The placental mammalian clade Afrotheria is now supported by diverse forms of genomic data, but interordinal relationships within, and morphological support for, the group remains elusive. As a means for addressing these outstanding problems, competing hypotheses of afrotherian interordinal relationships were tested through simultaneous parsimony analysis of a large data set  containing genomic data  and 400 morphological characters scored across 16 extant and 35 extinct afrotherians.Herodotius) never grouped with that clade and instead consistently joined pseudoungulates or paenungulates. When character transformations were optimized onto a less resolved afrotherian tree that reflects uncertainty about the group's interordinal phylogeny, a total of 21 morphological features were identified as possible synapomorphies of crown Afrotheria, 9 of which optimized unambiguously across all character treatments and optimization methods.Parsimony analysis of extant taxa alone recovered the interordinal topology , ))). Analysis following addition of extinct taxa instead supported Afroinsectivora (Afrosoricida + Macroscelidea) and Pseudoungulata (Tubulidentata + Paenungulata), as well as Tethytheria (Proboscidea + Sirenia). This latter topology is, however, sensitive to taxon deletion and different placements of the placental root, and numerous alternative interordinal arrangements within Afrotheria could not be statistically rejected. Relationships among extinct stem members of each afrotherian clade were more stable, but one alleged stem macroscelidean (Instability in afrotherian interordinal phylogeny presumably reflects rapid divergences during two pulses of cladogenesis – the first in the Late Cretaceous, at and just after the origin of crown Afrotheria, and the second in the early Cenozoic, with the origin of crown Paenungulata. Morphological evidence for divergences during these two pulses either never existed or has largely been "erased" by subsequent evolution along long ordinal branches. There may, nevertheless, be more morphological character support for crown Afrotheria than is currently assumed; the features identified here as possible afrotherian synapomorphies can be further scrutinized through future phylogenetic analyses with broader taxon sampling, as well as recovery of primitive fossil afrotherians from the Afro-Arabian landmass, where the group is likely to have first diversified. The monophyly of the supraordinal placental mammalian clade Afrotheria, whose living members include the endemic Afro-Arabian aardvarks (order Tubulidentata), elephant-shrews or sengis (order Macroscelidea), golden moles (family Chrysochloridae), tenrecs (superfamily Tenrecoidea), sea cows (order Sirenia), hyraxes (order Hyracoidea), and elephants (order Proboscidea), is now strongly supported by diverse forms of genomic data, including indels ,2, SINEsAn important first step in addressing the problem of morphological character support for Afrotheria will be resolution of the phylogenetic position of tenrecs and golden moles (order Afrosoricida) within that clade ,19. AfroAnother outstanding problem in afrotherian phylogenetics is the branching order among Hyracoidea, Proboscidea and Sirenia within Paenungulata. Various types of genomic data have been collected in an effort to resolve paenungulate relationships ,4,28,29,The extant members of afrotherian orders differ dramatically in their morphology and adaptations, and represent the tips of long branches that extend well back into the Paleocene and/or Late Cretaceous . ExtinctThis study includes 400 morphological characters scored across 16 extant and 35 extinct afrotherians, and is combined with chromosomal associations  to creatRegardless of how morphological characters were treated , simultaneous analysis of extant taxa alone recovered Paenungulata, Tethytheria, a Macroscelidea-Tubulidentata clade, and a Macroscelidea-Tubulidentata-Paenungulata clade to the exclusion of Afrosoricida Fig. . Aside fHerodotius, which in different equally parsimonious trees emerged as the sister taxon of either Pseudoungulata, Tubulidentata, Paenungulata, or Hyracoidea, but never as a sister group of crown Macroscelidea. The lower support for Tethytheria can be explained by the inclusion of primitive fossil proboscideans and sirenians, which reveal that a number of the apomorphies that were unambiguously optimized as tethytherian synapomorphies in the analysis of extant taxa alone are in fact more parsimoniously explained as homoplasies rather than homologies ..HerodotiArsinoitherium was excluded from the analysis, Afrosoricida was again placed as the sister group of a ) clade, within which Hyracoidea was placed as the sister taxon of Proboscidea to the exclusion of Sirenia )) were only two steps longer than the unconstrained tree, and could not be statistically rejected; nor could the alternative arrangement of a monophyletic Afroinsectiphillia containing Afroinsectivora Table . None ofia Table . Such a scideans  – some ogy Table , but afrgy Table . Interesgy Table .Arsinoitherium as a stem sirenian rather than as a stem proboscidean [Moeritherium, which is variously seen as a late-surviving basal form or as more deeply nested within the proboscidean radiation, sharing more recent common ancestry with Oligocene-Recent elephantiforms than with older taxa [Phosphatherium and Daouitherium [Numidotherium koholense [Moeritherium in a more nested position. Moeritherium's enigmatic contemporary Barytherium, which was recently placed with Daouitherium in a more restricted analysis [The phylogeny of the diverse Paleogene paenungulate radiation has never been analyzed within the context of afrotherian monophyly. One of the novel results of this analysis is the placement of enigmatic oscidean ,42,43 oroscidean . Within der taxa ,45. The itherium ,46,47) aoholense  as the sanalysis , was plaSeggeurius [Microhyrax [Geniohyus as the sister taxon of other hyracoid genera [Thyrohyrax and Saghatherium are consecutive sister taxa of large-bodied forms including Geniohyus. The genus Thyrohyrax was consistently found to be polyphyletic, with only the type species (Thyrohyrax domorictus) and Thyrohyrax meyeri forming a clade along with extant Procavia. Geniohyus was found to be nested deep within the Paleogene radiation in a clade containing species of Bunohyrax and Pachyhyrax. A clade containing Antilohyrax, Titanohyrax, and early Miocene Afrohyrax was also well-supported, and placed as the sister group of a Bunohyrax-Geniohyus-Megalohyrax-Pachyhyrax clade.The hyracoid phylogeny recovered in this analysis is very different from all previous estimates, none of which took into account multiple afrotherian outgroups -51. The ggeurius  and Micrcrohyrax ,51 as thd genera , in thisChambius and Herodotius are generally considered to be primitive macroscelideans [Metoldobotes, including cranial remains, helps to place that genus as the sister taxon of crown Macroscelidea with strong bootstrap support . A placement of both herodotiines as stem macroscelideans could not be statistically rejected, however  have undergone reversals to the dental character states observable in more primitive Cretaceous placentals.Considerable ambiguity is introduced by missing data and different methods for optimizing character states onto slightly different afrotherian phylogenies, but it remains distinctly possible that there are a number of morphological synapomorphies of crown Afrotheria, and that the ancestral crown afrotherian more closely resembled a "proto-ungulate" than an "insectivore". For instance, some of the only character transformations that consistently optimized unambiguously onto the afrotherian stem on the less resolved tree Table  are relaHerodotius, from the late Eocene of Egypt, which consistently groups with paenungulates or pseudoungulates to the exclusion of afroinsectivorans, while an older and very similar herodotiine (Chambius) is placed in Afroinsectivora with macroscelideans. Herodotiine diphyly is probably an artifact of missing data, but the distribution of herodotiines on both sides of the afrotherian tree again lends some support to the idea that the paenungulate-like dental morphology of herodotiines may be primitive within Afrotheria. The most likely explanation for herodotiine diphyly is that, in known parts, Chambius and Herodotius overlap solely in having very similar upper and lower molars and fourth premolars, but Chambius is now known to have macroscelidean-like astragalar and calcaneal morphology [Herodotius has somewhat paenungulate-like anterior premolars and incisors . If some uniformity of herodotiine morphology is assumed and Chambius and Herodotius are assigned the character states of the herodotiine taxon that preserves those parts, then these taxa together join Tubulidentata (OSA) or Pseudoungulata (UA Adams consensus), but never Macroscelidea, in parsimony analyses of the data set presented here. Additional cranial or postcranial morphology of herodotiines should play a key role in future efforts to tease apart homology and homoplasy among early afrotherians: if herodotiines are in fact stem macroscelideans within Afroinsectivora, then their detailed dental resemblances to paenungulates will either not be present in older and more primitive stem macroscelideans, or these features will emerge as plesiomorphic within Afroinsectivora and Afrotheria and will support a proto-ungulate origin for both clades.Of interest in this regard is the phylogenetic placement of the alleged stem macroscelidean rphology , whereasArsinoitherium, and aardvarks as well as some macroscelideans, tenrecs, golden moles, and fossil hyracoids, is here identified for the first time as another possible morphological synapomorphy of crown Afrotheria.Of the remaining morphological features that support afrotherian