Evolution has led to diversification of all living organisms from a common ancestor. Consequently, all living organisms use a common method to duplicate their genetic information and thus pass on their inherited traits to their offspring. To duplicate chromosomal DNA, double-stranded DNA must first be unwound by helicase, which is loaded to replication origins and activated during the DNA replication initiation step. In this review, we discuss the common features of, and differences in, replicative helicases between prokaryotes and eukaryotes. All organisms must copy their genetic information faithfully to their offspring. The copy process, called chromosomal DNA replication, is very sophisticated and is regulated strictly within every single cell. Differences in cell structures and organization of genomic DNA between prokaryotic and eukaryotic cells suggest that their DNA replication processes must also exhibit differences, although more mechanisms that are common may also be shared.Chromosomal DNA replication initiates from origins in a chromosome that an initiator specifically recognizes. Other replisome components are recruited to the initiator-associated origins in a coordinated manner. The latter step includes loading of the replicative DNA helicases onto the origin region. DNA helicases are enzymes required for both DNA replication initiation and elongation steps, where they catalyze the unwinding of duplex DNA strands and translocate along the bound DNA strands using the energy provided by ATP hydrolysis, thereby providing DNA templates for DNA polymerases to synthesize new strands . The keyIn this review, we focus on the initiation step of DNA replication, especially loading of replicative DNA helicase onto origins and discuss its diversity as well as general features in various organisms.Primase synthesizes primer RNA, and DNA polymerases extend the DNA strand from the primer on a single-stranded template DNA. To form single-stranded DNA, helicase uses the energy from ATP hydrolysis to break the hydrogen bonds that connect two single strands together in duplex oligonucleotides. The replicative helicases specialize in DNA replication events where they unwind the double helix to provide single strands as the templates for primase and DNA polymerase to synthesize new DNA strands. Replicative helicases are ubiquitous, evolutionarily conserved proteins. Their loading and activation at chromosomal replication origins are the most important events in replication initiation. The replicative helicases perform activities such as binding to DNA strands, ATP-binding and ATP hydrolysis activities, double-strand unwinding and translocation in a specified direction coupled with ATP hydrolysis. Although there are some sequence differences, the unwinding activity of the helicase catalytic core is not sequence specific.Escherichia coli DnaB homohexamer. The N-terminal domain of the DnaB protein acts in primase binding, and the C-terminal domain with Walker A and B motifs, and arginine finger inside engages with ATP and associates with the DnaC helicase loader  and the DnaA-binding region  1–467) has four domains: I (aa 1–86), II (aa 87–134), III (aa 135–373) and IV (aa 374–467)  (+ (ATPases associated with diverse cellular activities) ATPase sequence  . Domain itiation . Domain on (oriC . Other p E. coli  (Kurokaw E. coli .Saccharomyces cerevisiae (in vivo (see below)  and DnaI that have been suggested to participate in helicase loading at the replication initiation , MCM2–7 seals the gap and changes the configuration to the notched state, but only for a small percentage. This suggests more factors are involved in the sealing and activation process. Subsequently, Cdc45 and GINS together seal off the open ring with a discontinuity between Mcm2 and Mcm5 of MCM hexamer, thereby reinforcing a more planar configuration with a large interior channel . Further study will uncover the mechanism responsible for this simultaneous activation.Why do eukaryotes need complicated steps to activate helicase at the initiation of DNA replication? There are several explanations. First, these steps may regulate replication of local chromosomal DNA. Huge chromosomal DNA spreads throughout the nucleus and origins fire at different times during the S phase. As origins bind to ORC ubiquitously, helicase loading and its activation are regulated in the S phase. In prokaryotes, the level of ATP (ATP–DnaA) affects the association between the initiator protein and origin. However, it is not suitable for the regulation of the local initiation of chromosomal DNA in the eukaryotic multiple-origin system because of the free diffusion of intracellular ATP. Protein phosphorylation, instead, may occur locally because protein kinase can be localized by its affinity to chromatin proteins. Thus, phosphorylation-dependent reactions may regulate helicase loading and subsequent origin firing on chromosomes locally. Second, eukaryotes regulate the initiation of chromosomal DNA replication strictly to keep genome integrity in the cell cycle. The pre-RC is formed from late M to G1 phase in the absence of high CDK activity. Subsequent activation of CDK promotes formation of the CMG complex and inhibits formation of the pre-RC . This meE. coli DnaB and B. subtilis DnaC assemble into homohexameric rings, each with a central channel that is sufficient for inducing their activities. These helicases unwind DNA progressively in the 5′–3′ direction. Interestingly, MCM2–7 heterohexamer serves only as a catalytic core and requires Cdc45 and GINS to exert its full activity. The activated helicase CMG exerts unwinding activity in the 3′–5′ direction. These differences underline the diversity in the detailed mechanisms and strategies that various species have adapted in the processes that duplicate their genomes. Recent advances in research have identified more variations in functional orthologues in sequences or structures between species. At present, we do not know much about DNA replication and replication factors in plant cells. Further investigation of DNA replication in various organisms will identify the ultimate and integral similarity.ATP binding and hydrolysis seem to be the common processes used to regulate bacterial replicative helicase loading and activation. Eukaryotes use finer regulatory systems, including control of stepwise DDK and CDK activities, in addition to ATP binding and hydrolysis. Compared with bacterial helicase, in which unwound origin DNA is loaded directly and is followed immediately by its activation, the eukaryotic replicative helicases take a detour. The catalytic unit MCM2–7 first loads onto double-stranded DNA without helicase activity and then switches to tracking along single-stranded DNA, which coincides with the active CMG formation. Bacterial helicases including


Helical ventricular myocardial band (HVMB) is global three-dimensional anatomical model which defines principal, cumulative vectors, integrating tissue architecture and net forces developed within the ventricular mass. Objective of this study is to demonstrate functional macroscopic anatomy of the interventricular septum (IVS) and to emphasize its clinical relevance.Three bovine and two porcine hearts ware prepared according to Torrent-Guasp’s technique. Special dissection technique was undertaken with non- toothed forceps, scalpel and scissors. Blunt dissection, using finger peeling technique was applied to identify predominant direction of the linear (fiber) and laminar (layer) fiber pathways within the myocardial mass.IVS displays significant fiber disarray at the boundaries of LV and RV free walls, and contains an intriguing structure that may be freshly examined by the HVMB dissection. These dissections contradict the concept that the interventricular septum belongs to the LV, since both ventricles participate in its formation. Ascending and descending segments od the HVMB provide the origin and significance of mayor “septal fiber crossing”. Conventional low resolution ultrasound imaging of the ventricular septum previously identified the border of this crossing as hyper-echogenic “septal line”. We suspect the overlap of the crossing of descending and ascending segments creates this “bright line”. Histological analyses have shown that septal RV and LV fibers create a connective tissue true space. Coronary artery septal branches run through this space, a fact, which may be useful in Ross’ procedure.IVS belongs to both ventricles. Anatomical dissections of the IVS and boundaries of LV and RV free walls have demonstrated significant changes in principal fiber orientation, explaining IVS three-layered functional anatomy. The functional significance of this septal fiber organization has to be further examined.


