Picornaviridae and Reoviridae are assembled in the cytoplasm and are generally released by cell lysis. However, recent evidence suggests that some non-enveloped viruses exit from infected cells without lysis, indicating that these viruses may also utilize alternate means for egress. Moreover, it appears that complex, non-enveloped viruses such as bluetongue virus (BTV) and rotavirus interact with lipids during their entry process as well as with lipid rafts during the trafficking of newly synthesized progeny viruses. This review will discuss the role of lipids in the entry, maturation and release of non-enveloped viruses, focusing mainly on BTV.Non-enveloped viruses such as members of  As an example, in this report we will discuss mainly the interaction of Bluetongue virus (BTV) proteins that form a separate genus in the non-enveloped Reoviridae family with membrane lipids both during the virus entry and the exit.The replication cycle of viruses involves entry into host cells, synthesis of viral genes and proteins, assembly of progeny virus particles and their subsequent egress or release. Along with the plasma membrane, viruses also have to interact with the endosomal and vesicular membranes during their replication in host cells. All cellular membranes are composed of lipids and proteins that are usually arranged in various micro domains. During infection of cells by enveloped viruses, the lipids present in both viral and cellular membranes mediate fusion and fission reactions to facilitate virus entry and egress. Since non-enveloped viruses do not have a lipid envelope, it is generally believed that their entry mechanism does not involve membrane fusion activity and that these viruses are mainly released by cell lysis. Usually, non-enveloped viruses enter the cells by penetrating the membrane barrier, either via the endocytic pathway using clathrin-coated vesicles or caveolae, or by the formation of a pore at the cell surface –4. Recen2.Reoviridae family, infect animals, plants and insects, and are transmitted by arthropod vectors. An important factor in the distribution of BTV worldwide is the availability of suitable vectors, usually biting midges (gnats) of the Culicoides genus. BTV infects wild and domestic ruminants, particularly sheep often with high morbidity and mortality. Bluetongue disease (BT) was first observed in domestic animals in Africa in the late 18th century. Currently, BTV (24 serotypes) is endemic in many parts of the world, particularly in tropical and subtropical countries. As a result of its economic significance, BTV has been the subject of extensive molecular, genetic and structural studies and is now the most well characterized orbivirus.Orbiviruses (some 140 members) which form a genus within the The virion particle is composed of seven discrete proteins (VP1-VP7) that are organized into two concentric shells (capsids); an outer shell and an inner shell or ‘core’ and a genome of 10 dsRNA segments . The outIn mammalian cells, BTV entry proceeds via virus attachment to the cell, followed by endocytosis and release of a transcriptionally active core particle into the cytoplasm. Of the two outer capsid proteins, the larger VP2 (110 kDa) is the most variable and is the serotype determinant of the virus. Indeed, VP2 is responsible for eliciting neutralizing antibodies and possesses haemagglutination activity of BTV. Further, VP2, which oligomerizes as a trimer, binds to receptor(s) on the plasma membrane of mammalian host cells and is also responsible for virus internalization .et al. [4Cl) inhibited BTV replication. The pH-dependent character of BTV entry was further examined more recently by monitoring the effect of bafilomycin A1 in BTV infectivity [+-ATPase inhibitor, and is responsible for the specific inhibition of endosomal acidification, blocking endosomal traffic during endocytosis. From our studies, it was clear that the effect of bafilomycin A1 in BTV replication was strictly correlated with the reduction of virus infection in a dose-dependent manner. In parallel, confocal microscopy supported the hypothesis that the endosomes are the perfect location for BTV activation leading to virus uncoating. To further prove that BTV is internalized by the endosomal vesicles and to understand the internalization route followed by the virus, specific endosomal markers were used to investigate the presence of the virus in the early and late endosomes. The results obtained from various studies demonstrated that in the early stages of the infection BTV is present in the early endosomes for at least 30 minutes before further progression of the virion to the late endosomes [Some limited information regarding BTV entry was initially generated based on ultra-structural electron microscopy studies, which indicated that BTV entry utilizes clathrin coated vesicles . Therefoet al. , who shoectivity . This chndosomes . Since tThe 60 kDa protein, VP5, shares certain secondary structural features with the fusion proteins of enveloped viruses, indicating that it may be responsible for membrane penetration activity . Two dis3.Culicoides vector.The BTV outer capsid has an icosahedral configuration with a diameter of ∼880 Å . A serieThe structural data revealed that the globular VP5 is very rich in helices and possesses only one β sheet . The N-tDensity maps have also revealed that the VP2 trimers interact  with VP5 trimer very weakly, and similarly the interactions between VP5 and VP7 trimers are also very weak. These weak interactions would permit conformational changes of VP5 during the penetration process and during shedding of the outer coat. In comparison, both the VP2 tip and hub domains connect to their underlying VP7 trimers by stronger forces.4.Removal of the outer capsid proteins activates the virion transcriptase ,34 in thi.e., the viral ssRNAs, transcriptase complex (TC) proteins  as well as VP3 that encapsidates the TC and the viral genome [EM analysis of thin sections of BTV infected cells have revealed the presence of small numbers of intracellular virus particles and large numbers of juxtanuclear fibrillar networks referred to as virus inclusion bodies (VIBs). These structures are composed predominantly of non-structural phosphoprotein NS2 that recruits all the components of subviral particles (subcore), l genome –47. The l genome . The subl genome . Similarl genome –52. Howel genome ,47. Both5.In infected cells at later times post-infections, both VP2  and VP5 The association of VP5 with raft domains is quite convincing, since depletion of cellular cholesterol with methyl beta cyclodextrin not only altered the association of VP5 with raft domains, but also decreased the relative BTV viral titer, indicating the importance of raft domains in BTV replication . In addiin vivo [Similar interactions have been observed for rotavirus and lipid rafts during the later stages of virus replication. Although rotavirus specifically infects highly polarized intestinal cells in vivo , the majin vivo . NSP4 hain vivo .In contrast to VP7, the other outer capsid protein of Rotavirus, VP4, is cytosolic. However, it associates with lipid rafts in both MA 104 cells and Caco 2 cells but this association is sensitive to cholesterol extraction by methyl beta cyclodextrin only in one cell line but not the other . Since Vin vitro and in vivo [In infected Caco-2 cells, VP4 associates with cellular lipid raft domains early in infection . Since o in vivo . Studies in vivo .6.BTV infects and replicates successfully both in mammalian and insect cells. Although BTV is released from infected mammalian cells mainly by cell lysis, it also egresses either by budding or protrusion and the nature of release depends on the particular cell type . In infeMore recently using a reverse genetic system we confirmed that viruses containing mutations of the Tsg101 binding motif had altered patterns of virus egress and left nascent particles tethered to the cellular membrane, indicating that Tsg101 binding of NS3 is directly involved in pinching off the newly assembled viruses from the plasma membrane . FurtherIn subcellular fractionation experiments, not only does NS3 co-fractionate with raft domains in virus infected cells but cholesterol-dependent raft integrity is also essential for NS3 segregation with rafts . Since cSimilar to BTV, rotavirus release also depends on cell type. While the virus is released by lysis in MA104 cells, it exits by non-lytic means from the apical surface of differentiated cells –91. Sinc7.Membranes provide a physical barrier both in cell entry and egress of viruses from infected cells. Compared to the enveloped viruses that have a lipid bilayer that enables fusion of virus and cellular membrane, the non-enveloped naked capsids must adopt alternate strategies to penetrate through the cellular membrane lipid bilayer. In BTV this function is achieved by VP2 and VP5, the outer most virus structural proteinsStructural studies of VP2 suggest that following the attachment of the tip domain to the cell surface, secondary binding to sialic acid via the hub domain might cement the interaction and lead to cell ingress . BTV theTraditionally, it has been believed that non-enveloped viruses are released by cell lysis. However, it has been shown that along with BTV, rotavirus, simian virus 40, poliovirus, and parvovirus can be released without lysis of the infected cells [ . This inDuring BTV assembly, recent studies have shown that along with VP2 and VP5, the non-structural protein NS3 also associates with lipid rafts. The fact that extraction of cellular cholesterol decreases viral titer is consistent with the hypothesis that this interaction is important for the assembly of the outer capsid proteins onto the core particles to form the mature virus particle.The presence of a transient envelope like structure associated with BTV in EM sections of infected cells has led to the hypothesis that BTV may acquire an envelope via NS3 during virus release. It is noteworthy that BTV and related orbiviruses, seadornaviruses and coltiviruses are the only groups of arboviruses that lack a lipid envelope. It is possible that orbiviruses were originally also enveloped but lost their stable lipid membrane at some point during evolution.