monophyly on the less resolved tree, none clearly point to either a "proto-ungulate" or "insectivore" origin for the clade. Under delayed transformation, at least one morphological feature that was previously thought to be a synapomorphy of Paenungulata (loss of lunar-unciform contact)  instead There are a few obvious deficiencies of the present study, some of which should be improved upon in future analyses. One obvious improvement that can be made is greater taxon sampling within Placentalia , including a greater diversity of Cretaceous mammals, as all such taxa should help to clarify ancestral character states for crown Placentalia. One obvious criticism of the equally-weighted total evidence approach taken here is that "rare genomic changes" (RGCs) such as retroposons and chromosomal syntenies have been given equal weight to point mutations in DNA sequences, the latter of which are surely much more prone to homoplasy . UnfortuSimultaneous analysis of ≤ 4,590 parsimony informative genomic and morphological characters scored across 16 extant and 35 extinct afrotherians recovers a monophyletic Afroinsectivora (Afrosoricida + Macroscelidea), Pseudoungulata (Tubulidentata + Paenungulata), and Tethytheria (Proboscidea + Sirenia) within Paenungulata. None of these supraordinal clades are well-supported, however, and phylogenetic alternatives such as Afroinsectiphillia, a ) clade, an Afrosoricida-Tubulidentata clade, afrosoricid diphyly, a Hyracoidea-Proboscidea clade, and a Hyracoidea-Sirenia could not be statistically rejected.Divergences among Afrosoricida, Macroscelidea, Paenungulata, and Tubulidentata must have occurred very rapidly in the Late Cretaceous, and unambiguous morphological evidence for afrotherian supraordinal clades aside from Paenungulata either does not exist or has been overwritten by subsequent evolution through the Cenozoic. On the optimal topologies derived from these analyses, identification of unambiguous morphological support for afrotherian monophyly is dependent on optimization method, but on a less resolved interordinal phylogeny a total of 21 unambiguous afrotherian synapomorphies are identified, 9 of which appear as such across all character treatments and optimization methods.Arsinoitherium as a stem sirenian, and the placement of the alleged macroscelidean Herodotius with pseudoungulates. Additional cranial and postcranial material of Eocene Chambius, Herodotius, and other enigmatic early African taxa will be of great importance for understanding afrotherian interordinal phylogeny, and will help to test competing hypotheses of a "proto-ungulate" versus "insectivore" origin for that clade. Of utmost importance, however, is the recovery of even earlier fossil afrotherians from previously unsampled early Paleocene and Late Cretaceous horizons in Afro-Arabia.Relationships among early fossil members of each afrotherian order are generally consistent with stratigraphic succession; novel results are the placement of The 400-character morphological partition draws heavily on previous phylogenetic investigations of afrotherian orders -44,60-63Dasypus) to join Afrotheria or Boreoeutheria in secondary analyses.The nucleotide partition employed in this analysis is essentially that of Amrine-Madsen et al. , combiniCharacter data were compiled in the program Nexus Data Editor . See addArsinoitherium, which has been placed as a stem proboscidean, stem tethytherian, or stem paenungulate [Myorycteropus) consistently emerged as the sister taxon of the armadillo (Dasypus), in large part because both taxa retain single-rooted anterior premolars that have been lost (and thus cannot be scored) in the extant aardvark Orycteropus. As Myorycteropus otherwise exhibits a number of potentially informative character states that have either been lost or transformed in the highly derived genus Orycteropus, the two were constrained as sister taxa in all analyses. Outgroup taxa include the extant marsupial Didelphis, the Early Cretaceous stem placentals Montanalestes [Prokennalestes [Dasypus, the extant euarchontans Lemur (order Primates) and Tupaia (order Scandentia), and the extant eulipotyphlans Condylura  and Erinaceus (family Erinaceidae). While it is not necessarily the case that the extant outgroups adequately capture the primitive morphotype for Boreoeutheria, when compared with stem placentals these taxa are, in many ways, more generalized than the highly specialized members of other boreoeutherian clades that could have been sampled .The taxon set includes extant representatives of all afrotherian orders and samples much of the morphological diversity observable within ordinal crown clades. As one of the primary goals of this study was to break up the "long branches" that separate autapomorphic extant taxa from supraordinal nodes, the oldest and most basal extinct stem taxa of each afrotherian order were sampled, most of which are Eocene in age . An attempt was also made to sample most of the morphological diversity among Paleogene afrotherians, because the intraordinal relationships among such taxa remain controversial ,49,50. Aungulate ,42,43. Inalestes  and Proknalestes ,79, the Hyopsodus, Meniscotherium, and Phenacodus form a sister clade of Cetartiodactyla rather than of Paenungulata (or any other afrotherian clade). These enigmatic fossil taxa are not included in the current analysis because competing hypotheses of their afrotherian versus laurasiatherian placement can only be tested through broader analyses that include more morphological characters and a large sample of living and extinct boreoeutherians (in particular perissodactyls and artiodactyls) [A number of extinct taxa from the Paleocene and Eocene of Laurasia  have recently been identified as afrotherians in phylogenetic analyses with relatively limited character and/or taxon sampling ,38,80, adactyls) .Kelba from east Africa and late Eocene Widanelfarasia from Egypt – have recently been identified as possible or probable afrotherians, respectively, based on new material [et al. [Kelba shares with aardvarks, while Seiffert et al. [Widanelfarasia represents a tribosphenic stem tenrecoid. Both genera exhibit unique constellations of morphological characters when compared with other living and extinct afrotherians, and as these character distributions could prove to be of use in helping to unravel afrotherian phylogeny, Kelba and Widanelfarasia were added to the matrix for secondary analyses in order to determine 1) where these taxa are placed if they are in fact afrotherians, and 2) whether their unique morphological features have an impact on afrotherian phylogeny.In contrast, two enigmatic African fossil mammals with no clear link to historically Laurasian clades – early Miocene material ,81. Cotet et al.  have morList of morphological characters. Document contains detailed descriptions of each morphological character and character state.Click here for fileCharacter-taxon matrix. Character-taxon matrix, in Nexus format, containing all nucleotide, genomic, and morphological characters used in the phylogenetic analysis.Click here for file


Anastrozole (Arimidex™) is a third-generation aromatase inhibitor which has been shown to possess superior efficacy and tolerability over established endocrine agents in advanced breast cancer. Inhibition of aromatase prevents the conversion of androgen substrates to oestrogen, its sole source in postmenopausal women, thereby leading to regression of hormone-sensitive breast carcinomas. Clinical pharmacology data indicate that anastrozole is a potent aromatase inhibitor, providing near-maximal suppression of serum and intratumoural oestrogens to below detectable levels. Anastrozole may offer greater selectivity compared with other aromatase inhibitors, being without any intrinsic endocrine effects and with no apparent effect on the synthesis of adrenal steroids. It is well tolerated and has a convenient once-daily dosing regimen, ensuring maximum patient compliance. A major clinical programme has demonstrated that anastrozole is superior to the standard endocrine therapy, tamoxifen, for the first-line treatment of postmenopausal women with hormone-sensitive advanced breast cancer. Its superior efficacy in advanced disease, together with its improved tolerability and convenient dosage, make it a suitable agent to be assessed for the treatment of early breast cancer in postmenopausal women. This was investigated in the largest single adjuvant breast cancer study ever to be carried out, the ATAC  trial, which has now completed recruitment, with the first efficacy and safety data awaited. © 2001 Cancer Research Campaign


Sir,We would like to make comment on the above article, which as its main conclusion found that abnormal p53 was associated with failure of response to radiotherapy in patients with rectal cancer. Our department has recently conducted a similar analysis focusing purely on rectal cancer undergoing neoadjuvant treatment and the conclusions that we drew were somewhat different.p53 gene directly, four using single-strand conformational polymorphism analysis (ssCP) (p53 gene sequencing (DGS)  and only four (18%) showed that it could predict response. A further six studies assessed the Firstly, patients in both DGS studies underwent short-course radiotherapy, whereas there is an increasing use of long-course neoadjuvant radiochemotherapy (RCT) as standard of care in many institutions (We therefore feel that the conclusion of this study, based principally on the results of two papers, is a little premature. What does seem apparent, however, is that IHC and ssCP analysis of p53 yield unreliable and heterogeneous results. While DGS may hold promise, the question of whether such an expensive and labour-intensive technique will become useful in the clinical setting still remains to be answered.