The aim of this systematic review is to synthesize existing evidence about treatment satisfaction among clients with substance misuse and mental health co-morbidity .We examined satisfaction with treatment received, variations in satisfaction levels by type of treatment intervention and by diagnosis (i.e. DD clients vs. single diagnosis clients), and the influence of factors other than treatment type on satisfaction. Peer-reviewed studies published in English since 1970 were identified by searching electronic databases using pre-defined search strings.Across the 27 studies that met inclusion criteria, high average satisfaction scores were found. In most studies, integrated DD treatment yielded greater client satisfaction than standard treatment without explicit DD focus. In standard treatment without DD focus, DD clients tended to be less satisfied than single diagnosis clients. Whilst the evidence base on client and treatment variables related to satisfaction is small, it suggested client demographics and symptom severity to be unrelated to treatment satisfaction. However, satisfaction tended to be linked to other treatment process and outcome variables. Findings are limited in that many studies had very small sample sizes, did not use validated satisfaction instruments and may not have controlled for potential confounders. A framework for further research in this important area is discussed.High satisfaction levels with current treatment provision, especially among those in integrated treatment, should enhance therapeutic optimism among practitioners dealing with DD clients. The evidence base regarding best practice for the treatment of clients with co-occurrence of substance misuse and mental health problems  remains ambiguous. While some studies have found promising client outcomes after integrated treatment (simultaneous care for both problem areas by the same provider) -3, severWhile most DD studies evaluate treatment effectiveness in terms of improvements in clinical outcomes (i.e. severity of substance misuse and/or psychiatric symptoms), recent research has also started to focus on client perceptions of treatment. Clients' views towards their care have been commonly subsumed under the term of 'treatment satisfaction', which refers to "the extent to which a programme is perceived as having met an individual's treatment wants and needs" (p. 456) . ExaminiThe aim of this review is to synthesize existing evidence about treatment satisfaction among DD clients. The following four key questions have guided the review:1) How satisfied are DD clients with treatment they receive?2) Do satisfaction levels among DD clients differ according to whether they receive integrated or standard care?3) Do DD clients report lower treatment satisfaction levels compared to single diagnosis clients when treated in the same clinical setting?4) Do studies identify other factors related to treatment satisfaction in DD clients?We considered all quantitative studies that assessed treatment satisfaction among adult clients with co-existing drug/alcohol misuse and mental health problems, without placing restrictions on the clinical setting in which the study was carried out or the type of diagnosis procedure used. Studies were excluded if sample sizes were smaller than N = 10, treatment provision comprised self-help groups only or was limited to a single treatment session. Furthermore, studies had to provide basic information about the satisfaction assessment used and to report results of DD clients' satisfaction ratings separately from any other groups that may also have been investigated. The electronic databases PsycInfo, Medline, Academic Search Premier and ProQuest were searched using pre-defined search strings to identify studies published in English-language peer-reviewed journals between January 1970 and October 2010  were screened based on study titles by the first author. Studies were excluded if titles indicated that the focus was on populations with other co-morbidities  or non-treatment contexts  and seven studies compared such ratings by type of treatment intervention provided (research question 2). Only three studies could be found that investigated whether or not satisfaction ratings differ among clients with or without DD problems when treated in the same setting (research question 3). Nine studies reported testing for links between additional factors (e.g. client demographics) and treatment satisfaction in DD clients (research question 4). Of the 27 studies, two represented updates or extensions of earlier studies conducted in the UK ,29 and tSample sizes ranged from 17 to 2,729 clients  assessed clients' treatment satisfaction at a single point in time only  with standard care models without specific DD focus ,41,44,52The earliest of these seven studies compared satisfaction ratings of 42 male clients treated in a residential integrated programme with 93 clients receiving residential addiction treatment only . ParticiThe same author was part of a research team that replicated the above-mentioned study in Honduras . Here, 4In a large multi-site study  1,415 feSimilar findings were shown by a smaller recent study , which cThe other three studies that compared satisfaction levels between DD clients who were provided with either integrated or standard care were conducted in outpatient treatment settings. The most recent study took place in the UK and had the advantage of using two different satisfaction measures  or suffering from mental illness only (n = 68) . Several4 or suffSeveral studies reported investigating associations between satisfaction, client and treatment-related factors in their DD samples. The selection of test variables  differed across studies hence complicating direct comparisons. Studies that examined client socio-demographics found no link between gender, age, education, employment, marital status or ethnicity and treatment satisfaction ,34,41,54A number of studies examined associations between satisfaction and other treatment-related variables. One study found a weak but significant positive relationship between greater satisfaction levels, clients' own outcome ratings  and case managers' evaluations of clients' progress  . AnotherNevertheless, the results above are partially supported by a well-controlled study  which foMoving from treatment process to outcome variables, one study demonstrated the positive effect of client satisfaction on clinical outcomes, including reduced substance misuse problems and psychiatric symptom severity at both 1- and 5-year follow-ups, after controlling for a range of potential confounders .Over the last four decades, 27 studies meeting our inclusion criteria could be identified that examined treatment satisfaction in DD clients. This review shows that most DD clients report being satisfied with their treatment experience, reflected by average ratings close to the "satisfied" end of the scales used. This applied regardless of the differences in study location , treatment settings and types of interventions delivered. When comparing satisfaction ratings of dual and single diagnosis clients treated in the same setting , a large and well-designed study found that DD clients were significantly less satisfied than single diagnosis clients . Two smaIf replicated in future studies, a finding that DD clients are less satisfied with standard  treatment than single diagnosis clients would support the common understanding that disease-specific treatment is inadequate to address the complex needs of the DD population. An integrated treatment model is usually favoured in discussions about which approach is the most beneficial for co-morbid clients e.g. ,58. The Nine studies investigated which factors - other than treatment type - are associated with satisfaction among DD clients. Studies that examined client