S1Real-time observation of influenza virus on the cell. Movie showing the movement of a DiI-labeled influenza virus particle on an H292 cell following manipulation with optical tweezers.(MOV)Click here for additional data file.


A336C/A336T/T337C variations in HBV core gene were demonstrated to relate to the decreases in serum HBV DNA levels and HBV replication in chronic hepatitis B patients. Usually the drastic decrease in serum HBV DNA levels correlates with spontaneous HBeAg loss during the course of chronic HBV infection. The aim of the present study was to investigate whether there was correlation between A336C/A336T/T337C variations and spontaneous HBeAg lossA modified PCR-RFLP assay and ELISA were adopted to determine A336C/A336T/T337C variations and serum HBeAg levels in chronic hepatitis B patients without any antiviral therapy, respectively, whereas G1896A variation and HBV genotype were detected using Taqman-PCR assay. RFLP pattern C, E, G, C/G mixture and a new pattern C' were found in this study. A336C/A336T/T337C variations occurred in 40/166(24.1%) chronic hepatitis B patients. Chi-square test showed that C336/T336/C337 variants was more frequent in chronic hepatitis B patients with A1896 variants than those with the wild type G1896 , and moreover, patients with C336/T336/C337 variants had a significantly lower HBeAg-positive percentage than those with the wild type A336/T337. Binary logistic regression identified genotype B , the presence of C336/T336/C337 variants  and A1896 variants  as independent factors associated with spontaneous HBeAg loss.A336C/A336T/T337C were naturally occurring polymorphisms in HBV core gene, and moreover, the presence of C336/T336/C337 variants was first demonstrated to be an independent factor associating with spontaneous HBeAg loss in chronic hepatitis B patients. Infection with the hepatitis B virus (HBV) remains a very important human disease, with an estimated 400 million people infected chronically worldwide. It is reported that about 1.5 million people die annually as a result of infection, most of them in Asia and Africa. In China, chronic HBV infection is also a major public health problem and associated with a wide range of clinical states, from an asymptomatic carrier state with a normal liver function to severe liver disease, which includes liver cirrhosis and hepatocellular carcinoma.Spontaneous hepatitis B e antigen (HBeAg) loss during the course of chronic HBV infection is usually accompanied by the disappearance of the biochemical markers of hepatitis and a drastic decrease in viral replication. Factors associated with a higher rate of spontaneous HBeAg loss include certain HBV genotypes and certain HBV genetic diversities. Currently, HBV is classified into eight genotypes (A-H) based on an intergroup divergence of 8% or more in the complete nucleotide sequence of viral genomes. Genotypes B and C are prevalent in China. Previous cross-sectional and retrospective studies showed that genotype B patients had a significantly lower prevalence of HBeAg at presentation and a significantly higher rate of spontaneous HBeAg loss than genotype C-6. In adTsp509I, were demonstrated to correlate with the decreases in serum HBV DNA levels and HBV replication in chronic hepatitis B patients, and moreover, A336C/A336T variations caused the substitution of Glu-83 with Asp in HBcAg[A336C/A336T/T337C variations in HBV core gene, destroying the cleavage sites of  in HBcAg. HBeAg i in HBcAg,15. In a in HBcAg. TherefoThis is a cross-sectional study using stored serum samples from 166 chronic hepatitis B patients with genotype B and genotype C in Union Hospital of Huazhong University of Science and Technology in 2009. Chronic HBV infection was defined as persistent positivity for hepatitis B surface antigen (HBsAg) for at least six months at presentation. Patients with malignancy, liver cirrhosis, anti-HCV positivity, anti-HIV positivity and autoimmune disease were excluded. Before collecting blood samples, all patients had never been treated with any antiviral therapy. Informed consent was obtained from all of the patients. The study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Serum samples were stored at -80°C before they were tested. HBV DNA was extracted from 100 μl of serum with extraction solution in a PCR-fluorescence quantification kit for HBV  by the protocol described by the manufacturer.HBsAg, HBeAg, anti-HBs antibodies, anti-HBc antibodies, anti-HBe antibodies, anti-HCV and anti-HIV antibodies were determined with commercial assay kits .Quantification of serum HBV-DNA was carried out by real-time PCR assay using the method described before. HBV-DNATsp509I and primers for amplification and sequencing were in agreement with those used in reference[The presence of HBV core gene A336C/A336T/T337C variations in patient's sera were determined by a modified PCR-RFLP assay and cleaved PCR products were electrophoresed on 2.5% agarose gel,17, and reference.