Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM's resistance to X-rays in a clinical setting.Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with  Radiation therapy with surgery and chemotherapy is the standard treatment for glioblastoma multiforme (GBM) . However10B) releases alpha (4He) and 7Li particles through 10B 7Li reaction. The key players in the anti-tumor effects of BNCT are these high linear-energy-transfer (LET) particles. With BNCT, good results have already been achieved for patients with newly diagnosed GBM and recurrent malignant glioma . The difficulty of treating GBM may be attributed to the existence of GSCs in GBM, judging from the numerous published findings about GSCs.In previous reports, GSCs were isolated from glioma tissues as spheres cultured in SFM containing stem-cell mitogens, epidermal growth factor and fibroblast growth factor, which is the same method used to isolate neural stem cells from brain tissue –4, 17. B1H1H) or nitrogen capture reaction 14C) at irradiation, and these particles exhibited high-LET radiation. The LET of proton particles produced by the former reaction was about 50 keV/μm, and that produced by the latter reaction was about 35 keV/μm, whereas the gamma rays exhibited low-LET radiation. Therefore, it can be concluded that high-LET radiation can better overcome the radioresistance of GSLCs in comparison with low-LET irradiation. Ionizing radiation produces a broad spectrum of molecular lesions in DNA, including single-strand breaks, DSBs, and a great variety of base damages. DSBs are the most toxic form of DNA damage, because a single unrepaired DSB can lead to abnormal mitosis with losses of large fragments of DNA [To evaluate the difference in radiosensitivity between GSLCs and CCs, we irradiated these cells with gamma rays or neutron beams, and found that the latter could overcome the radioresistance of GSLCs to gamma rays Fig. . To obtas of DNA . Furthers of DNA , 24. In To clarify the response to DNA DSBs induced by gamma rays or neutron beams, we employed a gamma-H2AX assay. From a previous report, we judged the persistence of gamma-H2AX foci 24 h after treatment as unrepairable DSB . GSLCs h2). It is speculated that the neutron beams from KUR formed a wide-range beam that included gamma rays and secondary gamma rays. At the absorbed dose of 4 Gy, the compositions of fast, epithermal and thermal neutrons as well as of gamma rays were 25.5%, 2.5%, 22% and 50%, respectively. Almost half of the neutron beam components of the absorbed dose were induced by gamma rays, which could explain why small foci were induced mainly by gamma rays, even under neutron-beam irradiation. As described above, Fig. Indeed, under both gamma-ray and neutron-beam irradiation, more than half of all gamma-H2AX foci were small  (23390355) from the Japanese Ministry of Education, Culture, Sports, Science and Technology to S-IM.


MicroRNAs (miRs), a class of small non-coding RNA molecules, act as posttranscriptional regulators and are involved in a plethora of cellular functions. miRs have attracted a great deal of attention as potential therapeutic targets, as the sequence-specific mode in which they act, allows the simultaneous targeting of multiple target genes, often members of the same biological pathway(s) . PrevioumiR expression in SFs from TghuTNF and WT control mice were determined by deep sequencing and the arthritic profile was established by pairwise comparisons. qRT-PCR analysis was utilised for profile validation, miR and gene quantitation in patient SFs. Dysregulated miR target genes and pathways were predicted via bioinformatic algorithms.Deep sequencing demonstrated that TghuTNF-SFs exhibit a distinct pathogenic profile with 22 significantly upregulated and 30 significantly downregulated miRs . qRT-PCR validation assays confirmed the dysregulation of miR-223, miR-146a and miR-155 previously associated with human RA pathology, as well as that of miR-221/222 and miR-323-3p. Notably, the latter were also found significantly upregulated in patient RASFs, suggesting their association with human RA pathology. Bioinformatic analysis suggested Wnt/Cadherin signaling as the most significant pathway targets of miR-221/222 and miR-323-3p and CSNK1A1 and BTRC, the negative regulators of β-catenin, amongst predicted gene targets. qRT-PCR assays confirmed the downregulation of these genes in RASFs, validating our hypothesis that the newly identified miRs may function to modulate Wnt/Cadherin signaling.In this study, by performing comparative analyses between an established mouse model of arthritis and RA patient biopsies, we identified novel dysregulated miRs in RASFs potentially involved in pathways important for the pathogenic phenotype of these cells and highlighting the value of such cross-species comparative approaches .


Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Information remains limited about the comparative protein expression of host cells in response to TGEV infection. In this study, cellular protein response to TGEV infection in swine testes (ST) cells was analyzed, using the proteomic method of two-dimensional difference gel electrophoresis (2D DIGE) coupled with MALDI-TOF-TOF/MS identification.33 differentially expressed protein spots, of which 23 were up-regulated and 10 were down-regulated were identified. All the protein spots were successfully identified. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and the mitochondrial pathway. Western blot analysis was used to validate the changes of alpha tubulin, keratin 19, and prohibitin during TGEV infection.To our knowledge, we have performed the first analysis of the proteomic changes in host cell during TGEV infection. 17 altered cellular proteins that differentially expressed in TGEV infection were identified. The present study provides protein-related information that should be useful for understanding the host cell response to TGEV infection and the underlying mechanism of TGEV replication and pathogenicity. Coronaviridae family -1-propanesulfonate; DTT: Dithiothreitol; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TGEV: Transmissible gastroenteritis coronavirus; IEF: Isoelectric focusing; IPG: Immobilized pH gradient; MALDI-TOF-TOF/MS: Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; RT-PCR: Reverse transcriptase-polymerase chain reaction; PMF: Peptide mass fingerprinting; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.The authors declare that they have no competing interests.LF designed the study. LF and XZ were responsible for data analysis and interpretation and drafted the manuscript; HS, JC, and DS carried out the 2D DIGE gel analysis and excised the protein spots; HL and ZW were responsible for mass spectrometry analysis and manuscript editing. All authors read and approved the final manuscript.MS and MS/MS spectra of the identiifed protein spots.Click here for fileCell proteins identified from the differential 2D DIGE analysis after TGEV infection.Click here for file


HTLV-1 expression is generally extremely low in freshly isolated ATL cells, suggesting that gene silencing has occurred. The silencing of viral genes in ATL cells could be due to not only host epigenetic mechanism, but also consequences of natural selection of ATL cells to evade the host immune response. Recently, it has been reported that stromal cells could contribute to the regulation of HTLV-1 expression. In this study, we investigated whether stromal cells could be involved in reactivation of viral gene expression in HTLV-1 infected cells using co-culture systems. We used CR cells, a viral gene silenced ATL cell line, and human HEK-293T epithelial cells as stromal cells. To reactivate viral gene expression in CR cells, the histone deacetylase inhibitor, Trichostatin A (TSA) was employed. CR cells co-cultured with HEK-293T cells dramatically escaped TSA-induced apoptosis, while CR cells cultured alone underwent apoptosis by TSA in a dose-dependent manner. Moreover, the transcriptional activity of NF-κB in CR cells co-cultured with HEK-293T cells was decreased by TSA, whereas the activity in CR cells cultured alone was increased. These results suggest that stromal cells might allow HTLV-1-infected cells to escape from apoptosis induced by activation of HTLV-1 expression. This study provides an insight into the potential role of stromal cells in the maintenance of latency in HTLV-1 infected cells.