pre-treatment factors  found no association with satisfaction ratings. In contrast, a number of treatment process and service-related variables were identified that appeared linked to satisfaction . In some studies though, it remained unclear whether or not potential confounders were taken into account, which needs to be addressed in future studies. Moreover, it would be important to examine the effect of variables that have been found to be associated with treatment satisfaction among single diagnosis samples in the past  ,59-65.In terms of rigor, the 27 studies were diverse, and some had important methodological shortcomings. Only 13 studies used standardised measures to assess treatment satisfaction, and while the selected instruments have shown acceptable psychometric properties when used with single diagnosis treatment populations e.g. ,55,66,67Secondly, studies were restricted in their examination of potential confounders of satisfaction ratings. Only five studies reported explicitly that they controlled for any links between client characteristics and satisfaction levels ,36,41,54A third methodological difficulty concerns possible time-in-treatment effects on satisfaction ratings. In most of the reviewed studies, clients were at different treatment stages when satisfaction was assessed, with only ten studies taking the length of treatment exposure into account. Two of these reported client satisfaction at different treatment stages, with one showing stable high ratings throughout  and the The current review has highlighted some important gaps in our knowledge of treatment satisfaction among DD clients such as the influence of practitioner characteristics and treatment process variables as well as the effect of client satisfaction on different treatment outcomes. Clients' subjective evaluations have been recognised in both mental health and addiction treatment populations as key indicators of treatment quality and effectiveness e.g. ,21,36,69A limitation of the current review is that no meta-analysis could be carried out. A quantitative synthesis of data could have taken into account small sample sizes and moderate - if not significant - effects thus providing further insight into the current evidence base. Depending on the growth of studies in this field, future reviews should include such analyses where possible.Our review shows that dually diagnosed clients are, on the whole, satisfied with current treatment provision, despite the common notion that individuals with co-morbidity are the most difficult-to-treat clients e.g. . IntegraThe authors declare that they have no competing interests.SJS carried out the literature search, examined all records obtained, interpreted the data and drafted the manuscript. PSM assisted in the literature search and made substantial contributions to the evaluation of selected articles and manuscript draft. JS was involved in revising the draft in several stages. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/64/prepubStudies on dual diagnosis clients and treatment satisfaction that were excluded after full-text retrieval. Shows full citations for those studies that did not meet the review's eligibility criteriaClick here for fileOverview of studies assessing treatment satisfaction among dually diagnosed clients. Shows key characteristics of all studies included in the reviewClick here for file


Patients with advanced or metastatic non-small cell lung cancer (NSCLC) can develop acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. Here, we report the successful treatment with alternating chemotherapy and TKIs of two cases of advanced NSCLC who developed resistance to TKI.Two patients with advanced or metastatic NSCLC were treated with palliative chemotherapy followed by erlotinib/gefitinib. When TKI therapy failed, two cycles of chemotherapy were provided, which were followed by re-challenge with erlotinib or gefitinib.NSCLC patients with acquired TKI resistance should be managed aggressively whenever possible. Subsequent chemotherapy and target treatment is one of the reasonable choices for those with an initial dramatic clinical response with erlotinib/gefitinib treatment. Further studies are warranted to substantiate the association of erlotinib /gefitinib treatment with the efficacy of NSCLC patients with acquired TKI failure. Lung cancer is a leading cause of cancer-related death throughout the world and its death toll reached 7.4 million or approximately 13% of all deaths worldwide in 2004. Early diagnosis of lung cancer is difficult due to the insidious nature of symptoms and most patients have progressed to an advanced stage at the time of diagnosis. Multiple studies have clearly shown that chemotherapy is important in the palliative care of advanced non-small cell lung cancer (NSCLC) even compared with the best supportive treatment. Chemotherapy as the main treatment approach for advanced lung cancer can improve patient survival and their quality of life [Erlotinib and gefitinib, as oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), are commonly used as second- or third-line drugs ,3, and aIn June 2007, a 70-year-old non-smoking female was seen at our hospital because of a 3-month history of progressive dysponea at rest. CT scan revealed a mass at the right lower lobe and moderate pleural effusion on the right side. Metastasis to right adrenal gland was observed on abdominal CT. There was no evidence of extra thoracic metastasis on brain MRI and bone ECT scans. Lung needle aspiration revealed adenocarcinoma and the patient was diagnosed with NSCLC stage IV. She started 4 cycles of systemic chemotherapy with cisplatin and gemcitabine in Jul 2007. CT scan showed a stable disease of the carcinoma and marked clinical improvement was noted as dyspnoea disappeared and the patient reported a general feeling of wellness. The patient then received two cycles of docetaxel for maintenance therapy. In the following six months, the patient was well without any evidence of local or systemic recurrence. In June 2008 a routine follow-up bone ECT and brain MRI revealed bone and brain metastatic lesions, and the patient commenced erlotinib (150 mg daily ), which she tolerated well and only experienced grade 1 skin rash without requiring dose adjustment. After 4 weeks of erlotinib, the patient showed complete response in her intracranial disease and a partial response in her lung disease. After 12 months of erlotinib therapy, tumor at the right lower lobe progressed, and two cycles of carboplatin and paclitaxel were administered. CT imaging confirmed stable disease in the right lower lobe tumor. However, the patient refused further cytotoxic chemotherapy because of severe treatment-related diarrhea. She was re-challenged with erlotinib (150 mg daily ) in October 2009, and experienced grade 3 skin rash without dose modification. Fortunately, she improved clinically with her right lower lobe tumor showing partial response after 4 weeks of erlotinib treatment and the treatment was continued for eleven more months as third-line therapy in May 2007. She tolerated the regimen well and only experienced grade 1 diarrhea. After 4 weeks of gefitinib, the patient showed partial response in her lung disease. After 29 months of gefitinib, the patient developed brain metastases and the tumor also spread to the right lung. Brain radiotherapy (25 Gy/five fractions) was administered followed by two cycles of carboplatin and paclitaxel. Chemotherapy, however, was terminated because of severe hematological toxicity. She was re-challenged with gefitinib (250 mg daily) in Nov 2009 and experienced grade 1 skin rash and diarrhea. After 4 weeks of gefitinib, a CT scan showed a marked regression in the right lung tumor and intracranial disease, Stable disease was noted in the left lung tumor. So far, the patient continued gefitinib