Solid tumors are known to recruit new blood vessels to support their growth. Therefore, unique molecules expressed on tumor endothelial cells can function as targets for the antiangiogenic therapy of cancer. Current efforts are focusing on developing therapeutic agents capable of specifically targeting cancer cells and tumor-associated microenvironments including tumor blood vessels. These therapies hold the promise of high efficacy and low toxicity. One recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics is to encapsulate anticancer drugs into targeting liposomes that bind to the cell surface receptors expressed on tumor-associated endothelial cells. These anti-angiogenic drug delivery systems could be used to target both tumor blood vessels as well as the tumor cells, themselves. This article reviews the mechanisms and advantages of various present and potential methods using peptide-conjugated liposomes to specifically destroy tumor blood vessels in anticancer therapy. One of the primary goals of a successful cancer treatment regimen is to deliver sufficient amounts of drug to tumors while minimizing damage to normal tissues. Most chemotherapeutic agents enter normal tissues in the body with indiscriminate cytotoxicity and do not preferentially accumulate at tumor sites. At times the dose reaching the tumor may be as little as 5% to 10% of the doses accumulating in normal organs . One reaSeveral approaches have been developed to improve the ability of anticancer drug to more specifically target tumors and avoid normal organs. One of the most effective strategies is to encapsulate drugs in particles that deliver them preferentially to tumor sites. For example, liposome particles have been found able to deliver radionuclides, genes, and chemotherapeutic agents to tumor sites. –10. AnotVirtually every conventional cytotoxic drug has been found to be antiangiogenic in in vitro and in vivo models . One treThe targeting of proliferating endothelial cells in the blood vessels of tumors has several advantages. First, endothelial cells in malignant tumors are genetically stable, nonmalignant, and rarely drug resistant, compared to the cancer cells , 21. HowIn addition, we can take advantage of the differences between endothelial cell plasma membrane proteins  to develop drug delivery systems capable of guiding therapeutic or imaging agents to a particular organ or tumor , 35. EndThe key to delivering drugs specifically to these targets is to identify and use ligands that specifically bind to and that can be internalized by endothelial cells in tumors. Combinatorial peptide libraries displayed on microorganisms have become a research tool for identifying cell surface-binding peptides that can become targets for antitumor treatment. Of the many molecular display techniques, phage display has been the most popular approach. Phage display is a selection technique in which a peptide or protein is fused with a coat protein of bacteriophage and displayed on the surface of the virion. Phage-displayed random peptide libraries have helped researchers map B-cell epitopes –40, discRecently, using affinity selection (biopanning) of phage-displayed peptide libraries, researchers have discovered molecules that are expressed on tumor blood vessels exclusively , 34–36. 2 of doxorubicin [2. Colbern et al. found that the activity of PEGylated liposomal doxorubicin 1-2 mg/kg was almost equivalent to that of free doxorubicin 9 mg/kg in mouse Lewis lung carcinoma [Most of the drug delivery systems approved for marketing are liposomal- or lipid-based formulations or therapeutic molecules linked to polyethylene glycol (PEG) , 55, 56.orubicin . Free doarcinoma . One cliarcinoma , suggestThe hyperpermeability of tumor vasculature is a key factor for the success of liposome-delivered chemotherapy agents. The “leakiness” of the angiogenic tumor vasculature is estimated to have an average pore size of 100–600 nm . These pPassively targeted liposomal drug delivery systems have some disadvantages. Normal organ uptake of liposomes leads to accumulation of the encapsulated drug in mononuclear phagocytic system cells in the liver, spleen, and bone marrow , which mThe disadvantage of the passive PEGylated liposomes can be overcome by creating ligand-mediated targeting liposomes with more selective anticancer activity. The activity of anticancer drugs can be enhanced by coupling targeting moieties to the surface of liposomes to promote selective binding to tumor-associated antigens and facilitate the delivery of drug-containing liposomes to the intended cellular sites. This drug delivery system has a higher drug-to-carrier ratio than immunoconjugates and multivalent presentation of ligands, which increases their binding avidity . Antibodies that bind to tumor-specific antigens have so far yielded little success as a drug delivery system for solid tumors, which make up more than 90% of all cancers in humans. Although monoclonal antibodies have shown clinical potential as tumor targeting agents, they are limited by their large molecular size and poor tumor penetration , by the Peptide-conjugated liposomes have three main components: anticancer drug, a liposome carrier, and targeting peptide . Remote The bioavailability and pharmacodynamics of liposome-encapsulated chemotherapeutic drugs must be considered in developing these delivery systems. To take advantage of the EPR effect, liposomes need to have long half-lives so that the drug stays within the carrier as long as possible in blood circulation until it accumulates in diseased tissues . Once liThe use of drug-encapsulated liposomes with ligands to target tumor blood vessels allows us to destroy both tumor blood vessels and tumor cells. In mice bearing human cancer xenograft, low dose of peptide-conjugated liposomal doxorubicin has been found to markedly inhibit vascularization and reduce total volume and weight of tumors , 16, 17.One peptide-conjugated liposome can deliver over ten thousand anticancer drug molecules directly into target tumor cells efficiently and effectively. The targeted and sustained release of the drug molecules can increase the maximum tolerated dose (MTD) of the cytotoxic drugs and dramatically lower dose-limiting toxicities, and in turn prevent treatment delay or discontinuation. The affinity of targeting ligands may allow the liposomes to move past the high IFP barrier within tumors , 13, 16.Advances in nanotechnology and molecular biology are moving us closer to developing an ideal “multifunctional smart nanodrug delivery system” using various types of ligands and drugs based on the kinds of diagnosis, imaging, or therapy needed. Such smart nanodrug delivery systems will allow accurate, specific, and noninvasive disease treatment, early diagnosis, and monitoring. In the future, combining ligands that specifically bind to cancer cells (including cancer stem cells) and tumor blood vessels with multifunctional liposomal drug delivery systems may help improve the effectiveness of cancer treatment and minimize the side effects traditionally associated with chemotherapy.The development of highly selective anticancer drugs that can discriminate between tumor cells and normal cells is the most important goal of current oncology research. The potential use of ligand-conjugated liposome-encapsulated drugs to target tumor cells and vasculature is very promising. Peptides that specifically bind to tumor targets can be coupled to the PEG terminus of sterically stabilized liposomes and subsequently precisely deliver chemotherapeutic agents to tumor cells or blood vessels. Peptide-mediated liposomes that target vasculature are a new generation of chemotherapy delivery systems with superior pharmacokinetics, controlled biodistribution, efficacy, and safety profiles.


The majority of viruses infecting animals and plants today are RNA viruses cis-acting signals in the viral RNA template have been implicated in regulating the relative levels of genomic and antigenomic RNAs.For many (+)RNA viruses, RNA replication requires viral enzymes such as RdRp, helicase, capping enzymes, and NTPase, as well as non-enzymatic proteins that participate in the assembly of the viral replication complex. Host cell proteins often play essential roles in (+)RNA virus replication as well. (+)RNA virus replication is asymmetric: the synthesis of (+)RNA, the one that is packaged into the progeny virus, is up to ∼100 times greater than that of (−)RNA Host membranes play an important role in (+)RNA virus replication, as the RNA replication complexes released from membranes during purification generally lose the ability to catalyze true RNA replication, although a limited activity may be retained. In general, the intracellular membranes of cells infected with (+)RNA viruses rearrange to form anchor sites for viral RNA replication complexes Although little is known about the structure of the membrane-associated RNA replication complexes, (+)RNA virus RdRps have been well characterized to have an overall right-handed shape with three domains, the fingers, palm, and the thumb RNA template is selected from the dsRNA genome during transcription. A 5′-cap binding site, identified on the reovirus polymerase, provides a neat solution for some dsRNA viruses of high eukaryotic hosts (−)RNA viruses have a helical-shaped, protein-coated genome, also known as the ribonucloeprotein complex (RNP), which functions as the sole template for viral RNA replication and transcription . Newly mThe composition of the RNA replication machinery differs between segmented and non-segmented (−)RNA viruses. The influenza A virus, a segmented (−)RNA virus, contains eight vRNAs, each packaged as a double helical-shaped RNP with multiple nucleoproteins (NPs) and a heterotrimeric polymerase  that is bound to the conserved vRNA termini . Each NPRNA replication of non-segmented (-)RNA viruses, such as the rabies virus and the vesicular stomatitis virus (VSV), requires an intricate interplay of the nucleoprotein N, the large protein L, a non-enzymatic P, and the N-enwrapped genome, also called the nucleocapsid Different replication strategies are used by (+), (−), and dsRNA viruses to limit dsRNA exposure, to regulate (+)RNA versus (−)RNA synthesis, and to modulate transcription versus replication throughout the infection cycle. The differences in the functionalities of the replication complexes are reflected in their structural composition, their subcellular localization, and in the interaction of the complex with viral RNA templates. It is expected that studies of RNA virus replication machineries will have a large impact on antiviral drug development due to their specific activities in virus replication.


