AbstractTetrasticta gnathasp. n., collected under the bark of a rotten fallen tree in Peninsular Malaysia, is described. A habitus photograph, line drawings of diagnostic characters, and a diagnosis are provided. The new species is readily distinguished from all known congeners by having long mandibles, and long, curved maxillary palpi. PageBreakTetrasticta Kraatz, 1857 (Aleocharinae: Aleocharini) is a small genus that currently contains ten species from the Oriental and Afrotropical regions , anterior margin widely emarginate. Mandibles slightly asymmetric, strongly curved, pointed apically ; surface sparsely covered with yellow setae, and with approximately five bristles along lateral margin; each depression bearing a small bristle. Elytra: wider than long, subparallel-sided, rounded posterolaterally; surface moderately punctured and covered with yellow setae, and with four bristles laterally. Legs: rather short in length; relative lengths of tarsomeres from base to apex: 7:5:5:6:10.5 in foretarsus; 10:6:7:7:11 in midtarsus; 11:11:11:11:19 in hindtarsus. Abdomen: flattened, subpararell-sided, widest around segments IV and V; tergites III–VII sparsely covered with small setae. shining . Color: 10:10:22 . Mouthpaapically . Maxillaapically . Mentum Male: tergite VIII generalized in shape; surface smooth, with approximately 5–6 macrosetae ; HL, 0.70–0.91 (0.79±0.06); HW, 0.99–1.15 (1.07±0.06); AL, 1.68–1.97 (1.84±0.09); PL, 0.71–0.84 (0.77±0.05); PW, 1.12–1.36 (1.25±0.07); EL, 0.61–0.78 (0.70±0.05); EW, 1.22–1.56 (1.38±0.09); FTL, 0.64–0.81 (0.72±0.05); MTL, 0.78–0.97 (0.89±0.06); HTL, 0.89–1.16 (1.04±0.07); HW/HL, 1.22–1.52 (1.34±0.09); PW/PL, 1.58–1.68 (1.63±0.03). N = 12.Tetrasticta species by having a large head, which is as wide as pronotum, flattened body with unique mouthparts . Furthermore, the species lacks a coiled flagellum inside the median lobe of the male aedeagus.This new species is easily distinguished from other Dr. T. Tsuru collected all the specimens from a rotten log about 50 cm in diameter lying in the rainforest.PageBreakMalaysia (Peninsula).PageBreakPageBreakTetrasticta belongs to the Tetrasticta genus group, which comprises the genera Creochara Cameron, 1931, Cratoacrochara Pace, 1986, Ilarochara Pace, 1993, Aleonictus Kistner, 1997, Formicaenictus Kistner, 1997, and Myrmecosticta Maruyama, 2011  long mandibles; 2) long, 3) curved segment II of the maxillary palpus; and 4) short, 5) simple flagellum of the median lobe of the aedeagus. Of these, states 2) and 3) are probably correlated with long mandibles, as also observed in the other aleocharines with long mandibles . All other known species of Tetrasticta share a long, coiled flagellum of the median lobe of the aedeagus. The other character states fully coincide with those of Tetrasticta. A short, simple flagellum is also observed in Aleonictus and Formicaenictus in the same genus group, which are closely allied to Tetrasticta, but this state is apparently plesiomorphic, and cannot support a relationship between Tetrasticta gnatha and these genera. Although Tetrasticta gnatha is unique within the Tetrasticta genus group at first glance, we do not erect a genus for it. All of the type specimens of Tetrasticta gnatha were found under bark. Since no behavioral observations were made, termitophily of Tetrasticta gnatha remain uncertain. The long mandibles in both sexes and the flattened body suggest a predatory life under bark.ma, 2011 . Pace (2gnathos means jaw, for the exceptionally long mandibles.The Greek


Success in cancer immunotherapy depends on the identification and efficient targeting of specific tumor-associated antigens. Two pivotal strategies to prime patients’ immune system against malignant cells are tumor-specific adoptive T-cell therapy and tumor-specific vaccination. Here, we will focus on immunotherapeutic vaccination and discuss the advantages and disadvantages of different strategies to deliver tumor-specific T-cell epitopes. A particular focus will be put on virus-like particles (VLPs) as vehicle to deliver tumor-specific epitopes in the context of full-length proteins, as multi-epitope constructs or as individual tumor-associated T-cell epitopes. VLPs represent non-infectious and non-replicating antigen delivery systems devoid of any nucleic acid. They constitute innovative immunotherapeutic agents against cancer due to their superior, adjuvant-like antigenicity. We will present various tumor-associated antigens currently in different stages of development including survivin, as promising candidates for targeted tumor therapies. Antigen-derived peptides associated with major histocompatibility complex (MHC) class I molecules are presented on the surface of antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). To stimulate CD8 T, cells soluble exogenous antigens can access the cytosol of these APCs through the endosome-mediated internalization pathway or alternatively through the TAP-dependent MHC class I-specific antigen processing and presentation pathway , but also to the tumor-associated self antigens displayed by the particles. Based on the inherent capacity to break self-tolerance, VLPs represent ideal antigen delivery devices for immune intervention in preventive and therapeutic settings . , 9. 4, 9settings .In an early example human papillomavirus (HPV)-based VLPs induced protective CD8 T cell responses in an HPV16 tumor model . HPV VLPE. coli was inserted into a surface exposed loop of murine polyomavirus VP1  from VP1 Fig. . The forVP1 Fig. . FurtherVP1 Fig. . The indTo bypass these structural limitations of the delivery of antigens in the form of complete proteins by VLPs, murine polyoma VLPs harboring a protein-binding domain (WW domain) of the mouse formin binding protein 11 (FBP11) were employed. These WW domains interact with proline-rich ligands containing a PPLP motif . PPLP-taAnother way to hide foreign proteins within the cavity of polyomavirus VLPs utilizes the naturally occurring hydrophobic interaction between the capsid proteins VP1 and VP2/VP3 Fig. . SeveralS. cerevisiae, due to the simplicity of the manufacturing process.Whether structurally complex interplay between heterologous ligands and binding domains or the native self-assembly of VP1 and VP2/VP3 capsid proteins – it needs further clarification what is best suited for the interior packaging of protein antigens into, or surface loading onto polyoma VLPs. And in the end, it still may be determined by the character of the tumor-associated antigen. In regard of the industrial manufacturing of VLPs in yeast, the latter notion may represent the most plausible strategy for accomplishing high yields of heterologous particles directly isolated from Escherichia coli-based expression system Abbing and colleagues (anchor). A GFP-VP2anchor fusion protein was co-assembled in vitro into VP1 VLPs reaching 89% of the theoretical packaging efficiency, assuming 72 VP1 pentamers per nanoparticle. After subcutaneous (s.c.) tumor implant with 105 B16-GFP melanoma cells, mice were s.c. treated with 50 μg VLPs VP1/GFP-VP2anchor at day 4 and 11 leading to a 60% survival rate , was fused to the C-terminus of VP1 resulting in a VP1-OVA252-270 fusion protein  was packaged into each 45 nm VP1-particle. These VP2-HER2/neu1-683-containing VLPs were manufactured in insect cells using a baculovirus expression system.Likewise, this delivery concept was used to package the tumor antigen Her2/neu into murine polyomavirus VLPs, again exploiting the natural hydrophobic interaction Fig. . VLPs weSurvivin belongs to a family of proteins, known as inhibitor of apoptosis protein (IAP), which plays a key role in the regulation of apoptosis and cell division . As compE. coli as C-terminal translational fusion of the polyoma VP1 capsid protein. Pentameric structures that are known to be formed in E. coli were assembled into VLPs (Fig. In order to evaluate survivin as tumor antigen delivered by VLPs, full-length murine survivin was expressed in LPs Fig. . VLP preLPs Fig. , which iLPs Fig. , 30, 31.Prior to analyzing the therapeutic potency of VP1-survivin VLPs, naive expression of survivin and expression of survivin after induction of apoptosis in two melanoma cell lines, was investigated. To induce apoptosis B16F10 and MO5 cells are irradiated. Preparations of the cells were analyzed with anti-survivin and anti-tubulin antibodies Fig. .Subsequently, C57BL/6 mice were subcutaneously immunized with VP1-survivin VLPs. Controls were mock-immunized with buffer or vaccinated with VLPs lacking the tumor-associated antigen. Seven days later these mice were inoculated with MO5 melanoma cells. The survival rate of the animals was monitored over a period of 50 days. Mice that had received VP1-survivin VLPs showed significantly increased survival within the observation period (Fig. In a similar experiment C57BL/6 mice were charged s.c. with B16-F10 melanoma tumor cells and subsequently immunized with VP1-survivin pentamers. Immunization with antigen-presenting pentamers significantly increased the survival rate of the animals in this therapy approach Fig. . As expeThis demonstrates that particulate VLP structures displaying the full-length murine survivin antigen as C-terminal VP1 fusion have beneficial immunotherapeutic effects under early onset of treatment measures in a murine melanoma model.The most important concerns with novel CTL-stimulating vaccines capable of introducing antigens into MHC class I processing and presentation pathways is represented by safety issues seen with replicating vectors. Therefore, the development of non-replicating antigen delivery systems is an important advance in the design of efficacious and safe CTL-priming cancer vaccines. In this regard VLPs receive growing attention due to their capability to elicit CMI with emphasis on CD8 T cell responses in the absence of additional immunostimulatory substances.in vivo (The fact that chimeric VLPs, which represent important antigen sources for MHC class I-specific processing and presentation pathways, are efficiently taken up by DCs in vivo , 32, 33