treatment(CT scans were shown in Figure Systemic chemotherapy has played an important role in the palliative care of advanced NSCLC. Both quantitative and qualitative benefits have been noted with platinum-based 2-drug chemotherapeutic regimens in advanced NSCLC patients with a good performance status ,7.Patients with advanced NSCLC, however, eventually relapse or become refractory to first-line treatment. Acceptable toxicity and improved quality of life are especially important for those patients in second-line therapy. Docetaxel and pemetrexed, have demonstrated efficacy and been approved as second-line therapy for patients with advanced or metastatic NSCLC -10. MeanErlotinib and gefitinib are EGFR TKIs that suppress intracellular signaling pathways, which normally promote cell growth and proliferation . They haCurrently, there is no clear evidence suggesting an optimal treatment strategy for patients with acquired TKI failure. Some trials are investigating the role of anticancer therapies in the third- or fourth-line setting after EGFR TKI failure. An irreversible TKI targeting the EGFR, HER2, and vascular endothelial growth factor receptor was evaluated in the third- and fourth-line settings after EGFR TKI failure . A smallOur current two cases highlighted good responses to erlotinib/ gefitinib, and had obtained long-term stable disease with the second or third-line therapy initially. When the patients developed acquired resistance to EGFR TKIs, two cycles of a taxane-based chemotherapy was selected, after which erlotinib/gefitinib was administered again, which yielded more than ten months progression-free survival. Restored sensitivity to EGFR-TKIs after chemotherapy may be due to the following mechanisms: first, the proportion of sensitive and resistant cells in the tumor could have been modified by the treatment as chemotherapy may have killed erlotinib/gefitinib resistant cells, and erlotinib/gefitinib sensitive cells have become dominant. Secondly, chemotherapy may induce novel genetic mutations in EGFR or other unknown associated genes that regulated resistance to TKI.Recently, some Asian cases have been described in whom chemotherapy can restore gefitinib efficacy after gefitinib resistance -18, indiBased on the data herein, it is suggested that acquired TKI resistance be managed aggressively whenever possible. Subsequent chemotherapy and target treatment are one of the reasonable choices. Since TKI resistance becomes increasingly important in the treatment of NSCLC, our results provide promising insight for future clinical practice and future investigations. Erlotinib/gefitinib could be re-considered for those NSCLC patients who had already benefited from prior erlotinib/gefitinib treatment. Further studies are warranted to substantiate the association of erlotinib /gefitinib treatment with the efficacy of NSCLC patients with acquired TKI failure.Written informed consent was obtained from the two patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.RHG and XFC analyzed the data and wrote the manuscript. TSW, ZYZ, JS made substantial contributions in data acquisition. YQS participated in study design and coordinate among the authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/90/prepub


P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1–7 nights. At 1–6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70–100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2′,3′-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating ( Since the recognition of acquired immune deficiency syndrome (AIDS), animal models have been developed to study various aspects of human immunodeficiency virus (HIV) infection, pathogenesis and control. The most common route of HIV transmission, sexual transmission , has beeOur animal model of HIV infection employs EcoHIV, chimeric HIV clones in which the gp120 coding region is replaced by the coding region of the ecotropic murine leukemia virus (MLV) envelope, switching the viral tropism from human to rodent ; all othBackgroundHuman immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), is primarily transmitted via heterosexual intercourse. Constituents of seminal fluid drive cytokine responses in the female reproductive tract that can amplify HIV infection. Moreover, levels of immunity in the female reproductive tract vary over the course of the menstrual cycle and susceptibility to virus infection is influenced by sex hormones, demonstrating the importance of the physiological environment. Although HIV infection and pathogenesis have been modeled fairly extensively in animal models, the currently available models do not allow the dynamic features of viral sexual transmission to be examined. To address this limitation, the present study describes a system for HIV transmission during mating.ResultsTo generate a system for viral sexual transmission, chimeric HIV molecular clones (EcoHIV) were constructed and used to infect rodents. The authors demonstrate that EcoHIV-infected male mice efficiently transmit the virus by mating to uninfected females. They show that infection of females can be detected by the presence of antibodies against HIV, or of HIV DNA or RNA in lymphocytes and macrophages. In addition, exposure of females to antiretroviral drugs around the time of mating prevents their infection. Finally, the authors report that female mice in estrus lose susceptibility to EcoHIV sexual transmission yet retain susceptibility to infection via injection.Implications and future directionsThe findings reported here indicate that HIV sexual transmission can be modeled by mating conventional uninfected mice to mice infected with EcoHIV. This novel approach creates the possibility of studying the requirements for sexual transmission of HIV using the extensive repertoire of available genetically engineered and mutant mice while integrating the influences of the male and female mammalian reproductive tissues on infection. Highlighting this potential, the authors demonstrate that the rate of viral transmission declines sharply during estrus, indicating that the local environment in the female reproductive tract influences viral infectivity. Most importantly, the work provides a simple, small animal platform to investigate interventions to prevent the most frequent route of HIV transmission.Given the ability to investigate HIV replication in conventional mice and the pivotal observation that male to female transmission of retroviruses in mice is almost exclusively through sexual transmission , this stgag RNA burden in the vas deferens and spleen 2 weeks after mouse infection by injection   . Seven oes (PMs) . Female es (PMs) .gag RNA was detected in all females (P<0.002) in infected, mated female mice. Thus, female mice can be infected by mating through limited exposure to infected males; they produce EcoHIV/NDK proteins and mount antiviral responses.Because mice infected by injection of EcoHIV/NL4-3 seroconvert , detecti females . At 6 we females . The antgag burden was measured in PMs (P<0.05). We then limited antiretroviral treatment to two injections and also limited female exposure to infected males to 1 night. Females were treated with tenofovir or vehicle and 12 hours later were housed with EcoHIV/NDK-infected males overnight. The next morning females received another injection and were caged separately from males. At 1 week after exposure to males, females were euthanized and virus burden was measured in PMs  in human beings can prevent HIV sexual transmission  and antid in PMs . EcoHIV d in PMs . Pericoie humans .Foxn1nu) male mice, in which T cell development is impaired  mice from 6–16 weeks of age were either purchased from the Jackson Laboratory  or bred in our mouse colony. All use of mice was approved by the St Luke’s-Roosevelt Institutional Animal Care and Use Committee (IACUC). Most experiments reported here employed C57BL/6 mice; the use of nude mice is explicitly noted in the text.Male and female C57BL/6 mice and 6 pg HIV p24, ddC or tenofovir administration by IP injection, and real-time PCR amplifying HIV gag DNA or gag or vif RNA, standardized by amplification of glyceraldehyde phosphate dehydrogenase, were as described previously (Escherichia coli and purified by nickel-NTA affinity chromatography. HIV burden in groups of mice were compared using Student’s t-test.EcoHIV/NDK construction, preparation, mouse euthanasia and tissue collection were as described previously . Plasmideviously . An effeeviously  and tenoeviously . Every aeviously , except Female mice were caged in groups of five unless otherwise indicated. At 1 week after EcoHIV/NDK infection, individual male mice were housed overnight with two uninfected female mice. Males were removed the next morning and were returned to cages for 1 or more nights as indicated in the text. Synchronization of females in estrus was achieved through the Whitten effect : females


Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell.Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes.Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.The organic matrix of  Lotgi1|, 2009]  .2 and water. The first carbonic anhydrase isolated from a mollusc shell and characterized at the molecular level was nacrein [Pinctada fucata shells, contained two carbonic anhydrase domains separated by a Gly-X-Asn repeat domain. The same protein was also identified in the prismatic layer [One of the most important enzymes in biomineralization events is carbonic anhydrase, which catalyzes the formation of hydrogen carbonate from CO nacrein . This pric layer . Since tic layer ,10,57,58Lottia shell matrix contained three entries that showed some similarity to nacreins  and only 23% to the sequence of Mytilus californianus nacrein-like protein [Lottia shell matrix contained two other predicted carbonic anhydrases apparently completely unrelated to nacreins . Lotgi1|66515 contained another predicted carbonic anhydrase, which was a moderately abundant protein in the acid-soluble matrix proteome ( In addition to nacrein-like carbonic anhydrases we identified two putative carbonic anhydrases without obvious similarity to nacrein in sequence similarity searches Table . Lotgi1|roteome  . The lacDaphnia pulex protein (E9GAB5_DAPPU). The regulatory properties of this protein in vertebrates may implicate this minor shell protein in Lottia shell production.FAM20C, also known as dentin matrix protein 4, was first detected in mouse dentin matrix  and may Lottia osteonectin was a major protein in the acid-soluble shell matrix proteome and a minor one in the acid-insoluble fraction  and the sequences were included in the sequence alignment  and the domain distribution in these sequences (B).Click here for fileSequence analysis of Pif-relatedLottiaproteins. Doc-file showing the alignment of Pif-related protein sequences to Pinctada fucata Pif (A) and the domain distribution in these sequences (B).Click here for file


Recent advances in spring arrival dates have been reported in many migratory species but the mechanism driving these advances is unknown. As population declines are most widely reported in species that are not advancing migration, there is an urgent need to identify the mechanisms facilitating and constraining these advances. Individual plasticity in timing of migration in response to changing climatic conditions is commonly proposed to drive these advances but plasticity in individual migratory timings is rarely observed. For a shorebird population that has significantly advanced migration in recent decades, we show that individual arrival dates are highly consistent between years, but that the arrival dates of new recruits to the population are significantly earlier now than in previous years. Several mechanisms could drive advances in recruit arrival, none of which require individual plasticity or rapid evolution of migration timings. In particular, advances in nest-laying dates could result in advanced recruit arrival, if benefits of early hatching facilitate early subsequent spring migration. This mechanism could also explain why arrival dates of short-distance migrants, which generally return to breeding sites earlier and have greater scope for advance laying, are advancing more rapidly than long-distance migrants. IdentifLimosa limosa islandica, have advanced significantly, at rates similar to other short-distance (within-continent) migrants [The dates of spring arrival into Iceland of a wide range of migratory bird species have advanced significantly over the last two decades and these advances have coincided with rising temperatures . Among tmigrants . Iceland2.(a)The Icelandic black-tailed godwit population numbers approximately 50 000 individuals  and, ove(b)Limosa limosa, snipe Gallinago gallinago, redshank Tringa totanus, golden plover Pluvialis apricaria, oystercatcher Haematopus ostralegus and whimbrel Numenius phaeopus) have been located through intensive surveys of study locations throughout south Iceland. The laying date of each nest is estimated by conventional egg-floating techniques [Since 2001, approximately 100 nests per season of lowland-breeding waders . Repeatability of laying dates is estimated here for females; few mate changes have occurred as godwits are strongly mate-faithful .(c)Since 1999, over 740 black-tailed godwit chicks (average per year = 57 ± 34.2 s.d.) have been caught at locations throughout Iceland, between mid-June and mid-July. Chicks are individually colour-ringed and biometrics are recorded. To assess annual variation in godwit hatch dates, mean relative chick size  of ringing) was calculated for each year, as chicks will be larger on any given day in years in which hatching is earlier.(d)www.vedur.is). Mean April temperatures at Reykjavík , a major arrival location, were used to explore the influence of temperature on spring arrival dates. Mean June temperatures at Hæll , the closest station to the breeding study locations, were used to explore the influence of temperature on nest-laying dates. In addition, values of the North Atlantic Oscillation (NAO) index were extracted for March and April (http://www.cru.uea.ac.uk/cru/data/nao/), the months prior to and during migration.Mean monthly temperature data were extracted for two weather stations of the Icelandic Meteorological Office ((e)Annual, individual/pair and temperature-related variation in arrival and laying dates  were explored using general linear models (GLMs) with normal error distributions, with year or temperature fitted as covariates and individual or pair as fixed factors. Annual variation in arrival dates of known-age individuals and in mean relative chick size were explored with GLMs with year fitted as a covariate. Repeatability of individual arrival dates between years was calculated following .3.(a)F53,230 = 6.6, p < 0.001; figure 2a). There is no significant trend in individual arrival dates over time  and individual arrival dates are not significantly earlier in warmer springs (table 1a).The timing of arrival of black-tailed godwits in Iceland has advanced significantly, as shown by the date on which the first marked individuals have been recorded at coastal arrival sites since 1999, and the date of the first godwit arrival at an inland site in Iceland since 1989 . Howeverb). Arrival dates do not appear to change with age, as three of these known-age individuals were first recorded in their second calendar year (the likely recruitment year), and the average difference between their arrival dates in this year and subsequent years was 1 day for two individuals seen in 2 years and 3 days for one individual seen in 3 years.If individuals are consistent in their migratory timings but populations are advancing, the advances must result from new recruits migrating earlier, on average, than recruits from earlier years. Arrival dates of individual godwits hatched in recent years are indeed significantly earlier than arrival dates of their predecessors b. Arriva(b)Identifying the drivers of advances in the arrival dates of recruits is key to understanding the links between changes in migration timing and population status. Several mechanisms could potentially alter recruit timings, and thus the frequency of individuals with different migratory timings, within this population.