The present study examined the ability of human monocytes to produce reactive oxygen intermediates after a contact with tumour cells. Monocytes generated oxygen radicals, as measured by luminol-enhanced chemiluminescence and superoxide anion production, after stimulation with the tumour, but not with untransformed, cells. The use of specific oxygen radical scavengers and inhibitors, superoxide dismutase, catalase, dimethyl sulphoxide and deferoxamine as well as the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide, indicated that chemiluminescence was dependent on the production of superoxide anion and hydroxyl radical and the presence of myeloperoxidase. The tumour cell-induced chemiluminescent response of monocytes showed different kinetics from that seen after activation of monocytes with phorbol ester. These results indicate that human monocytes can be directly stimulated by tumour cells for reactive oxygen intermediate production. Spontaneous monocyte-mediated cytotoxicity towards cancer cells was inhibited by superoxide dismutase, catalase, deferoxamine and hydrazide, implicating the role of superoxide anion, hydrogen peroxide, hydroxyl radical and hypohalite. We wish to suggest that so-called ‘spontaneous’ tumoricidal capacity of freshly isolated human monocytes may in fact be an inducible event associated with generation of reactive oxygen intermediates and perhaps other toxic mediators, resulting from a contact of monocytes with tumour cells. © 1999 Cancer Research Campaign


Managed care organizations use a variety of strategies to reduce the cost and improve the quality of medication use. The effectiveness of such policies is not well understood. The objective of this research was to update a previous systematic review of interventions, published between 1966 and 2001, to improve the quality and efficiency of medication use in the US managed care setting.We searched MEDLINE and EMBASE for publications from July 2001 to January 2007 describing interventions targeting drug use conducted in the US managed care setting. We categorized studies by intervention type and adequacy of research design using commonly accepted criteria. We summarized the outcomes of well-controlled strategies and documented the significance and magnitude of effects for key study outcomes.We identified 164 papers published during the six-year period. Predominant strategies were: educational interventions ; monitoring and feedback ; formulary interventions ; collaborative care involving pharmacists (n = 15); and disease management with pharmacotherapy as a primary focus . Overall, 51 studies met minimum criteria for methodological adequacy. Effective interventions included one-to-one academic detailing, computerized alerts and reminders, pharmacist-led collaborative care, and multifaceted disease management. Further, changes in formulary tier-design and related increases in copayments were associated with reductions in medication use and increased out-of-pocket spending by patients. The dissemination of educational materials alone had little or no impact, while the impact of group education was inconclusive.There is good evidence for the effectiveness of several strategies in changing drug use in the managed care environment. However, little is known about the cost-effectiveness of these interventions. Computerized alerts showed promise in improving short-term outcomes but little is known about longer-term outcomes. Few well-designed, published studies have assessed the potential negative clinical effects of formulary-related interventions despite their widespread use. However, some evidence suggests increases in cost sharing reduce access to essential medicines for chronic illness. Managed care organizations (MCOs) are the predominant form of health insurance coverage in the United States. MCOs provide health care to over 160 million enrollees and almost 90% of physicians have at least one managed care contract [Healthcare payers, including MCOs, grapple with the challenge of providing access to essential care that improves health outcomes in the face of increasing need to control healthcare costs. Growth in pharmaceutical spending over the past decade is partly due to increased ingredient costs per prescription, higher levels of utilization, and changes in the drugs being prescribed . A varieDrug formularies are a common cost containment strategy used by healthcare payers. Formularies are lists of preferred pharmaceutical products covered by an institution within various therapeutic categories . Health The validity and reliability of information about the effectiveness and unintended consequences of drug-related interventions depends heavily on the strength of the research design ,8. By crThe purpose of this review is to update our previous systematic review of interventions to improve medication use in MCOs . SpecifiWe performed a systematic review of published interventions conducted in MCOs to reduce pharmaceutical costs or improve use of medicines. We searched MEDLINE and EMBASE from 2001 through January 2007 using a combination of search terms describing the study setting , methodology , intervention types , drug use , and clinician practice patterns . The reference lists of studies identified in this way were reviewed to identify studies that our search strategy may have missed. The search retrieved all studies published since January 2001 including some of those analyzed in our previous review.A total of 2899 articles were identified by the search. Two investigators  each independently reviewed half of the study abstracts. Studies were included in the review if they met the following criteria: were published between July 2001 and January 2007 (inclusive), conducted in the US managed care setting including all forms of HMOs, PPOs, and independent practice associations (IPAs); described intervention(s) targeting medication use, including over-the-counter medications, herbals, or vitamins; included a clear description of methods; and measured drug-related outcomes. Disease management interventions focusing on pharmacotherapy were also considered. We have also included in the current review the four methodologically adequate formulary interventions not reported in our previous review. Due to the rapid growth in research on this topic since 2001, there are now sufficient studies to draw more valid conclusions. Clinical effectiveness trials and cost effectiveness studies of medications, descriptive studies, and those examining vaccinations were excluded.We reviewed the methodological adequacy of studies identified by our search. Based on the ability of specific research designs to control adequately for common threats to internal validity ,11,12, oWe extracted key features of the characteristics, methods, and outcomes of methodologically adequate studies using a standardized data extraction tool. Key features included: setting, objective/s, health condition/s addressed, intervention components, predominant intervention strategy, intervention target/s, and up to three primary outcome measures reported by the authors. To check reliability of data extraction, two investigators  independently extracted data from the included studies and calculated effect sizes of outcome measures for one-third of the studies in this review.One hundred and fifty one published studies met our initial inclusion criteria, of which 31% met our criteria for minimally acceptable methodology. The majority of these studies (45%) were conducted in staff, group and mixed model settings and 6 studies were conducted in IPAs, network model organizations, and Medicare or Medicaid MCOs; the remaining studies (40%) did not provide detailed information on the type of managed care setting.We classified studies according to predominant strategy and key intervention components Table . In ordeThe primary interventions tested in studies that met criteria for methodological adequacy were: educational interventions (7), monitoring and feedback 16), formulary-related interventions (15 including 4 published prior to July 2001), collaborative care involving pharmacists (5), and disease management (8). We categorized methodologically adequate interventions involving financial incentives as formulary-related interventions because the predominant focus of these studies was to assess the effects of differential patient copayment associated with tiered formularies. Additional file 6, formulThe dissemination of educational materials alone  is used commonly as a behavior change strategy, although this approach has been shown repeatedly to be ineffective as a stand-alone intervention ,9. This One-to-one educational outreach (often referred to as academic detailing) has been shown to be an effective technique to change many aspects of physician behavior, particularly medication use ,15. SimoTwo studies evaluated the impact of one-to-one patient counseling by pharmacists. One intervention successfully decreased mortality rates in patients using drugs with a narrow therapeutic index; hospitalizations were also reduced by both brief and detailed pharmacist counseling, although the effects were not significant . ResultsGroup education relies on either didactic or problem-based approaches to influence behavior . In our Audit and feedback interventions report physicians' past or current clinical practice, usually in relation to the practice of peers or accepted standards. Audit and feedback can achieve small to moderate improvements in physicians' practice ,23. FourTwo studies targeted physicians and patients using educational materials and feedback. The first achieved a small reduction in antibiotic prescribing for acute respiratory tract infections by delivering practice guidelines and performance feedback to physicians, combined with mailing educational materials to patient households and placing information in waiting and examination rooms . In the The common types of information technology used in clinical care include electronic medical records, computerized physician order entry, and real-time decision support (such as reminders and prompts). Computerized order entry with clinical decision support has been demonstrated to improve prescribing and reduce medication error rates . Smith eTwo studies in this review combined computerized alerts and group academic detailing ,32. InteBambauer et al  evaluateThe effectiveness of computerized reminders has also been demonstrated to improve rates of recommended laboratory drug monitoring. In two studies by Raebel et al ,35 dailyA study by Feldstein et al  comparedTelephone outreach to patients by pharmacists was evaluated in three papers. In one study ,39 threeTiered formularies with different levels of patient copayment to encourage use of preferred drugs are used increasingly to enhance cost-effective use of medicines . Six papChanging from a single copayment to a three-tier structure reduced continuation of the original medications, increased switching to agents in a lower tier, discontinued use of all drugs in the affected class in some patients, and decreased plan spending while increasing enrollee spending ,43. SimiPatient cost-sharing  is an important factor influencing medication use. Roblin et al  showed tPrior authorization (PA) policies are commonly used to control drug utilization and cost . In a heAfter vaginal anti-fungal products became available over-the-counter, Gurwitz et al  showed aTwo studies by Delate et al ,56 evaluCollaboration of pharmacists in the care process has emerged as a promising approach to improve quality of care and patient