TNF-α may play a key role in bone metabolism and is important in inflammatory bone diseases such as rheumatoid arthritis. Cells directly involved in osteoclastogenesis include macrophages, which are osteoclast precursor cells, osteoblasts, or stromal cells. These cells express receptor activator of NF-κB ligand (RANKL) to induce osteoclastogenesis, and T cells, which secrete RANKL, promote osteoclastogenesis during inflammation. Elucidating the detailed effects of TNF-α on bone metabolism may enable the identification of therapeutic targets that can efficiently suppress bone destruction in inflammatory bone diseases. TNF-α is considered to act by directly increasing RANK expression in macrophages and by increasing RANKL in stromal cells. Inflammatory cytokines such as interleukin- (IL-) 12, IL-18, and interferon-γ (IFN-γ) strongly inhibit osteoclast formation. IL-12, IL-18, and IFN-γ induce apoptosis in bone marrow cells treated with TNF-α  in vitro, and osteoclastogenesis is inhibited by the interactions of TNF-α-induced Fas and Fas ligand induced by IL-12, IL-18, and IFN-γ. This review describes and discusses the role of cells concerned with osteoclast formation and immunological reactions in TNF-α-mediated osteoclastogenesis in vitro and in vivo.Tumor necrosis factor- TNF-α-iammation . Osteoclsorption . Bone desorption . This boeability . Furtherpoptosis . TNF-α atruction . Thereforthritis .κB ligand (RANKL) was essential for osteoclast differentiation [α was also reported to induce the formation of osteoclastic cells from bone marrow macrophages in vitro [α, osteoclast formation is induced by low concentrations of RANKL, and TNF-α increases the effects of RANKL. In the absence of RANKL, osteoclast differentiation does not occur [α is considered to enhance RANKL signaling. Subsequently, TNF transgenic (TNF-Tg) mice × RANK−/− mice were created, and lack of RANK resulted in no changes in osteopetrosis even in the presence of TNF-α. This suggested that TNF-α-induced osteoclastogenesis is dependent on RANKL [β (TGF-β), osteoclastogenesis is induced by TNF-α alone. Furthermore, TNF-α could induce osteoclast differentiation in osteoclast lacking mouse models, including RANKL−/−, RANK−/−, and TNF receptor-associated factor-6 (TRAF6)−/− mice [α-induced osteoclastogenesis is independent of RANKL. Further studies are necessary to clarify these events. In 1998, two different research groups noted that receptor activator of NF-ntiation . RANKL intiation . TNF-α win vitro , 13. Altot occur . Thus, Ton RANKL . However−/− mice . This suα plays a central role in inflammatory osteoclastogenesis. Therefore, a better understanding of the contribution of these target cells in vivo may provide important therapeutic implications. It was reported that macrophages are direct targets of TNF-α in vitro [α-induced osteoclastogenesis in vivo using bone marrow transplants to determine the in vivo contribution of each cell type and whether they were a direct or indirect target of TNF-α. When a lethal dose of radiation was administered to mice, hematopoietic cells including macrophages were destroyed, but stromal cells survived. Donor myeloid cells were then transplanted into the irradiated recipients. The resultant chimeric mice contained recipient-derived stromal cells and donor-derived macrophages. Using this technique, 4 types of chimeric mice were created using wild type (WT) and both 55 kDa TNF receptor-1 (TNFR1) and 75 kDa TNF receptor-2 (TNFR2) deficient mice (KO). Each irradiated mouse underwent bone marrow transplant, and 4 groups were prepared as follows: (1) WT marrow transplanted into WT mice as a positive control for the administration of TNF-α, (2) WT marrow transplanted into KO, (3) KO marrow transplanted into WT, and (4) KO marrow transplanted into KO transplants as a negative control. Thus, groups of mice contained both TNFRs-bearing macrophages and stromal cells, TNFRs-bearing macrophages alone, TNFRs-bearing stromal cells alone, and those with TNFRs-deficient macrophages and stromal cells. After the marrow transplant, recipient T cells were blocked with anti-CD4 and anti-CD8 antibodies. These mice were injected with TNF-α in the calvariae and examined for osteoclastogenesis. The number of osteoclasts in KO to WT was less than that in WT to WT transplants. This suggested that TNF-α directly induced bone macrophages to undergo osteoclast differentiation. However, the osteoclast numbers in KO to WT were greater than those in WT to KO transplants indicating that while both bone marrow macrophages and stromal cells participate as direct cytokine targets in TNF-α-induced osteoclastogenesis, stromal cells are dominant. Analysis of RANKL and RANK expression by myeloid cells demonstrated that RANKL expression was increased in the WT to WT and KO to WT transplants. Thus, TNF-α may act directly on stromal cells to increase RANKL expression. Expression of RANK increased in the WT to WT, WT to KO, and KO to WT transplants. RANK expression also increased in mice with TNFRs-deficient macrophages. Considering these results, TNF-α may act by directly influencing macrophages to increase RANK expression and inducing stromal cells to increase expression of RANKL [α increases the number of bone marrow macrophages in WT to WT and KO to WT but not WT to KO transplants. M-CSF increases the proliferation of macrophages. Expression of M-CSF also increased in WT to WT and KO to WT transplants, suggesting that M-CSF was expressed in response to TNF-α produced by stromal cells. Therefore, TNF-α-induced M-CSF may increase the number of bone marrow macrophages. TNF-α directly increased RANK expression by influencing macrophages. Furthermore, the expression of RANK also increased in KO to WT transplants as described earlier. In addition, it was reported that RANK expression increased in vitro when macrophages were incubated with M-SCF. Thus, TNF-α may induce RANK expression via TNF-α-induced M-CSF from stromal cells. It was suggested that M-CSF also plays an important role in TNF-α-induced osteoclastogenesis in vivo [Cells directly involved in osteoclastogenesis include macrophages, stromal cells that express RANKL and induce osteoclastogenesis, and T cells that express RANKL and promote osteoclastogenesis , 18. TNFin vitro . Teitelbof RANKL , 20. TNFκB) are activated by RANKL–RANK-induced stimulation [κB and c-Fos deficient mice suggest that these molecules are essential for osteoclastogenesis [κB, which activate nuclear factor of activated T-cell c1 (NFATc1), the master transcription factor in osteoclast differentiation. NFATc1 is required for osteoclast differentiation [RANK, a receptor of RANKL, is expressed in osteoclast precursors. Mitogen-activated protein kinase (MAPK) pathways such as TNF receptor-associated factor (TRAF), family-mediated c-jun N-terminal kinase (JNK), p38, and nuclear factor-kappa B  activates the IκB kinase (IKK) complex. The IKK complex phosphorylates IκBα that ubiquitinates and degrades IκBα. This subsequently releases NF-κB subunits, which translocate into the nucleus and promote gene transcription [α-induced NF-κB activation in macrophages can be mediated by c-Src, which is a nonreceptor tyrosine kinase [α-induced signaling pathways, depending on the cell type. Meanwhile, two types of TRAF-6-deficient mice have been reported. One contained only few weak TRAP-positive mononuclear cells [α signaling in osteoclastogenesis remains poorly understood, and further studies are required to elucidate the relationship between TNF-α and osteoclast differentiation.It was reported that TRAF6-deficient mice develop osteopetrosis with defects in bone remodeling caused by impaired osteoclast formation. TNF-in vitro . Howeverent mice . These sormation . It may ntiation . The intntiation . Subsequcription –36. TNF-e kinase . Therefoar cells . Therefoar cells . Therefoα has an inhibitory effect during various stages of osteoblast differentiation and can act on osteoblast precursor cells during the early stages of differentiation to inhibit insulin-like growth factor 1, which increases the differentiation of osteoblast precursor cells from stem cells [α acts on osteoblasts to inhibit the transcription of runt-related transcription factor 2 (RUNX2), the master transcription factor for osteoblast differentiation, by promoting the degradation of RUNX2 mRNA [α also inhibits MAPK-mediated osterix expression and promoter activity [α increased IL-1 expression and IL-1-induced RANKL expression in bone-marrow-derived stromal cells, which promoted osteoclast differentiation [Osteoblasts differentiate from mesenchymal stem cells. TNF-em cells . FurtherNX2 mRNA . TNF-α aactivity , increasntiation .In vivo immune and inflammatory responses are regulated by a complex network of cytokines. In rheumatoid arthritis, TNF-α, IL-1, IL-6, and IL-17 produced by synovial macrophages and T cells act on osteoblasts to promote RANKL expression [β [γ [γ, IL-12, and IL-18 are acted during TNF-α-mediated osteoclastogenesis in myeloid cells, TNF-α induces Fas expression and IFN-γ, IL-12, and IL-18 induce FasL expression leading to apoptosis of osteoclast precursors [Inflammatory cytokines have multiple effects on bone resorption. pression . Thus, Ipression , IL-6 [4pression , and IL-pression , as wellssion [β , promotession [β –49, IL-1ssion [β , IL-12 [ssion [β –54, IL-1ssion [β , IL-18 [ssion [β –58, and on [β [γ  inhibit ecursors –59 , is an active immunotherapy targeting TNF-α. However, TNF-K induced anti-TNF antibody production [α recognition loop on TNFRI, and it prevents the interactions of TNF-α with TNF receptors. The peptide inhibited osteoclast formation in vitro and in vivo in mice [TNF-rthritis –62. Rheurthritis . TNF-α ial fluid , 65. TNF in mice , 67. Ostreatment , 68. TNFr, c-Fms . The relrthritis , 71. Howoduction . The cyc in mice , 74. α plays a key role in inflammatory arthritis; TNF-α-targeting biological drugs are highly effective in the treatment of inflammatory arthritis. It was reported that administration of antibodies to c-Fms (anti-c-Fms antibody) completely blocked osteoclastogenesis and bone erosion induced by TNF-α administration. Furthermore, in the study on the efficacy of anti-c-Fms antibodies injected into the experimental arthritis model using the serum of K/B × N mice [α and IL-1, from macrophages or other cells in inflammatory tissues [in vivo and in vitro studies [in vivo [α in osteoclastogenesis in arthritis, M-CSF blocking may be effective. Although treatments targeting TNF-α are highly effective in inhibiting the progression of bone destruction caused by inflammatory arthritis, other cytokine inhibitors that effectively target bone destruction should be developed and considered for concomitant usage.TNF-× N mice , there w× N mice . Lipopol× N mice –78. LPS  tissues . These c studies –82. It w[in vivo . With reα is a key molecule for inflammatory osteoclastogenesis and bone destruction during inflammatory arthritis. Furthermore, the role of cells involved in TNF-α-induced osteoclast formation and the interactions between TNF-α-induced osteoclast formation and cytokines and their signaling pathways have gradually become clear. Currently, TNF-α is the major target of highly effective biological drugs for the treatment of inflammatory arthritis. The progress of studies for TNF-α-induced osteoclast formation and bone destruction has been accelerated. However, there is still much to learn. In addition, TNF-α-targeting therapies have several drawbacks. Therefore, further studies are required to fully understand TNF-α-induced osteoclast formation and bone destruction. Many studies have indicated that TNF-


MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases , whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that DOI:http://dx.doi.org/10.7554/eLife.00825.001 Blood cells are produced within bone marrow by specialized stem cells and progenitor cells. Abnormalities in this process lead to a group of diseases known as myeloid malignancies, which include acute myeloid leukaemia—in which the bone marrow produces abnormal white blood cells—and myelodysplastic syndromes, which are caused by too few mature blood cells being produced.Many individuals affected by these disorders possess a shortened form of chromosome 20 that lacks a number of genes. This deletion is only ever seen in one of their two copies of the chromosome—suggesting that at least some of these genes are essential for survival—but the identity of the gene(s) that are associated with the increased risk of myeloid malignancies is unknown.MYBL2, encodes a transcription factor that helps to control the cell division cycle. Myeloid malignancy patients lacking one copy of this gene showed levels of MYBL2 expression that were less than 50% of those in healthy individuals. This suggests that additional mechanisms must be acting to reduce expression of their remaining copy of the gene. Surprisingly, MYBL2 levels were also reduced in myeloid malignancy patients who possessed two intact copies of chromosome 20, indicating that loss of a single copy represents only one mechanism to reduce MYBL2 expression, i.e., the ‘tip-of-the-iceberg’. Hence, this finding reveals a more general role for MYBL2 as it indicates that more patients are likely to be affected by altered expression of this gene.Now, Heinrichs et al. have uncovered a key tumor suppressor among those genes frequently lost on chromosome 20. The gene, which is called MYBL2 in mice and showed that this induced symptoms of myeloid malignancies in the animals. Moreover, injection of modified cells from these animals into healthy mice also induced symptoms in the recipients. The modified cells are able to expand more robustly than normal cells, and this dominance induced by downregulation of the tumor suppressor increases the risk of malignancy.To confirm their findings from studies in patients, Heinrichs et al. used gene silencing techniques to reduce the expression of In addition to revealing a new tumor suppressor gene and its contribution to myeloid malignancies, the study by Heinrichs et al. highlights the importance of gene dosage in mediating the effects of tumor suppressors.DOI:http://dx.doi.org/10.7554/eLife.00825.002 The molecular changes underlying human myeloid malignancies remain difficult to unravel, posing major obstacles to the development of effective countermeasures. Although the silencing of tumor suppressor genes by chromosomal deletions, point mutations, or other mechanisms is an acknowledged factor in myeloid cell transformation, the specific involvement of gene dosage is not well understood. In broadest terms, single-copy loss of a suppressor gene can be sufficient to modify gene function and promote tumorigenesis , while in other tumors, the loss of two alleles is required (two-hit paradigm of Knudson) . Recent CTNNA1 in myeloid malignancies with deletions of chromosome 5q  is detected in about 4% of myelodysplastic syndromes (MDS) , and thiosome 5q .MYBL2, which encodes a transcription factor with functions in checkpoint control of the G2 cell cycle phase, as a key tumor suppressor gene in over one half of all MDS cases, whether characterized by a 20q deletion or a normal karyotype. Reductions of MYBL2 dosage to levels below those commensurate with single-copy loss conferred a competitive advantage to hematopoietic progenitor cells in both primary and secondary transplantation assays and were associated with histopathologic changes typical of myeloid neoplasia. These findings implicate aberrantly low levels of MYBL2 expression as a central mechanism in the development of clonal dominance in MDS and other myeloid malignancies.Here we identify MYBL2, which resides near the center of this region  was less than that of normal CD34+ cells . These microarray data were confirmed by QRT-PCR analysis of MYBL2 expression levels in 10 MDS cases and eight normal CD34+ bone marrow samples, including four additional normal control CD34+ samples that were not part of the original microarray analysis (MYBL2 levels measured by QRT-PCR in MDS samples were highly correlated (r = 0.966) with those determined by microarray analysis , we used a set of eight shRNA-expression vectors in the MDS/AML cell line SKM1, which reduced MYBL2 expression to 5–30% of the endogenous MYBL2 levels  , the early mitotic regulator FBXO5 and the coregulator of chromosome architecture CDCA2   identifiansition   classifier and Euclidean distance (k = 3) to predict the class label by a majority vote. 10 of these cases (56%) were classified as MYBL2 low, while the remaining eight cases had an MYBL2 signature that was statistically similar to that of normal CD34+ controls (MYBL2-low signature in 36 cases (38%), most of which (30 cases) were identified at a confidence level of 1.0. 58 cases (62%) had a normal MYBL2 signature . This finding is supported by a recently published study that did not identify methylation affecting the MYBL2 gene in cells from 83 MDS cases  by analyzing the expression levels of mir-29a and mir-30e. Both of these miRNAs target MYBL2  to 33% (M5) of those expressed by control shRNA-expressing vectors in 32D cells, a murine myeloid cell line , or with two different control shRNA vectors coexpressing a red fluorescent protein (RFP) . By contrast, in mice injected with control/GFP vs control/RFP cells, the median frequencies of both RFP- and GFP-expressing cells were low  , we washted mice . A seconted mice . Analysited mice  showed a− cells with single hairpins specific for Mybl2. For this experiment (− bone marrow cells with either the M3 shRNA vector (n = 7 mice) or the M4 shRNA vector (n = 8 mice). These two shRNAs were chosen because QPCR analysis of the mice injected with pools of cells transduced with each of the five shRNAs  at 6 to 7 months post-transplantation, and studied their bone marrow cells by flow cytometry.The marked competitive expansion of Mybl2-knockdown cell population compared to control bone marrow cells  bone marrow cells of the affected mice (n = 12) in comparison to controls. Within the LSK population of GFP+ or RFP+ Mybl2-knockdown bone marrow cells, HSCs (CD150+/CD48−) were diminished, MPPs (CD150−/CD48−) were maintained and LRPs (CD150−/CD48+) were increased compared to unlabeled, non-transduced cells. Thus, the clonal dominance due to low Mybl2 levels is first evident as an increased percentage of cells in the LRP fraction.  of 26 human MDS cases. This result is somewhat surprising given that MYBL2 has been implicated as a gene that is typically overexpressed (rather than downregulated) in several types of cancer and whose elevated expression correlates with a poor prognosis in human breast cancer and other malignancies  of 18 of our normal-karyotype MDS cases, and 38% of those reported by Pellagatti et al. , and were depleted of lineage-marked cells using sheep anti-rat IgG-coupled Dynabeads  together with affinity-purified rat anti-mouse antibodies  against CD3, CD5, B220, Gr1, Mac1, Ter119. Cells were cultured for 24 hr in StemSpan SFEM  supplemented with 100 U/ml penicillin/streptomycin, 2 mM glutamine, 80 ng/ml Scf, 40 ng/ml TPO and 40 ng/ml Flt3l. Cells were aliquoted and transduced in separate wells with single shRNA- or control shRNA-expressing vectors, extensively washed and cultured separately for an additional 24 hr before transplantation. Single vector transduced cells were pooled according to the scheme in PAPOLA, UBQLN1 and VPS39 (housekeeping genes) and scaled to the mean of the controls. Sequences of primer sets are available in RNA was extracted from Trizol  according to the manufacturer’s instructions, and 500 ng were used for reverse transcription . Real-time PCRs were performed on an ABI PRISM 7700 system using SYBR green-based assays with AmpliTaq Gold (Life Technologies). All reactions were performed in triplicate. Quantitative data were calculated from the Ct-values for each reaction using the mean reaction efficiency for each primer pair. Data were normalized to expression levels of Total RNA was isolated from cells lysed in Trizol (Life Technologies). 100–500 ng of each sample was converted into fragmented, biotinylated cDNA hybridized to a microarray chip (Gene 1.0 ST) and fluorescently labeled according to the standard protocol  at the Dana-Farber microarray core facility. Raw data were processed in Expression Console (Affymetrix) using RMA normalization. Expression values for each gene were annotated by mapping all probe sets to the human genome version hg19. Data complexity was reduced to one canonical transcript per gene, resulting in a single identifier per gene . The expression data were processed in Gene Pattern  Broad I. Data arMYBL2  based on the short hairpin knock-down experimental data were reduced to a 59 gene signature positively correlated with MYBL2 by removing 14 genes which hierarchically clustered with the 8 negatively correlated genes and 9 genes having low expression (≥3 out of 4 controls) in the CD34+ control samples in the 30 patient samples arrays. This signature was also examined in an independent cohort of 94 normal karyotype patients using KNN.A k-nearest neighbor (KNN) algorithm was used to classify normal-karyotype MDS cases by using Euclidean distance and k = 3 to predict the class label by a majority vote. To generate a training set, the 89 genes most correlated with Protein extracts were prepared by SDS lysis and normalized by total protein content (BCA assay). Total protein (5–25 µg) was separated by SDS polyacrylamide gel electrophoresis and blotted on PVDF membranes. Specific proteins were visualized by chemiluminescence using antibodies for MYBL2  or Tubulin .Cryopreserved human bone marrow CD34+ cells were obtained from Lonza and cultured in StemSpan SFEM (Stem Cell Technologies) supplemented with 100 U/ml penicillin/streptomycin, 2 mM glutamine, 50 µg/ml lipoproteins , 80 ng/ml SCF, 40 ng/ml TPO, 40 ng/ml FLT3L, 10 ng/ml IL3 and 10 ng/ml IL6. SKM1 cells were obtained from the DSMZ  and maintained in RPMI medium supplemented with 10% fetal bovine serum.EGFP or DsRedExpress2 (MYBL2 cDNA including a 3xFLAG tag was cloned into derivate of the pLenti6.3 (Life Technologies) vector under control of the SFFV promoter. Lentiviral particles were produced in 293T cells by cotransfection of pMD2G, pCMV-dR8.9 and the lentiviral vector. Cells were spin-transduced in the presence of polybrene (4 µg/ml) and selected at 24 hr post-transduction with puromycin (1.5 µg/ml) or blasticidin (3 µg/ml). Growth curves were obtained with the CellTiter-Glo luminescent assay .Oligonucleotides encoding shRNAs were cloned into pLKO1. pLKO1 derivatives were obtained by replacing the puromycin resistance marker with Express2 . LentiviA FACSCanto II machine  equipped with a 407 nm, 488 nm and 633 nm laser was used. Staining was performed with the following antibodies (clone id in parentheses), all purchased from eBioscience: Mac1 (M1/70), Gr1 (RB6-8C5), B220 (RA3-6B2), CD3 (145-2C11), CD71 (R17217) and Ter119 (Ter119).C57BL/6 mice, purchased from Jackson Laboratories, were housed in individually ventilated cages in the Dana-Farber Cancer Institute animal facility in accord with guidelines of the Institutional Animal Care Use Committee. Transplanted mice received Baytril-containing water  to reduce the probability of infection from opportunistic pathogens in first 30 days after transplantation. For peripheral blood analysis, tailvein blood was collected and RBCs were lysed with ammonium chloride buffer prior to staining for flow cytometry. For bone marrow analysis, cells were isolated from femurs and tibias with RBCs lysis buffer used for all subsequent procedures except analysis of erythropoiesis by Ter119/CD71 flow cytometry.Cytospin slides were prepared from isolated mononuclear bone marrow cells and stained with Wright-Giemsa stain. Bone and spleen samples were fixed in 10% formalin, washed with PBS and stored to 80% ethanol. Sections were stained with hematoxylin