New Frontiers in Personalized Medicine: Cardiovascular Research and Clinical Care conference held the previous day and cosponsored by the NHLBI, Personalized Medicine Coalition, American College of Cardiology, American Medical Association, and Cheney Cardiovascular Institute at George Washington University. The conference brought together leaders from academia, industry, and government to (1) discuss personalized medicine's current and potential impact on cardiovascular patient outcomes; (2) review emerging technologies and applications that may shape the field in the future; (3) discuss the results of an American College of Cardiology survey examining personalized medicine adoption rates among US cardiologists; (4) identify the barriers to adoption of pharmacogenetics-based personalized medicine in cardiovascular practice; and (5) develop recommendations for next steps with an emphasis on actions and evidence generation that are needed for adoption and improved quality of cardiovascular patient care.The National Heart, Lung, and Blood Institute (NHLBI) convened a Working Group on January 7, 2011, at George Washington University in Washington, DC, to provide recommendations to the NHLBI that would guide informed decisions on research directions and priorities in the field of cardiovascular pharmacogenomics. This meeting was timed to follow the New Frontiers in Personalized Medicine: Cardiovascular Research and Clinical Care conference; to identify areas and challenges that should be addressed to further the field of cardiovascular pharmacogenomics and increase its adoption into the clinical setting; to develop recommendations and priorities for implementing current pharmacogenomics-based treatment modalities; and to identify future research needs related to cardiovascular pharmacogenomics.The goals of the NHLBI Working Group were to review the discussion and recommendations from the New Frontiers in Personalized Medicine: Cardiovascular Research and Clinical Care conference brought together leaders from academia, industry, and government to address the critical issues facing the future of cardiovascular research and clinical care in science, business, and policy.Cardiovascular disease remains the number one cause of death in the United States and will likely soon become the number one cause of death globally. At the same time, new understandings of individual characteristics including genetic variations are increasingly reshaping our ability to treat various aspects of cardiovascular disorders. The Four major goals for the next 5 to 10 years were outlined as follows: (1) to establish standards of quality for the research enterprise, (2) to establish robust systems for more rapid evidence generation, (3) to harmonize regulatory and reimbursement standards, and (4) to develop innovative partnerships to accelerate the development and implementation of personalized medicine applications. Three examples of personalized medicine that are primed for widespread clinical use were highlighted as follows: (1) the use of pharmacogenomic algorithms to guide the dosing of warfarin therapy; (2) the use of blood cell gene expression profiles to detect or rule out cardiac transplant rejection; and (3) the targeted sequencing of areas of genes (resequencing) implicated in long-QT syndromes to identify causal mutations in affected individuals and to screen family members for the same mutations.Several new technologies with the potential to result in new tests and new drugs were discussed, including deep sequencing of DNA and RNA, biomarker panels, polymerase chain reaction technology, proteomics, and metabolomics. In addition, strategies to bridge the much-cited “valley of death” between basic science discoveries and development of successful drugs included the use of genomics data to establish the relevance of drug targets in humans before starting clinical trials, the use of intermediate biomarker or imaging endpoints to make the decision to proceed with or terminate drug candidates early in the development process, and early identification of subpopulations most likely to benefit from therapy, for example, by using common genetic polymorphisms  or gene expression profiles.It was noted that few cardiologists report feeling knowledgeable with respect to personalized medicine, highlighting the particular need for education aimed not only at patients but also at providers if widespread adoption of a test is to be achieved. A number of potential solutions to overcoming clinical and regulatory barriers to personalized cardiovascular care were addressed, including the education of providers in principles of basic science that are relevant to personalized medicine; integrating personalized medicine practices into the clinical workflow using electronic platforms; “real-world” studies to assess whether personalized medicine improves outcomes when used directly in a clinical practice context; and incentives for providers and patients to improve adherence to personalized medicine practices.New Frontiers in Personalized Medicine: Cardiovascular Research and Clinical Care conference, the NHLBI Working Group characterized and discussed challenges for cardiovascular pharmacogenomics in 5 domains as follows: clinical needs, clinical validation, information delivery, education and compliance, and cost-effectiveness. For the purpose of discussion, the Working Group adopted the International Conference on Harmonization E15 harmonized definition of pharmacogenomics as “the study of variations of DNA and RNA characteristics as related to drug response.” The majority of the subsequent discussion focused on the study of DNA sequence variation as related to drug response.Guided by the proceedings of the The Working Group sought to identify and prioritize the most pressing clinical needs to focus research and translational efforts. In particular, 3 areas of emerging pharmacogenomic applications were reviewed: anticoagulation (warfarin), antiplatelet therapy (clopidogrel), and lipid-lowering therapy (statins).CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex subunit 1), account for more than one third of the interindividual variation in stable therapeutic dosing of warfarin.5Warfarin, used widely for the prevention and treatment of thromboembolic disease, is challenging to use because of highly variable responses among patients and even within an individual patient. Patients receiving warfarin anticoagulation require frequent monitoring of blood clotting activity as measured by the prothrombin time  particularly in the immediate period after the initiation of warfarin therapy—with significant risk of either thromboembolism if the warfarin dose is too low or bleeding if the dose is too high. Polymorphisms in 2 genes, CYP2C9 and VKORC1 polymorphisms in an attempt to better predict the optimal starting warfarin dose.6 When compared with the usual practice , the pharmacogenomic algorithm did not improve the safety of warfarin initiation—the number of out-of-range international normalized ratios during the initiation period was unchanged—although it did reduce the dosing changes needed to achieve stable anticoagulation.6 Subsequent small prospective clinical trials suggested that addition of genetic information could improve the safety and efficacy of warfarin use, particularly in guiding the choice of maintenance dosing.9An early small clinical trial evaluated an algorithm that incorporated the 10 It was performed in a community practice setting rather than as a randomized prospective study. In this “real-world” study setting, CYP2C9 and VKORC1 genotypes were determined in about 900 patients in 23 prescription benefit plans who were starting warfarin therapy, the genotype data were made available to their providers (with no subsequent communication with the providers), and outcomes were compared with those in 2700 historical controls in the same 23 plans for whom genotypes had not been determined. Two external control groups from a different set of 56 plans, one concurrent with the genotyped group and the other concurrent with the historical control group, were also followed. Within a 6-month follow-up period, there was a 31% reduction of hospitalization in the genotyped patients compared with the controls, with a 28% reduction of hospitalization due to bleeding or thromboembolism; there were no significant differences between the external control groups.10A large warfarin pharmacogenomics study, the Medco-Mayo Warfarin Effectiveness Study with almost 4000 individuals, was designed to test whether the use of genotype information could reduce the incidence of hospitalizations from warfarin-related adverse effects.CYP2C9 and VKORC1 genotyping, with providers receiving this data several weeks after initiation of warfarin. Of note, the delay in obtaining the genotyping information indicates that the benefits of the data came in guiding maintenance dosing, rather than the initial dosing of warfarin. Studies of alternative designs are underway, such as the Clarification of Optimal Anticoagulation Through Genetics study, an NHLBI-sponsored prospective randomized clinical trial that will enroll up to 1200 participants and compare a clinical algorithm for determining the dosing for warfarin initiation to a pharmacogenomic algorithm using CYP2C9 and VKORC1 genotypes.11The study design of Medco-Mayo Warfarin Effectiveness Study has been criticized for using historical controls instead of contemporaneous controls, and for the relatively slow turnaround time of CYP2C9 and VKORC1 genotypes. The availability of alternatives to warfarin, such as the newly approved dabigatran, also makes possible a therapeutic strategy whereby a patient with CYP2C9 and VKORC1 genotypes and other clinical characteristics that presage higher risk for warfarin-related adverse effects might be prescribed a different anticoagulant, assuming the patient does not have contraindications to that anticoagulant .The results from Medco-Mayo Warfarin Effectiveness Study and other warfarin trials suggest that anticoagulation has the potential to be an early widely adopted pharmacogenomic application in cardiovascular medicine. Of note, the US Food and Drug Administration—approved label for warfarin has been revised to include specific dosing guidelines for patients with known 13 it is clear that patients display variable responses to clopidogrel therapy. A major contributor to this variability is the common *2 loss-of-function variant in CYP2C19, encoding a hepatic cytochrome P-450 2C19 enzyme important for clopidogrel bioactivation.16 Three large studies of mostly post—acute coronary syndrome and/or post-PCI patients on clopidogrel therapy  