Yersinia has features of both apoptosis and pyroptosis. Cell death and caspase-1 processing in Yersinia-infected cells occur in response to inhibition of NF-κB and MAPK signaling by the Yersinia virulence factor YopJ. However, the molecular basis of YopJ-induced cell death, and the role of different death pathways in anti-Yersinia immune responses remain enigmatic. Here, we discuss the role that cell death may play in inducing specific pro-inflammatory signals that shape innate and adaptive immune responses against Yersinia infection.Cell death plays a central role in host-pathogen interactions, as it can eliminate the pathogen's replicative niche and provide pro-inflammatory signals necessary for an effective immune response; conversely, cell death can allow pathogens to eliminate immune cells and evade anti-microbial effector mechanisms. In response to developmental signals or cell-intrinsic stresses, the executioner caspases-3 and -7 mediate apoptotic cell death, which is generally viewed as immunologically silent or immunosuppressive. A proinflammatory form of cell death that requires caspase-1, termed pyroptosis, is activated in response to microbial products within the host cytosol or disruption of cellular membranes by microbial pathogens. Infection by the bacterial pathogen  Vaccinia and CMV viral infections  and virulence factors, known as Yersinia outer proteins (Yops) . Nevertheless, macrophages primed by inflammatory stimuli still undergo pyroptosis in response to YopJ-deficient Yersinia (Bergsbaken and Cookson, Pathogens express pore-forming toxins and virulence proteins that disrupt membrane integrity and modulate signaling networks (Vance et al., Salmonella infection (Broz et al., Y. pestis infection, and both NLRP3 and NLRP12 contributed to host defense against Yersinia infection, presumably via induction of caspase-1-dependent IL-1β and IL-18 (Vladimer et al., Yersinia host defense remains unknown.Interestingly, YopJ-dependent apoptosis is also associated with caspase-1 activation (Lilo et al., Yersinia virulence. Oral infection with Y. pseudotuberculosis and Y. enterocolitica demonstrate that YopJ/P contributes to systemic disease and barrier dysfunction (Monack et al., Yersinia had significantly reduced levels of spleen colonization (Monack et al., + TUNEL+ and total TUNEL+ cells compared to YopJ-deficient bacteria, consistent with the role of YopJ in apoptosis in vivo. Furthermore, in competitive index experiments, YopJ-deficient Yersinia showed colonization defects in PPs, mLNs, and spleen. YopJ-deficient Yersinia were not defective for splenic replication following intraperitoneal infection, indicating that YopJ primarily regulates dissemination from mucosal tissues, rather replication at systemic sites (Monack et al., Y. pestis are still able to cause systemic infection in a rat model of bubonic plague, despite a defect in induction of apoptosis and cytokine inhibition (Lemaitre et al., Yersinia to eliminate immune cells and dampen anti-Yersinia immunity during infection of peripheral or mucosal tissues.A number of studies indicate that YopJ/P promotes Y. enterocolitica in Y. pseudotuberculosis results in its attenuation in oral mouse infection (Brodsky and Medzhitov, Y. pseudotuberculosis and Y. enterocolitica cause cell death in cultured macrophages and infected tissues, infection with YopJ- vs. YopP-expressing Y. pseudotuberculosis showed a significant increase in TUNEL+ CD11b+, CD11c+ and B220+ cells in mLNs in mice infected with the YopP-expressing strain (Brodsky and Medzhitov, Y. pestis strains expressing YopP had higher cytotoxic potency than strains expressing YopJ, both in vitro and in tissues of infected mice; furthermore, expression of YopP in Y. pestis also resulted in lower virulence following subcutaneous, but not intranasal or intravenous routes of infection (Zauberman et al., Y. pestis expressing YopP protected against infection with virulent Y. pestis, regardless of the route of challenge. These observations suggest that YopJ contributes to dissemination of Yersinia from barrier surfaces, but may be less important once bacteria have spread to systemic sites. Whether YopJ or additional immunosuppressive virulence mechanisms play a role in dampening the early inflammatory response to Yersinia infection in pneumonic plague (Lathem et al., Paradoxically, ectopic expression of a hypercytotoxic YopP from Y. pseudotuberculosis, due to a loss of TLR2-dependent Reg3β expression in the gut epithelium (Dessein et al., Yersinia infection remains to be further dissected. Notably, IL-1α is associated with pathological intestinal inflammation and increased dissemination of Y. enterocolitica (Dube et al., Consistent with observations that YopJ promotes systemic dissemination following oral infection, YopJ contributes to gut barrier disruption (Jung et al., + cytotoxic T cells also induce death of Yersinia-infected cells, and are important for control of Yersinia infection, as demonstrated by the more severe disease in infected β2m−/−, anti-CD8α-treated, or perforin-deficient mice (Bergman et al., + T cell-mediated killing of bacteria-associated cells targeted them for phagocytosis by uninfected macrophages, and could bypass the anti-phagocytic activity of Yersinia Yops (Bergman et al., + T cells were not responsible for resistance to YopP-expressing Y. pestis, (Zauberman et al., + T cell-mediated killing due to elevated cytotoxicity induced by the bacteria themselves. These studies collectively suggest that regulation of cytotoxicity during Yersinia infection impacts virulence, and that a balance between the cytokine-blocking and death-inducing functions of YopJ is required for optimal virulence. Specifically, absence of YopJ results in failure of Yersinia to suppress cytokine production or induce cell death and causes a defect in dissemination. However, Y. pseudotuberculosis expressing YopP, which enables stronger inhibition of cytokine production and elevated levels of cell death, are also significantly attenuated in vivo (Figure Yersinia infection in vivo are not yet defined, activation of cell death in vivo either in response to YopJ activity, or as a consequence of T-cell-mediated cytotoxicity likely promotes immune responses against Yersinia.In contrast to cell death induced by the activity of a bacterial virulence factor, CD8in vivo (Kepp et al., + DCs to CD8+ T cells in both non-infectious and infectious settings (Sancho et al., + T cells into TH17 cells (Torchinsky et al., Yersinia infection may provide new insights into both anti-Yersinia immunity and potential bacterial evasion strategies (Lin et al., Apoptotic cell death is generally viewed as non-inflammatory or immunosuppressive, and YopJ-dependent cell death has been characterized as apoptosis (Mills et al., Understanding the control of programmed cell death remains essential for understanding anti-bacterial immunity. Appropriately distinguishing pathogens and non-pathogens is critical both for host defense and the maintenance of tissue homeostasis. The ability to detect conserved bacterial virulence activities, such as the disruption of actin cytoskeleton or inhibition of core signaling pathways, provides a mechanism for rapidly sensing the presence of pathogens. Induction of cell death in response to microbial virulence activities is an evolutionarily conserved response from plants to higher eukaryotes that limits the spread of infection. In higher organisms, distinct forms of programmed cell death, including apoptosis, pyroptosis, and necrosis, can have differential effects on downstream responses. Various cell types may have differing thresholds for activation of these distinct death pathways due to differential expression of pro-survival, pro-apoptotic, and immune sensor proteins. Thus, understanding how cells die and how cell death influences both the local microenvironment and ensuing systemic response may provide key insight into new approaches to modulate both antimicrobial immune responses and immunopathologies that result from dysregulation of these death pathways.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many studies have demonstrated the ability of perioperative hemodynamic goal-directed therapy (pGDT) to decrease postoperative morbidity in patients undergoing medium-to-high-risk surgery . As a reThe largest and most recent meta-analysis  on pGDT The 2011 meta-analysis , they weDepending on the pre-implementation morbidity rate, the degree of pGDT-induced morbidity reduction and the country, cost-savings ranged between $808 and $13,434 per patient. This large variability suggests that local/hospital estimations are desirable before starting pGDT implementation. These tailored evaluations would also allow more precise cost-saving estimations by taking into account the actual and expected pGDT compliance rates.


Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. Throughout the planet, environmental temperatures can reach low to freezing levels. Organisms indigenous to these habitats are presented with potential desiccation, which can lead to potentially detrimental challenges such as decreased enzymatic rates, freezing, and aggregation of endogenous proteins , 2. BesiThe temperature at which water freezes varies based on solution homogeneity . Pure waCellular cryodamage incurred from freezing is dependent on freezing rate and ice crystal location . For intOne line of defense against cold temperature stress is the production of small molecules that can act as cryoprotectants , 7. CommAn alternate strategy of cryoprotection is the production of antifreeze and ice nucleation proteins , 8. BothAcinetobacter or Bacillus. On the other hand, antifreeze proteins (AFPs) are a class of proteins characterized by the ability to bind to embryonic ice crystal and inhibit further growth , 7229, 7Antifreeze proteins have been proposed to inhibit ice growth through an adsorption inhibition mechanism based on the Kelvin effect . AccordiHowever, the adsorption inhibition mechanism has one major assumption: irreversible binding of antifreeze proteins . The proHowever, Pertaya and colleagues  argue thα-helices to large ordered β-solenoids [The efficient inhibition of ice crystal growth requires a large variety of ice binding surfaces complementary to the many ice crystal planes. This diversity in binding can provide ideal coverage of the ice crystal . Adaptinolenoids , 77.