and eosin.http://www.graphpad.com). p-values (two-tailed) for competitive reconstitution were calculated by a paired t-test.All data were analyzed with GraphPad Prism, version 5.04 for Windows ( review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  The reviewers are unconvinced by the argument that Mybl2 hairpins demonstrated a clonal growth advantage, recipient mice appeared well with no evidence of overt hematologic disease for XX months” (if not). Along these lines, what were the peripheral blood counts and spleen sizes of the recipient mice shown in 2) The phenotypic description of the mice shown in  1) The reviewers are unconvinced by the argument that MYBL2 levels must be reduced to sub-haploinsufficient levels to cause myeloid malignancy. After all, Clarke et al show that deletion of a single copy of MYBL2 is sufficient to cause myeloproliferation in mice. The authors do not test an shRNA that only reduces MYBL2 levels by 50%, so they cannot rule out the possibility that MYBL2 could function as a typical haploinsufficient tumor suppressor. It would seem best to tone down this aspect of the Discussion.MYBL2 could function as a haploinsufficient tumor suppressor .We agree with the reviewers and we have revised the Discussion to indicate that we cannot rule out that 2) The phenotypic description of the mice shown in  Mybl2 hairpins demonstrated a clonal growth advantage, recipient mice appeared well with no evidence of overt hematologic disease for XX months” (if not). Along these lines, what were the peripheral blood counts and spleen sizes of the recipient mice shown in ?We agree that this information is important and we have included new data in the figures, with new descriptions in the Results section to address these issues.First, we extended our description of the phenotype by including the peripheral blood counts and spleen weights at the time of euthanasia in Our strategy was to evaluate all of the mice at 6–7 months post-transplant, which is a time at which long-term hematopoietic reconstitution has been achieved in competitive repopulation assays. Thus, a Kaplan–Meier curve can’t be included as the mice were electively euthanized and the majority of the primary recipients appeared well at 6–7 months post-transplant.The circulating blood abnormalities in the secondary animals appeared much more pronounced as illustrated in


Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have been shown not only to produce substantial increases in lifespan but also to ameliorate several age-related diseases. In C. elegans, the multitude of phenotypes produced by the reduction in IIS are all suppressed in the absence of the worm FOXO transcription factor, DAF-16, suggesting that they are all under common regulation. It is not yet clear in other animal models whether the activity of FOXOs mediate all of the physiological effects of reduced IIS, especially increased lifespan. We have addressed this issue by examining the effects of reduced IIS in the absence of dFOXO in Drosophila, using a newly generated null allele of dfoxo. We found that the removal of dFOXO almost completely blocks IIS-dependent lifespan extension. However, unlike in C. elegans, removal of dFOXO does not suppress the body size, fecundity, or oxidative stress resistance phenotypes of IIS-compromised flies. In contrast, IIS-dependent xenobiotic resistance is fully dependent on dFOXO activity. Our results therefore suggest that there is evolutionary divergence in the downstream mechanisms that mediate the effects of IIS. They also imply that in Drosophila, additional factors act alongside dFOXO to produce IIS-dependent responses in body size, fecundity, and oxidative stress resistance and that these phenotypes are not causal in IIS-mediated extension of lifespan.The insulin/insulin-like growth factor-like signaling (IIS) pathway in metazoans has evolutionarily conserved roles in growth control, metabolic homeostasis, stress responses, reproduction, and lifespan. Genetic manipulations that reduce IIS in the nematode worm  Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse not only increase lifespan but also delay the onset of age-related pathology and disease  proteins, a highly conserved family of transcription factors. Phosphorylation of FOXOs by the insulin-activated protein kinases PKB/AKT and SGK leads to their sequestration within the cytoplasm and, as a result, transcriptional inactivation of target gene expression (The insulin/insulin-like growth factor (IGF)-like signaling (IIS) pathway of metazoans regulates such diverse processes as growth, developmental timing, body size, metabolism, stress responses, reproduction, and lifespan . Geneticpression . Severalpression . Hence, C. elegans are completely dependent upon the activity of the worm FOXO transcription factor, DAF-16  and adult-specific ubiquitous expression of a dominant negative form of PI3 kinase, all of which show phenotypes typical of reduced IIS. We found that mutation of dfoxo almost completely blocked lifespan extension in these IIS-compromised flies. However, removal of dfoxo failed to rescue IIS-mediated developmental delay, small body size, reduced egg laying, and resistance to paraquat. In contrast, increased resistance to the xenobiotic toxin, dichlorodiphenyltrichloroethane (DDT), was completely dependent on dFOXO activity. Our results show that, unlike in C. elegans, where all phenotypic traits produced by reduced IIS are DAF-16 dependent, additional factors besides dFOXO have evolved in Drosophila to mediate the full IIS response.In this study, we have examined whether several IIS-dependent phenotypes, including increased lifespan, reduced fecundity, increased stress resistance, developmental delay, and growth inhibition, are dependent on dFOXO activity in Drosophila dfoxo transcription factor have already been described. These include dfoxo21 and dfoxo25, both of which contain chemically induced nucleotide transversions within the dfoxo coding region, resulting in premature stop codons within the proposed DNA-binding domain of the protein  spans over 20 kb of the dfoxo locus, removing part of the predicted promoter region as well as several coding exons. Homozygotes for the deletion were adult viable, and neither dFOXO protein expression nor DNA-binding activity was detected in these flies ED5624/dfoxoΔ94 flies using a deficiency that removes the entire dfoxo locus  under the control of the ubiquitous and constitutive daughterless-GAL4 driver (daGAL) or late ablation of the mNSCs by the expression of UAS-reaper (UAS-rpr) under the control of the InsP3-GAL4 driver (InsP3GAL). In addition, we examined adult-onset ubiquitous expression of UAS-InRDN as well as adult-onset ubiquitous expression of a catalytically inactive, dominant negative form of PI3 kinase (UAS-Dp110DN) using the inducible daughterless-GeneSwitch (daGS) driver. daGS only drives transgene expression in the presence of the RU486 steroid drug. Treatment with RU486 had no effect on the lifespan, fecundity, or stress resistance of daGS/+ flies themselves  . However, no significant differences in survival were apparent between groups during the first 50 days of the experiment , whereas survival at ages beyond 50 days was increased in daGAL > UAS-InRDN dfoxo− flies compared with both controls   for daGAL > UAS-InRDN dfoxo− flies  relative to both genetic controls . This apparent age-related difference in survival was observed in a second, independent experiment, suggesting that ubiquitous and constitutive expression of UAS-InRDN can increase survival later in life and hence extend maximum lifespan even in the absence of dFOXO activity.In the presence of lifespan . In a df dfoxo−) . When alnk test) . Hence, InsP3GAL > UAS-rpr females also lived significantly longer than both genetic controls (InsP3GAL/+ and UAS-rpr/+) in the presence of dfoxo with approximately 30% increase in median and approximately 20% in maximum lifespan  and both daGS > InRDN dfoxo− and daGS > Dp110DN dfoxo− females treated with RU486 laid significantly fewer eggs than their uninduced controls , suggesting that loss of dFOXO activity and reduced IIS in these flies acted additively to reduce egg laying.kgrounds . Similarkgrounds . Ubiquitckground . Taken tdfoxo can reverse the reduction in GSC proliferation caused by the loss of chico function, demonstrating that dFOXO is required in the GSCs for at least some of the effects of lowered IIS on reproduction  in daGAL4 > UAS-InRDN females, in either a wild-type or dfoxo− background, indicating that YP2 expression is unresponsive to both IIS itself and dFOXO activity. In contrast, transcription of the yolk protein receptor, yolkless, whose expression is normally restricted to the oocyte, was significantly increased in daGAL4 > UAS-InRDN females compared with controls , we observed a complete rescue of growth inhibition in the dfoxoΔ94 mutant background  are expressed in the MNCs of the Drosophila brain. Ablation of the MNCs or genetic deletion of all three MNC-expressed dilps produces small flies owing to systemic effects on IIS-mediated growth  has been well documented as a direct target of dFOXO , in response to low TOR signaling .Several dFOXO target genes have been well characterized in C. elegans, where all of the phenotypic effects of reduced IIS are dependent on DAF-16 activity, in Drosophila, several IIS-dependent phenotypes appear to be regulated, at least in part, through dFOXO-independent mechanisms. These results therefore have important implications when analyzing the requirements for IIS in particular phenotypic traits. In worms, the standard protocol would be to remove DAF-16 activity and look for abrogation of the response or phenotype. In flies, and possibly higher organisms, such experiments may prove misleading. For example, dfoxo mutants display a normal response to DR, but overexpression of the dominant negative insulin receptor or removal of dilps-2, -3 and -5 almost completely blocks the DR response , UAS-InRDN (K1409A), UAS-Dp110DN (D954A), eyeless-GAL4 (ey-GAL4), and mata-GAL4 stocks were obtained from the Bloomington Stock Centre. daughterless-GeneSwitch (daGS) was kindly provided by Veronique Monnier (InsP3-GAL4 was kindly provided by Michael Pankratz (whiteDahomey (wDah) stock. wDah was derived by backcrossing white1118 into the outbred wild-type Dahomey background. Flies were raised and maintained on standard sugar/yeast medium , allowed to mate for 24 h, sorted by sex, and then transferred to experimental vials at a density of ten flies per vial. Flies were transferred to fresh vials three times a week, and deaths were scored during transferral.For fecundity measurements, eggs were collected over a 24-h period and counted. Data are reported as the mean number of eggs laid per female ± SEM over this 24-h period.m paraquat or 0.03% (w/v) DDT.Flies were reared and housed as for lifespan experiments until 7 days of age, and then flies were starved for 5 h on 1% agar before being transferred to fly food containing either 20 mn = 10 for each genotype) were measured using a precision balance. Wing areas were measured as previously described  protocols. cDNA was prepared using oligod(T) primer and Superscript II reverse transcriptase according to the manufacturer's protocol (Invitrogen). Quantitative RT-PCR was performed using the PRISM 7000 sequence detection system and Fast SYBR® Green PCR Master Mix . Relative quantities of transcripts were determined using the relative standard curve method and normalized to Chromatin immunoprecipitations were carried out essentially as described  software.Immunohistochemical analysis of dFOXO protein overexpression in whole mount ovaries of 7-day-old females was performed using the anti-dFOXO antibody at a dilution of 1:250 followed by Alexafluor 488 labeled anti-rabbit secondary antibody (Invitrogen). Nuclei were visualized using DAPI. Images were acquired using a Zeiss LSM 700 confocal microscope and jmp (version 7) software . Lifespan data were subjected to survival analysis (Log-rank tests). Maximum lifespans were calculated as the median of the last surviving 10% of the population. Other data were tested for normality using the Shapiro–Wilk W test on studentized residuals (anova) were performed, and planned comparisons of means were made using Tukey–Kramer HSD (Honestly Significant Difference) or Student's t-test.Statistical analyses were performed using