® Systems for real-time PCR . For each sample, real-time PCR reaction A and B were performed in two different capillary tubes according to the instructions of the manufacturer. In brief, the real-time PCR reaction was carried out in a final volume of 20.4 μl, consisting of 18.4 μl of master reaction mixture A or B and 2 μl of DNA template. Thermal cycling conditions were as follows: One cycle of 37°C for two minutes and 94°C for two minutes, followed by 35 cycles of 93°C for five seconds and 62°C for 30 seconds at a programmed temperature transition rate of 20°C/s, then cooled at 40°C for less than one second. Monitoring of fluorescence intensity occurred at regular intervals during the annealing-extension phase. The recombinant plasmids including wild type G1896(or A1896 mutant) were used as negative control(or positive control). Ct value obtained from real-time PCR was adopted to identify wild type G1896 and A1896 mutant and the identification standards were list in Table G1896A variation in HBV core gene was detected with PCR-fluorescence detection kit based on Taqman MGB (minor groove binder) probes . The amplification and detection were carried out using the LightCycler® real-time thermocycler . Real-time PCR reaction was performed in eppendorf tube according to the instruction provided by the manufacturer. In brief, the real-time PCR reaction was carried out in a final volume of 30 μl, consisting of 26 μl of master reaction mixture and 4 μl of DNA template. Real time PCR program consisted of heating at 50°C for 2 minutes and 94°C for 5 minutes, followed by 40 cycles of a two stage temperature profile of 93°C for 15 seconds and 60°C for 45 seconds at a programmed temperature transition rate of 3°C/s. Fluorescence emitted from reporter dyes FAM and Hex™ was monitored at regular intervals during the annealing-extension phase respectively. Ct value obtained from real-time PCR was adopted to identify HBV genotypes and the identification standards were list in Table HBV was genotyped with PCR-fluorescence diagnosis kit based on Taqman probes . The amplification and detection were carried out with the MXP3000Statistical analysis was carried out with the SPSS 15.0 software package. Chi-square test was used for comparison of categorical data. Binary logistic regression analysis was adopted to estimate the independent factors associating with spontaneous HBeAg loss. During statistical analysis, serum ALT levels, serum HBV DNA levels and age were transformed into categorical variables, respectively: (1) serum ALT levels were categorized into normal group(≤50 U/L) and abnormal group (>50 U/L); (2) serum HBV DNA levels were categorized into mild elevated group (<6.0) and severely elevated group(≧6.0); (iii)age were grouped into <35 years and ≧35 years. The results were presented as odds ratios(OR), 95% confidence intervals (CI), wald values and P values. P ≤ 0.05 was considered statistically significant.Tsp509I, namely RFLP C, C', E, G and C/G mixture were found. RFLP C' was a new pattern found in this study chronic hepatitis B patients in this study. C336/T336/C337 variants were found in 24/75 (32.0%) chronic hepatitis B patients with A1896 variants and 16/91(17.6%) chronic hepatitis B patients with the wild type G1896. Chi-square test showed C336/T336/C337 variants were more frequent in chronic hepatitis B patients with A1896 variants than those with the wild type G1896 , suggesting that A336C/A336T/T337C variations correlated closely with G1896A variation  were infected with HBV genotype B and the remaining 79 (47.6%) were infected with genotype C. A336C/A336T/T337C variations occurred in 26/87 (29.9%) chronic hepatitis B patients with genotype B and 14/79 (17.7%) patients with genotype C, and Chi-square test showed that there was no significant difference in the rate of A336C/A336T/T337C variations between HBV genotype B and C  chronic hepatitis B patients. Chi-square test showed chronic hepatitis B patients with C336/T336/C337 variants had a significantly lower HBeAg-positive percentage than those with the wild type A336/T337, indicating that the presence of C336/T336/C337 variants was closely associated with spontaneous HBeAg loss in chronic hepatitis B patients  and 47.6% (79/166) of chronic hepatitis B patients respectively. HBV genotype B was thought to be related to spontaneous HBeAg loss. One retrospective study of 146 Taiwanese adult HBeAg-positive hepatitis B carriers showed genotype C patients had a significantly lower rate of spontaneous HBeAg loss than genotype B patients. In agre/166 and In conclusion, A336C/A336T/T337C were naturally occurring polymorphisms in HBV core gene, and the presence of C336/T336/C337 variants was first demonstrated to be an independent factor associating with spontaneous HBeAg loss. This study also demonstrated that the presence of A1896 variant and HBV genotype B were also independent factors related to spontaneous HBeAg loss in chronic hepatitis B patients.The authors declare that they have no competing interests.WF performed most of the experimental work. LH participated in specific PCR-RFLP assay. ZZ participated in the detection of G1896A variation in HBV core gene. YL was responsible for the planning of the study, data analysis, and drafted the manuscript. All authors have read and approved the final manuscript.