(i)F14,19 = 0.92, p = 0.56) and their laying dates have advanced significantly in recent years and are significantly earlier in warmer springs (figure 3a and table 1b). In addition, the size of chicks caught on any given date throughout Iceland has increased over the last 13 years and is greater in years with warmer mean temperatures in May , again suggesting that hatching dates are advancing (and/or that chick growth rates are increasing) and the frequency of early fledged chicks is increasing (figure 3b). If advances in timing of migration are related to advances in nest-laying dates and associated benefits of early hatching, the greatest advances in migratory timings would be expected in species with a greater capacity to advance nest-laying. Across six wader species breeding in Iceland, rates of advance in arrival dates are significantly lower for species with smaller time-gaps between arrival and laying, with the shortest arrival–laying gap occurring in the species (whimbrel) that travels furthest (to sub-Saharan Africa) and has advanced migratory timings the least  interval between arrival and laying provides an opportunity for pairs to respond to annual variation in the timing of local environmental conditions. Pairs of individually marked godwits for which nests have been located in multiple years have low repeatability of laying dates b suggests both an increase in the frequency of early arriving recruits and a decrease in the frequency of later arriving recruits , thus this mechanism would also require recruits arriving later  to be subjected to increasing mortality rates.Changes in the mortality patterns of early arriving recruits could drive population-level advances, for example if more benign weather conditions in recent years increased survival rates of early arriving recruits. However, figure 2(iii)a and table 1a), this mechanism could only operate during the year of recruitment, and arrival dates of the 46 known-age individuals are not significantly related to the NAO index in March  or April , or to mean daily temperature in Iceland during April  in their likely year of recruitment .Recruiting individuals may respond to weather conditions during migration in the year in which they recruit, resulting in greater numbers of earlier arriving recruits in warmer years. However, as individual arrival dates are highly consistent between years , and subsequent earlier arrival of recruits hatched earlier in season, could also explain the widely reported differences between short- and long-distance migrants in shifts in arrival ,5,17. Lo


Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals. Pulmonary fibrosis is a severe, chronic lung disease, which is associated with high mortality rates, afflicting more than five million patients worldwide . Its mosOver the past few years, it has become evident that animal model systems to study the pathogenesis of lung fibrosis in rodents need to be more carefully evaluated with respect to their applicability to human lung fibrosis . As an ein vivo. However, systematic micro-computed tomography-based analysis of developing lung fibrosis induced by gene transfer of biologically active transforming growth factor-β1 as compared to classical histopathological approaches to monitor fibrotic disease progression in mice using both visual assessment and semi-automated quantification algorithms has not been reported so far.Diagnostic tools to assess the degree of lung fibrosis in humans currently include lung CT scans, lung function tests and histological examinations of lung tissue biopsies. In mice, where complete lungs can be removed, particularly histological and biochemical approaches are employed to assess the degree of fibrosis ,7. In reTherefore, in the current study, pulmonary fibrosis was induced in mice by gene transfer of biologically active transforming growth factor-β1, and fibrotic disease progression was evaluated by micro-computed tomography and then correlated to histological scoring of lung fibrosis as pioneered by Ashcroft .223-225 contains the cDNA of the coding region of full-length porcine TGF-β1, while containing a mutation of cysteine to serine at positions 223 and 225, rendering expressed TGF-β1 biologically active  per mouse) suspended in a total volume of 50 μl of phosphate-buffered saline (PBS). At 1, 2, 3, and 4 weeks after adenoviral transfer, mice  were subjected to micro-computed tomography  were either infected with AdTGF-β1n detail ,15. Briein situ with PBS-buffered  formalin , and were then carefully removed and immersed in PBS-buffered formalin. Lung tissue samples were paraffin-embedded, and sections of 5 μm were prepared and stained with haematoxylin/eosin (H/E) and Masson's or Elastica-van-Gieson staining. The degree of lung fibrosis was determined according to the method described by Ashcroft and colleagues [Complete lungs were inflated lleagues , employilleagues  was calcA GE Explore Locus cone-beam micro-CT  was used for data-acquisition in prone position under isoflurane inhalation anaesthesia  with and without respiratory gating . Scanning took approximately 10 and 20 min without and with respiratory gating, respectively. Respiratory monitoring was performed using a pressure transducer pad under the animal's chest. Images were reconstructed and assessed at a constant window width/window level (5000/2000).Three radiologists blinded to the experimental groups and respiratory gating strategies, independently assessed image qualities of all individual entire micro CT scans, using a rating scale ranging from 1 (very good) to 5 (insufficient) and the amount of lung consolidations using a rating scale ranging from 1 (no consolidation) to 5 (very pronounced consolidation).Due to the diffuse nature of fibrotic consolidations, direct quantification of the pathology of lung fibrosis by measurement of focal fibrotic lesions was not feasible. Therefore, quantification of aerated lung areas known to be compromized by developing lung fibrosis was used as a surrogate marker for progressive lung fibrosis. A region-growing segmentation algorithm was applied on each individual entire micro CT scan using the MevisLab software package  . The algAll statistical computations have been performed using the statistical program SPSS 17  The significance level of all tests has been set to 0.05 (5%). Because most of the observed variables were ordinal scaled we used non-parametric tests for statistical analysis. Significance of differences in image quality values were tested for all three observers in respiratory gated and respiratory ungated exams using a Wilcoxon signed ranks test. Furthermore, significance of differences in consolidation assessment values for respiratory gated and respiratory ungated exams was tested using a Wilcoxon signed ranks test for all three observers. Differences between the assessment of the 3 observers were evaluated comparing the 95% confidence interval for the mean for assessment values of the image quality for respiratory gated/ungated exams and the assessment values of the consolidation assessment for respiratory gated/ungated exams.Correlation of the Ashcroft histology scores (assessing the degree of fibrosis) and the lung consolidation values (obtained by assessment of the images by three