outcomes . Five stFinley et al  showed iDisease management programs are structured, population-based approaches for identifying, treating, and monitoring persons with chronic illness. Generally, these are multidisciplinary, integrated approaches aimed to improve processes of care and patient outcomes . Seven pDepression is a common medical condition that is associated with significant social and functional impairment as well as high direct and indirect health care costs . ResearcIn one study, psychiatrists reviewed patient psychosocial history and current depressive episodes over multiple sessions. The intervention, which also included books and videotapes for patient education and pharmacologic treatment where necessary, effectively increased antidepressant adherence at 12 months with modest improvements in depression symptoms among severely depressed patients . Katon eRay et al  examinedLozano et al  evaluatePublished guidelines recommend testing for HP in patients receiving long-term acid suppression therapy for acid-related disorders, and antibiotic treatment for those with HP-positive peptic ulcer disease ,74. A teThere has been a substantial increase in published research evaluating interventions targeting medication use in managed care settings since our previous review . This reSeveral findings from this report are consistent with our previous review and the results of other systematic reviews on the impact of interventions designed to change medication use. We once again confirmed that dissemination of educational materials alone is ineffective and one-to-one educational outreach visits are effective in increasing adherence to prescribing guidelines ,10,15,78The current analysis, like other reviews, suggests group education interventions may be beneficial but the findings are inconclusive ,79. We fIntervention approaches involving monitoring and feedback have been shown previously to produce small to moderate effects on medication use ,23,80, aOur review provides evidence that tiered formulary and patient copayment interventions decrease non-preferred drug use, reduce overall insurer costs, and increase patient out-of-pocket expenses as intended. The use of prior authorization policies resulted in similar effects. However, our findings concur with previous reviews in managed care and other settings that these interventions may also be associated with undesirable effects such as increased rates of switching to other medications or discontinuation of essential and cost-effective medications ,48,82,83Evidence from the literature suggests that coordinating pharmacist services as a component of patient care improves quality of care -86. ThisFinally, our findings confirm that disease management interventions are associated with improvements in the process and quality of care, both short and longer term . HoweverThere are several limitations to this review which need to be acknowledged. Despite our intensive efforts to identify all of the published literature on interventions to improve the quality and efficiency of medication use in US managed care settings, our search strategy may have missed published articles. The full range of interventions currently used by managed care programs to influence use of medicines is not covered by the studies in our review. For example, no studies evaluated the impact of physician incentives on prescribing, despite that fact that this strategy is commonly used by MCOs. Further, our findings are likely to be subject to under-reporting bias with interventions showing no or negative effects, which are less likely to be reported in the peer-reviewed literature. Even when studies are published, many are methodologically inadequate; 69% of the studies identified in this review were not included in the detailed analyses because of design flaws. There are also fundamental difficulties in comparing interventions with diverse objectives, intervention targets, measurement methods, and outcomes. Finally, it is difficult to reach definitive conclusions about the contribution of individual components to the overall effect of multifaceted interventions.Despite the substantial number of interventions to improve drug use in managed care, our understanding of the impacts of these interventions is still limited.First, a large proportion (40%) of the reviewed studies did not detail the specifics of the managed care setting within which the study was conducted. We found few studies conducted in PPOs despite their prominent role in the US managed care industry or in other "lightly" managed care settings. The majority of studies in our review were conducted in staff, group, or mixed-model HMOs, which represent more heavily managed settings. Future research should establish whether intervention approaches that are successful in such settings are also effective in more lightly managed settings. Second, most studies in this review used common surrogate outcome measures, such as prescribing rates or proportions of patients achieving specific medication use practices. Few studies reported medication adherence or patient-relevant outcomes such as clinical status, hospitalization rates, or quality of life measures. Furthermore, evidence about the cost-effectiveness of most interventions is still quite limited. Cost-effectiveness data are needed to select among many possible strategies for improving medication use and associated costs. Third, there is still a lack of publicly available, high quality evidence concerning the effects of interventions involving formulary changes or financial incentives, which are commonly used to influence medication use. Although the increased reporting of formulary-related interventions is encouraging, the high prevalence of inadequate designs  among studies of formulary-related interventions (77%) is striking. An important conclusion to be drawn from this review is that improvements in research methodology will be essential in order to produce valid and reliable study results about cost, quality of care, and patient outcomes to inform managed care policy decisions. It is highly likely these interventions do reduce costs to the payer as they continue to be dominant in the managed care environment. Evaluation of the patient-relevant outcomes of formulary-related interventions is a research priority. Finally, the durability of most of the intervention effects reported in this review is uncertain because few studies extend beyond two years follow-up and many cover even shorter periods. In particular, further research is needed to determine if computerized clinical support systems are associated with improved patient outcomes over the long-term.There is good evidence for the effectiveness of several strategies in changing drug use in the US managed care setting. Policy makers have a solid and expanding knowledge base to employ in the design of educational, monitoring and feedback, and collaborative care interventions to improve medication use. Computerized alerts also show promise in improving short-term outcomes. Despite the wide use of formulary-related approaches to managing pharmaceutical use, there are still few well-designed studies assessing their effects on patient outcomes. In order to maximize health care cost-effectiveness while containing costs, it is crucial to continue to promote rigorous testing of interventions to improve medication use in managed care settings in order to expand available high quality evidence about their clinical and economic impacts.The author(s) declare that they have no competing interests.Conception and design: CYL, SAP, DRD, SBS. Administrative support: CYL, SAP. Collection and assembly of data: CYL, SAP. Data analysis and interpretation: CYL, SAP. Manuscript writing: CYL, SAP, DRD, SBS. Final approval of manuscript: CYL, SAP, DRD, SBS.The pre-publication history for this paper can be accessed here:Key features of 51 methodologically acceptable studiesClick here for file


Digital ischemic loss is a cause of significant morbidity in patients with systemic sclerosis (SSc). Microvascular disease with intimal proliferation and luminal narrowing of small digital arteries, as well as macrovascular disease with narrowing or occlusion of larger digital arteries, contribute to the perfusion defects involved in digital ischemic loss. Immediate clinical evaluation and treatment are mandatory at the onset of critical digital ischemia to prevent digital loss. Hospitalization for medical therapies including intravenous prostacyclin therapy should be considered for all SSc patients who present with critical digital ischemia. Surgical interventions are typically reserved for patients who fail medical therapies and for those with late stage, necrotic tissue. This paper summarizes the current knowledge regarding the risk factors, pathogenesis, evaluation, and treatment of digital ischemic loss in SSc. Systemic sclerosis (SSc) is a disease of unknown etiology characterized by immune activation, tissue fibrosis, and vasculopathy. Peripheral vascular involvement manifesting as Raynaud's phenomenon (RP) affects almost all patients. In a subset of SSc patients, episodes of progressive digital ischemia can result in a digital ulcer—a denuded area with a defined border with loss of epithelialization and loss of epidermis and dermis. Sustained reduction in digital perfusion with impaired tissue viability can lead to critical ischemia, in some cases resulting in gangrene necessitating amputation. On a microvascular scale, episodes of digital ischemia are thought to be due to neuroendothelial imbalance of vasoconstriction and vasodilatation, structural abnormalities of the vasculature, and intravascular factors such as platelet activation, procoagulants, and oxidative stress. Recent studies on macrovascular disease in SSc suggest that ischemic demarcation and loss of digits occurs secondary to narrowing or occlusion of larger digital arteries  or medium-sized and large arteries in the lower extremities. This paper will summarize the current knowledge regarding the risk factors, pathogenesis, evaluation, and treatment of digital ischemic loss in SSc.SSc has a prevalence of 1–50 cases per 100,000 people worldwide . A more P < .0001). There was no correlation between disease duration and severity of digital vasculopathy [Predictors of ischemic digital loss in patients with SSc have been identified from data collected from observational studies . Patientulopathy .P = .01). There was also a trend for an association with the presence of the anti-topoisomerase antibody, in that six patients with severe ischemia had anti-topoisomerase antibodies compared with two patients without ischemia (P = .08). The authors concluded that complications from severe digital vasculopathy are more likely in patients with scleroderma specific antibodies [An initial observation by Herrick et al. found weakly positive anti-cardiolipin (aCL) antibodies in four of eight patients with SSc who had severe digital ischemia requiring amputation . Howevertibodies . Another review of a prospective cohort from the UK also found that anti-centromere antibody positivity is a predictor for amputation. Of 171 patients, 75% of whom had lcSSc, 37% were anti-centromere antibody positive and 16.4% were identified as having amputations. Of patients who had undergone amputations, 60.7% were anti-centromere antibody positive (OR 3.12). Smoking was also found to be an independent risk factor for amputation  [Other studies have also investigated whether smoking is a risk factor for poorer outcomes related to digital ischemia. Previously, Wigley