identified at least one copy of CYP2C19*2 in ≈30% of individuals. In all 3 studies, reduced-function allele carriers experienced significantly higher rates of cardiovascular death, myocardial infarction, and stroke.19 Considering the totality of evidence, the US Food and Drug Administration—approved clopidogrel label was updated to include a “boxed warning” to underscore that individuals carrying 2 reduced-function CYP2C19 alleles—so-called “poor metabolizers”—experience diminished effectiveness of the drug at standard dosing and that alternative therapeutic strategies should be considered in these patients.Although dual antiplatelet therapy with aspirin and clopidogrel is now standard therapy for acute coronary syndrome patients, particularly those undergoing percutaneous coronary intervention (PCI),CYP2C19 alleles suffered a 57% increase in risk of cardiovascular death, myocardial infarction, or ischemic stroke compared with noncarriers.20 The increased risk affected carriers of 2 reduced-function CYP2C19 alleles (76% increase) as well as carriers of 1 reduced-function CYP2C19 allele (55% increase). Notably, there was an almost tripling of risk of stent thrombosis in reduced-function CYP2C19 allele carriers.Subsequent studies have begun to clarify when clopidogrel pharmacogenomics may be helpful in guiding therapy. A meta-analysis of 9 clopidogrel studies with a combined 10 000 participants—comprising mostly PCI patients, with over half being treated for an acute coronary syndrome—found that carriers of reduced-function CYP2C19 alleles and noncarriers.21 However, very few of the patients in either trial underwent PCI with stent placement (14.5% in CURE). For participants in the PLATO (Platelet Inhibition and Patient Outcomes) trial, in which 64% underwent PCI with stent placement, reduced-function CYP2C19 allele carriers experienced a higher event rate on clopidogrel than noncarriers within 30 days of initiation of therapy, but in the long term there was no difference in the event rate on clopidogrel.22 Together, these studies suggest that (1) the effect of reduced-function CYP2C19 alleles may be more relevant in the acute setting rather than the long term, and (2) CYP2C19 genotyping may be more useful in higher-risk patients  than in lower-risk patients.In contrast, genotype data from participants in the CURE (Clopidogrel in Unstable Angina to Prevent Recurrent Events) and ACTIVE A  trials indicated that the relative cardiovascular risk reduction seen with clopidogrel treatment (vs placebo) was similar for carriers of reduced-function CYP2C19 genotypes improves clinical outcomes. Therapeutic strategies that await testing include (1) giving carriers of reduced-function CYP2C19 alleles a higher-than-standard dose of clopidogrel or (2) giving carriers alternative thienopyridines such as prasugrel and ticagrelor. Several hospitals have already begun planning for on-site, point-of-care CYP2C19 genotype testing of PCI patients in anticipation of implementing one of these strategies. The relative merits of focused point-of-care testing of acute patients versus routine pretesting for CYP2C19 genotype (as well as other pharmacogenomic data) in all at-risk patients in the outpatient setting  remain to be determined.Unlike with warfarin, there have not yet been any trials assessing whether clinical decision-making informed by knowledge of KIF6 (kinesin-like family 6) gene  was identified in a small case-control study of myocardial infarction.23 Follow-up studies suggested that the variant might have value for a pharmacogenomic application. In a subset of the WOSCOPS (West of Scotland Coronary Prevention Study) trial, carriers of the KIF6 variant experienced greater protection against coronary heart disease with pravastatin therapy compared with noncarriers.24 In the CARE (Cholesterol and Recurrent Events) trial, there was a smaller difference in response to pravastatin therapy between KIF6 variant carriers and noncarriers, with the trend favoring the carriers.24 A similar trend favoring carriers was observed in the PROSPER (Prospective Study of Pravastatin in the Elderly at Risk) trial in subjects with a prior history of vascular disease.25 Finally, in the PROVE IT-TIMI 22  study, KIF6 variant carriers obtained significantly greater benefit from intensive statin therapy (atorvastatin 80 mg daily) compared with moderate statin therapy (pravastatin 40 mg daily) than did noncarriers.26Statins are among the most widely prescribed drugs in the world, used for the reduction of plasma low-density lipoprotein cholesterol levels and the prevention of cardiovascular disease. Development of pharmacogenomic tests that could predict response to statin therapy might therefore have significant clinical implications. The Trp719Arg (W719R) variant of the KIF6 variant carriers and noncarriers in their response to therapy (simvastatin vs placebo), with both groups receiving significant benefit from statin therapy.27 A similar observation was made in a study from the JUPITER  trial, where KIF6 W719R variant noncarriers experienced as much protection from statin therapy (rosuvastatin vs placebo) as did carriers.28 As has been observed for clopidogrel use in reduced-function CYP2C19 allele carriers (as discussed previously), differences in study designs, study endpoints, risk characteristics of the study populations, and the statin drugs and dosages used in the various trials may account for the conflicting findings with the KIF6 W719R variant. Another possibility is that the later, larger studies signify that the original findings with the KIF6 W719R variant were simply the play of chance.Published subsequent to the meeting of the Working Group, an analysis of the HPS (Heart Protection Study) clinical trial in subjects with a history of coronary or peripheral vascular disease found no difference between SLCO1B1, which encodes a hepatic uptake transporter, has been associated with a 17-fold increased risk of myopathy with high-dose (80 mg/d) simvastatin (see later); the finding was replicated albeit with lower odds ratios at a dose of 40 mg/d.29 Thus, the SLCO1B1 variant could potentially be used to identify susceptible individuals in whom to avoid the use of high-dose simvastatin; the recent US Food and Drug Administration relabeling of the drug  should also reduce population risk.A single variant in Besides the 3 areas addressed previously—anticoagulation, antiplatelet therapy, and lipid-lowering therapy—the Working Group noted additional areas in cardiovascular medicine for which pharmacogenomics could potentially be of significant clinical impact, including treatment of hypertension, treatment of heart failure, and prediction of the development of cardiomyopathy with chemotherapeutic drugs.31 blood pressure,33 and coronary artery disease.35 GWASs have also been successfully been performed on medication responses, for example, the finding of the statin myopathy—associated SLCO1B1 variant described earlier.29 A GWAS on the response of platelet aggregation to clopidogrel therapy identified variants near the CYP2C19 gene,36 the same gene known to harbor reduced-function alleles that affect clinical responses to the drug, as described previously; conditioning the analysis on the CYP2C19*2 allele eliminated the GWAS signal, indicating that this variant drives the result. In both of these cases, the GWASs pointed to genes already functionally linked to the drugs of interest. It can be anticipated that future GWASs on medication responses will implicate novel genes with unclear function, understanding of which might ultimately lead to new pharmacogenomic applications and therapeutic strategies. Accordingly, investment in GWASs and, in particular, follow-up studies on GWAS discoveries—identifying causative genes, DNA variants, and biological mechanisms using cellular and organismal model systems, systems biology approaches, integration with bioinformatics databases, targeted candidate gene resequencing, and other methodologies—is warranted.The Working Group also recognized that recent advances in human genetics have made it possible to identify many DNA variants with potential pharmacogenomic relevance. During the past few years, genome-wide association studies (GWASs) have been enormously successful in identifying variants associated with cardiovascular phenotypes, including blood lipid levels,The Working Group noted that the gold standard for pharmacogenomic applications  is a prospective trial with treatment determined by genotype and with a clinical endpoint as the primary outcome, but it also recognized that it would be very difficult to sustain and fund a large number of such trials, and that in some cases the weight of evidence from observational studies may be so compelling as to render prospective trials unnecessary. Thus, there is a need to carefully choose which pharmacogenomic applications warrant funding of definitive prospective randomized trials. This would be best served by convening expert panel meetings or conferences to periodically evaluate the latest evidence for several cardiovascular pharmacogenomic clinical applications and recommend whether to provide funding for large clinical trials. The Working Group also acknowledged that large trials will not always be practical or optimal to test many potential pharmacogenomic applications, highlighting the need for innovative clinical trial designs to test pharmacogenomic strategies. One such example is provided by the Medco-Mayo Warfarin Effectiveness Study warfarin trial, as described previously. Another example is to compare outcomes between community practice groups cluster randomized to pharmacogenomic strategies and groups that maintain the usual practices. To promote innovation in clinical trial design, the Working Group envisions expert panel meetings or conferences with the express purpose of devising clinical trial designs specifically suited to the testing of pharmacogenomic strategies.CYP2C19 and KIF6 W719R variants, as described previously, highlight the need for a substantial evidence base comprising numerous studies in order to define whether a DNA variant truly predicts a clinical outcome and, if so, which patient populations are most likely to benefit from a proposed pharmacogenomic application using the variant. Recognizing the need for validation studies for many future pharmacogenomic applications, the Working Group discussed means by which to promote the inclusion and utilization of DNA collections in as many clinical trials and clinical populations—that is, large cohorts with close surveillance—as possible.The examples of the reduced-function One mechanism would be for funding agencies to underwrite the