observers) was calculated for respiratory ungated/respiratory gated exams and all three observers using Spearman's rank correlation coefficient for ordinary scaled variables.Correlation of the Ashcroft histology scores and the lung segmentation values (obtained by the semiautomated segmentation routine described above) was calculated for respiratory ungated and respiratory gated exams using a Pearson correlation for continuous variables. To assess differences between the 4 time points for histology scores, visual consolidation assessment and segmentation of aerated lung areas 95% confidence intervals were plotted against the 4 time points and differences were tested for significance.As shown in Figure SD 2.70, MVCVF 2.33, MVTR 2.27) as compared to ungated exams of fibrosing lungs . Moreover, all three observers determined similar consolidation assessment values for respiratory gated  as compared to ungated exams .We next questioned whether detection of consolidations in fibrosing mouse lungs would differ between observers. However, initial comparative image quality assessment between three different observers revealed only slightly increased mean image quality values in respiratory gated  and ungated exams (p = 0.699).Overall, these data illustrate that visual assessment of micro-CT based lung fibrosis in mice was independent of the observer.We next compared classical Ashcroft scoring with micro-CT based evaluation of TGFβ-1 induced lung fibrosis in mice. Ashcroft histology scores and lung consolidation values showed significant correlations for all observers and respiratory gated as well as respiratory ungated exams using Spearman's rank correlation coefficient , we were not able to detect significant differences between different time points for visual consolidation assessment and segmentation of aerated lung areas. However, to better exemplify the time course of fibrosis in mice on days 7, 14, 21, and 28 post treatment, mean values and standard deviations for Ashcroft histology scores and visual assessment of all three observers for ungated exams are given .In this study we evaluated the use of micro-CT for assessment of pulmonary fibrosis induced by adenoviral gene transfer of biologically active TGF-β1 in mice. We found micro-CT imaging to be a highly valuable tool to study pulmonary fibrosis progression in mice using both visual inspection of lung images and a semi-automated segementation algorithm that estimates aerated lung areas as a surrogate marker inversely correlating with developing lung fibrosis in mice.18F-fluoro-deoxyglucose micro-PET [Micro-CT allows a detailed assessment of lung morphology due to high density differences and well defined borders between air and lung tissue. However, one limitation of this method is that the activity of inflammatory processes can only be assessed by secondary characteristics by micro-CT, such as affection of adjacent strucutre or pleural effusion. Other imaging techniques such as magnetic resonance imaging (MRI), micro-positron emission tomography (PET) and optical imaging could provide functional data, with micro-PET beeing an especially promising candidate to robustly assess inflammatory activity by increase of glucose metabolism as detected by icro-PET -19, whicImage quality is of equal importance in small animal imaging as in human imaging. However, data on imaging protocols are still not extensive. One major issue addressed in previous studies relates to respiratory gating. Various gating techniques, including prospective and retrospective as well as intrinsic and extrinsic techniques have revealed that mostly, gated imaging resulted in increased image quality ,21. In tA further important aspect of micro CT imaging of normal or fibrosing mouse lungs is the time-point in the respiratory cycle when images are acquired. Due to the typical respiratory pattern in anesthesia, with a short breath followed by a long expiratory plateau, imaging is mostly performed in expiration. Under those experimental conditions, where intratracheal intubation and ventilation is performed, micro-CT imaging can also be performed in inspiration, as a different and consistent respiratory pattern can be created . WithoutThe semi-automated segmentation routine described in this study was found to assess consolidations in a quantitative way, as proven by the significant correlation of segmentation values with Ashcroft-based histological grading. To a minor degree, observer interaction is possible, as the observer has to place its own seed points for the region growing algorithm. As seed points have to be placed carefully within aerated lung areas, inadvertent positioning of seed points within non-aerated lung areas may increase the likelihood that consolidated aereas will be included in the segmentation volume and thus may cause assessment bias, which however, according to the presented data, appears to be very low.In the current study, segmentation volumes were not correlated with total lung volume or body weight of individual mice. However, when considering that differences in expiratory total lung volumes between individual mice of the same age and strain are neglectable, it may be largely excluded that this aspect may have confounded the reported correlations. Future algorithms might take segmentation assessments of the entire lung into account, thereby allowing the correlation of measured consolidations with total lung volumes, or simply by employing a quotient taking the body weight into account.in vivo imaging of small laboratory animals.Additionally, different techniques of fibrosis assessment could be applied. A promising approach is the measurement of CT densities expressed by Hounsfield Units and assessing fibrosis not only by consolidation but also by evaluation of density distribution . MorpholIn another study we evaluated the applied radiation dose for the protocols used in this study applying phantom and cadaver thermoluminescence dosimetric measurements (own unpublished observations). The expected mean doses in an average C57BL/6 mouse for the respiratory gated and ungated protocols used in this study were 201 mGy and 194 mGy respectively. The expected mean value for an estimated time of 1 minute fluoroscopy to determine the scan field of view was 22 mGy. Day et al. reported that high radiation doses of 7 - 9 Gy did not result in significant lung fibrosis in mice after 90 days, just few microscopically small collagen-rich foci were detected subpleurally . TherefoHuman lung fibrosis differs from the animal model reported here, as it is irreversible and shows typical morphological patterns in computed tomography representing definite fibrotic changes of the lung. The application of the methods reported for imaging in human lung fibrosis therefore is not intended. We believe that the currently applied technique will primarily be helpful to better assess e.g. novel therapeutic strategies for the treatment of pulmonary fibrosis in rodent model systems.In conclusion, the current study shows that respiratory gated/ungated micro-CT allows valid in vivo evaluation of the degree of pulmonary fibrosis in mice using both visual assessment and semi-automated quantification algorithms. Micro-CT thus offers a non-invasive imaging tool for evaluation of novel therapeutic strategies for the treatment of pulmonary fibrosis in mice.The authors declare that they have no competing interests.TR, CvF and UAM designed the study. TR, RM, RH and UAM carried out the preparation of the animals and micro-CT exams. TR, CvF and SD read and assessed the imaging studies. FL performed the histopathological workup. LH performed the statistical analysis. KA, MK, JG, TW, MG and JB participated in its design and coordination and helped to draft the manuscript. TR and UAM wrote the manuscript. All authors read and approved the final manuscript.