et al. concluded that smoking was not a risk factor in 98 patients in their SSc cohort at a US tertiary care center [P = .017). The IgA subtype showed the strongest association . However, there was no difference in aCL antibody frequency between the two groups, as has been previously reported. In addition, after adjusting for demographics, disease type, smoking status, and anti-centromere antibodies, anti-beta2-glycoprotein I antibodies positivity was significantly associated with active digital ischemia , elevated estimated right ventricular systolic pressure (RVSP) , and increased mortality . They also noted that patients with a history of ischemic digital loss have more severe pulmonary vascular disease with overall a higher RVSP, worse lung severity scores, and lower diffusing capacity of carbon monoxide (DLco) which might be contributing to the higher mortality. All in all, anti-beta2-glycoprotein I antibodies is likely important in scleroderma vascular disease; however, further research is necessary to determine whether these antibodies are directly involved in the pathogenesis of disease or represent an epiphenomenon [A recent study investigated whether anti-beta2-glycoprotein I (anti-beta2-glycoprotein I antibodies) and aCL antibodies are correlated with macrovascular disease, including digital loss in SSc patients. Seventy five SSc patients with a history of ischemic digital loss were matched to 75 SSc patients without a history of digital loss. Anti-beta2-glycoprotein I antibodies was significantly more frequent in SSc patients with digital loss than in patients without digital loss: 27/75 (36%) in the digital loss group had these antibodies compared with 14/75 (19%) in the group without digital loss  lcSSc patients with ischemic digital loss immunoblotted for autoantigens to granzyme B compared with 6/15 (40%) lcSSc patients without ischemic digital loss . The risk of anti-granzyme B antibodies persisted even when controlling for the presence of antiphospholipid antibodies . This inThe pathogenesis of SSc is complex and incompletely understood. The current view of this disease includes the development of vasculopathy, activation of the cellular and humoral immune responses, and progressive fibrosis. The pathological hallmark of SSc is an obliterative vasculopathy combined with interstitial fibrosis in target organs. Raynaud's phenomenon, a reversible process in primary disease, is due to dysregulation of the peripheral and autonomic nervous systems leading to vasospasm. However, in SSc, ischemia can progress due to irreversible changes within the endothelium associated with decreased production and responsiveness of endothelial derived vasodilators (nitric oxide and prostacyclins) and increased production and responsiveness of vasoconstrictors (endothelin-1). In addition, microvessels have increased permeability, increased leukocyte extravasation, and activation of coagulation and fibrinolytic factors with platelet aggregation, eventually leading to thrombosis. This vasculopathy affects capillaries, arterioles, and even large vessels and progresses to luminal occlusion due to intimal/medial hypertrophy and adventitial fibrosis with persistent endothelial damage and apoptosis. The process of revascularization is also defective in SSc, affecting both angiogenesis, in which new vessels arise from preexisting vessels, and vasculogenesis, in which new vessels derive from endothelial progenitor cells to replace damaged or senescent blood vessels, despite elevated levels of angiogenic factors. Therefore, in SSc there is a widespread obliterative vasculopathy associated with the failure to repair and replace damaged vessels, thus resulting in poor digital perfusion and the potential for ischemic digital loss .P = .002) and smoothly thickened (P < .0001) than those of non-SSc controls, while other arterial beds were not significantly different [P < .01) [Evidence has shown that both proximal and distal arteries are affected in SSc-related vasculopathy resulting in digital ischemic loss. Stafford et al. evaluated Doppler studies of arteries in the limbs, neck, and abdomen of 20 SSc patients compared with controls who were non-SSc rheumatology patients. They found that the ulnar arteries in SSc patients were significantly narrower (ifferent . AnotherP < .01) . Hasegawa et al. studied macrovascular disease in patients with SSc with digital ulceration or gangrene using catheter arteriography of the upper and/or lower extremities. Seven of eight patients in this study were found to have macrovascular occlusion in the upper extremities. Of these, three had occlusion limited to digital arteries, three had obliteration of the ulnar and superficial palmar arch, and one had a radial artery occlusion. Of five patients who underwent lower extremity angiography, one had limited digital artery occlusion, one had occlusion of the posterior tibial artery, one had dorsalis pedis and arcuate occlusion, and two had occlusion of the plantar arch .The initial clinical evaluation for the signs of digital ischemia includes assessing for persistent discoloration , increased pain, digital ulceration, extreme tenderness, or frank gangrene. Nailfold capillaroscopic changes with dilatation, irregularity, megacapillaries, and drop-out are often present in SSc patients, and progression of these features may be predictive of the development of digital ischemia . The difAngiographic techniques to evaluate for digital occlusions include conventional angiography, magnetic resonance angiography (MRA) or computed tomography (CT) angiography . ConventThe main indication for imaging is to identify proximal lesions amenable to angioplasty or surgery in cases of severe digital ischemia. It should be noted that MR and CT angiography are still investigational techniques and should be reserved for patients who have contraindications to conventional angiography. Conventional angiography is still considered the gold standard to visualize more distal arteries, especially if arterial reconstruction is being considered .Digit-threatening ischemia warrants prompt and aggressive treatment to control symptoms and prevent digital loss. As this is considered a medical emergency, hospitalization should be considered for all patients to expedite interventions. Nonpharmacologic treatments such as a warm environment and bed rest to decrease activity or possible injury to the affected limb are essential. A simple xeroform dressing with an antibiotic ointment can be used to prevent a superinfection and allow wound healing. Intravenous antibiotics should be used if overlying infection or osteomyelitis is suspected, especially in patients who are being considered for surgical debridement or if there is any collection of purulent material or necrotic tissue. Surgical debridement is generally reserved for patients with purulent drainage, necrotic/late stage ischemic tissue, or severe structural arterial disease who do not respond to medical therapies.Analgesia with opioids is of utmost importance as pain due to critical digital ischemia is extremely intense. Local anesthetic blocks with lidocaine or bupivicaine without epinephrine may be helpful for pain control, but these interventions have not been studied in clinical trials. Temporary chemical sympathetic block should strongly be considered for patients with severe pain before surgical sympathectomy .Anticoagulation is recommended for patients with rapidly advancing ischemic tissue who do not have contraindications. Intravenous heparin for 24–72 hours is typically used; however, no double-blind clinical trials have been performed . TheoretAggressive vasodilatation is thought to improve bloodflow to ischemic areas. Firstly, oral calcium channel blocker doses should be titrated to maximum tolerated doses. However, intravenous prostacyclins, which not only vasodilate but also inhibit platelet aggregation, are considered the mainstay of management for acute digital ischemia. Intravenous epoprostenol or iloprost (0.5–2 ng/kg/min) daily infusions for 1–3 days, each infusion lasting 6 hours, is the recommended regimen. However, for patients with severe progressive ischemia, continuous prostacyclin infusion and higher doses may be required as tolerated. Studies have shown that intravenous iloprost was effective in reducing both the frequency and severity of ischemic attacks and in the healing of digital ulcerations , 19. EpoIf symptoms are persistent and medical therapy fails, proximal or digital sympathectomy, microsurgical revascularization of the hand, and digital arterial reconstruction have been reported to improve digital vascular perfusion, heal digital ulcers, and substantially relieve or eliminate pain from one to 46 months postoperatively . A proximal sympathectomy involves resection or ablation of a section of the cervical or thoracic sympathetic chain. This treatment is now only rarely performed due to its significant associated risks such as permanent Horner's syndrome, persistent neuralgia, and decreased localized cutaneous sweating; however, an endoscopic thoracic approach may be safer. Sympathectomy is less effective in patients with secondary than primary Raynaud's phenomenon and the duration and degree of improvement is variable .Digital sympathectomy was introduced in the 1980s as an alternative to proximal sympathectomy. A recent study in 20 patients primarily with SSc, who had 42 ulcerated digits due to ischemia, found that periarterial sympathectomy led to complete healing or decrease in ulcer number in 28 of 42 digits after a mean of 96 months of follow-up. In addition, the rate of amputation in the treated patients with underlying autoimmune disease was half of that for patients with underlying atherosclerotic disease, indicating that patients with autoimmune-induced ischemia may be more amenable to treatment with digital sympathectomy . HoweverIf there is larger vessel occlusive disease, especially at the level of the ulnar or radial artery, successful reconstruction can be performed with vein grafts . Of 15 SA recent case report describes successful vascular reconstruction, with improved digital temperatures, pain and cold sensitivity, and health-related outcomes with peripheral artery bypass for vasoocclusive disease of the superficial palmer arch and tarsal arch in two SSc patients with severe digital ischemia . AnotherSystemic sclerosis is a complex autoimmune fibrosing disease with major vascular complications. Digital vasculopathy with critical ischemia is one of the more challenging complications and carries significant morbidity in a substantial proportion of patients. Identified risk factors for ischemic digital loss include the diffuse subtype of SSc, SSc specific antibodies (anti-centromere antibodies and anti-topoisomerase antibodies), current smoking, anti-beta2-glycoprotein I antibodies, and anti-granzyme B antibodies. Ischemic digital loss is related to both macrovascular disease with involvement of proximal and larger digital arteries, as well as microvascular involvement caused by neuroendothelial, structural, platelet, and procoagulant effects. The medical treatment algorithm for critical digital ischemia includes urgent evaluation for reversible causes, analgesia, intravenous vasodilator therapies, angiography, and possibly anticoagulation. Surgical therapies such as sympathectomies and vascular reconstructions are considered in patients who fail medical therapy. Further research is necessary to identify specific biomarkers for digital ischemia in patients with SSc, and to develop more effective treatments.