α -helix or extensive hydrogen bonding network in the protein core to retain rigidity [There are some similarities amongst antifreeze proteins. One similarity is a flat, hydrophobic ice binding surface that is a common antifreeze protein identifier. With this surface, antifreeze proteins may even be globular . In orderigidity , 29.A second common feature amongst antifreeze proteins is the presence of amino acid tandem repeats in the protein sequence. These repeats are non-specific but are structured so that ice binding residues are aligned on one side to create an ice binding face , 29, 76.With these similarities in mind, Doxey et al.  were ablMpAFP from Marinomonas primoryensis [MpAFP consists of five distinct domains, of which two (Regions II and IV) contain repetitive sequences. Region II has 120 perfectly conserved 104 amino acid tandem repeats. Region IV is a smaller repeat region comprised of 13 tandem repeats of 19 amino acids each and was the domain confirmed with antifreeze activity.Currently, the most well characterized bacterial antifreeze protein structure is oryensis . MpAFP cβ-roll [β-roll loop each, these tandem repeats form a calcium binding turn along the length of the domain when X-Gly-Thr-Gly-Asn-Asp is aligned , and a C terminal domain . These pPseudomonas syringae was destabilized by ozone treatment, the resultant ice nucleation activity was lowered from −2.8°C to −7.3°C [P. syringae with lipases, detergents, and organic solvents. However, ice nucleation activity could be recovered with phospholipid supplements. Yu and colleagues [Pantoea ananatis ice nucleation protein was expressed in Escherichia coli missing FabH, a protein responsible for fatty acid elongation, there was reduced ice nucleation activity. This activity loss was also recoverable by overexpressing FabH.The relatively hydrophobic and globular N-terminal domain, comprising 15% of the protein, is hypothesized to bind to lipids, polysaccharides, and other ice nucleation proteins . Interaco −7.3°C . A similo −7.3°C  treated lleagues  also demInvestigating the N-terminal domain specifically in InaQ, Li and colleagues  were ablPseudomonas syringae, the consensus is Ala-Gln-Glu-Gly-Ser-Asn-Leu-Thr-Ala-Gly-Tyr-Gly-Ser-Thr-Gly-Thr-Ala-Gly-Ala-Asp-Ser-Ser-Leu-Ile-Ala-Gly-Tyr-Gly-Ser-Thr-Gln-Thr-Ser-Gly-Ser-Glu-Ser-Ser-Leu-Thr-Ala-Gly-Tyr-Gly-Ser-Thr-Gln-Thr. This fragment can be broken into three mid-fidelity fragments of 16 residues each. In PbINP from Pseudomonas borealis, the consensus of these fragments is Gly-Tyr-Gly-Ser-Thr-X-Thr-Ala-X-X-X-Ser-X-Leu-X-Ala [Xanthomonas campestris, Kumaki and colleagues [β-turns. These 16 residue fragments can be further broken down into two low fidelity octapeptides, which are speculated to influence the bends in CRD folding.As the bulk of the protein, the central repeating domain is proposed to be the site of ice interaction . There aeu-X-Ala . When thlleagues  found thβ-conformation with alternating β-strands and random coils linked by interpeptide bonds. They speculated that stacking of β-structures in a hairpin manner produced a rigid structure with alternating patterns of rectangular and trapezoidal structures. Unlimited aggregation was believed to occur by interdigitization of similar sides of another ice nucleation protein . More recently, Graether and Jia [β-helix using a Thr-X-Thr motif for ice binding similar to antifreeze proteins. An ice nucleation protein from X. campestris was speculated to form a β helix structure based on 16-residue loops allowing the alignment of Thr-X-Thr motifs [Two decades ago, Kajava and Lindow  proposed and Jia  argue thr motifs . Howeverr motifs , 42.PbINP as a right handed β-helical structure that is stabilized by inward facing Ser and Gly ladders. The protein dimerizes via desolvation of the aromatic side chains from an exposed Tyr ladder on each monomer (Further supporting Graether and Jia's  hypothes monomer . DimerizIn the last 15% of the protein, the function of the globular C-terminal domain remains undetermined and unexplored . Li et aIce binding depends on the ability to recognize and interact with ice crystal planes. Typically, ice crystal controlling proteins are comprised of a flat, hydrophobic ice binding surface , 72, 77.Currently, a detailed mechanism for adsorption inhibition is continuously evolving. Initially, hydrogen binding between the complementary ice binding surface and ice crystal planes was hypothesized to arrest ice growth . HoweverA secondary hypothesis relies on entropy, van der Waals forces, and hydrophobic interactions for ice binding. It has been speculated that non-anchored clathrate waters within the troughs of ice binding surface are released immediately prior to adsorption , 72. To An alternate hypothesis proposes that anchored clathrate waters influence the hydration layers of the ice crystal controlling proteins . These lIce nucleation activity does not utilize the adsorption inhibition mechanism. However, Garnham and colleagues  speculatPseudomonas putida GR12-2 remained consistent following entry into the stationary phase, a growth stage associated with stress and cell maintenance. Similarly, ice nucleation proteins have also been reported to be expressed at high levels under conditions of cold temperature [Pseudomonas syringae occurred from a combination of cold temperature and nutrient starvation, which is indicative of the stationary phase. However in another case, Chen and coworkers [Pseudomonas fluorescens MACK-4 showed optimal expression following cold temperature growth with sorbitol, mannose or starch, and peptone. This suggests that ice crystal controlling protein expression may be dependent on the bacterium studied, with cold temperature being a common inducer. Understandably, cold expression is required as some ice crystal controlling proteins have been reported to be thermolabile, becoming inoperable at regular bacterial growth temperatures [Knowledge of the regulation of ice crystal controlling protein expression is limited. Typically, these proteins are induced under cold stress and can be expressed seasonally , 7, 27. perature , 88. In perature  shows theratures .However, this temperature-based induction is not very well understood. One model proposes that ice crystal controlling protein induction occurs as a response to upregulation of cryoprotective genes by a cold sensor . A cold Regulation of ice crystal controlling proteins also occurs at the posttranslational level. N-terminal acetylation and/or C-terminal amidation have been found for some ice crystal controlling proteins . In otheAntifreeze and ice nucleation protein production is a common strategy for the survival of various organisms in cold environments. The diversity between organisms suggests that due to convergent evolution the antifreeze protein expressed in a particular system may be unique to that organism or environment. Regardless, the ice crystal controlling nature of these proteins has been exploited in many biotechnical applications over the last two decades.The most common application for antifreeze protein is the enhancement of cryopreservation of biological materials. Antifreeze protein treatments protect against freezing damage to cell membranes and their associated proteins by preventing large ice crystals or spicules development , 91. In Recently, antifreeze proteins have been considered for non-preservation based applications including use as hydrocarbon hydrate inhibitors and biopolymers , 98. MosPseudomonas syringae, have been used in the recreation and food industries. These environmentally safe and efficient proteins have replaced inorganic equivalents such as silver iodide [Ice nucleation proteins, mainly from r iodide . Able tor iodide .ipdC and iaaM genes [Outside of the industry, ice nucleation proteins have research applications as well. Exploiting the cell surface anchoring capabilities, recombinant proteins linked to ice nucleation proteins have been expressed as fusion proteins to be displayed on the cell surface of the expression host . Only liaM genes . TranscraM genes .Enterobacter cloacae expressing an ina gene from Erwinia ananas was shown to increase the freezing temperature of corn borer and cotton bollworm following ingestion [Instead of purified proteins, viable ice nucleation active bacteria are being considered for agricultural use as biocontrol agents for insect pests. Transgenic ngestion . Tang anngestion  report sBoth ice crystal controlling proteins are prevalent globally in a wide variety of psychrophilic bacteria. With a focus on freeze tolerance, bacterial antifreeze proteins are known for a low level of non-colligative freezing point depression with greater emphasis on ice recrystallization inhibition. Superior ice recrystallization inhibition is utilized for improved survivability against freeze-thaw stress common to temperate, non-Antarctic environments. On the other hand, bacterial ice nucleation proteins induce ice crystal formation at high subzero temperatures and can influence precipitation in the atmosphere. For both antifreeze and ice nucleation proteins, only a small number of bacterial sources of these enzymes have been studied in any detail and very few of these bacterial enzymes have been isolated and characterized at the protein structure level. However, the suggested mechanism for both protein classes features the same ice crystal interactions, using an ordered ice-like hydration layer, with protein functions differentiated based on protein size.


Seizure is a common presenting symptom of primary brain tumors. There are no published studies regarding the roles of MicroRNA (miRNA) in tumor-related epilepsy. The authors set out to correlate miR-196b expression in low-grade glioma patients with pre-operative seizures and post-operative seizure control.Twenty-three patients with WHO grade II astrocytomas and 83 similar patients for independent validation were included. Follow-up visits regarding seizure prognosis were scheduled at 6 months. MiRNA profiling was used to identify differentially expressed miRNAs. The most important miRNA was determined by quantitative reverse-transcriptase polymerase chain reaction (q-PCR) in the validation cohort. Gene ontology (GO) analysis and whole genome mRNA profiling was performed to investigate the underlying biological processes.Array results showed that 30 miRNAs were overexpressed and 10 miRNAs were underexpressed (with more than 2 fold change) in patients with pre-operative seizures. MiR-196b was validated in the independent validation cohort. Patients with good seizure prognosis exhibited low levels of miR-196b expression compared with those who had poor seizure prognosis in the group without pre-operative seizures. Biological processes that relate to transcription and cell cycles were over-represented in the miR-196b-associated gene expression signature. MiR-196b-associated gene expression profiling was characterized by enrichment of genes usually involved in cell proliferation.We have provided the first evidence that expression of miR-196b was associated with the occurrence of pre-operative seizures in low-grade gliomas, and may predict seizure prognosis in patients without pre-operative seizures. Targeted treatments that decrease endogenous levels of miR-196b might represent novel therapeutic strategies. Epilepsy is a common presenting symptom of primary brain tumors, particularly low-grade gliomas (LGGs). Most patients with LGGs experience epileptic seizures at disease onset MicroRNAs (miRNAs) are single-stranded non-coding RNAs recognized as endogenous regulators of post-transcriptional gene expression. These small RNAs are capable of controlling gene expression by mediating either mRNA degradation or translation inhibition Previous studies have indicated that miR-196b may be a prognostic predictor in glioblastoma (GBM) patients This study was approved by the Ethics Committee of Beijing Tiantan Hospital and written informed consent was obtained from all the patients included. Between September 2005 and June 2009, 1134 patients with LGGs were treated at the Glioma Treatment Center of Tiantan Hospital . Of these patients, 508 (>16-years-old) underwent primary resection of supratentorial LGGs. All patients were pathologically confirmed and diagnosed as having WHO Grade II gliomas, including 48 oligodendrogliomas (O), 229 astrocytomas (A) and 231 oligoastrocytomas (OA). Tissue samples were recovered immediately after surgery, snap-frozen using liquid nitrogen vapors and stored at −80°C until needed. For the purpose of creating more homogeneous sample conditions, only samples that strictly met the following criteria were selected: 1) age <55-years-old; 2) no medication involving AEDs before admission; 3) a single lesion mainly focused at the frontal lobe as determined by MRI; and 4) pure WHO grade II astrocytomas by histopathology. We excluded older patients (≥55-years-old), patients on AEDs medication before admission, patients with partial or occipital lobe or 3 lobes of tumor location, as well as those with secondary resection or needle biopsy only. To make a comparison between seizure and non-seizure tissue, we defined the 2 groups as the seizure  and non-seizure (patients who demonstrated non-seizure symptoms for more than 6 