costs of storage of sample collections from clinical trials as well as large healthcare systems  in which electronic record-keeping and close clinical follow-up occur, which would empower future large-scale pharmacogenomic observational studies. Another mechanism would entail funding agencies requiring DNA banking as a condition for future clinical studies to receive funding; an alternative would be for proposed clinical studies that include DNA banking to receive priority over clinical studies that do not. A third mechanism would be for funding agencies to provide extra funding for studies using DNA collections through existing ancillary studies programs. Finally, it could be helpful to convene an expert panel meeting or conference in which participants would seek to achieve consensus on the optimal use of DNA collections in pharmacogenomics research.Finally, the Working Group recognized that, in the interest of developing a pipeline of new pharmacogenomic applications, funding would be needed for small-to-medium size proof-of-concept clinical studies, numbering several dozen to several hundred participants, to validate preliminary pharmacogenomics findings from basic science studies or post-hoc analyses of clinical trials. This would help to screen out false-positive findings before much effort and resources are devoted to larger clinical studies.The Working Group identified barriers to implementation, such as the need for fast delivery of pharmacogenomic data in the point-of-care setting in order to enable clinical decisions, the reliance on clinical workflow rules to streamline a busy clinical schedule, and lack of utilization even when providers are aware that pharmacogenomics may be helpful. Pharmacogenomic guidelines, reference databases, and electronic platforms to integrate personal pharmacogenomic data into the clinical workflow would all be helpful in overcoming these barriers. Innovative strategies are needed to promote the widespread adoption of pharmacogenomic clinical applications by cardiovascular providers.CYP2C19 alleles and, if not, would be prompted to consider ordering a genotyping test. Extending this model, if the computerized provider order entry were linked to the patient's medical record and found that the patient was known to have reduced-function CYP2C19 alleles, the system would prompt the provider to consider using an alternative antiplatelet medication. Thus, preemptive genotyping—placing data in computerized provider order entry—enabled electronic medical records—is another strategy that needs further evaluation.One such strategy would be the use of information technology to integrate pharmacogenomics into the clinical workflow, which would follow a “learning by doing” model. For example, a provider using a computerized provider order entry system to prescribe clopidogrel for a patient would be asked whether the patient is known to have reduced-function http://www.ncbi.nlm.nih.gov/omim), that would curate functional and pharmacogenomic data as well as evaluate the evidence related to specific DNA variants. The Pharmacogenomics Knowledge Base , initially founded as part of the National Institutes of Health's Pharmacogenomics Research Network, includes as a mission to serve as a central repository from which pharmacogenomic guidelines, reference databases, and electronic platforms could all draw. The Pharmacogenomics Research Network and PharmGKB have also organized the Clinical Pharmacogenomics Implementation Consortium37 to provide specific evaluations of the strength of evidence for specific drug-gene variant relations that might support their implementation in practice; this represents another critical need in the field.More generally, information delivery would be facilitated by the development of a publicly available database, modeled on the highly regarded Online Mendelian Inheritance in Man database , it is to be expected that inclusion of pharmacogenomics recommendations into clinical guidelines would, in time, result in broad adoption of pharmacogenomic practices.Another priority should be the establishment of training programs in pharmacogenomics—incorporating formal training in clinical pharmacology, genetics, biostatistics, informatics, epidemiology, and clinical trial design—that could be supported by mechanisms such as the National Institutes of Health T32 type grants. This would serve 2 objectives: to promote pharmacogenomics research by developing investigators with the optimal set of skills needed to perform the research, and to create a cohort of experts widely distributed across many institutions who could serve as local leaders to educate others in the use of pharmacogenomic applications.Finally, efforts should be made to promote the training of established clinicians as well as clinical trainees in the principles of genetics and pharmacogenomics—the former through continuing medical education activities, the latter through curricula introduced in residency and fellowship programs.The Working Group recognized that pharmacogenomic testing would add costs that are easy to assess but would also yield cost savings, clinical benefits, and other types of “value” that are harder to quantify. The Working Group noted that there are no established models to use in this relatively new field, requiring the recruitment of expertise to develop such models. This process could be initiated by convening expert panel meetings or conferences aimed at achieving consensus on the “best practices” for the use of biostatistics and cost-effectiveness analyses in pharmacogenomic studies. These models will be essential to persuade various stakeholders to support the implementation of specific pharmacogenomic applications and to ensure that resources and efforts in clinical development of pharmacogenetic tests  are appropriately allocated.CYP2C9 and VKORC1 genotype-guided dosing of warfarin found that, with a cost of $400 and a 3-day delay for genotyping, the marginal cost-effectiveness would be $170 000 per quality-adjusted life-year; with a cost of $200 and a 24-hour turnaround time, the marginal cost-effectiveness would fall to $50 000 per quality-adjusted life-year, and genotyping would be cost-saving if the cost fell below $40 per test.8 Thus, with cost-effectiveness improving as the costs associated with pharmacogenomic tests fall, it will be critical to develop inexpensive, rapid, and extremely accurate genotyping and targeted resequencing assays for a variety of clinical applications. To date, the dramatic decrease in genotyping and sequencing costs over the past 15 years—such that the much-heralded “$1000 genome” is likely to become reality within a few years—has largely been driven by high-volume research applications  funded by public agencies like the National Institutes of Health and charitable foundations like the Wellcome Trust. It can be expected that similar sources of funding will be needed to stimulate the development of technology for low-cost pharmacogenomic tests that are appropriate for widespread, routine clinical use or for preemptive testing coupled to sophisticated electronic medical record systems.One analysis of The Working Group identified a set of research and policy priorities designed to facilitate the development and adoption of cardiovascular pharmacogenomics in patient care. High-priority research programs and infrastructure needs identified include the following:Research projects targeted to high-need areas, including the following:Small-to-medium size proof-of-concept clinical studies  to validate preliminary pharmacogenomics findings from basic science studies or post-hoc analyses of clinical trials.Studies to reap the benefit of GWAS locus discovery by identifying causative genes, DNA variants, and biological mechanisms using cellular and organismal model systems, systems biology approaches, integration with bioinformatics databases, targeted candidate gene resequencing, and other methodologies.The development of inexpensive, rapid, and extremely accurate genotyping and targeted resequencing assays for clinical use, acknowledging that current methods are geared toward research applications rather than clinical use.Training programs for young investigators as well as established investigators to develop skills in cardiovascular pharmacogenomics. Such programs could include formal training in clinical pharmacology, genetics, biostatistics, informatics, epidemiology, clinical trial design, and health outcomes and cost-effectiveness research.Promotion of the inclusion and utilization of DNA collections in clinical trials and for clinical populations for pharmacogenomics analyses by:Providing funding for storage of sample collections.Requiring or incentivizing DNA banking for funded clinical studies.Providing funding for studies using DNA collections via the ancillary studies program.Expert panel meetings or conferences to:CYP2C19 genotypes to guide clopidogrel therapy.Periodically evaluate the latest evidence for several cardiovascular pharmacogenomic clinical applications and recommend whether to provide funding for large clinical trials. An example of a clinical application that is a high priority for evaluation is the use of Achieve consensus on the optimal use of DNA collections in pharmacogenomics research.Propose innovative clinical trial designs to test pharmacogenomic strategies, acknowledging that large prospective randomized clinical trials will not always be practical or optimal to test many potential pharmacogenomic applications. An example is to compare outcomes between community practice groups cluster randomized to pharmacogenomic strategies and groups that maintain the usual practices.Achieve consensus on the “best practices” for the use of biostatistics and cost-effectiveness analyses in pharmacogenomic studies.Propose innovative strategies to promote the widespread adoption of pharmacogenomic clinical applications by cardiovascular providers. An example is the use of information technology to educate providers and to integrate pharmacogenomics into the clinical workflow.Inclusion of cardiovascular pharmacogenomics experts on guidelines committees to facilitate the inclusion of pharmacogenomics recommendations in clinical guidelines. Such committees include the following:American College of Cardiology/American Heart Association Task Force on Practice Guidelines .National Cholesterol Education Program .Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure .Engagement with the US Food and Drug Administration and other regulatory agencies to develop guidance documents on the types of evidence required to gain approval of diagnostic biomarkers and pharmacogenomically targeted therapies.