The current issue is the 2013 issue which includes 21 interesting papers.Acupuncture stimulation elicits Deqi, a composite of unique sensations that is essential for clinical efficacy according to traditional Chinese medicine. In recent years, clinical trials of acupuncture have paid increasing attention to the evocation of Deqi. The physiological mechanism that produces the effect of Deqi has also been explored in several studies but is not well understood.As mentioned above, this special issue contains 21 papers, of which two papers are related to the characterization of the Deqi during acupuncture treatment. Deqi sensation often occurs during acupuncture treatment and is believed to be important for a successful acupuncture therapy. So far there exist no questionnaires for children. E. Anders et al. created a sentence based questionnaire for children on the basis of the Southampton Needle Sensation questionnaire (SNSQ). Three papers study the interaction between Deqi and acupuncture by neuroimaging technology. They provide evidence to understand neural mechanism underlying acupuncture. Four papers are related to the physiological mechanism of Deqi. These papers describe current knowledge in understanding of Deqi from a physiological aspect. Three reviews are related to the recent advances in Deqi and acupuncture effects. The current evidence base is not solid enough to draw any conclusion regarding the predicative value of natural Deqi for clinical efficacy or the therapeutic value of manipulation-facilitated Deqi. Six papers focus on Deqi in manual acupuncture compared with other types of acupuncture, of which 4 papers introduce the influence of the Deqi sensation by suspended moxibustion stimulation. Two papers adopt randomized controlled clinical trial and multicenter prospective cohort design to compare the clinical effectiveness of Deqi sensation.Deqi should be taken into account in clinical trials, and more researches are required to understand the underlying mechanisms, as described in this special issue.Cun-Zhi LiuCun-Zhi LiuGerhard LitscherGerhard LitscherFan-Rong LiangFan-Rong LiangJian KongJian KongLin-Peng WangLin-Peng WangLu WangLu Wang


The promoter -1031(T/C) polymorphism in the tumor necrosis factor-alpha gene was reported that it plays a part in reproduction-related diseases. Among these, polycystic ovary syndrome (PCOS) is known to be a common gynecological disease of women in reproductive age women. Here, we performed a comparative study of -1031(T/C) polymorphism of TNF-alpha gene with PCOS in a Korean population.A TNF-alpha gene was analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in a total of 217 PCOS patients and 144 matched female controls of healthy women. And statistical analysis was performed using HapAnalyzer. X2 test and logistic regression were utilized analyze the association between two groups. A p-value under 0.05 was considered statistically significant.The -1031(T/C) polymorphism of TNF-alpha gene and PCOS  = 2.53). In addition, the frequency of C allele was significantly higher in PCOS patients compared with controls. Sequence analyses also showed the -1031(T/C) polymorphism of TNF-alpha gene.The genotype and allelic frequencies were in Hardy-Weinberg equilibrium (HWE). There was strong association between the -1031(T/C) polymorphism in the promoter region of TNF-alpha gene in PCOS. We concluded that the -1031(T/C) polymorphism of TNF-alpha gene is associated with PCOS in a Korean population. Therefore, it is possible that it may be considered as a clinical biomarker to diagnose for PCOS, and is helpful in understanding the etiology for the pathogenesis of PCOS.This is the first study on the -1031(T/C) polymorphism of  Polycystic ovary syndrome (PCOS) is one the most common gynecological disorder and affects up to 5% women in reproductive ages -4. Generα (TNF-α) are related with gynecological diseases including pre-eclampsia, endometriosis [α may be considered as an immunological and molecular indicator for gynecological-related diseases. A previous study showed that -857(C/T) and -863(C/A) polymorphisms in TNF-α gene showed no effect on endometriosis patients in a Japanese population [TNF-α gene have no association with endometriosis in a Caucasian population [α gene plays a part of endometriosis in Asian populations [TNF-α gene may affect PCOS. Here, we investigated the relationship between SNP in -1031(T/C) of TNF-α gene and PCOS to investigated -1031(T/C) polymorphism in a TNF-α gene is associated with PCOS.Up to now, several association studies were reported that the some of polymorphisms of tumor necrosis factor-etriosis . It is aetriosis ,14. Manypulation . Additiopulation . And -30pulation . In contulations ,18. EvenAll samples were recruited from at Fertility Center of CHA General Hospital in Seoul, Korea. The study appraised 217 PCOS patients and 144 healthy Korean women as case and control groups based on the revised diagnostic criteria according to the 2003 ASRM/ESHRE Rotterdam consensus.Written informed consent was obtained from all of the participating women. Blood samples were collected in tubes containing EDTA as an anti-clotting factor and stored at -20°C until use. Genomic DNA was isolated from the blood samples of PCOS patients and controls. PCOS patients and controls in this study were all Korean women and this study with human blood samples was authorized by an Institutional Review Board (IRB).2, TSH, testosterone, and DHEA-S were measured as indicators of distinction.The clinical and biochemical characteristics of the PCOS patients and controls are indicated in Table TNF-a gene and PCOS, we collected 217 PCOS patients sample and 144 control samples, and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis was performed. The -1031(T/C) polymorphism of TNF-α gene was amplified by PCR with a set of primers, 5'-TAT GTG ATG GAC TCA CCA GG-3' and 5'-CCT CTA CAT GGC CCT GTC TT-3'. The 30 cycles of PCR was perform at 96°C for 5 min, 94°C for 30 sec, 63°C for 40 sec, and 72°C for 1 min and final cycle of 72°C for 10 min. Amplified PCR products were purified using AccuPrep Bioneer's PCR purification kit , and digested with Bbs I  for 14 hrs at 37°C.To investigate the association between the -1031(T/C) polymorphism of DNA fragments were electrophoresed on 2% agarose gels containing ethidium bromide in 0.5× Tris-Borate-EDTA buffer. Stained fragments were visualized under ultraviolet transilluminator. The fragments of 251- and 13-bp revealed homozygosity for the T allele and 180-, 71- and 13-bp fragments indicated homozygosity for the C allele , waist/hip ratio (WHR), glucose, and hormone levels including FSH, LH, ETNF-α gene, we recruited 217 patients and 144 control samples. In the present study, the frequency of T/T, T/C, and C/C genotypes of -1031(T/C) polymorphism in TNF-α gene was confirmed by sequence analysis and it showed different proportion  = 2.53) between PCOS and control groups . The frequency of T/T genotype between PCOS and control groups was 66.3% and 84.7%, respectively. The frequency of T/C genotype between PCOS and control groups was 32.7% and 15.3%, respectively. And, the frequency of C/C genotype between PCOS and control groups was 0.9% and 0%, respectively. Our results demonstrated that there was significant difference between PCOS and control groups, indicating that there is strong association between the -1031(T/C) polymorphism in the promoter region of TNF-α gene and PCOS.For the RFLP analysis for the -1031(T/C) polymorphism of α is a multifunctional proinflammation cytokine and plays an important role in wide-range of various diseases [α was shown to increase in patients with PCOS. The TNF-α gene is known to cause a decrease in insulin receptor tyrosine phosphorylation and an increase in serine phosphorylation of insulin receptor substrate, leading to inhibition of downstream insulin signaling and glucose uptake [TNF-diseases ,20. It ediseases -24 and gdiseases -28, but diseases -31. It idiseases ,33. Furte uptake .α expression is regulated at both the transcriptional and post-transcriptional levels. Moreover, TNF-α gene transcription is regulated by the promoter region which consists of an 1100 base pair of DNA [TNF-r of DNA ,36.TNF-α were reported including Behcet's disease [TNF-α on PCOS by biochemical, clinical, and molecular genetic approaches. Our data showed that the -1031(T/C) polymorphism in the promoter region of TNF-α gene is associated with PCOS in a Korean population. Our study indicated that the C allele may provide protection from PCOS, and this promoter polymorphism has been associated with the number of other diseases including inflammatory bowel diseases [TNF-α gene and PCOS. Genetic predisposition can be investigated with association studies, and our results suggest that it could be one of the etiological factors for PCOS and that the -1031(T/C) polymorphism may play an important role for the progression of PCOS.Up to now, several studies of the -1031(T/C) polymorphism of  disease , large j disease , and Cro disease . Crohn's disease ,40. In tdiseases , rheumatdiseases , and brediseases . FurtherTNF-α gene. It is very important to investigate the molecular mechanism of the polymorphism for PCOS. Further studies are required for the functional significance of the polymorphisms in TNF-α and may be suggested to be a credible molecular and immunological marker. And further association studies of PCOS with related genes required for finding the pathogenesis of PCOS and SNPs, which have association with PCOS important for prediction and suggestion as a biomarker for diagnosis of the disease.Up to now, there is no transcriptional factor binding to the -1031(T/C) polymorphism of α gene with PCOS. These data might be very informative for the advancement of future genetic treatment for PCOS patients.In conclusion, this is the first report on the association of -1031(T/C) polymorphism of TNF- The authors declare that they have no competing interests.JY designed the study and drafted the manuscript. JY and JC performed molecular genetic studies. KL and JS designed the study and offered the blood samples. KB designed the study, contributed the data analysis, and wrote the manuscript. All authors read and authorized the final manuscript.