Chronic kidney diseases share common pathogenic mechanisms that, independently from the initial injury, lead to glomerular hyperfiltration, proteinuria, and progressive renal scarring and function loss. Inhibition of the renin angiotensin system (RAS) has been consistently found to reduce or halt the progressive deterioration of renal function through reduction of blood pressure and proteinuria, the two main determinants of renal function decline. In few instances, RAS inhibition may even promote amelioration of the glomerular filtration rate. Animal data suggest that chronic therapy with angiotensin-converting enzyme inhibitors or angiotensin II receptor type I blockers promotes regression of glomerulosclerosis, even in later phases of the disease. In humans, studies investigating the effect of angiotensin II inhibition on renal structural changes have shown inconsistent results, possibly due to small numbers and/or short duration of follow-up. Whether regression of glomerulosclerosis relies on a direct regenerative effect of RAS inhibition or on spontaneous kidney self-repair after the injury has been removed is still unknown. Improved understanding of mechanisms that promote renal regeneration may help in designing specific therapies to prevent the development of end-stage renal disease. This is a desirable goal, considering the economic burden of chronic kidney diseases and their effect on morbidity and mortality. Chronic kidney disease (CKD) represents a major health problem worldwide. It has been estimated that approximately 830,000 deaths every year are linked to renal diseases, but the scale of the problem is probably largely underestimated  Costs foIndependently from the initial insult, chronic nephropathies seem to share common pathogenic mechanisms leading to progressive renal function loss and fibrosis . TargetiExperimental and clinical research has clearly documented that, independently from the initial injury, renal disease progression is sustained by common mechanisms that, starting from nephron loss, lead to compensatory glomerular hemodynamic changes. In the experimental model of renal mass reduction by five-sixths nephrectomy, resembling advanced phases of CKD, the remnant glomeruli undergo hypertrophy, and the tone of afferent arterioles drops more than that of efferent arterioles . This inA key player in these glomerular hemodynamic changes, crucial to progressive renal injury, is angiotensin II . Indeed,Podocyte injury secondary to glomerular hypertension and the direct effects of angiotensin II leads to increased protein ultrafiltration in the urinary space. When proteinuria is highly selective, that is, when albumin represents its major component, tubulointerstitial damage and renal function loss is rather infrequent. Conversely, when larger proteins also pass through the glomerular barrier into the urinary space, tubulointerstitial damage takes place and renal function progressively declines . ConsistProtein overload in the tubules induces tubular cells to release cytokines, chemokines, growth factors and vasoactive substances, which leads to abnormal interstitial accumulation of inflammatory cells, ECM collagen, fibronectin and other components that are responsible for interstitial fibrosis . NotablyFiltered oxidized lipoproteins can promote lipid accumulation in glomerular, tubular and interstitial cells, which in turn promotes progressive renal function loss . MoreoveThe aforementioned mechanisms sustain progressive renal function loss and scarring, which, if left untreated, inexorably leads to end-stage renal failure (ESRF). Thanks to compensatory adaptations by the high number of unimpaired nephrons, this does not immediately translate into changes in the renal function parameters most commonly used in the clinical practice, such as serum creatinine and urea. Beyond a certain level of injury, however, compensatory adaptations no longer keep pace with nephron loss, and glomerular filtration rate declines.Given the important role of angiotensin II in inducing and sustaining glomerular hypertension and proteinuria and its deleterious consequences, clinical studies have focused on the renoprotective effects of angiotensin II inhibition.The Angiotensin-Converting Enzyme Inhibition in Progressive Renal Insufficiency (AIPRI) study showed that angiotensin-converting enzyme (ACE) inhibitor therapy reduced the risk of serum creatinine doubling in 583 mainly non-diabetic patients with chronic proteinuric nephropathies . HoweverAlthough the responsiveness to antiproteinuric therapy may vary between the different non-diabetic glomerulopathies, the aforementioned studies were not designed to detect such differences. However, efficacy of angiotensin II inhibition has been reported for diverse proteinuric diseases including IgA nephropathy , membranpost hoc analysis of the REIN study showed that the efficacy of ACE inhibitor therapy over placebo in prevention of ESRF was consistent across all levels of baseline GFR [et al., who randomly assigned 224 non-diabetic patients with an estimated GFR < 30 mL/min to benazepril or placebo [P = 0.02), despite similar BP control. Thus, RAS inhibitors should be continued up to the very advanced phases of renal failure. However, when GFR declines to < 15 mL/min, RAS inhibitor discontinuation may be considered, in order to increase GFR and retard the need for renal replacement therapy[Of note, current evidence supports the benefit of RAS inhibition even in the advanced phases of renal failure. A line GFR . Even mo placebo . After 3t therapy.In (incipient) diabetic nephropathy, ACE inhibitors and angiotensin II receptor type I blockers (ARBs) also exert their renoprotective effects. In hypertensive T1DM or T2DM patients with microalbuminuria or overt nephropathy, both ACE inhibitors and ARBs protected against the progression of renal disease independent of BP reduction -37. Morevice versa. In the Renoprotection of Optimal Antiproteinuric Doses (ROAD) study, non-diabetic proteinuric patients were randomized to either benazepril or losartan in the conventional dose or in a dose that was titrated upwards until the maximum antiproteinuric efficacy was reached [Only few studies have addressed the question of whether ACE inhibitors are better than ARBs or  reached . This st reached . Other s reached ,44. Thes reached .Double RAS inhibition may have a superior antiproteinuric effect to single inhibition with either agent. In patients with persisting proteinuria, the addition of an ARB to an ACE inhibitor counteracts the effects of angiotensin II that is produced via ACE-independent pathways, whereas the addition of an ACE inhibitor to an ARB limits compensatory angiotensin II production. Direct comparisons of combined RAS blockade versus single blockade are scarce, and the only moderately large study reported to date has recently been retracted because its validity could not be proven in a subsequent institutional investigation . HoweverConcerns have been raised about double RAS inhibition because combined treatment of ramipril and telmisartan increased the risk of the combined primary renal outcome of death, serum creatinine doubling or dialysis compared with either agent alone in a large study comprising 25,620 patients with atherosclerotic disease and/or diabetes with end-organ damage . HoweverThe studies mentioned above have predominantly used prevention of progression of albuminuria or deterioration of kidney function as the outcome of interest. Not many studies have addressed the possibility of long-term remission or even regression of proteinuric kidney disease with angiotensin II inhibition Table . Precede2/year after at least 5 years of continued ramipril use [For non-diabetic patients, long-term follow-up of the REIN study showed that the rate of measured GFR decline progressively improved to a level of about 1 mL/min/1.73 mpril use , which april use . Moreovepril use . Taken tet al. developed a technique for three-dimensional reconstruction of the glomerular capillary tuft, thus permitting a more accurate estimation of the extent of glomerulosclerosis, which is underestimated by currently employed two-dimensional imaging methods. Using this method, it was possible to show that in rats with advanced non-diabetic proteinuric nephropathy, administration of high-dose ACE inhibitors reduced the volume of sclerosis in most glomeruli , and increased the volume of normal capillary tissue by up to 40% renin receptor in glomeruli, tubules and cortical vessels. Other sFinally, growing evidence is accumulating of an antiproteinuric and nephroprotective effect of vitamin D, possibly acting again, at least in part, through RAS inhibition. Indeed, vitamin D receptor-null mice show increased levels of renin, whereas administration of vitamin D to wild-type animals suppresses renin mRNA levels in the kidney . In remnTwo main, non-mutually exclusive mechanisms may be advocated to explain improved renal function and regression of histological lesions with angiotensin II inhibition. First, angiotensin II inhibition itself may favor kidney repair beyond reduction in BP and proteinuria, by affecting ECM deposition and podocyte structure and function. Alternatively, the kidney has an inherited capability to repair itself once the chronic insult (for example proteinuria) has been eliminated or attenuated. The evidence supporting these two hypotheses will be described below.Several mechanisms have been proposed by which angiotensin II inhibition may directly promote regression of renal injury. A prominent feature of glomerulosclerosis is increased deposition of ECM, which is brought about by a change in the balance between breakdown proteinases and their inhibitors . ResultsSeveral studies have suggested that angiotensin II has a direct effect on glomerular permselectivity ,95. In mFinally, in cell cultures, proliferation of glomerular mesangial and endothelial cells is stimulated by angiotensin II . ConsistRegression of glomerulosclerosis and neoformation of glomerular tissue has been linked to progenitor cells in the bone-marrow or inside the kidney ,99-102. Stem cells have also been identified in proximal and distal tubuli and peritubular capillaries, although their role in and relative effect on kidney regeneration still has to be defined . FinallyDifferent renal injuries eventually lead to reduced nephron numbers and increased intraglomerular pressure in the remaining nephrons. This results in increased urinary protein losses that, through a toxic effect on tubular cells, promote progressive renal scarring and function decline. Experimental studies and clinical trials clearly showed that strict BP control and maximal inhibition of RAS can reduce proteinuria and the rate of renal function decline. Notably, these therapies may halt and even promote regression of renal injury in some patients. Kidney repair induced by angiotensin II antagonists may either result from inhibition of the direct effects of angiotensin II on podocytes, glomerular permselectivity and fibrogenesis, or from the intrinsic capability of the kidney to repair itself once elements sustaining disease progression, such as hypertension and proteinuria, are controlled. A more in-depth understanding of the mechanisms by which RAS inhibition may retard or even reverse renal disease progression may significantly improve the morbidity and mortality associated with chronic kidney disease.The authors declare that they have no competing interests.All authors have been involved in the drafting of the manuscript and have revised it critically for important intellectual content. All authors read and approved the final manuscript.