FH), a tumor suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumors. Loss of FH enzymatic activity results in accumulation of intracellular fumarate which has been proposed to act as a competitive inhibitor of 2-oxoglutarate-dependent oxygenases including the hypoxia-inducible factor (HIF) hydroxylases, thus activating oncogenic HIF pathways. Interestingly, our studies have questioned the role of HIF and have highlighted other candidate mechanisms, in particular the non-enzymatic modification of cysteine residues (succination) that could lead to disruption or loss of protein functions, dysfunctional cell metabolism and cell signaling. Here, we discuss the evidence for proposing fumarate as an onco-metabolite.The drive to understand how altered cellular metabolism and cancer are linked has caused a paradigm shift in the focus of cancer research. The discovery of a mutated metabolic enzyme, isocitrate dehydrogenase 1, that leads to accumulation of the oncometabolite 2-hydroxyglutarate, provided significant direct evidence that dysfunctional metabolism plays an important role in oncogenesis. Striking parallels exist with the Krebs cycle enzyme fumarate hydratase ( Cancer cells exhibit characteristic “hallmarks” of malignancy including increased proliferation, survival, and in particular dysregulated metabolism . There iR(-)-2-hydroxyglutarate ((R)-2HG), the reduced form of 2-oxoglutarate (2OG). (R)-2HG is a by-product produced by gain-of-function mutations of IDH1 and IDH2, which normally catalyze the reversible NADP+-dependent oxidative-decarboxylation of isocitrate to produce 2OG in the cytoplasm and mitochondria, respectively (R)-2HG accumulation caused by oncogenic IDH mutations enhances DNA methylation and epigenetic remodeling, which stalls cell differentiation and thereby primes cells for malignancy (The term oncometabolite has only recently been coined and assigned with confidence to ectively . IDH mutectively . 2HG actectively . In gliolignancy .R)-2HG as an example, one could propose that an oncometabolite is a small molecule component (or enantiomer) of normal metabolism whose accumulation causes metabolic dysregulation and consequently primes cells allowing future progression to cancer. There are likely to be numerous and complex interacting steps in this process including inhibition, disruption or activation of pathways each of which will require detailed investigation. Nevertheless, the concept of oncometabolites is novel and exciting and offers a real and innovative route into therapies for a variety of cancers. Here we will discuss evidence implicating fumarate as an oncometabolite in FH-deficient cells. Furthermore, we will highlight where these studies have provided useful insights into cell metabolism.How should we define an oncometabolite? Using . To address this question we have used a conditional Fh1 (the ortholog of human FH) knockout mouse model , and Fh1-deficient MEFs in which there is stable re-expression of either full length, mitochondrial-targeted FH (Fh1-/- + FH), or cytoplasmic FH , although the defect in aerobic metabolism is not corrected  inhibit the activity or function of other members of the 2OG oxygenase superfamily, including histone demethylase enzymes (HDMs) and TET proteins which are critical in epigenetic regulation of gene expression . DespiteS-(2-succinyl) cysteine (2SC), a process termed succination (+ ratio, leading to feedback inhibition of oxidative phosphorylation and accumulation of mitochondrial intermediates including fumarate, which in turn causes protein succination (In addition to its role as an allosteric regulator of 2OG-dependent oxygenases, fumarate is also an endogenous electrophile and reacts spontaneously with cysteine residues in proteins by a Michael addition reaction to form cination . Accumulcination . Mechanicination . Targetscination .FH inactivation and an increase in 2SC proteins, which is absent in non-HLRCC tumors and normal tissue controls and has provided a potentially robust diagnostic biomarker for FH-deficiency in cells and tissues (NRF2 mutations and inactivating KEAP1 mutations are prevalent in many cancer types (Compared to the situation in diabetes, protein succination is predictably more severe in FH-deficiency due to the significantly higher levels of fumarate accumulation. Immunohistochemical analysis of FH-deficient tumors and cysts has shown a striking direct relationship between  tissues . We hypo tissues . NRF2 co tissues . In the  tissues . KEAP1 h tissues . This al tissues . The acter types  and oncoer types . NRF2 maSuccination may result in the disrupted function of multiple proteins and offers a unique mechanism by which fumarate may lead to dysregulated cellular metabolism and act as an oncometabolite. Clearly screens need to be undertaken to identify other candidate succination targets which have cysteine residues critical for their function and are associated with oncogenic signaling or metabolic pathways.in vitro and in vivo and in combination with metabolite and transcriptome profiling. Clearly, however, the adaptive response to fumarate accumulation of FH-deficient cells through alterations of primary metabolism may contribute to oncogenic transformation. Glycolysis, glutaminolysis, anaplerosis and the urea cycle may all be relevant for FH-deficiency and a greater understanding of these and their inter-relationships in normal and dysregulated cell metabolism are vital.The Krebs cycle dysfunction caused by loss of FH activity poses significant challenges to cells in meeting energy requirements, in the generation of macromolecular precursors and in survival. Studies, in part contradictory, using a number of cellular models, have identified a variety of mechanisms by which FH-deficient cells may deal with these problems. Impaired respiration and upregulation of aerobic glycolysis have been observed in FH-deficient cell lines and tissues, presumably as an adaptation to meet cellular energy requirements by producing ATP independently of the TCA cycle . ElevateDespite evidence for fumarate as an oncometabolite, in other circumstances this metabolite has been shown to exhibit cytoprotective roles. For example, it has been reported that in yeast cytoplasmic FH translocates to the nucleus following DNA damage. There it activates the damage response to double strand breaks, a process that can be complemented by high concentrations of fumarate in the absence of FH enzymatic activity. Elevated nuclear FH has also been detected in HeLa cells following irradiation damage suggesting that human cytosolic FH may have a similar function . AdditioThe shift in focus of cancer research to one of trying to understand how altered cellular metabolism and cancer are linked has highlighted how woefully ignorant we are about the complexities and interrelationships of cellular metabolic pathways and how these are altered under conditions of a variety of stress agents. However, studies into rare genetic disorders associated with metabolism are beginning to provide real insights into the adaptive responses of cells and dysregulated metabolism associated with cancer.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.