months) groups. Twenty-three samples were eventually included for whole genome miRNA microarray analysis . Each ofEach patient was routinely prescribed sodium Valproate (0.6–1.2 g/day) at admission regardless of their symptoms. After surgery, patients who presented with or without preoperative seizures were discontinued the same AED after 3 months or 2 weeks, respectively, upon demonstration of seizure-free conditions. For outcome analysis, Engel classification was dichotomized as Class I (completely seizure-free) versus Class II–IV (not seizure-free) Total RNA from frozen tumor tissues was extracted using the mirVana miRNA Isolation kit  according to the manufacturer's protocol. RNA was quantitated using a ND-1000 UV-VIS Spectrophotometer (NanoDrop Technologies) and the integrity of RNA was assessed with a RNA 6000 Labchip kit in combination with an Agilent 2100 Bioanalyzer (Agilent) according to the manufacturer's instructions. RNA samples used in this study all had an OD 260/280 ratio above 1.9 and an RNA integrity number (RIN) greater than 7.0.Total RNA was polyadenylated and then converted to cDNA using a biotin-labeled oligonucleotide dT primer with a universal PCR sequence. After cDNA synthesis, miRNA were individually detected using specific oligonucleotides. A single miRNA-specific oligonucleotide (MSO) was designed against each mature miRNA sequence, and miRNA-specific primers were extended using DNA polymerase. Universal primers were used to amplify the cDNA templates and the primer complimentary to the array was fluorescently labeled. The labeled, single-stranded PCR products were hybridized to a Human v2.0 miRNA Expression BeadChip  with 1,146 human miRNAs (97% coverage of the miRBase 12.0 database).Quantitative RT-PCR was performed using a standard TaqMan PCR kit on an ABI 7900 real-time PCR system . All the primers and probes for hsa-miR-196b and U6 rRNA endogenous controls for TaqMan microRNA assays were purchased from Applied Biosystems. Thermal cycling for the quantitative RT-PCR was carried out according to the manufacturer's recommendation and the relative expression levels were calculated using the comparative Ct method.Microarray analysis was performed using an Agilent Whole Human Genome Array according to the manufacturer's instructions. The integrity of total RNA was checked using an Agilent 2100 Bioanalyzer . The cDNA and biotinylated cRNA were synthesized and hybridized to the array; data were acquired using an Agilent G2565BA Microarray Scanner System and Agilent Feature Extraction Software (v9.1). Probe intensities were normalized using GeneSpring GX 11.0.Associations for miR-196b expression, if considered as a continuous variable, with baseline clinical features were analyzed with Student's t-test using GraphPad Prism 4.0. Univariate analyses were carried out using the Chi-square test for dichotomous variables and the Mann-Whitney U-test for continuous non-parametric data. Logistic regression models were constructed to analyze factors related to the probability of post-operative seizure control using SPSS 13.0 for Windows. Gene ontology (GO) analysis was performed using David To underscore the differentially expressed miRNAs between patients with and without pre-operative seizures, 23 samples were subjected to whole genome miRNA profiling . The difIn the validation cohort of our study, all patients were subjected to sodium Valproate monotherapy pre- and post-operatively. As shown in To further understand the potential functional role of miR-196b expression in low-grade gliomas, we performed a GO analysis. Biological processes that relate to transcription and cell cycles were over-represented in the miR-196b-associated gene expression signature . To gainTumor-related epilepsy is among the most common types of symptomatic epilepsy. Many recent studies have demonstrated dysregulated miRNAs in human TLE and post status epilepticus rats Currently, microarray profiles of miRNA expression patterns have shown that the highest expression levels of miR-196b were found in human bone marrow and spleen cells The mechanisms of action for miR-196 in tumor-related epilepsy are still unknown. These may depend on the biological processes underlying miR-196. In the present study, firstly, miR-196b correlates with seizure occurrence. Now, some studies have demonstrated that patients with epilepsy are more likely to have worse seizure control after surgery than those without pre-operative epilepsy We observed a positive correlation of miR-196b expression with the expression of proliferation-associated genes. This further suggests that the proliferation of tumor cells could possibly play a part in the pathogenesis of tumor-related epilepsy. GO analysis showed that many cell cycle-associated gene sets were over-represented in the miR-196b-associated gene expression signature. The expression of PCNA, a proliferation marker, was also positively correlated with miR-196b expression. These results suggest that miR-196 may obtain its biological role by regulating cell proliferation.Valproate acid (VPA), a stabilizer of the cell membrane and an inhibitor of GABA receptors controlling epileptic discharge, has the potential for apoptosis, growth arrest, and cell differentiation of tumor cells through inhibition of histone deacetylase Currently, changes in the expression of miR-146a have been implicated in both the development of multiple cancers and in the regulation of inflammation induced via the innate immune response MiR-15b, another miRNA found in our microarray data, is also reported in several similar studies on GBM Additionally, as shown previously, we demonstrated a relationship between extent of resection and seizure prognosis in patients without a history of seizure. It is generally accepted that minimizing the residue volume of tumors with gross total resection could increase the possibility of removing the epileptogenic zone. Thus, patients are more likely to achieve favorable seizure control.The current study has some limitations. Six or 12 months follow-up is a relatively short period of time for estimating seizure frequency, a longer follow-up would have been preferable when survival information is available. We did not use a control group to evaluate the response to another or newer AEDs such as levetiracetam. Further confirmation on large-scale prospective data would be advantageous.In vivo experiments to determine the exact role of miR-196b in tumor proliferation could contribute to explaining epileptogenesis and developing new treatment strategies aimed at improving patients' quality of life.Our data suggest that high miR-196b expression levels are a novel and valuable marker for the molecular diagnosis and prediction of glioma-related epilepsy. Prospective controlled studies are needed to address this issue. Table S1Differently expressed miRNAs between seizure group and non-seizure group.(DOC)Click here for additional data file.Table S2Genes significantly correlated with miR-196b expression .(DOC)Click here for additional data file.


Treg are currently classified, according to their origin, into thymus-derived Treg (tTreg) or peripherally induced Treg (pTreg) cells. Controversy exists over the prevalent mechanism accounting for Treg expansion in tumors, since both tTreg proliferation and de novo pTreg differentiation may occur. Since tTreg and pTreg are believed as preferentially self-specific or broadly directed to non-self and tumor-specific antigens, respectively, the balance between tTreg and pTreg accumulation may impact on the repertoire of antigen specificities recognized by Treg in tumors. The prevalence of tTreg or pTreg may also affect the outcome of immunotherapies based on tumor-antigen vaccination or Treg depletion. The mechanisms dictating pTreg induction or tTreg expansion/stability are a matter of intense investigation and the most recent results depict a complex landscape. Indeed, selected Treg subsets may display peculiar characteristics in terms of stability, suppressive function, and cytokine production, depending on microenvironmental signals. These features may be differentially distributed between pTreg and tTreg and may significantly affect the possibility of manipulating Treg in cancer therapy. We propose here that innovative immunotherapeutic strategies may be directed at diverting unstable/uncommitted Treg, mostly enriched in the pTreg pool, into tumor-specific effectors, while preserving systemic immune tolerance ensured by self-specific tTreg.The expansion of regulatory T cells (T The firwas CD25 . Few yeasyndrome , 7, and syndrome , both chhylation .reg are classified into two principal subsets based on their developmental origin: thymus-derived Treg (tTreg) develop in the thymus, while peripherally induced Treg (pTreg) develop in vivo in the periphery from conventional T cells (Tconv), through a process called “conversion”  under TGF-β and/or retinoic acid exposure, but their complete commitment into fully differentiated Treg is still under debate  into pTreg , a co-inhibitory member of the CD28/B7 family, endowed with strong immune-regulatory properties  , expressed on Treg membrane, which ensures the stability of Treg-DC interaction and allows Treg to efficaciously suppress DC  , a homolfunction , 21. Trepress DC . A studyng Nrp-1 . The rec mammary  and pros mammary .reg are also able to control the proliferation and function of different immune cells via the Perforin pathway , a transcription factor active in the IL-10 promoter  and IL-12α (p35) , further stimulating the new angiogenesis process and the establishment of a tolerogenic microenvironment  . In two ing Treg . It is wscovered . In the ironment .reg were considered one of the main obstacles for tumor clearance, according to their tolerogenic properties and their accumulation along tumor progression. In this view, several anti-tumor immune-therapies focus on Treg depletion or inhibition, in order to “contrasuppress” Treg inhibitory functions and to block the conversion of non-regulatory cells (non-Treg) into regulatory cells  reduced tumor growth and prolonged mice survival   –52. In tnal CD4+  and CD8+ CD4+   . Univariltration . Howeverlunteers .reg in tumor outcome, the new studies should take into account the tumor stage and the related inflammatory features, depending on the anatomical localization. In general, those tumors arising from chronic inflammation, almost at their initial stage, can benefit from the suppressive properties of Treg. In fact, during the inflammatory process, Treg highly accumulate in the site of inflammation such to prevent exacerbated immune responses and tissue damage, which are the prelude to neoplastic transformation. On the contrary, in the presence of an established tumor, Treg may reduce anti-tumor immunity thus favoring tumor escape. A more specific definition of Treg contribution in tumor development and progression is desirable for the design of new and more effective immunotherapies, allowing the discrimination among tumors that will benefit or not from Treg depletion/inhibition.The discrepancies observed in these studies may be due to the number of patients included, different strategies of analysis and non-homogeneity of tumor samples . Certainly, to properly define the role of Treg and pTreg, besides their site of origin  is involved in the stable commitment of the Treg lineage, and controversy still remains on whether iTreg or pTreg can efficiently demethylate this region and become fully committed Treg  in tumor-bearing mice or cancer patients, thus indirectly supporting the idea that expansion of pre-existing tTreg might prevail over pTreg differentiation in building the tumor-associated Treg pool. For instance, Treg have been shown to incorporate high levels of BrdU in tumor-draining lymph nodes and at cancer sites in several experimental