Animals often settle near competitors, a behavior known as social attraction, which belies standard habitat selection theory. Two hypotheses account for these observations: individuals obtain Allee benefits mediated by the physical presence of a competitor, or they use successfully settled individual as a source of information indicating the location of high quality habitat. We evaluated these hypotheses experimentally in two species of shrikes. These passerine birds with a raptor-like mode of life impale prey to create larders that serve as an indicator of male/habitat quality. Thus, two forms of indirect information are available in our system: a successfully settled shrike and its larder. Typically these two cues are associated with each other, however, our experimental treatment created an unnatural situation by disassociating them. We manipulated the presence of larders of great grey shrikes and examined the settling decisions of red-backed shrikes within and outside the great grey shrike territories. Male red-backed shrikes did not settle sooner on plots with great grey shrikes compared to plots that only contained artificial larders indicating that red-backed shrikes do not use the physical presence of a great grey shrike when making settling decisions which is inconsistent with the Allee effect hypothesis. In contrast, for all plots without great grey shrikes, red-backed shrikes settled, paired and laid clutches sooner on plots with larders compared to plots without larders. We conclude that red-backed shrikes use larders of great grey shrikes as a cue to rapidly assess habitat quality. According to habitat selection theory the fitness of animals in a habitat patch is assumed to be negatively density-dependent, i.e., fitness is reduced by the presence of competitors Two general hypotheses explain why individuals might benefit by settling near competitors. The first posits that individuals obtain direct fitness benefits such as reduced predation risk, better defense against intruders, increased feeding benefits and/or social stimuli which improves their mating opportunities The second hypothesis suggests that animals use the presence of a successfully established individual as an indicator of the location of high quality habitat We examined heterospecific attraction in shrikes to test these hypotheses. Shrikes, small raptor-like passerine birds, create temporary caches by impaling prey on thorns or in forked branches throughout their territory because, unlike raptors, they do not have talons and strong feet, so impaling and wedging is a necessary adaptation for dismembering and handling large prey items. Scattered caches are distributed over a shrike's territory, most frequently in distinctive places, such as at the tops of trees or shrubs on dry or broken sprigs, but also on artificial objects, such as on barbed wire fences Lanius collurio) and great grey shrikes (Lanius excubitor) share similar breeding habitats: a mix of farmland and natural meadows with dispersed shrubs and trees for nests, perches and cache sites, and often nest near one another Red-backed shrikes , red-backed shrikes did not settle earlier on plots with great grey shrikes compared to plots without great grey shrikes (+L+G and C+G vs. +L−G)  and for plots with great grey shrikes, more red-backed shrikes settled on plots with supplemented larders (+L+G) compared to those without (C+G) .Experimental treatments also affected the total number of red-backed shrikes that settled on the plots , Poland, in an agricultural landscape containing arable fields, meadows, small woodlots and tree rows During early spring, the study area was searched for great grey shrike territories and all nests were mapped. In April, we randomly established 40 300×300 m experimental plots in locations where red-backed shrikes as well as great grey shrikes had established territories in previous years. All plots were separated by at least 500 m. Twenty plots contained a great grey shrike territory and 20 did not. To minimize potential pseudoreplication, only one experimental plot was established in each great grey shrike territory. The great grey shrikes from the studied population have a high reoccupation rate of breeding territories, up to 70% of territories are occupied in the following seasons st, all plots were intensively monitored each day for the arrival of red-backed shrikes, as well as the initiation of mating and nesting behaviour, over the next five weeks. Over the past 20 years, May 1st was the earliest recorded arrival date for red-backed shrikes Beginning May 1All statistical tests were two-tailed, except where indicated. We used Kruskal-Wallis ANOVA with Newman-Keuls post-hoc tests to examine treatment effects on dates of settling, pair bond formation and egg laying.


As part of the PLoS Epigenetics Collection, Caroline Relton and George Davey Smith discuss the potential of epigenetics for the treatment and prevention of common complex diseases, including cancer. The epigenome records a variety of dietary, lifestyle, behavioral, and social cues, providing an interface between the environment and the genome. Epigenetic variation, whether genetically or environmentally determined, contributes to inter-individual variation in gene expression and thus to variation in common complex disease risk.Interventions based upon epigenetic agents, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, have been in clinical use for many years, but their role outside treatment of specific cancers is not established.Epigenetic therapies will only be fruitful if epigenetic mechanisms are causally related to the disease being treated. Evidence linking epigenetic variation to specific disease phenotypes to date is lacking.Epidemiological approaches can be applied to help separate causal from non-causal associations.We propose the development of a Mendelian randomization approach , which could help overcome the problems of confounding and reverse causation .There is considerable anticipation of future improvements in disease prevention and treatment following recent advances in genomics Genes are packaged into chromatin and dynamic chromatin remodeling processes are required for the initial step in gene expression (transcription), achieved by altering the accessibility of gene promoters and regulatory regions In rare developmental disorders, the role of aberrant epigenetic processes is well established There is an increasing body of evidence to demonstrate that epigenetic patterns are altered by environmental factors known to be associated with disease risk  Epigenetic patterns, including histone modifications, microRNA (miRNA), and DNA methylation, can be assessed in a range of tissue types. As DNA methylation assays on stored DNA samples are straightforward, this has been extensively studied The tissue specificity of epigenetic patterns is a well-established phenomenon, with variation between tissues within individuals being greater than variation between individuals Epigenetic patterns are heritable across cell divisions (mitosis) Several other factors beyond tissue type and age The most widely studied lifestyle influence on epigenetic patterns is smoking. It has been associated with global hypomethylation in DNA Animal studies have shown that chronic alcohol consumption is associated with reduced genomic DNA methylation in the colon Air pollutants such as air particulate matter and airborne benzene exposure levels have been associated with changes in DNA methylation in genes involved in inflammation and carcinogenesis Helicobacter pyloriSeveral infectious agents, including Perhaps the most widely celebrated example of the influence of environmental conditions (other than diet) on the epigenome relates to maternal postnatal nurturing and epigenetically mediated alterations to the hypothalamic-pituitary-adrenal response to stress IGF2 (insulin-like growth factor 2) locus could be explained by five single nucleotide polymorphisms (SNPs) cis and trans genetic effects upon DNA methylation (cytosine guanine dinucleotide [CpG]) sites, the predominant influences being in cis, defined as SNPs influencing methylation at CpG sites within 1 Mb of themselves cis effects have been reported in whole blood DNA Twin- and family-based studies have demonstrated that variation in epigenetic patterns, including both chromatin states Despite the relatively large body of evidence that disease-related environmental exposures are associated with epigenetic alterations, there remains little compelling data to support the link between epigenetic variation and common complex disease phenotypes (other than cancer). Investigation of parent-of-origin effects on risk of common complex disease have suggested a role of perturbed DNA methylation Using germ-line genetic variation as a proxy for environmental exposures provides a route to strengthening causal inference within observational data Mendelian randomization methods can be applied to many categories of environmentally modifiable exposures to help define whether their relationship with phenotype is causal. For example, with respect to behavioral factors, it has been used in a proof-of-principle manner to demonstrate associations of alcohol intake with esophageal In a similar fashion, genetic variants related to body mass index and obesity have been shown to influence a wide variety of metabolic, cardiovascular, and bone-related traits, strengthening evidence on the causal influence of adiposity in these cases cis effects on gene expression, which allows isolation of specific loci influencing transcript level. The identification of strong cis effects in a genome-wide association study analysis of methylation patterns In the field of gene expression studies, identifying causal processes within a multitude of associations is at least as problematic as in observational epidemiological studies. For example, the majority of gene expression signatures in adipose tissue, and in high proportions (up to 10%) in blood, have been found to be related to obesity As a hypothetical example of how this approach could be applied, we will consider alcohol intake and HNSCC. It is likely that alcohol intake would be associated with a wide range of epigenetic changes, although at least some (and probably many) of these associations could reflect confounding by the many other factors related to alcohol consumption. Similarly, HNSCC could be related to a multitude of epigenetic changes, which could arise through reverse causation (the disease influencing the epigenetic patterns) or confounding (factors associated with HNSCC risk influencing the epigenetic patterns). If the epigenetic processes are to be targeted as a component of disease prevention they must be causally associated with HNSCC, and for them to mediate the effect of alcohol intake on HNSCC risk they need to be responsive to changes in alcohol intake. Observational data demonstrating an association of alcohol intake with a particular epigenetic profile exists, but the association of this profile with HNSCC risk does not, of course, establish causality. As depicted in It can be argued that mitotically stable changes in gene expression are very likely to underlie the development of virtually all disease  and ultimately exploring the causal impact of epigenotype on disease outcomes (epigenotype/disease) using genetical epigenomics and other methods, progress towards epigenetic interventions can be made. As genome-wide association studies and other approaches identify robust associations between genetic variants and epigenetic patterns, possibilities for elucidating causal pathways and predicting the effect of manipulation—through environmental (including lifestyle) modification or pharmacotherapeutic means—is considerable. In this way, epigenetic markers may become targets for modification as well as biomarkers for exposure and disease risk. The International Human Epigenome Consortium is poised to invest millions of dollars to map 1,000 reference epigenomes in a range of normal tissues and define the level of variation that exists between individuals Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, et al. (2004) Epigenetic programming by maternal behaviour. Nat Neurosci 7: 847–854. This landmark paper demonstrated that the epigenomic state of a gene can be altered through behavioural programming and that this environmentally induced modification is potentially reversible.Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, et al. (2005) Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci U S A 102: 10604–10609. This article describes how epigenetic patterns in monozygotic twins become more discordant with advancing age. This epigenetic drift is postulated to be invoked through differences in environmental exposures.Bjornsson HT, Sigurdsson MI, Fallin MD, Irizarry RA, Aspelund T, et al. (2008) Intra-individual change over time in DNA methylation with familial clustering. JAMA 299: 2877–2883. This study showed greater than 10% methylation change over time, that individuals within families showed both gain and loss of methylation, and that this change in methylation showed familial clustering indicative of a genetic basis.Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, et al. (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462: 315–322. This paper reports the first genome-wide, single base-pair resolution map of methylated cytosines in the mammalian genome from embryonic stem cell and fetal fibroblasts, showing widespread differences between the tissue types.cis (correlation within 1Mb of a CpG site) with only a handful of SNPs determining trans methylation (distant regulation effects).Zhang D, Cheng L, Badner JA, Chen C, Chen Q, Luo W, et al. (2010) Genetic control of individual differences in gene-specific methylation in human brain. Am J Hum Genet 86: 411–419. This study demonstrated that DNA methylation is a heritable trait, determined in part by common genetic variation. The vast majority of genetically determined variation was observed to be in