An extraordinary progress has been made over the last two decades on understanding the components and mechanisms governing plant innate immunity. After detection of a pathogen, effective plant resistance depends on the activation of a complex signaling network integrated by small signaling molecules and hormonal pathways, and the balance of these hormone systems determines resistance to particular pathogens. The discovery of new components of hormonal signaling pathways, including plant nuclear hormone receptors, is providing a picture of complex crosstalk and induced hormonal changes that modulate disease and resistance through several protein families that perceive hormones within the nucleus and lead to massive gene induction responses often achieved by de-repression. This review highlights recent advances in our understanding of positive and negative regulators of these hormones signaling pathways that are crucial regulatory targets of hormonal crosstalk in disease and defense. We focus on the most recent discoveries on the jasmonate and salicylate pathway components that explain their crosstalk with other hormonal pathways in the nucleus. We discuss how these components fine-tune defense responses to build a robust plant immune system against a great number of different microbes and, finally, we summarize recent discoveries on specific nuclear hormonal manipulation by microbes which exemplify the ingenious ways by which pathogens can take control over the plant’s hormone signaling network to promote disease. In nature, plants live in complex environments in which they intimately interact with a broad range of microbial pathogens with different lifestyles and infection strategies. To defend themselves against all these different types of pathogens, plants have evolved sophisticated strategies to perceive their attacker and to translate this perception into an effective immune response. Two tiers of recognition by the innate immune system have been defined . The firvice versa  in phytohormone signaling . In factHere, we review the most recent and outstanding examples regarding hormonal crosstalk at the molecular level, focusing on the jasmonate and salicylate pathways and how the newly identified nuclear components fine-tune defense responses to build a robust plant immune system against a great number of different microbes. We also describe some of the best characterized molecular examples of specific nuclear hormonal manipulation by microbes, which exemplify the ingenious ways by which pathogens can take control over the plant’s hormone signaling network to suppress host immunity.Plant immunity strongly relies on two plant mutually antagonistic hormones, JA and SA . Both hoArabidopsis  TFs MYC2, MYC3, and MYC4 . Fernandsyringae , indicatsyringae . HoweverPseudomonas syringae  genes  3, 6, and 7  genes . PR geneactivity . NPR1 exactivity . Howeveractivity . NPR1 mo6, and 7 . In the 6, and 7 . Strikin6, and 7 . Thus, iThe activation of plant defenses implies allocation and ecological costs . For exaArabidopsis, mitogen activated protein kinase 4 (MPK4) acts as a negative regulator of SA signaling and positive regulator of JA signaling  and ORA59 , including GRX480 and several others of the ROXY class . These pnd ORA59 . For exand ORA59  and has  pathway , indicatAnother important regulatory component is the SA master regulator NPR1 itself, which interacts with TGA TFs that are involved in the activation of SA-responsive PR genes . NuclearErysiphe cichoracearum but repressed the expression of JA-responsive marker gene PDF1.2 and compromised resistance to the necrotrophic pathogen Alternaria brassicicola . The ERF branch is regulated by members of the AP2/ERF family of TFs, such as ERF1 and ORA59 , a gene encoding an isochorismate synthase required for SA biosynthesis , RGA (REPRESSOR OF GA1-3), RGL1 (RGA-like1), RGL2, and RGL3  serve as CK receptors , a mimic of the bioactive jasmonate hormone JA-Ile, by some strains of syringae . COR, assyringae . This lesyringae , indicatsyringae , facilitsyringae , 2008 an pathway . Interes pathway . This inPseudomonas effector AvrPtoB stimulates ABA biosynthesis and ABA responses, which in turn antagonize SA biosynthesis and SA-mediated defenses  metabolism  provides a paradigm to explain the rapid and appropriate level of defense responses to specific signals. Moreover, key hormones regulating growth, such as GA and auxins, are involved in the orchestration of the plant immune response suggesting that developmental and defense signaling networks are closely interconnected. This would explain why activation of costly defenses is often accompanied by significant growth inhibition. Thus, hormone homeostasis and cross-talk seem to be a dominant feature to maximize defenses and to fine-tune growth and protection. Not surprisingly, pathogens and beneficial microbes have learned the necessity of manipulation of plant hormonal pathways to rewire the immune signaling circuitry for their own benefit. Indeed, the importance of hormones in plant immunity is highlighted by the increasing number of pathogens that are predicted to produce phytohormones or phytohormone mimics, and the recent findings indicating that microbial effectors also targets hormonal pathways to promote disease or establish beneficial interactions . Due to 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.


Intriguingly, hairy2 represses the mesoderm transcription factor brachyury (bra) throughout its domain in the marginal zone. Here we show that in early gastrula, bra and hairy2 are expressed in complementary domains. Overexpression of bra repressed hairy2. Interference of bra function with a dominant-negative construct expanded the hairy2 domain and, like hairy2 overexpression, promoted ectopic expression of dorsal axial markers in the ventral side and induced secondary axes without head and notochord. Hairy2 depletion rescued the ectopic dorsal development induced by interference of bra function. We concluded that an intact bra function is necessary to exclude hairy2 expression from the non-organiser field, to impede the ectopic specification of dorsal axial fates and the appearance of incomplete secondary axes. This evidence supports a previously unrecognised role for bra in maintaining the dorsal fates inhibited in the ventral marginal zone, preventing the appearance of trunk duplications.We have previously shown that the member of the HES family  Brachyury (bra) is highly conserved in terms of sequences and expression patterns between vertebrates bra. The allotetraploid genome in Xenopus laevis resulted from the hybridisation of two similar but nonidentical ones. Thus, this species contains the homeologs  Xlbra-a and Xlbra-b , and in addition, the separate gene bra3Xlbra-a (bra).The Brachyury or T mutation was first described in 1927 and the mouse gene was isolated in 1990. Notochord differentiation was severely impaired in homozygous mutant embryos, which lacked mesoderm posterior to the 7th somite bra was characterised as a transcriptional activator of mesoderm specific genes Bra is one of the first genes known to directly respond to mesodermal inducers and is only activated at intermediate Activin concentrations, which induce posterolateral and chordal mesoderm, while the high ones induce dorsoanterior organiser mesoderm but are unable to activate braThe T-box DNA-binding protein encoded by Xenopus, bra expression begins at mid-blastula transition. Although it is initially expressed in a widespread fashion, when gastrulation begins the transcripts are detected only in the marginal zone (MZ), in the prospective mesoderm. By the end of gastrulation, they only persist in the notochord and in the circumblastoporal mesoderm bra is sufficient to specify posterior mesoderm, including somites, but it requires at least the transcription factor foxA4a or the BMP antagonist noggin to promote notochord development In Xenopus with an interfering bra construct (braEnR), in which its transcription activation domain was replaced with the repressor domain of Drosophila engrailed (EnR), resulted in loss of posterior structures, indicating that bra is essential for posterior mesoderm development Bra promotes the convergence-extension movements that elongate the notochord and is necessary but not sufficient for its differentiation Loss-of-function experiments in Xenopus laevis has two homeologs of hairy2, a member of the HES family of transcription factors bra at gastrula stage hairy2a on later development were not previously studied, hairy2b overexpression was able to induce an incomplete secondary axis hairy2a increased bra expression in the gastrula organiser, and an anterior expansion of the notochordal bra domain was observed in neurulae depleted from hairy2bhairy2 regulates bra expression on the dorsal midline (DML) structures or their precursors in the organiser.Hairy2 belongs to the family of bHLH-Orange transcriptional repressors hairy2 in controlling tissue demarcation, for example, favouring the FP fate at the expense of the notochord and maintaining the identity of the anterior PM by repressing specific genes of neighbouring tissues bra target genes produce strikingly similar phenotypes to those of hairy2a overexpression, including the induction of a secondary trunk. The ectopic dorsal development