The incidence of dengue, an infectious disease caused by dengue virus (DENV), has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs) has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated.By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses.Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection. Aedes aegypti and Aedes albopictus [Flaviviridae, genus Flavivirus. The virion contains a positive-sense single-strand RNA genome with a long open reading frame coding for capsid (C), premembrane(prM), and envelope(E) structural proteins, as well as seven non-structural(NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5[Dengue viruses (DENV) are transmitted among humans by mosquitos, such as bopictus . DENV inbopictus , mainly Because of the widespread geographical distribution and the severe clinical symptoms, dengue vaccine is urgently needed. However, licensed vaccine is not currently available for prevention of DENV infection. One major reason is the phenomenon of antibody dependent-enhancement (ADE), which is known as that a subsequent infection with an alternate serotype can enhance severity of dengue disease . One expTo develop an effective and safe dengue vaccine, we tested the effect of recombinant dengue virus-like particles (VLPs). Virus-like particle vaccine has shown considerable promise as vaccine candidate for many viral diseases -13. VLPsIn this study, four serotypes of dengue virus-like particles containing recombinant prM and E proteins were generated in mammalian cells, and their immunogenicity was evaluated in BALB/c mice. The results showed that monovalent VLPs of each serotype could stimulate specific IgG and neutralizing antibody against homotypic virus, and tetravalent VLPs could induce specific IgG and neutralizing antibodies against all four serotypes of dengue virus. Moreover, vaccination with monovalent or tetravalent VLPs also resulted in the induction of specific cellular responses. Therefore, dengue VLPs can be a potential vaccine candidate for the prevention of dengue infection.2. C6/36 Aedes albopictus cells (ATCC No.CRL-1660) were grown at 28°C without CO2 in Eagle's Minimum Essential Medium  supplemented with FBS, penicillin and streptomycin as well.293T cells (ATCC No.CRL-11268) were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% CO14-14-2 was also propagated in C6/36 cells and mainly used for cDNA cloning.Each serotype of dengue virus was passaged and propagated in C6/36 cells. The DENV-1 strain GZ01/95 and DENV-2 strain ZS01/01 were supplied by the Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, China. DENV-1 strain Hawaii, DENV-2 strain NGC, DENV-3 strain H87 and DENV-4 strain H241 were preserved by our laboratory. Strain GZ01/95, ZS01/01, H87 and H241 were used for RNA extraction and then VLPs expression plasmids construction while strain Hawaii, NGC, H87 and H241 were used for neutralization analysis. Japanese encephalitis virus (JEV) strain SAThe QIAamp Viral RNA Kit  was used to extract genomic RNA of DENV1-4 and JEV from 140 μl C6/36 cells culture supernatant infected with each virus. The extracted RNA was subjected to RT using Transcriptor High Fidelity cDNA Synthesis Kit  to generate cDNA templates for amplification of target genes. The PCR segments were digested with NheI and NotI enzymes and inserted into NheI and NotI sites of pcDNA5/FRT vector.14-14-2 strain. To further compare the impact of E protein transmembrane and cytoplasmatic domains, the third type of plasmids was designed as chimeric constructs pJD1-D4prMEΔ20%JEV, which contained JESS and full length of prM, but replaced the 3' terminal 20% region of E gene with the corresponding sequence of JEV (strain SA14-14-2).Three types of expression plasmids were constructed for each serotype of DENV Figure . The fir293T cells were prepared in wells of 6-well plates one day earlier and were transfected with pD1-D4prME, pJD1-D4prME or pJD1-D4prMEΔ20%JEV for each well using lipofectamin2000  according to instructions supplied by the manufacturer. Briefly, for each plasmid transfection, 8 μl lipofectamin2000 was diluted in 200 μl Opti-MEM (Gibco), and after incubating for 5 min at room temperature the diluted lipofectamin2000 was combined with diluted plasmid DNA (4 μg diluted in 200 μl Opti-MEM). After 20 min's incubation at room temperature, the mixture was added to each well with 80%-90% confluence of 293T cells. 48 hours post-transfection, cells and supernatants were harvested for future use.Concentrated culture supernatants were applied to a NuPAGE 4-12% Bis-Tris gradient gel, and followed by electroblotting onto PVDF membrane. Non-denatured proteins were then probed with E protein specific rabbit polyclonal sera which were produced in our laboratory. A goat anti-rabbit IgG conjugated to HRP was used as the secondary antibody. The reactions were detected by 3,3' diaminobenzidine (DAB) reagent according to the manufacturer's instructions.For DENV1-4 VLPs purification, 293T cells in T175 flasks were transiently transfected with optimized plasmids of each serotype. The culture supernatants containing extracellular VLPs were harvested routinely every 2 days, continuously repeated 3 times. To purify DENV1-4 virions which were used for electron microscopy and mice immunization, C6/36 cells in T175 flasks were infected with DENV strain Hawaii, NGC, H87 and H241 respectively. At 7 days after infection, the supernatants were harvested and inactivated with 1:2000 β-propionolactone.Cell supernatants collected from dengue VLPs production cells or dengue virus infected C6/36 cells were clarified by centrifugation at 10 000× g for 30 min followed by concentration using ultrafiltration system Vivaflow 200 (Sartorius stedim biotech). The concentrated culture supernatants were then purified by a two-step ultracentrifugation. The first step was a rate zonal centrifugation. Samples were added on the top of a 15-60% sucrose gradient and ultracentrifuged in a SW41 rotor (Beckman Coulter Inc.) at 38,000 rpm at 4 °C for 4 h. About 1 ml fractions were collected and pelleted by a second ultracentrifugation and then resuspended in PBS. The total protein concentrations of authentic virions and dengue VLPs were determined by Pierce BCA Protein Assay Kit.Purified dengue VLPs and dengue virions from sucrose density-gradient fractions were fixed with 2% glutaraldehyde. Small droplets of fixed samples were placed on copper formvar-coated grids for 1 min, then the grids were stained with sodium phosphotungstate for 1 min (excess samples of each step were removed). At last, the grids were visualized by TEM.Four to six-week-old female BALB/c mice were purchased from Chinese Academy of Medical Sciences Breeding Laboratories and were intraperitoneally (i.p.) inoculated with monovalent DENV VLPs (100 μg per dose) or a tetravalent combination (25 μg of each serotype per dose) in Freund's complete adjuvant (Sigma) for priming and in Freund's incomplete adjuvant for two times of boosting at an interval of 2 weeks. Equal amount of DENV virions  were used as controls with the same regimen. On days 0, 14 and 28, blood samples were collected through tail vein for measurement of serum IgG. At 2 weeks after the last inoculation, mice were sacrificed to collect serum for the neutralizing antibodies assay and separated splenocytes for testing cytotoxic T cell responses.VLPs specific serum IgG antibodies were titred by the binding capacity with rEIII protein, a recombinant protein that chimericly expressed DENV1-4 EIII domains in a certain order and previously produced in our laboratory. IgG titers were measured using enzyme-linked immunosorbent assay (ELISA). Briefly, 200 ng purified rEIII per well was coated on 96-well plates at 4°C overnight. Then, the plates were blocked with 5% skimmed milk in PBS for 1 h, and incubated with 2-fold serial diluted serum samples (starting from 1:50) at 37°C for 1 h. Bound IgG was detected by HRP-conjugated goat anti-mouse IgG (Sigma). After addition of 3,3', 3,5'-tetramethylbenzidine (TMB), absorbance was measured at 450 nm. The value which exceeds the mean+2 S.D. of negative control was considered positive.2 incubator at 37°C for 7 days. The neutralization titer was expressed as the maximum serum dilution at which the CPE of the virus was not observed in all four wells.The neutralization ability of serum antibodies against DENV was determined using CPE-determination assays. Briefly, mice sera from all groups were heat-inactivated at 56°C for 30 min, then the sera were two-fold serial diluted from 1:5 to 1:160 in Eagle's medium supplemented with 1% heat-inactivated FBS, penicillin and streptomycin and mixed with 100TCID50 virus. After 1 h incubation at 37°C, 100 μl of virus-serum mixture was inoculated to the confluent monolayer of BHK-21 cells in 96-well plates. Every dilution of each serum was performed in quadruplicate. The plates were then incubated in a CO5 cells) from the immunized mice were added to each well and stimulated with DENV VLPs at 37°C for 40 h. After cells were washed out, biotinylated anti-mouse IFN-γ was added to each well and incubated for 2 h at room temperature. Thereafter, the plates were washed and incubated for 1 h at room temperature with streptavidin-HRP. Finally, AEC substrate solution (BD) was added and spots were counted by ImmunoSpot® Analyzer (Cellular Technology Ltd.).The ELISPOT 96-well plates (BD) were coated with 100 μl of anti-mouse IFN-γ (5 μg/ml in coating buffer) at 4°C overnight. The following day, plates were washed and blocked with blocking solution for 2 h. Then, 100 μl freshly isolated splenocytes  Figure . All fouTo evaluate humoral responses induced by recombinant VLPs, BALB/c mice were immunized with monovalent dengue VLPs of each serotype or their corresponding virion counterparts for three times at two-week intervals. Serum samples were collected after 2 weeks of each immunization, and analyzed for IgG antibodies specific against rEIII, a recombinant protein that expressed chimerical EIII domains of all four DENV serotypes. EIII domain of DENV contains several neutralizing epitopes and host cell receptor recognition sites. As shown in Figure The results that monovalent dengue VLPs could efficiently enhance specific IgG and develop neutralization antibodies, suggested that the tetravalent formulation of DENV VLPs, which was constituting of four types of DENV VLPs at equal amount, might be capable to stimulate neutralizing antibodies against all four serotypes of DENV. As generating balanced neutralizing antibodies to each serotype of a tetravalent dengue vaccine is desired for its safety and efficacy, a tetravalent dengue VLPs combination was applied to BALB/c mice for all priming and boosting immunizations as the regimen used for monovalent VLPs vaccination. Tetravalent dengue virions and PBS were again used as controls. The serum samples collected at Day 14, 28, and 42 post the initial tetravalent VLPs vaccination, were analyzed for rEIII specific IgG antibodies. The data showed that tetravalent virions could stimulate IgG antibodies against rEIII protein. Intriguingly, tetravalent VLPs could even achieved to a higher level than tetravalent virions could induce Figure . MoreoveTherefore, similar as DENV virions, both monovalent and tetravalent formula of DENV VLPs could effectively induce virus specific IgG and produce high titer of protective neutralizing antibodies in vaccinated mice.in vitro stimulated with corresponding DENV1-4 VLPs and monitored for IFN-γ production. As depicted in Figure in vitro stimulation with each serotype of DENV1-4 VLPs, and with no significant differences to the homotypic virions  assay showed all constructs could express intracellular VLPs (data not shown). However, the western blot analysis of culturing supernatant showed that, pD1-D4prME constructs without signal peptide could not secret dengue VLPs. And except for pJD1prME, the constructs simply adding a JEV signal sequence could not secret dengue VLPs. All constructs which replaced the carboxy-terminal 20% of DENV E protein with corresponding JEV E protein could secret VLPs into cell culturing supernatant. Based on data from these express plasmids, pJD1prME and pJD2-D4prMEΔ20%JEV were identified as optimized VLP formation constructs for each DENV serotype. These data from different express plasmids indicated that i) signal peptide was one of the most important factors that influence downstream protein translocation and topology, thus dictating correct processing of dengue virus prM and E proteins by the host encoded signalase and endopeptidase ,31, replThis was different from previous studies ,32, whicin vivo development of neutralizing antibodies. Furthermore, the tetravalent formula of VLPs was able to simultaneously induce balanced neutralizing antibodies against all four serotypes. All these results confirmed that all four serotypes of DENV VLPs prepared in this study preserved the antigenicity of prM and E proteins.The immunogenicity of dengue VLPs was evaluated using BALB/c mice. The analysis of humoral immune responses revealed that dengue VLPs, in either monovalent or tetravalent formula, induced high levels of rEIII-specific IgG. Because dengue virus-induced neutralizing antibodies can bind to virus and prevent virus from binding to host cell receptors, therefore inducing neutralizing antibodies is particularly important to block virus entry into target cells . In thisin vitro stimulation with dengue VLPs.One of the marked advantages of VLPs is their ability to induce cellular immunity ,35. SincIn conclusion, by optimizing the expression plasmids, we successfully generated recombinant DENV1-4 VLPs in mammalian cells. Furthermore, the vaccination effect of VLPs in mice showed that either monovalent or tetravalent formula of dengue VLPs could efficiently elicit virus specific humoral and cellular immune responses. These results supplied evidence that VLP vaccine may serve as a promising strategy for dengue vaccine development.The authors declare that they have no competing interests.ZS performed most of the experiments and involved in manuscript preparation. LM coordinated laboratory manipulation and edited the manuscript. GW participated in mice immunization and detection of humoral immune responses. LC, MF, WX and ZQ were involved in cells culture, virus infection and VLPs purification. JC participated in editing of the manuscript. ZL and ZF participated in the detection of cellular immune responses. JL and LM provided Chinese strains of dengue viruses and gave advices for the project. LD is the project leader and was involved in project design, manipulation, data analysis and finalization of the manuscript. All authors read and approved the final manuscript.