models (reg (identified as CD4+CD25high cells) than CD4+CD25− or CD25low cells, suggesting that the Treg pool mainly derived from peripheral expansion rather than recent thymic emigration .One of the first attempts to distinguish between pT sarcoma . The anaecursors , 71, thuto tTreg . Many stl models , 74. A sigration . Howeverreg conversion actually occurs in tumor-bearing hosts at higher efficiency than in physiological conditions, even if controversy still exists on whether pTreg may prevail numerically over tTreg at the tumor site. We have in the past demonstrated that thymectomized and CD25-depleted mice, subsequently transplanted with carcinoma cells, showed a significantly higher Treg recovery in tumor-draining than in contralateral lymph nodes, suggesting that in tumor-bearing mice the Treg pool might be replenished mostly by newly derived pTreg than by proliferation of residual Treg. To prove this possibility, CD25-depleted CD4-purified T cells were transferred into immunocompetent, Thy1.1-congenic, CT26 tumor-bearing mice. In this setting, we could show that the transferred cells acquired CD25 and Foxp3 at significantly higher levels in draining lymph nodes, compared to contralateral lymph nodes of tumor-bearing mice, or to the lymph nodes of tumor-free mice  compatible with the pTreg identity , but that the extent of pTreg induction was not affected by the concomitant presence of tTreg, either exogenous (adoptively co-transferred) or endogenous (pre-existing in the host)  , 82. Thireg accumulation in tumors should take into account, beside de novo conversion, the active proliferation of not only tTreg but also pTreg. Proliferation plays opposite roles in the differentiation of Tconv into pTregversus the expansion of already differentiated pTreg. Regarding the former aspect, we have demonstrated that Tconv proliferation was not required for their conversion into pTreg, since CD25+Foxp3+ cells could develop in tumor-bearing mice from CD25-depleted cells treated with an anti-proliferative agent  compared to a CNS1-sufficient (containing pTreg) counterpart, suggesting that not only the tTreg pool, but also the pTreg niche, may be controlled by autonomous homeostatic mechanisms  self-antigens physiologically expressed as in the tissue of origin, (ii) self-antigens aberrantly expressed, in terms of expression level, developmental stage, or histotype , and (iii) neo-antigens uniquely expressed by tumor cells, mostly derived from oncogenic mutations (named tumor-specific antigens or TSAs). Based on the above considerations, self-antigens and TAA should be recognized by tTreg have been shown to promptly proliferate in response to the cognate antigen specifically expressed by tumor cells, suggesting that Treg can undergo tumor-antigen-driven activation and expansion , and suppressing the cognate antigen-directed T cell response, have been obtained from tumor-draining lymph nodes and tumor biopsies of cervical cancer patients (reg clones specific for antigens of the Epstein–Barr virus (EBV), associated to several hematological and solid malignancies, can be generated from the peripheral blood of healthy donors  by the immune system as a self-antigen and elicited the generation of a pool of memory Treg specific for that antigen  showed that Treg infiltrating murine transplanted tumors displayed a biased TCR repertoire toward “public” CDR3 sequences , suggesting Treg intra-tumor clonal expansion driven by the recognition of dominant antigens  cells than tTreg.Few data exist on the extent of TSDR demethylation in tumor-associated Themistry . Of note studies , 111. Threg in some tumor conditions and therapies. An increased frequency of epigenetically committed (TSDR-demethylated) Treg has been determined in tumor-infiltrating cells of ovarian, colorectal, and bronchial cancers compared to non-tumoral tissue counterparts , CD45RA−FOXP3low non-Treg, and CD45RA−FOXP3high (CD45RO+) activated Treg (aTreg), endowed with different potentials of proliferation, suppression, stability, and plasticity  and pTreg (derived from non-Treg or Tconv). The CD45RA−FOXP3low non-Treg subset may represent a mixture of activated Tconv (promiscuously and unstably expressing FOXP3), latent Treg (transiently downregulating FOXP3), and recently converted pTreg   adapt their molecular programs to optimize their in vivo suppressive function in distinct inflammatory milieus, which may be alternatively dominated by Th1, Th2, Th17, or TFH responses. Strikingly, these Treg specialized programs are orchestrated by the same transcription factors that drive the polarization of the targeted T-helper subset: therefore, T-bet, IRF4, Stat3, and Bcl6 expression are respectively and selectively required for the Treg specialized suppression of Th1  can express not only T-helper-related transcription factors and migratory molecules, but also cytokines such as IFN-γ or IL-17, thus turning from specialized suppressors into so-called Th1-like or Th17-like Treg that may rather contribute to inflammation  when cultured ex vivo with IL-2; under similar conditions, tumor-infiltrating Treg were prone to FOXP3 downregulation, attenuation of suppressive function, and prompt IL-17 production , and function specifically during type-1 inflammation  antigen  non-Treg subset, mostly including activated Tconv and/or uncommitted Treg. Conversely, in ovarian cancer, tumor-infiltrating Treg were enriched in CXCR3+ cells, highly expressing T-bet but poorly producing IFN-γ, and strongly suppressing Th1 response ex vivo  under Th1-polarizing conditions  endpoint; on the other side, pTreg, containing less committed and foreign antigens-specific cells, may be more prone to the complete reprograming into pro-inflammatory (T-bet+ IFN-γ+) cells. Future studies will elucidate the mechanisms by which different growing tumors may favor the expansion of pro-tumoral specialized Th1 suppressors or the induction of Th1-like plastic Treg.Some therapeutic interventions can force tumor-associated Tum IL-12 . On the reg as potent immune suppressive cells hampering the establishment of anti-tumor immunity, it soon became clear that anti-tumor vaccination might fail to elicit an effective immune response and to achieve successful tumor eradication, because of the immune suppressive barrier created by Treg. In addition, since Treg may recognize TAA and TSA at higher frequency than Tconv, tumor-antigen-based vaccines may expand/induce Treg rather than effector cells, thus inhibiting rather than boosting the anti-tumor response. Indeed, Zhou et al. first demonstrated that TCR-transgenic CD4 T cells specific for a TAA, adoptively transferred into mice bearing TAA-expressing tumor cells, proliferated extensively after administration of a therapeutic tumor vaccine (in the form of a recombinant vaccinia virus encoding the antigen), but tumor-antigen-experienced cells were mostly regulatory cells, ex vivo suppressive, and anergic to subsequent stimulation  or not (low-dose cyclophosphamide), failed to consistently eliminate more than 50% of committed Treg, as identified by TSDR demethylation  could result in enhancing Treg propensity to reprograming. In line with this idea, treatment of murine melanomas with a GITR agonistic antibody resulted in the accumulation of exTreg at the tumor site  Helios- and TNFR2-expressing cells, and contain the precursors of specialized T-bet+ Th1-suppressing cells, thus representing not only the guardians of systemic immune homeostasis but also the “hard core” of tumor immune escape. On the other side, pTreg may mostly develop following local encounter with TAA or TSA antigens, possibly represent a mixed population of committed (TSDR-demethylated) and uncommitted (TSDR-methylated) cells, and are more prone to be reprogramed into Th1-like or Th17-like effector cells. We envisage that future successful immunotherapies may not only target committed Treg but also favor “recycling” uncommitted Treg into prompt anti-tumor effectors.Even though discrimination between pTThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This review summarizes the application of the divinylcyclopropane–cycloheptadiene rearrangement in synthetic organic chemistry. A brief overview of the new mechanistic insights concerning the title reaction is provided as well as a condensed account on the biological relevance of the topic. Heteroatom variants of this rearrangement are covered briefly. The elusive cis-divinylcyclopropane (9) was characterized ten years later by Brown and coworkers , an, an21, sheromone .25, see Thuja koraiensis, farnesyl pyrophosphate (22) is supposed to undergo ring closure and the intermediate carbocation is trapped by hydroxide to give hedycaryol (23)  was was50) a55, see 59) , an, an55, s see 59) , prezizaene (64) . The syn terreus –53 startreus  cycloaddition -cycloaddition with an intermediate DVCPR . S. Sepi-viof TMSCl  yielded  variant –109 of t126, see Schisandra wilsoniana -cycloaddition  was usedxylation  resultedperature  resultedlate 155 –135, foltic acid .146, see 160 , i, i197, soworkers –154. Stareaction  took plasolution , albeit xide 187 –158. Forng TMSCN –161, folnt (193)  was used reagent –164. Dec reagent –166 was ing TMSI –168. The206, see 198 a Corey–Chaykovsky cyclopropanation reaction was achieved to yield cyclopropane 199. The keto-group was removed using a three step sequence, with concomitant reduction of the ester to give alcohol 200. Oxidation and Wittig olefination gave olefin 201. Lithium bromide exchange followed by 1,2-addition to ketone 202 yielded tricyclic 203, which was immediately subjected to acidic hydrolysis to give the desired tricyclic core skeleton 205 in 51% yield through transition state 204.The group of Wender applied the DVCPR in an approach towards the core skeleton of tiglianes  or via ionic intermediate 208''' and fragmentation to give 211. The different product distribution can be controlled by the choice of the solvent. Notably cycloheptadiene 210 arising from the DVCPR contains two as well as the related trans-silylated compound 209. This demonstrates the huge driving force of the DVCPR, as two highly strained olefins are favoured over a cyclopropane-moiety.The group of Davies  demonstr218, see 213 to copper catalysis yielded vinylcyclopropane 214. Deprotonation and silyl ether formation resulted in intermediate cis-divinylcyclopropane 215, which smoothly underwent DVCPR to give cyclohexane 216. Tsuji–Trost allylation [217 under forcing conditions.Wood and coworkers  applied lylation –177 furnlylation –179 finicis-divinylcyclopropane rearrangement to build up the diterpenoid cyanthin skeleton [2 iron complex 229. Oxidation led to the formation of the desired divinylcyclopropane, followed by reduction of the ester to the desired alcohol to give compound 230. Subjection of the trans-cis-divinylcyclopropane mixture to elevated temperature smoothly formed the desired bicycle 231. The less hindered double bond was removed using Wilkinson’s catalyst, followed by standard functional group interconversions to yield epoxide 232. When this compound was subjected to oxidative conditions both alcohols were oxidized to the corresponding aldehyde/ketone. Base-induced epoxide opening led to the formation of a double bond (C4/C5) and concomitant lactol-formation. The lactol was oxidized to the corresponding lactone under the same reaction condition. Final reduction gave core skeleton 233.Donaldson and coworkers  used the227 see  formatio234 methane resulted in the formation of cis-divinylcyclopropane 246 as the corresponding bis-zinc-enolate species. DVCPR occurred at ambient temperature, final acidic workup provided cycloheptadione 247 in excellent yield  catalyst, which was used to selectively cyclopropanate the less hindered double bond of dienes like 263. Spontaneous DVCPR provided cycloheptadiene 265 via 264 in good yields.Nevado and coworkers  applied Further contributions to the topic have been put forward by the group of Echavarren  and Gung274, see 5(tol) upon irradiation to give annulated tricycle 275. The common mechanism for this type of reaction proceeds via endo-dig cyclization of enynes like 266 to give zwitterionic intermediate 267. Metal-carbenoid formation with subsequent cyclopropane formation gives rise to either cis- or trans-divinylcyclopropanes 268/271. cis-Divinylcyclopropane 271 can readily undergo DVCPR to give bicycle 272. 