induced after interfering with bra function was rescued by hairy2 depletion. Thus, we conclude that normal development requires an intact bra function to exclude the hairy2 domain from the non-organiser field in order to maintain the inhibition of dorsal axial fates in the ventral marginal zone, thus impeding the formation of spurious incomplete secondary axis.The basic body plan of the vertebrates is generated according to early events triggered by the Wnt/β-catenin pathway. After fertilization, the stabilization of the maternal β-catenin protein at the site opposite to the sperm entry point determines the future dorsal side of the embryo, where the Spemann-Mangold organizer will arise Xenopus laevis embryos were obtained using standard methods, staged and fixed with MEMFA bra and braEnR mRNAs were described elsewhere hairy2a cDNA construct fused to 6 myc-tag epitopes in pCS2+ (MT-hairy2a), kindly provided by Dave Turner, was digested with Not I and transcribed with SP6 RNA polymerase. Overexpression experiments for hairy2a were performed with MT-hairy2a mRNA. The hairy2a and hairy2b morpholino antisense oligonucleotides  used in this study were previously shown to specifically knock-down the corresponding homeolog nuc-lacZ mRNA or 10 ng of Dextran Oregon Green 488, MW 10000, anionic lysine fixable  were co-injected as tracers.All animal studies in this report followed the rules and protocols approved by the Institutional Review Board for the Care and Use of Laboratory Animals (CICUAL) in the School of Medicine, University of Buenos Aires, Argentina. Albino and wild-type hairy2 and bra, fluorescein-labeled antisense RNA probes were prepared with fluorescein-12-UTP (Amersham). We used either a mix of a hairy2-digoxigenin+bra-fluorescein probes, or a mix of a hairy2-fluorescein+bra-digoxigenin probes. After hybridisation, embryos were washed and blocked according to the standard protocol and incubated first with one of the antibodies . After revealing the first probe with either BCIP or Magenta Phos (Sigma), the corresponding antibody was removed with 0.1M glycine, pH 2.0 with 1% Tween-20 (40 minutes), followed by six 10 minutes washes in MAB buffer and 15 minutes incubation in blocking reagent before adding the second AP-conjugated antibody. In some double ISH we used a chordin probe labeled with biotin-16-UTP (Roche), as previously described The preparation of digoxigenin-labelled antisense RNA probes and the whole-mount in situ hybridisation (ISH) procedure were performed as previously described Embryos were exposed for 1 minute to UV light (254 nm), or were incubated for 1 hour with 0.1 M LiCl hairy2 and bra during the blastula-gastrula transition. Embryos were bisected in the animal-vegetal plane, and each hemisection was incubated with either a bra probe or with a hairy2 probe that recognises the transcripts of both homeologs hairy2 transcripts are present in an animal-vegetal gradient, with maximum levels in the animal hemisphere , with highest levels on the dorsal NIMZ (bra demarcates a ring of cells in the involuting MZ (IMZ) in a more vegetal location in relation to hairy2  and to the dorsal NIMZ (black arrowhead). On the other hand, the arc of lower bra expression corresponds to DML populations which are still segregating and show co-expression of both genes (white asterisk), and to cells of the posterior pre-involuted axial mesoderm, which mainly show bra transcripts (yellow arrowhead). bra in a complementary fashion to the strong hairy2 expression in the surrounding cells. This complementarity is also clearly observed in the ventral region, where the bra domain is larger than in the dorsal side , but hairy2a MO was more effective if the percentages of embryos with bra expansions were compared at the same doses  than that obtained with 5 ng of the mix containing 2.5 ng of each MO in the simultaneous knock-down (56%)  , indicathairy2 throughout the NIMZ confines bra to the IMZ, preventing its animal expansion, and also restricts bra expression in the organiser.Our results indicate that the expression of bra could affect hairy2 expression. Since the three Xenopus laevis bra genes have nearly identical expression patterns braEnR mRNA, a dominant-negative bra construct classically used to directly repress bra target genes, in which the transcription activator domain of Bra was replaced with EnR Xenopus laevis. When the EnR domain was fused to a particular T-box transcription factor, only the specific wild type protein, but not other members of the family, was able to rescue the phenotypic effect of the antimorphic construct. Moreover, embryos injected with the EnR domain alone were indistinguishable from uninjected controls Next, we wondered whether braEnR mRNA in 1 cell at the 4-cell stage and analysed hairy2 expression at gastrula and neural plate stages. In gastrulae, braEnR increased hairy2 transcripts and produced a vegetalward expansion of its domain, so that hairy2+ cells invaded the ring otherwise corresponding to bra in the MZ , either in ventro-lateral locations after ventral injections . At this stage, ventral injections of braEnR resulted in strong ectopic expression of hairy2 in ventro-lateral locations, including the circumblastoporal collar , and we previously showed that hairy2a overexpression induces ventral expression of DML markers braEnR. Remarkably, ventrolateral injections of braEnR mRNA induced ectopic chd, foxA4a and not I in the VLMZ in similar proportions than those obtained with hairy2a mRNA  or hairy2b overexpression bra target genes were repressed with braEnR or in which hairy2 was activated secrete a signal that, in turn, induces chd in neighbouring cells. While in the ventral side, both braEnR and hairy2 overexpression were able to induce ectopic chd expression, on the dorsal side, chd was repressed, and this occurred in the same cells where bra was blocked or in which hairy2 was overexpresed , in agreement with previous results of hairy2a or hairy2b overexpression It was interesting to notice that in the ventral region of embryos injected with bra function or overexpression of hairy2a activate the dorsal marker chd in a ventral context but repress it in a dorsal one. UV-irradiation of fertilised eggs destabilises maternal β-catenin, producing embryos with ventral character. LiCl stabilises β-catenin, resulting in completely dorsalised embryos We made use of UV-irradiation or LiCl-treatment to confirm whether impairing of Chd was drastically down-regulated in UV-treated, uninjected embryos . The most anterior marker otx2 was absent, indicating the lack of forebrain  was detected in the secondary axes , when they have already segregated from the chordal population braEnR or hairy2a mRNAs induced ectopic gsc in the VLMZ at stage 11  chd was detected in the ventral side, albeit in much fewer cells than in the endogenous center, ectopic Xnr3 was rarely and faintly detected  at the 16-cell stage.It was reported that ventral injections of hairy2a mRNA into the CD4 blastomere at the 16-cell stage  showed high levels of apoptosis (TUNEL+++) in the posterior region  hairy2a and braEnR mRNAs injections result in striking similar phenotypes; b) braEnR potently induces ectopic hairy2 expression; c) the knock-down of both hairy2 homeologs significantly rescues the ectopic expression of the dorsal marker chd and contributes to restore the normal development of embryos in which bra function was interfered.The following line of evidence supports that bra have been mainly focused on posterior mesoderm development, but there is also evidence for the involvement of bra in delimiting contiguous territories during Xenopus embryogenesis. For example, among the two subpopulations of the axial mesoderm, bra inhibits the active cell migration behaviour of the PM and promotes the convergent-extension movements of the notochordal cells bra is expressed in the animal half of the IMZ and restricts the expression of the ventral mesoderm inducer Xnr2  to the vegetal half bra (ntl) disfavours medial FP development but promotes the notochordal fate bra is also necessary to prevent the specification of DML fates within the non-organiser mesoderm field by precluding the vegetal expansion of the hairy2 domain into the ventrolateral IMZ, thus impeding the appearance of spurious, incomplete axes .All together, these results indicate that Xenopus embryos, where a group of ventral-posterior cells, in which bra target genes were repressed, were able to interact with a context with an intact bra function. The frog embryo offers the advantage to perform a more spatially controlled manipulation, and we could only obtain stronger and distinguishable secondary axes when we performed more localised injections of the antimorphic Bra in ventral-posterior cells.It is worthwhile noting that in mouse chimeric embryos in which embryonic stem cells mutant for the T-gene were injected into wild type blastocyst hosts, the posterior neuropore was unable to close, the mutant cells remained in the surface of the distant tail, and small lumps of tissue on the posterior region, tails with a branch or divided tails, or even a duplicated tail containing somites were observed bra is achieved through the release from repression in those regions where it is required rather than through specific activation bra promoter and