TNF-α as a pro-inflammatory cytokine plays a key role in host defense against tuberculosis (TB). Presence of mutation in TNF-α gene can influence the effectiveness, performance and capability of immune responses against this infection. The Aim of this study was to investigate the frequency of TNF-α alleles and its relationship with susceptibility to TB and TNF-α gene variations.A case-control study was conducted and 103 healthy controls and 93 TB patients were enrolled. Genotype of TNF-238, TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using PCR-RFLP method.TNF-857 and TNF-863 were in high frequency mutation regions in a population level, and a significant difference at TNF-857 was noticed between the two groups of case and control.Presence of mutation in TNF-857 region probably increases the host susceptibility to mycobacterial infection. Genotyping of these regions in combination with other factors can be used for screening of high risk persons. According to high distribution of mutations in TNF-857 and TNF-863 regions, further studies on association of these regions is suggested. Tuberculosis (TB) is now among the most common infections which causes death in more than 2.000.000 patients annually. ApproximNinety three pulmonary tuberculosis patients with positive sputum smears and cultures that referred to Masih Daneshvari Hospital, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran were selected for this study. Control group contained 103 healthy individuals who had no history of tuberculosis. The study was approved by Ethics Committee of National Research Institute of Tuberculosis and Lung Disease, Iran, and all subjects signed inform consents. Two milliliters of peripheral blood were collected in EDTA containing tube and stored at -4°C until DNA extraction.Genomic DNA was extracted from peripheral blood leukocytes (PBLs), using the standard phenol– chloroform procedure with slight modifications. The polyAll statistical analyses were performed using SPSS software . Chi-Square test was utilized to define any statistically significant differences among polymorphic allele or genotype between the two case and control groups. P values were calculated using Fisher Exact test . The data were analyzed for their fitness to Hardy-Weinberg equilibrium.The mean age was 50.04 years for patients and 38.75 years for controls, and the two groups were matched for gender. The mean age of cases was higher than controls but the difference was not significant. Genotype and allele frequencies in patients and healthy people were shown in Frequency of alleles of TNF-238A, TNF-308A, TNF-857T, and TNF-863A in the control group were 2.4%, 7.8%, 10.7%, and 14.1% respectively, and 4.8%, 4.8%, 22.6%, and 14.5% for case group, respectively. No change was observed in the region of TNF-244. Comparison of genotypes between the control and case groups showed a significant difference at region of TNF-857 ; the frequency of C/T at region-857 in control and case groups was 17.5% and 45.2% respectively .In regions of TNF-238, TNF-244 , TNF-308 and TNF-863, no significant relationship was observed between the two groups. The distribution of alleles of regions -238, -308, -857 and -863 in the control group showed a good fitness using the Hardy–Weinberg equilibrium.Many single nucleotide polymorphisms are detected in the TNF-α gene region but most of the effective polymorphisms on TNF-α gene expression are those which are located in promoter region and has been altered levels of circulating TNF-α.27] Duri Duri27] In this study, we found new association between TNF-857 and TB while the TNF-857 C/T genotype had significantly a higher frequency in TB patients. However, Delgado did not find any significant association between TB and TNF-857 in Cambodian population. We also The associations between TNF-a production and TNF promoter polymorphisms are controversial. Wilson's study showed that TNF-308A is a much stronger transcriptional activator than TNF-308G in the human B cell line, and the TNF-308 polymorphisms have direct effects on TNF gene regulation. However,These divergent findings may be due to ethnical differences in various populations that is why the studies that were performed in healthy controls showed allele diversities.35]36][[36][35][[[36][35]We can conclude that TNF-857C/T genotype may be considered as a genetic marker that influences the susceptibility to tuberculosis in studied Iranian populations. Therefore, more comprehensive studies on -857 and -863 allele diversity of TNF gene is necessary.


Recent studies of antibodies in human plasma from HIV-1-infected and immunized individuals have revealed an important role for the V1/V2 region of gp120 in an antiviral response, and recent evidence that protection in the RV144 vaccine trial correlated with the presence of V1/V2-specific antibodies suggests that the V1V2 region is an important target for candidate HIV-1 vaccines. However, the function of V1V2-specific antibodies is not well understood. In this study we present data about the development, subtype specificity, and neutralization activity of such antibodies present in plasma from several hundred infected North American and African subjects.Human plasma from infected individuals were screened for neutralization activity versus a panel of subtype B, subtype C, and other Tier 2 pseudoviruses, and were titrated for binding activity against the consensus subtype B, C and A/E V1/V2 fusion glycoproteins, expressed via fusion to a fragment of the MuLV gp70 sequence. This system expresses V1/V2 domains in their native glycosylated and conformational forms. V1V2-specific antibodies were isolated from selected plasma by immunoaffinity chromatography on gp70-V1/V2 antigen columns, and characterized for neutralizing activity against various HIV-1 pseudoviruses.50s generally in the 1-20 μg/ml range.Most (>80%) of the HIV-1 infected subjects possessed robust levels of V1/V2 binding activity versus the three antigens. Interestingly, the development of V1/V2-reactive antibodies tracked with the development of autologous neutralizing antibodies in several subjects infected with subtype C viruses. Immunoaffinity-purified V1/V2-specific antibodies from selected broadly neutralizing plasma samples also possessed broad neutralization activities, with ICHighly cross-reactive V1V2-specific antibodies were present in almost all broadly neutralizing human plasmas at large concentrations, and frequently possessed modest neutralizing activities against a range of isolates, including tier 2 viruses. Additional information about the nature of these antibodies and their target epitopes would help elucidate their potential roles in protection against infection.


Tumor necrosis factor-α (TNF-α) is a key factor for the pathogenesis of inflammatory bowel diseases (IBD), whose function is known to be mediated by TNF receptor 1 (TNFR1) or 2. However, the precise role of the two receptors in IBD remains poorly understood. Herein, acute colitis was induced by dextran sulfate sodium (DSS) instillation in TNFR1 or 2−/− mice. TNFR1 ablation led to exacerbation of signs of colitis, including more weight loss, increased mortality, colon shortening and oedema, severe intestinal damage, and higher levels of myeloperoxidase compared to wild-type counterparts. While, TNFR2 deficiency had opposite effects. This discrepancy was reflected by alteration of proinflammatory cytokine and chemokine production in the colons. Importantly, TNFR1 ablation rendered enhanced apoptosis of colonic epithelial cells and TNFR2 deficiency conferred pro-apoptotic effects of lamina propria (LP)-immune cells, as shown by the decreased ratio of Bcl-2/Bax and enhanced nuclear factor (NF)-κB activity. Tumor necrosis factor (TNF) plays a crucial role in immune response at physiological and pathological states, which is based on its pleiotropic function on differentiation, growth and apoptosis of both immune and non-immune cell populations +CD45hiTNF-R2−/− T cells into RAG−/− recipients leads to acceleration of the onset of overt disease and to aggravated severity of intestinal inflammation + T cells on the course of colitis. The paradoxicality on the role of TNF signaling via TNF-R1 in IBD also exists. Ebath et al. Increasing data have been shown to support the intimate relationship of TNF-R expression with the pathogenesis of IBD. During active stages of the disease, TNF-R2 expression by colonic epithelial cells is increased in patients suffering from IBD and in mice with experimental colitis The studies on the function of TNF signaling via TNF-R1 or 2 on the course of DSS-induced colitis, which is another widely used murine model of IBD and closely resembles UC, is relatively few. Recently, Stillie et al. reported that, although ablation of TNF-R1or 2 had an effect on some parameters of DSS colitis in C57Bl/6 mice, TNF signaling via either of its receptors appeared to play a redundant role in the pathology of intestinal inflammation −/− and TNFR2−/− mice from Dr. Zhihai Qin  were backcrossed for 12 generations onto the BALB/c background BALB/c wild type (WT) mice were purchased from Jackson Laboratory . TNFR1Acute colitis was induced in mice by adding 3% DSS  to their drinking water and allowing them drink ad libitum, starting from day 0 for 8 days. Weights were measured before induction of colitis and daily thereafter. At the end of the experiments mice were sacrificed by cervical dislocation under anesthesia.Colons removed from mice at indicated time of death were fixed in 10% neutral-buffered formalin solution and then embedded in paraffin, cun into tissue sections, and stained with hematoxylin and eosin. Stained sections were examined for evidence of colitis using as criteria: the presence of lymphocyte infiltration, elongation and/or distortion of crypts, frank ulceration, and thickening of the bowel wall. The degree of inflammation on microscopic cross-sections of the colon was graded semiquatitatively from 0 to 4 , no structural changes observed; 2, moderate lymphocyte infiltration with infiltration seen in 10–25% hpf, crypt elongation, bowel wall thickeningwhich does not extend beyond mucosal layer, no evidence of ulceration; 3, high level of lymphocyte infiltration with infiltration seen in 25–50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer; and 4, marked degree of lymphocyte infiltration with infiltration seen in >50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall thickening with ulceration).2+, Mg2+-free HBSS supplemented with 10% fetal bovine serum, 5 mM EDTA, 15 mM HEPES, 100 µg/ml gentamycin under shaking at 37°C for 20 min then repeated for 4 times. The supernatants containing colonic epithelial cells were centrifuged at 800 g for 10 min at 4°C. the deposit was collected as colonic epithelial cells . The remaining was washed with HBSS, followed by digestion in RPMI 1640 medium containing 100 U/ml collagenase D (Roche Diagnostics) under shaking at 37°C for 1 h then repeated for 2 times. The total of digested supernatants was centrifuged at 800 g for 10 min at 4°C and washed with PBS. The pellet was layered on a 40%/100% Percoll gradient (Pharmacia) and spun at 1,800 rpm for 5 min to collect the lymphocyte-enriched population at the 40%/100% Percoll interface as LP-immune cells.The entire length of colon was opened longitudinally, washed with PBS, washed with PBS containing 100 µg/ml gentamycin, and cut into small pieces. The dissected mucosa was incubated with Cain situ cell death detection kits  according to the manufacturer’s instructions. In this method, residues of digoxigenin-labeled nucleotides are added to the ends of DNA fragments by terminal deoxynucleotidyl transferase, then peroxidase-labeled antidigoxigenin antibodies and 3,3′-diaminobenzidine are added in turn; the latter are oxidized by the bound peroxidase to generate visible signals from the apoptotic