1,2-Migration of the migrating group G leads to final bicycle 273 after regeneration of the catalyst. Trans-divinylcyclopropane 268 can undergo DVCPR after 1,2-migration and subsequent formation of cis-divinylcyclopropane 269 yielding bridged bicycle 270.Iwasawa and coworkers  discover276 both possible cycloheptadienes (bridged 277 or annulated 278) could be accessed. The selective formation of annulated bicycle 278 in preference of the possible bridged variant underlined the prefered reactivity of their enyne system.Chung and coworkers  discover279 could be converted into bicyclic ketone 281 in good yields.Gagosz and coworkers  recentlyThe oxygen substituted versions of the DVCPR have been subject to more intense research in the covered time period than the corresponding nitrogen variants. In general there are two different modes for the heteroatom incorporation into the DVCPR, as part of the cyclopropane moiety or as part of one of the vinyl moieties. All variants are covered in the following.292, see Salvia leucantha [282, desilylation was affected using TBAF, followed by ruthenium-catalyzed cycloisomerization [283. The carboxylic acid was converted into the corresponding acyl chloride, followed by addition of diazomethane and subsequent Arndt–Eistert homologation [284. Claisen condensation furnished the intermediate β-cyano-ketone, which was subjected to diazotransfer conditions [285. This compound underwent smooth cyclopropanation with the adjacent benzene moiety to give cis-vinylcyclopropane cyanide 286. Conversion of the ketone to the corresponding triflate followed by mono-reduction of the nitrile furnished cis-vinylcyclopropane carbaldehyde 287, which underwent smooth, but undesired rearrangement to dihydrooxepin 288. This rearrangement has been observed to be reversible [289. Lactonization was achieved upon Pd-catalyzed CO-insertion to give 290. The desired natural product was obtained after oxidation of the tetrahydrofuran to the desired natural product salvileucalin B (292) using chromium trioxide·3,5-dimethylpyrazole [The Reisman group ,212 obseeucantha . Startinrization  and clealogation  to obtainditions  to obtaiversible . Treatmepyrazole .293 to 294, see 297, beginning with cis-selective Rieke-Zn reduction. Epoxidation followed by oxidation furnished cis-vinylketone-epoxide 298. Enolate formation and acetate trapping afforded an intermediate enol-acetate, which underwent high-yielding cis-divinylepoxide rearrangement at elevated temperatures to yield dihydrooxepine 299 in good yield. De Meijere and coworkers [300 to yield cis-divinylepoxide 301. Gentle heating provided bridged bicycle 302, containing two-anti-Bredt olefins in a stereospecific reaction. Doyle and coworkers [trans-divinylcyclopropanes like 304 via rhodium-catalyzed vinyldiazo decomposition (using compound 101) and subsequent epoxide formation in the presence of cinnamon aldehyde (303). These rather stable epoxides (see 304) were shown to undergo trans-cis isomerization followed by divinylepoxide rearrangement to give dihydrooxepine 305 at 125 °C. The necessary reaction temperature could be lowered using copper(II) catalysis, accelerating the reaction rate as well.The divinyloxirane rearrangement  has largely added to the scope of natural products that were synthesized using a DVCPR and shortened the overall synthetic routes.


Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. For secondary implants, primary tumors were minced and digested with collagenase II (Sigma-Aldrich) and dispase (Invitrogen). Following DNAse (Sigma-Aldrich) treatment, cells were resuspended in ice-cold DMEM/20% fetal bovine serum and filtered through 100-escribed , using thttp://bioinf.wehi.edu.au/software/elda/) [Analyses were performed using Prism  or Excel . As dnMAML was expected to have an inhibitory effect in all assays performed, one-tail tests were utilized. For calculation of CSC frequencies, web program of extreme limiting dilution was utilized (e/elda/) .HES1 was inhibited in the presence of dnMAML or the γ-secretase inhibitor DAPT  was overexpressed using a retroviral vector, whereas GFP alone was expressed as a control A. Exprestor DAPT B.HEY1, HEYL, and HES1 were induced in coculture with Jagged-1 overexpressing cells, whereas HEY2 was not induced. Expression of dnMAML or DAPT treatment inhibited the induction of HEY1, HEYL, and HES1 in cocultured MCF-7 cells , showing a 60% decrease in CSC following Notch blockade. Moreover, MCF7-dnMAML cells showed a significant delay in the formation of palpable tumors when fewer cells were implanted  were harvested from primary tumors and sorted for implantation. Twelve mice were implanted with MCF7-GFP cells implanted on the left flank and MCF7-dnMAML cells implanted on the right flank. For the MCF7-GFP cells, 7 of 11 implant sites formed tumors , whereas 5 of 12 MCF7-dnMAML implant sites formed secondary tumors, which is not significantly different .Although expression of dnMAML decreased the CSC frequency, it was clear that MCF7-dnMAML cells were capable of forming tumors. To determine if the cells derived from MCF7-dnMAML tumors were capable of forming secondary tumors, GFP+ cells was examined in freshly isolated secondary tumors  that regulates the Notch-independent pool of CSC. Nonetheless, our results suggest that it will be important in cancer treatment to use combination therapies even if the rationale is to treat the CSC population.


A discrepancy between oxygen availability and demand has been found in most chronic kidney diseases (CKD) irrespective of etiology. This results from a combination of structural and functional changes that are commonly associated with the development of fibrosis, which include a reduction in peritubular blood flow, luminal narrowing of atherosclerotic vessels, capillary rarefaction and vascular constriction due to altered expression of vasoactive factors and signaling molecules . Consistent with decreased renal oxygenation in CKD is the increased expression of the oxygen-sensitive α-subunit of hypoxia-inducible factor (HIF)-1. HIF transcription factors are members of the Per-ARNT-Sim (PAS) family of heterodimeric basic helix-loop-helix transcription factors and consist of an oxygen-sensitive α-subunit and a constitutively expressed β-unit, also known as the aryl-hydrocarbon-receptor nuclear translocator (ARNT) or HIF-β. Recent experimental evidence suggests that prolonged activation of HIF signaling in renal epithelial cells enhances maladaptive responses, which lead to fibrosis and further tissue destruction. Cell type-specific functions of individual HIF transcription factors and their relevant transcriptional targets are discussed in the context of renal fibrogenesis. Despite the very large blood flow , the kidneys, which carry out complex and energy consuming cellular transport functions, operate under markedly reduced oxygen tension, with regional oxygen levels ranging from 10 to 60 mmHg. A reduction in renal oxygenation occurs in most chronic kidney diseases (CKD) irrespective of etiology. This is due to a combination of several pathophysiological and morphologic changes, which are typically associated with chronic kidney injury. These include increased oxygen demand from hyperfiltration and tubular hypertrophy, capillary rarefaction, glomerular injury, luminal narrowing of atherosclerotic vessels, as well as vascular constriction due to altered expression of vasoactive factors and signaling molecules . The resulting reduction in renal oxygen availability is furthermore exacerbated by extra-cellular matrix (ECM) expansion, which limits oxygen diffusion, and by renal anemia ).Consistent with decreased renal oxygenation in CKD is the increased expression of the oxygen-sensitive α-subunit of hypoxia-inducible factor (HIF)-1 in renal biopsy material from patients with CKD ,6. The hHIF heterodimers activate gene transcription in response to hypoxia by binding to specific DNA sequences, which are known as hypoxia-response elements (HREs) and by recruiting transcriptional co-activators such as CBP/p300 Figure . While HWhile HIF has been shown to be cytoprotective in acute kidney injury , work byphosphoglycerate kinase-1 (PGK-1), chemokine receptor CXCR4, lysyl oxidase-like 2 (LOXL2) and phosphofructokinase (PFKFB3). A comparable increase in LOXL2 expression was also seen by real-time PCR in micro-dissected tubulointerstitium from patients with IgA nephropathy and hypertensive nephrosclerosis  modulators, such connective tissue growth factor (CTGF), tissue inhibitor of matrix metalloproteinase (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 -18. PAI-2) led to morphologic and gene expression changes that were consistent with EMT, and furthermore enhanced the migratory ability of PTECs in a HIF-1-dependent fashion. Since HIF-regulation of lysyl oxidase (LOX) was required for hypoxia-induced migration of breast cancer cells [LOX and its homologue LOXL2 was HIF-1-dependent, and then used pharmacological means to investigate the effects of lysyl oxidase inhibition on epithelial cell migration. Lysyl oxidase inhibitors β-aminoproprionitrile (BAPN) or bathocuproine disulphate (BCS) phenocopied the effects of HIF-1 inactivation, and attenuated hypoxia-enhanced migration in a scratch wound closure assay; complete closure was observed by 24 hours in hypoxic cells, whereas pre-treatment with either BAPN or BCS inhibited migration with the scratch not closing for at least 30 hours , augmentation of epithelial migration in hypoxia involved HIF-1-mediated induction of lysyl oxidases.To investigate the contribution of epithelial HIF-1 signaling to the development of kidney fibrosis in vivo, we used a model of renal fibrogenesis that is based on unilateral ureteral obstruction (UUO). In this model one of the ureters is ligated, blocking urine flow completely, which results in rapidly progressing fibrosis in the affected kidney. UUO kidneys develop hypoxia, as demonstrated by pimonidazole staining and epithelial HIF-1α stabilization. HIF-2α was detected in tubulointerstitial cells, which is consistent with immunohistochemical studies in hypoxic kidneys ,5.To assess the contribution of epithelial HIF-1 to fibrosis in this model, we compared wild type mice to animals that were HIF-1-defcient in the proximal renal tubule. Proximal tubule-restricted inactivation of HIF-1 was accomplished through use of a Cre-recombinase under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Inactivation of HIF-1 in proximal tubule epithelial cells resulted in reduced accumulation of collagen, reduced macrophage accumulation and reduced expression of FSP-1-positive cells following UUO , supportSince genetic inactivation of HIF-1α in the context of UUO resulted in attenuation of fibrosis and inflammation, we next investigated whether activation of epithelial HIF in a normoxic context could enhance fibrogenesis in vivo. For these studies we used Cre-recombinase transgenes that either were driven by the gamma-glutamyl transpeptidase (γ-GT) promoter or were tetracycline-inducible under the control of a Pax8-driven transactivator . InhibitRenal epithelial cells contribute to the development of kidney fibrosis, as they increase and remodel ECM when stimulated with TGF-β1, angiotensin II and other cytokines ,30, or wcollagen I, decreases