restrict bra expression in two aspects: a) SIP1 prevents its expansion to the other germ layers (ectoderm and endoderm) bra to the chordamesoderm within the population of DML cells segregating during gastrulation There is some evidence that the definitive spatial pattern of bra, since proteins that recognise the SIP1 binding sites seem to be functionally relevant only at the beginning of gastrulation, and bra was down-regulated in animal cap cells treated with high Activin concentrations even when gsc was inhibited hairy2a suggested that this gene is necessary to restrict bra expression within DML precursors hairy2 homeolog, concluding that hairy2 participates in both aspects of bra confinement. Within the MZ, hairy2 restricts bra to the prospective mesoderm layer in the IMZ, by preventing its expansion toward the prospective ectoderm in the NIMZ.However, other factors might contribute to confine hairy2a seems to be more relevant than hairy2b, as the comparison of the morphants and the simultaneous knock-down suggest  were similar  to those obtained after injections in one ventral cell at the 4-cell stage. Notably, when analysed at stage 28, chd+ cells were observed in the secondary axes in a lower proportion of the injected embryos and were detected in lower levels in comparison to earlier stages  resulted in ectopic expression of the PM marker gsc at stage 11. Although at this stage we probably still observe a co-ectopic expression of gsc and chd in the VLMZ corresponding to DML precursors that have not yet completely segregated, they might later be unable to adopt a notochordal fate but preferentially choose the other alternative fates of the DML populations, i.e., the PM or the FP, accounting for the lower levels of chd+ cells and the lower proportion of secondary axis with chd+ cells in the injected embryos. This hypothesis is supported by the following evidence: a) the secondary axes that we obtained have a widened FP (no tail (ntl), the homolog of bra, lack notochord but they contain a wider FP, indicating that bra antagonises the development of the FP in favour of the notochord chd is normally expressed in the notochord and in the PM hairy2a- or braEnR-injected embryos. Therefore, the scattered chd+ cells that they contain might have migrated haphazardly, according to the active migration behaviour of the PM, which was shown to be induced by gscgsc expression indicates that they were able to specify cells with PM characteristics.It was previously shown that, among the precursors of the embryonic DML in the Spemann’s organiser, dened FP . This ishairy2a or with braEnR mRNA into one ventral cell at the 4-cell stage, we observed high levels of apoptosis surrounding the unclosed blastopore, where the ectopic chd+ cells remained arrested. Although embryos injected into the CD4 blastomere showed significantly lower levels of apoptosis in the posterior region, a considerable proportion of them revealed moderate levels of TUNEL staining, when sibling controls presented low or none apoptotic cells in the same region (chd in the secondary axes, indicating that some chd+ cells occasionally migrate anteriorly and survive in these conditions.In addition, in embryos injected with e region . Therefosiamois and twin are able to induce secondary axes containing trunk and head. Both are the earliest genes known to be zygotically activated in the BCNE center, and in turn, they induce other BCNE or organiser factors like chd, noggin, foxA4a and gscchd and gsc, induce incomplete secondary axis similar to the ones we obtained braEnR or hairy2a mRNAs at gastrula stage, when the expression of chd is normally restricted to the organiser and becomes independent of β-catenin signalling chd was very faintly induced, and neither injection consistently induced ectopic Xnr3, which was proposed as a direct target of β-catenin hairy2 expression braEnR strongly activated hairy2 and besides, promoted chd expression in embryos depleted of β-catenin activity by UV treatments.Among the genes directly activated by maternal β-catenin, only bra is involved in a later zygotic checkpoint, down-stream of the earliest transcription event triggered by maternal β-catenin. Thus, in early embryogenesis, there are two critical times in which dorsal development must be repressed in the ventral side: one, under maternal control, which prevents the accumulation of nuclear β-catenin bra, through the repression of hairy2. It is likely that the mechanism by which bra prevents ectopic axis development extends beyond gastrulation, since we have occasionally observed secondary tail-like structures. In Xenopus, the tail develops from secondary neurulation as a continuation of events triggered during gastrulation in the late blastopore, and bra expression persists in the tailbud All this evidence suggests that braEnR- or hairy2-injected embryos ectopically express cer, and still they fail to develop head structures in the secondary axes. Since these injections were unable to consistently induce ectopic BMP inhibitors at stage 9, it is tempting to speculate that the ventral ectoderm in our experiments was incompetent to respond to Cer signalling.The complete development of the head requires the early expression of BMP inhibitors in the BCNE center, which predispose the prospective neuroectoderm to form the brain, but it also requires vertical signals from the anterior endoderm during gastrulation, among which Cer is found. Notably, Cer induces secondary heads which do not contain any trunk-tail axial structures such as somites or notochord braEnR injections, hairy2 overexpression induced programmed cell death at neurula stage in the posterior region. Apoptosis is an evolutionary conserved physiological mechanism fundamental to developmental processes, since it sculpts or deletes structures, controls cell number, and eliminates abnormal, misplaced, or nonfunctional cells hairy2 promotes cell survival in NCC progenitors, where it is normally expressed hairy2, bra prevents the activation of dorsal genes in ectopic regions, guaranteeing the survival of cells with ventral-posterior fates. However, if dorsal mis-specified cells happen to escape cell death, they are able to organise an incomplete secondary axis.Similarly to braEnR or hairy2Fetus in fetus and parasitic conjoined twins are human pathologies that consist in a defective fetus attached externally, or located within a relatively normal fetus, respectively. The worldwide incidence of the former is about 1∶500,000 births, while the latter occurs with an incidence of 1∶100,000 births. DNA and chromosomal analysis indicates that the host and the defective fetus are genetically identical. The classical fusion theory, based on the pattern of the anomalies but not on embryological models of their pathogenesis, proposes that these pathologies result from the secondary union of two monozygotic embryonic discs


The HIV-1 genome is subject to great variability. On the one hand, there are recognized pressures that target the virus and resulting in escape and adaptation. On the other hand, there is a requirement for sequence conservation because of functional and structural constraints. Mapping the sites of positive selective pressure on the viral genome generates a reference for understanding the limits to viral escape, and can serve as a template for the discovery of sites of genetic conflict with known or unknown host proteins.To build a thorough evolutionary, functional and structural map of the HIV-1 genome, full subtype B sequences were obtained from the Los Alamos database. We mapped sites under positive selective pressure (using the SLAC method in HYPHY), amino acid conservation, protein and RNA structure [gp120, where they were enriched for sites under positive selective pressure. Similarly, CD4 T cell epitopes were enriched for sites under positive selection genome-wide, but these epitopes were significantly more conserved in gag. Thus, the various constraints and selective pressures do not act evenly across the genome.Globally, 33% of amino acid positions were found to be variable and 12% of the genome was under positive selection. Because interrelated constraining and diversifying forces shape the viral genome, we included the variables from both classes of pressure in a multivariate model to predict conservation or positive selection: molecular structures (structured RNA and α-helix domains) predicted conservation while CD4 T cell and antibody epitopes were associated with positive selection. Gene-specific analyses showed instances of departure from the genome-wide estimates. For example, CD8 T cell epitopes were generally well conserved, except in Because the global map of the viral genome also identifies positive selected sites that are not in canonical CD8 T cell, CD4 T cell or antibody epitopes, it identifies a class of residues that may be targeted by other host selective pressures, such as innate immunity effectors. As an example, sequence adaptation has been observed in the viral capsid in rhesus macaques upon cross-species transmission of SIVsm, due to selective pressure provided by restrictive TRIMS alleles. In addition to informing the combined analysis of host and viral genetic information, the results of this study may therefore help reveal novel mechanisms of antiretroviral response.