cells.For detection of apoptotic cells in tissues, labeling of degraded DNA specific to apoptotic cells was performed using a modification of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) technique by application of the g for 10 min. The supernatants were stored at −20°C until used for ELISA analysis.A 1 cm segment was divided from the distal 4 cm of the harvest colon. Wet weight was recorded separately for the whole distal 4 cm and the portion taken for homogenation. Colon tissue samples were homogenized in PBS containing a cocktail of protease inhibitors  with a Polytron homogenizer and centrifuged at 12 000 Colons were dissected at indicated timepoint after TNBS. A piece of the affected colon was then collected, weighed, placed in an 80°C oven for 24 h, then reweighed, and the wet-to-dry weight ratio was determined as a measure of colon edema Tissue MPO activity was determined by a standard enzymatic procedure as described previously g for 15 min, 30 µg of the supernatants were separated on 10% SDS-polyacrylamide gel and transferred onto an Immunobilon-P Transfer membrane (Millipore). After being blocked with 5% skim milk, the membrane was incubated with antibodies to Bcl-2 (1∶1000), Bax (1∶1000), or IκBα (1∶1000). Rabbit anti-actin antibody (1∶1000) was used as an internal control. ImmunoPure peroxidase-conjugated anti-rabbit IgG were used as secondary antibodies. The blotted membrane was then treated with the Super Signal West Dura Extended Duration Substrate (Pierce Biotechnology Inc.) and signals were detected by LAS-3000 mini CCD camera (Fuji Film). Primary antibodies used for immunoblotting were purchased from eBioscience.Samples were homogenized and sonicated in RIPA lysis buffer (Santa Cruz Biotechnology Inc.), supplemented with protease inhibitors. After centrifugation at 20,000 eBioscience.Colonic IL-6, IL-1β, MCP-1, IL-17A were measured by ELISA according to the manufacturers’ instructions. ELISA kits were purchased from Single cell suspension was prepared from spleen by passing them through nylon mesh or colonic tissues as described above and stained with appropriate antibodies in PBS with 2% heat-inactivated fetal calf serum and 0.1% sodium azide on ice for 25 minutes. The antibodies used are following: fluorescein isothiocyanate (FITC)-labeled anti-murine Gr-1 , F4/80  antibody, phycoerythrin (PE)-labeled anti-murine TNF-R1 , TNF-R2  antibody. These cells were fixed using PBS with 1% paraformaldehyde. For detecting apoptosis, double staining with annexin V and propidium iodide (PI) (eBioscience) was performed according to manufacturer’s instructions. Data collection and analysis were performed on a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson).5′-GCG CCC AGA CAG AAG TCA TAG-3′ and reverse primer 5′-AGC CTT GCC TTT GTT CAG TAT C-3′; CCL-3 forward primer 5′-TGT ACC ATG ACA CTC TGC AAC-3′ and reverse primer 5′-CAA CGA TGA ATT GGC GTG GAA-3′; GAPDH forward primer 5′-TGA AGG TCG GAG TCA ACG GAT TTG GT-3′ and reverse primer 5′-CAT GTG GGC CAT GAG GTC CAC CAC-3′. All reactions were performed in triplicate. The data were analyzed using Q-Gene software and expressed as fold change mean normalized expression (MNE) from control value. MNE is directly proportional to the amount of RNA of the target gene relative to the amount of RNA of the reference gene, GAPDH.RNA extracted from colon tissues was reverse-transcribed into cDNA using the SuperScript III First Strand cDNA synthesis system (Invitrogen). cDNA was synthesized from 0.5 µg RNA using random hexamer primers and SuperScript III(Invitrogen). Real-time RT-PCR was performed on a Bio-Rad iCycler to quantify mRNA levels. The primers for real-time were as follows: CXCL-1 forward primer 5′-ACT GCA CCC AAA CCG AAG TC-3′ and reverse primer 5′-TGG GGA CAC CTT TTA GCA TCT T-3′; CXCL-2 forward primer t test and 1-way analysis of variance (ANOVA) was used to determine significance, with P<0.05 considered significant. Kaplan-Meier was used for survival analysis with log rank P<0.05 to determine significance. Statistics were performed using SPSS 10.0 for Macintosh, and graphs were made on Deltagraph .Student Acute colitis was established in wild-type (WT) and TNF-R-knockout mice by continuous drinking of 3% DSS for eight days, respectively. WT mice lost an average of 11.8±4.3% of their baseline weight 8 days after DSS. TNF-R1−/− and TNF-R2−/− mice lost 28.8±2.6% and 4.4±2.3% at the same timepoint, respectively . FurtherNext, the influence of TNF-R1 or 2 knockout on macroscopic and microscopic damage of colons after DSS was examined. WT mice displayed remarkable shortening and thickening of the colon 8 day after DSS . TNF-R1 ablation led to more severe pathology of the target tissues. In contrast, the colon from TNF-R2−/− mice did not show considerable injury, resembling the colon of control mice . MoreoveNeutrophil infiltrate is a key event for initiation and promotion of inflammatory responses in many inflammatory diseases including IBD To determine whether opposite effects of TNF-R1 or 2 deficiency on pathological changes after DSS is associated with the alteration of expression of colitis-related proinflammatory cytokines and chemokines, the section of colons was dissected, homogenized and assayed at day 8 after DSS. Compared to WT colitis mice, lack of TNF-R1 resulted in significant up-regulation of IL-6, IL-1β, MCP-1, IL-17A levels in the colon tissues . In sharIt has been recognized that systemic inflammatory response is hallmark of DSS-induced colitis +) was increased in WT, TNF-R1 or 2-knockout DSS-instilled mice compared to untreated controls (Next, we defined the characteristics of this systemic inflammatory response induced by DSS administration. FACS analysis for frequency of granulocytes and monocytes in spleen was performed. The percentage of granulocytes (Gr-1controls , which wcontrols . These fTo determine the mechanisms underlying aggravated or attenuated intestinal damage in absence of TNF-R1 or 2 when DSS colitis was established, we speculate that lacking TNF signaling via TNF-R1 or 2 leads to alteration of apoptosis of colonic epithelial cells and/or lamina propria-residing immune cells. To address this issue, we first examined the expression of TNF-R1 and 2 on epithelial cells and LP-immune cells respectively. The result showed that TNFR1 was expressed only on CEC but not on LP-immune cells . SubsequNext, we examine whether increase in apoptosis of colonic epithelial cells or LP-immune cells in TNF-R1or 2 knockout mice after DSS is associated with reduced anti-apoptotic signals. The results showed that DSS instillation drastically reduced the expression of anti-apoptotic factor Bcl-2 in CEC of WT mice . This efTNF plays a critical role in the immunopathogenesis of IBD. Blocking TNF using specific monoclonal antibody has been identified to be beneficial for treatment of patients suffering from IBD It is worthy to note that the data presented in our study are incompletely consistent with a previous report that TNF signaling via TNF-R1 or 2 is redundant for the pathogenesis of DSS colitis in C57Bl/6 mice The harmful or beneficial outcome of lack of TNF-R1 or 2 on DSS-induced colitis is tightly related to alteration of production of proinflammatory cytokines in the attacked tissues. This result indicates that interference with TNF signaling affects other putatively colitis-related cytokine expression, for example IL-6. Indeed, TNF can directly induce IL-6 production by NF-κB-, NF-IL-6-, and AP-1-dependent mechanisms One of the hallmarks of intestinal inflammation is the infiltration of neutrophils into colons and release of large amounts of oxgen radicals Mucosal damage is a key event for DSS-induced colonic inflammation. TNF is an important factor in this process, as in pathological settings TNF is released largely and has deleterious effects on epithelial cells In the current study, we demonstrated an opposite role of TNF-R1 or 2 in the course of DSS-induced colitis, which is associated with alteration in several parameters, including infiltration of granulocytes, release of proinflammatory mediators and apoptosis of epithelial cells as well as inflammatory immune cells. These data suggest that targeting TNF-R2 rather than TNF-R1 specifically might be an alternative approach to therapy for patients with UC, considering that TNF is a key factor in host defense against microbe infection


Pulmonary metastasectomy for colorectal cancer is a commonly performed and well-established practice of ∼50 years standing. However, there have been no controlled studies, randomised or otherwise. We sought to investigate the evidence base that has been used in establishing its status as a standard of care.Among 51 papers used in a recent systematic review and quantitative synthesis, a citation network analysis was performed. A total of 344 publications (the 51 index papers and a further 293 cited in them) constitute the citation network.The pattern of citation is that of a citation cascade. Specific analyses show the frequent use of historical or landmark papers, which add authority. Papers expressing an opposing viewpoint are rarely cited.The citation network for this common and well-established practice provides an example of selective citation. This pattern of citation tends to escalate belief in a clinical practice even when it lacks a high-quality evidence base and may create an impression of more authority than is warranted. There is a professional consensus regarding the effectiveness of pulmonary metastasectomy for colorectal cancer (PMCRC), and along with metastasectomy for other primary sites, this surgery has become a routine  were ideBritish Medical Journal showed how authors seeking a basis for their own practice may create a citation cascade ‘resulting in unfounded authority of claims’. New Scientist, concerned with bias in the citation of stem cell research, used citation network analysis to show that US scientists rarely cited non-US authors  ( authors . In the  authors . These p authors  and in lWe examined the reference lists of the 51 papers incorporated into a systematic review and quantitative synthesis (SRQS) (http://pajek.imfm.si/doku.php?id=pajek/), to perform visualisations of the citation networks , 19 were otherwise classified , and 21 further PMCRC papers were cited by SRQS papers . The citOur search of the literature for evidence regarding PMCRC was prompted in part by a NICE Cancer Services Manual update stating, ‘Surgery for metastases confined to the liver or lung can be curative…for a minority of patients, it can increase five-year survival rates from close to zero to over 30%’ . We specNew England Journal of Medicine is cited by 14 of 51 of the index papers and is the thirteenth most frequent of 334 cited papers. Typical citations to it are: ‘The role of pulmonary metastasectomy for colorectal carcinoma was first introduced in 1944.’ (The manner by which ‘history’ is cited is of interest . In our Blalock's account of a pneumonectomy has no relevance to the current practice of pulmonary metastasectomy, and is not even claimed to be a first; hence, why is this such a popular citation? A possible answer is that among surgeons, his name adds authority: he went on to become extremely famous for the Blalock-Taussig shunt to palliate cyanotic heart disease . His papIn marked contrast, Aberg's publications are barely mentioned. His paper in 1980 and editorial in 1997 which challenge the effect of pulmonary metastasectomy are cited by only two of the index papers, and yet, unlike Blalock's paper, Aberg's publications have metastasectomy in their title and could not be missed in a literature search. The failure to cite suggests bias against his paper rather than that it was not retrievable. In addition, US citations may have a higher perceived status . BlalockTo quote from a remarkable essay on the whole question of citation , ‘The cu