matrix-metallopeptidase 2 (MMP-2) in renal epithelial cells [PAI-1 [tissue-inhibitor of metalloproteinase-1 (TIMP-1) [connective tissue growth factor (CTGF) [vascular endothelial growth factor (VEGF) [endoglin [EPO [In the context of chronic renal injury the biological consequences of HIF activation are different, and negatively impact clinical outcome, that is, promote fibrosis, ultimately leading to the development of end stage renal disease. This may occur through increased expression of i) ECM-modifying genes, such as LOX and PAI-1, ii) functional co-operation with transforming growth factor (TGF)-β1, iii) promotion of epithelial to mesenchymal transition (EMT), and iv) through modulation of renal inflammation . Hypoxiaal cells , and incs [PAI-1 , tissue-(TIMP-1)  as well r (CTGF)  through r (VEGF) , endogliendoglin  and EPO lin [EPO . The notlin [EPO ,41.While hypoxia is the main stimulus for HIF activation, signaling molecules with key roles in the pathogenesis of CKD, such as angiotensin II (Ang II), have also been shown to activate HIF-1 via prolyl-4-hydroxylase inhibition. PHD inhibition in this setting results from increased ROS generation and diminished intracellular ascorbate levels, leading to the oxidation of a critical iron atom that is positioned in the center of the PHD catalytic domain . AlthougThere is growing experimental evidence that hypoxia via HIF promotes EMT. This occurs through i) modulation of activity of EMT inducers or their receptors , ii) reHIF-1 induction of LOX and LOXL-2 has been shown to promote migration in primary renal epithelial, and in breast and cervical cancer cells, which was associated with decreased E-cadherin expression ,25. AlthLOXL2 expression was found in renal biopsy tissues from patients with CKD underscoring its relevance to the pathogenesis of CKD irrespective of etiology (Figure Our finding that HIF activation in epithelial cells promotes renal fibrosis has immediate clinical implications, as it encourages therapies that aim at improving tissue oxygenation to retard disease progression. Because HIF regulates multiple biological processes, which include erythropoiesis and iron metabolism, its systemic pharmacological inhibition is not desirable. Certain HIF regulated proteins, however, may represent better therapeutic targets to slow progression of CKD. Pharmacological inihibition of lysyl oxidases phenocopied the effects of genetic HIF-1 inactivation on cell motility and fibrogenesis, suggesting that lysyl oxidases are important contributors to the pathogenesis of renal fibrosis. In keeping with this notion, increased y Figure . WhetherThe author declares that he has no competing interests.


As versatile drug delivery systems, polymeric micelles have demonstrated particular strength in solubilizing hydrophobic anticancer drugs while eliminating the use of toxic organic solvents and surfactants. However, the true promise of polymeric micelles as drug carriers for cancer therapy resides in their potential ability to preferentially elevate drug exposure in the tumor and achieve enhanced anticancer efficacy, which still remains to be fully exploited. Here, we review various micellar constructs that exhibit the enhanced permeation and retention effect in the tumor, the targeting ligands that potentiate the anticancer efficacy of micellar drugs, and the polyplex micelle systems suitable for the delivery of plasmid DNA and small interference RNA. Together, these preclinical studies in animal models help us further explore polymeric micelles as emerging drug carriers for targeted cancer therapy. Self-assembled from biodegradable and biocompatible amphiphilic block polymers and with sizes ranging 10–200 nm, polymeric micelles are promising nanocarriers for anticancer drugs, as attested by a number of such formulations currently in clinical trials .d,l-lactide) (PEG-PLA), PEG-poly(ε-caprolactone) (PEG-PCL), PEG-distearoylphosphatidylethanolamine (PEG-DSPE) and PEG-poly(propyl oxide)-PEG , as listed in A micelle consists of a corona formed by the hydrophilic blocks extending into the aqueous solution and a core formed by the hydrophobic segments. The hydrophilic block is almost exclusively poly(ethylene glycol) (PEG), because of its excellent biocompatibility and “stealth” property that minimize undesirable interactions with serum proteins and cellular components. On the other hand, the composition of the hydrophobic block varies enormously, which can be tailored to encapsulate drug molecules with a wide variety of structures, lipophilicity and charges, contributing to the versatility of polymeric micelles as drug carriers. The frequently studied block polymers include PEG-poly(amino acids), PEG-poly, a poorly water-soluble anticancer agent 3 (CP750), which self-assembled into stable micelles with the oligopeptides formed the hydrophobic core  (mPEG-b-p(HPMAm-Lacn)) with doxorubicin methacrylamide (DOX-MA) covalently loaded into the micellar core aspartamide} (PEG-SS-P[Asp(DET)]) were evaluated as a carrier system for pDNA encoding RGD4C-fused human tumor necrosis factor α (RGD4ChTNF-α) gene [In a study by Kumagai -α) gene . Disulfiet al. described another PEG-PLL-based polyplex micelle system for VEGF and VEGF receptor 2 (VEGFR2) siRNA, in which amines of PLL were modified with 2-aminothiolane (2IT) and the cRGD peptide was installed on the PEG terminus [Interestingly, Kataoka terminus . In contPark and his coworkers reported that by conjugating VEGF siRNA with PEG, they were able to prepare stable siRNA-PEG/PEI polyplex micelles (polyelectrolyte complex) and achieve efficient siRNA delivery . The siRet al. a polyplex micelle (micelleplex) system comprised of a triblock copolymer PEG-b-PCL-b-poly(2-aminoethyl ethylene phosphate) (PEG-PCL-PPEEA) was evaluated for siRNA delivery [®. Intriguingly, co-administration of micelles separately loaded with siRNA and paclitaxel showed much less inhibition of tumor growth with no synergistic effect. This study underscores the potential of polymeric micelles for combined drug and siRNA therapy. In a study by Wang delivery . Whereasdelivery . In miceThe superior flexibility in polymer design coupled with the general simplicity of physical drug entrapment has distinguished polymeric micelles as versatile and effective drug carriers for cancer therapy. Herein, we have briefly summarized the recent progresses (mostly within the past 5 years) that have explored the use of polymeric micelles in preclinical tumor models. The promising results gained from these studies highlight the increasingly sophisticated designs of micelle constructs to overcome the biological barriers and achieve targeted delivery of anticancer drugs to the tumor, which have provided crucial stepping stones to further develop micellar nanocarriers for targeted cancer therapy.


Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: μ-, δ-, κ-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of μ-opioids on homo- or hetero-oligomerized μ-opioid receptor and discuss potential mechanisms through which bivalent ligands exert beneficial effects, including adenylate cyclase regulation and receptor-mediated signaling pathways. Opioids are one of the most commonly prescribed pain relievers and have been used to treat pain for thousands of years. Considered as broad-spectrum analgesics acting at multiple sites in both the central and peripheral nervous systems, opioids also have multiple side effects and potential complications . Concern2+ channels enkephalin (DAMGO), traditionally classified as MOR selective agonists, selectively activate MOR-DOR heteromeric opioid receptors with greater efficacy than homomeric opioid receptors nonan-9-yl)piperidin-4-yl)indolin-2-one], a nonselective ORL1 and MOR partial agonist, has potent antinociceptive activity in acute thermal pain, as well as CPP, an effect mediated by its MOR activity . It is s in mice . SR14150 in mice .versus the classical opioid receptors and (2) functional efficacy, i.e., agonist or antagonist activity [3H]nociceptin, [3H]DAMGO, [3H]Cl-DPDPE -enkephalin) and [3H]U69593, for ORL1, MOR, DOR and KOR, respectively. Functional activity was determined by stimulation of [35S]GTPγS binding to cell membranes [versus MOR agonist potency using SAR approaches will be useful for developing bivalent ORL1-MOR ligands for medication development [The structure-activity relationship (SAR) for discovering bifunctional MOR-ORL1 ligands, starting from ORL1-selective scaffolds, has been explored . Based oactivity . Most ORembranes ,199,200.elopment .m-methoxy-MPEP (M-MPEP) [50 (effective dose in 50% of the population) ~9 fmol). Since other members with shorter or longer spacers have ≥500-fold higher ED50s, the exceptional potency of MMG22 may result from the optimal bridging of the MOR-mGluR5 heteromer. MMG22 possesses a >106 therapeutic ratio, suggesting that it might be an excellent candidate for the management of chronic, intractable inflammatory pain via spinal administration [The selective mGluR5 antagonist, MPEP, acts allosterically by binding to the 7TM domain of the receptor . Co-admi(M-MPEP) , linked stration .Bivalent ligands of the opioid receptors have been prepared to improve the pharmacological properties of opioid ligands, because ligand-induced clustering of opioid receptors occurs on the cell surface ,148,205.The oligomerization of GPCRs is dynamic and regulated. Microscopically, bivalent ligands bind to and thereby stabilize or de-stabilize pre-existing dimers. Specific ligands may promote ligand-induced dimerization or inhibit further dimerization, depending on the intrinsic properties of the ligands. The pharmacological difference between bivalent ligand binding and co-application of two monovalent ligands is that the former interact with two adjacent receptors simultaneously, but the latter bind to two distant receptors sequentially. The downstream signaling process of these two events could be different spatially and temporally. The advantages of using bivalent ligands as therapeutic tools are less undesired side effects, whereas the disadvantages are non-specific and unexpected effects where the targeted heterodimers do not colocalize. Whether both ligand binding sites are equal in a dimer or have a certain degree of cooperativity depends on the structures of heterodimers and the orientation of the linked pharmacophores on the bivalent ligands. We speculate that most of the binding sites are not equal in the heterodimers and are somewhat cooperative when interacting with the bivalent ligands.in vitro methods of overexpression receptors in cell lines. The overexpression system apparently will have artificial results by forcing receptors together. Co-immunoprecipitation methods contain detergent, which could disrupt protein-protein interactions, thus disrupting receptor oligomerization. Moreover, the in vivo data are not solid for receptor oligomerization, and most of the studies are based on a heterologous expression system. The observed pharmacological profiles of the bivalent ligands linking a relatively non-selective agonist and an antagonist in vivo might not always reflect the effects of the heterodimers. In areas where the distributions of the two receptors are non-overlapping, the effects of the ligands observed may be owing to the agonistic or antagonistic effects on one of the receptors alone. For example, Pickel and her co-workers reported that mu-receptors were located in 21% of the dendritic profiles and 3% of the axon terminals containing CB1 receptors in the rat nucleus accumbens shell [in vivo.There are caveats that one should be aware of when evaluating the data of the oligomerized opioid receptors. Most studies for oligomerization employ ns shell , a ratheThe high-resolution crystal structures of MOR, DOR, KOR and ORL1 in ligand-bound conformations have been resolved ,210,211.


Although different authors suggest that the B-lymphocyte activating factor (BAFF) may be involved in the selective loss of B-cell tolerance in human systemic lupus erythematosus (SLE), the mechanisms responsible for the deregulation of this molecule in SLE remain unclear -3.To investigate any associations between regulatory genetic polymorphisms of BAFF gene, disease susceptibility and serum BAFF (s-BAFF) levels in Tunisian systemic lupus (SLE) patients.The case-control study included 124 SLE patients and 152 healthy controls. Three single nucleotide polymorphisms (SNPs)  in the 5’ regulatory region of the BAFF gene were explored by PCR-RFLP [-3) and patients without anti-dsDNA antibodies . In contrast, no correlation was found between global disease activity registered in SLEDAI and s-BAFF levels (p: 0.7). Furthermore, no association was found between BAFF genotypes and susceptibility to SLE. Single allele, genotype and haplotype association analyses showed no significant association with s-BAFF values, clinical features or SLEDAI score in SLE.s-BAFF levels were elevated in SLE patients  and in anti-dsDNA positive antibodies patients  compared to both controls (665,82 pg/ml, p<10Polymorphisms in the regulatory region of the BAFF gene do contribute neither to increased s-BAFF levels nor to the susceptibility to SLE in Tunisian patients. Increased s-BAFF levels in anti-dsDNA positive antibodies SLE patients suggest the central role of this molecule in the inflammatory process involving in autoantibodies production.


Fos expression in D1 receptor-bearing neurons in mice results in no change in the induction but a significantly delayed extinction of cocaine-induced conditioned place preference. These findings suggest that D1 receptor-mediated and c-Fos-regulated changes in cell signaling and gene expression may play key roles in the extinction process, and they provide a foundation for further exploring mechanisms underlying extinction of cue-elicited cocaine seeking.A prominent feature of drug addiction is that drug-associated cues can elicit drug-seeking behaviors and contribute significantly to the high propensity to relapse. We have been investigating the notion that the dopamine D1 receptor and the immediate early gene product c-Fos expressed in D1 receptor-bearing neurons mediate the development of persistent neuroadaptation in the brain dopamine system by regulating cell signaling and gene expression. We generated and analyzed genetically engineered mouse models and found that the D1 receptor and c-Fos expressed in D1 receptor-bearing neurons mediate the locomotor sensitization and reinforcing effects of cocaine. Moreover, these molecules regulate cocaine-induced dendritic remodeling, electrophysiological responses, and changes in cell signaling and gene expression in the brain. Notably, a lack of  Drug addiction is a brain disease that is characterized by the compulsive seeking and taking of drugs despite known negative consequences . A key fDopamine (DA) pathways that project from the midbrain to the nucleus accumbens (NAc), amygdala (AMG), hippocampus (HIP), caudoputamen (CPu), and prefrontal cortex (PFC) play a key part in mediating the behavioral effects of drugs of abuse -7. Abusevia regulation of gene expression [Fos in the NAc, CPu, AMG and PFC [Fos expression in the NAc [Fos is selectively mutated in this group of neurons [Fos expression in D1 receptor-bearing neurons in mice results in no change in the induction but a drastically delayed extinction of cocaine-induced conditioned place preference (CPP) [The persistent nature of drug addiction suggests that repeated exposure to drugs leads to stable alterations involving long-lasting changes in neuronal circuits, cell signaling and gene expression in the brain DA system , 4, 6, 7pression , 27. Thepression . The mos and PFC , 20, 27. the NAc . c-Fos p neurons . During ce (CPP) . These fFos mutant mice and subsequent analyses, see Zhang et al. [For details of the generation of the g et al. , 31.Fos mutation in D1 receptor-bearing neurons [Fos mutant mice 7 weeks of age or older compared to that in wild-type mice [Fos can be significantly induced by haloperidol in both Fos mutant mice and wild-type. These results indicate that the Fos mutation is primarily limited to D1 receptor-bearing neurons in the age range we used. The Fos mutant mice appear healthy with no obvious abnormalities in the brain. Fos mutant and wild-type mice exhibit similar baseline motor activity, acute locomotor responses to SKF81297, quinpirole and haloperidol treatment, and baseline dendritic structures and dendritic spine density compared to wild-type mice [Fos mutation did not drastically affect the development of the DA system in Fos mutant mice.We previously used a Cre/loxP-mediated DNA mutation strategy to engineer a mouse with a selective  neurons , 31. We ype mice . Importaype mice . Basal gype mice . These rFos mutant mouse in response to cocaine challenges using a multi-faceted approach. We found that while there were no differences in baseline activity after saline injections between Fos mutant mice and wild-type mice, acute cocaine administration induced significant locomotion compared to saline injections and repeated cocaine injections induced behavioral sensitization in wild-type but not in Fos mutant mice [Fos expression in D1 receptor-bearing neurons affected FosB and ΔFosB levels and AP-1 transcription complex formation in the NAc and CPu following cocaine treatment [Fos mutation also affected the expression of at least two classes of target genes [To investigate how c-Fos may mediate cocaine-induced persistent neurobiological changes, we previously analyzed the ant mice . Whereasant mice . Medium ant mice . A lack reatment . The Foset genes . The firEnvironmental cues previously associated with reinforcing drugs can play a key part in relapse to drug seeking behaviors in humans , 2. ThisFos mutant mice to perform a CPP study. We found that neither Fos mutant nor wild-type mice showed bias in freely exploring the two compartments [Fos mutant mice and wild-type mice developed CPP at two cocaine doses whereas saline-paired groups did not [Fos mutant mice still exhibited CPP after 10, 12, 14, 16 and 18 days of extinction training and they did not exhibit extinction till after 22 days of training [Fos mutant mice exhibited similar cocaine-induced reinstatement of CPP [Fos mutation in D1 receptor-bearing neurons does not obviously affect acquisition and reinstatement but significantly delays extinction of cocaine-induced CPP, and they suggest that D1 receptor-mediated and c-Fos-regulated changes may play key roles in the extinction of cue-elicited cocaine seeking.We investigated whether c-Fos-mediated intracellular changes contribute to the development and extinction of cue-elicited cocaine seeking by using artments . Followi did not . Signifi did not . Wild-tytraining . Wild-tyt of CPP . These rvia DA D1 receptors. c-Fos is a transcription factor that is robustly induced by D1 receptor agonists and cocaine in D1 receptor-bearing neurons. By generating and analyzing a mouse in which Fos is mostly mutated in D1 receptor-bearing neurons, we found that c-Fos is an intracellular regulator of cocaine-induced alterations in gene expression and neuronal activity, reorganization of neuronal circuits, and the development of behavioral sensitization. Moreover, our findings suggest that c-Fos-regulated mechanisms within the D1 receptor-bearing neurons may play key roles in the extinction of cue-elicited cocaine seeking.Repeated exposure to cocaine induces long-lasting changes in behavior, neuronal circuits and gene expression Much has been learned about the basis of the development of cocaine-induced persistent changes. Mechanisms underlying the extinction of such long-lasting changes, however, remain much less investigated. Multiple DA signals exist during the extinction of cocaine self-administration . MoreoveFos mutant mouse model provide new opportunities to dissect D1 receptor-mediated and c-Fos-regulated electrophysiological changes following acquisition and especially extinction of cue-elicited cocaine seeking in the reward circuitry.The basolateral AMG (BLA) mediates both acquisition and extinction of conditioned fear, and the PFC, HIP and NAc modulate these processes , 37. TheCPP is thought to reflect aspects of cue-elicited drug seeking yet this paradigm may not fully model addicted states in humans as drugs are delivered non-contingently and only a few pairings are required to produce CPP. We will consider using the self-administration paradigm which is thought to reflect active drug seeking and taking as an alternative in the future , 34. We