Hepatic resection in metastatic disease from colorectal cancer offers the best chance in selected cases for long-term survival. Neoadjuvant chemotherapy (NACT) has been advocated in some cases initially deemed irresectable, with few reports of the efficacy of such a strategy and the influence of the response to chemotherapy on the outcome of radical hepatic resection.Between December 1995 and May 2005, 27 patients with colorectal liver metastases  were treated with neoadjuvant chemotherapy. A seven-year survival analysis was performed. Chemotherapy included mainly 5-fluorouracil, leucovorin and either oxaliplatin or irinotecan for a median of eight courses.A total of 16 patients (59%) had synchronous and 11 (41%) metachronous metastases. During pre-operative chemotherapy, tumour regression occurred in ten cases (37%), stable disease in a further ten patients (37%) and progressive disease developed in seven cases (26%). The five-year overall survival for NACT responders was 64% and only 15% for non-responders (p=0.044).The response to chemotherapy is likely to be a significant prognostic factor affecting survival after liver resection for cure. Surgical resection of colorectal liver metastases, where possible, still remains the only treatment ensuring long-term survival, when margin-free (R0) hepatic resection can result in 25–35% reported overall five-year survival –7. This The perioperative risks of liver resection have been substantially reduced in recent years. It is not, however, clear whether systemic pre-operative chemotherapy increases the morbidity of subsequent surgery through the induction of steatosis, sinusoidal congestion and centrilobular necrosis as has been reported with fluoropyrimidine analogues , 17 or aBetween April 1996 and February 2005, 27 consecutive patients affected by colorectal liver metastases received systemic chemotherapy prior to liver surgery . The rationale of this approach relies on the assumption that occult micro-metastases may be present and that neoadjuvant chemotherapy is likely to improve the performance of a radical (R0) liver resection and that metastases initially deemed irresectable may be rendered operable for resection with curative intent.The relevant clinical data and tumour characteristics are shown in The objectives in medical management were different according to the initial resectability of the metastases. For unresectable patients, chemotherapy was the only means to convert cases into a resectable state and was utilized for longer. For resectable patients, the first objective of the chemotherapy was to provide a time interval before surgery for assessment of the tumour biology, to treat potentially occult disease and to avoid surgery in those patients with rapidly progressive disease as a result of primary resistance to chemotherapy. A second objective in these resectable patients was to achieve cytoreduction both to limit the extent of liver resection and potentially post-operative morbidity as well as to facilitate a margin-free R0 liver resection. The median number of cycles of pre-operative chemotherapy per patient was eight (range: 2–12). In ten patients, systemic chemotherapy was continued post-operatively for a median of six cycles.The response to chemotherapy was evaluated from serial imaging studies (thoraco- and abdomino-pelvic CT scan or MRI scanning of the abdomen where indicated) and was based on the change in tumour diameter according to the World Health Organisation criteria . ResponsPatients were eligible for hepatic resection when the following conditions were met, namely: (1) no co-morbid conditions were present precluding liver resection, (2) all malignant liver disease was amenable to resection and ablative treatment whilst being able to retain at least 30% of non-tumoural liver parenchyma, (3) recurrence of the primary tumour was excluded and (4) either no non-resectable extrahepatic disease was detected by pre-operative serial imaging studies, or where potentially resectable extrahepatic tumour was detected, it was not considered a contraindication to sequential or synchronous surgery. The time interval between the final chemotherapy dose and hepatic surgery was usually 2–4 weeks to minimize the risk of tumour progression and to reduce perioperative morbidity. This approach was adopted in accordance with other similar reports , 25. ThePatients were followed up one month after surgery and then every four months thereafter with evaluation of tumour markers , liver function tests and by hepatic ultrasound. An abdomino-pelvic or thoraco-abdominal CT scan was performed every six months during the follow-up. In the case of resectable extrahepatic metastasis(es), sites were resected 203 months following definitive hepatic surgery, using systemic chemotherapy between operations in order to prevent tumour progression.Overall and disease-free survival probabilities were determined by the Kaplan-Meier analysis  and compLiver resection was performed following an objective tumour response in ten patients (37%), after stabilization in a further ten patients (37%) and after tumour progression in seven patients (26%). Major liver resections (≥ 2 segments). were all performed with curative intent and included two right hepatectomies, one left hepatectomy, one extended right hepatectomy, four bisegmentectomies, two bisegmentectomies plus one segmetectomy and two bisegmentectomies plus a wedge resection. For two patients (3%), liver resection was combined with a gastrointestinal resection (namely an anterior resection of the rectum). Perioperative mortality was nil (30 days following surgery). Eighteen patients (67%) were discharged without complications with one patient (3%) undergoing a right hepatectomy having mild post-operative reversible liver failure. Among the minor complications in nine patients (33%), there were three pleural effusions, two abdominal collections, two wound infections and two central venous catheter (CVC) infections. All patients underwent hepatic resections with curative intent (R0 resections) with no evidence of microscopic involvement of the surgical margin. The patient and tumour characteristics according to response to pre-operative chemotherapy regimens utilized are shown in Following a mean follow-up of 36 months (range: 8–110 months), 18 patients suffered from recurrence (67%) amongst the 27 patients. Liver recurrence was isolated in eight patients (30%) and associated with extrahepatic recurrence in four cases (15%). Of the 12 patients with hepatic recurrence, two underwent a repeat hepatectomy. Of the 25 patients initially free of extrahepatic disease, ten subsequently developed extrahepatic recurrences (37%). Of these latter four patients underwent one or more re-operations for extrahepatic recurrence.The overall survival (OS) of the 27 patients was 93%, 57% and 34% at one, three and five years, respectively, with a median survival of 30 months . At lastOn univariate analysis, the only significant factor positively predicting survival was the response to neoadjuvant chemotherapy . This effect is shown graphically in This small study shows that liver resection combined with pre- and post-operative (sandwich) chemotherapy offers the possibility of long-term survival of patients with chemoresponsive liver metastases  from colorectal cancer. This benefit can be obtained only when the disease confined to the liver is controlled by chemotherapy prior to completely resectional surgery. Tumour progression whilst on pre-operative chemotherapy is associated with a poor outcome, even when hepatectomy is performed with curative intent.This neoadjuvant approach has been reported to be associated with prolonged OS by others where it has been used for potentially resectable liver lesions, with demonstration of progression-free survival advantage over unresected cases . It is, Available data would suggest that initial perioperative morbidity following hepatic resection appears to adversely affect the longer-term cancer-specific survival  but thatIt should be recognized that complete chemo-responsiveness on imaging may frequently be associated both with macroscopic and microscopic evidence of residual disease that may account for the relatively high incidence of hepatic in-situ recurrence found in our study as well as by others . Tumour In this study, the number of nodules was not a contraindication to resectional surgery. The five-year survival following such R0 liver resection was 34% in our cases: data that compare favourably with other surgical series reporting three-year and five-year survival of 21%  and 23%,In conclusion, the response to neoadjuvant chemotherapy plays a key role in the potential benefit offered by radical liver resection in selected patients with hepatic metastatic disease from colorectal cancer. The poor results obtained by surgery in patients with tumour progression suggest that control of the disease with a modern combination regimen is preferable to immediate surgery. This delayed approach awaiting initial chemo-responsiveness might also prove to be relatively liver sparing and better defines those patients who are potentially advantaged by a formal hepatic resection as opposed to a tailored segmentectomy. Moreover, in those cases where larger metastases completely respond in one area of the liver, resection of the contralateral lobe may become an operative option . This viOverall, the data concerning neoadjuvant benefit to radical hepatectomy in colorectal hepatic metastatic disease are hard to interpret. This is as a result of variable histopathology in the liver, differing chemotherapy schedules, varying types of liver resections and different ischaemia/reperfusion cycling techniques during hepatic parenchymal transection. In this setting, it is not surprising that patients with multiple metastases tend to have more prolonged chemotherapy exposure, more extended resections and larger tumour/remnant liver ratios. Resections for hepatic colorectal metastases are increasing in incidence  and it i


Endoscopic retrograde cholangiography (ERCP) is widely used as a first-line therapy for biliary drainage. ERCP occasionally fails owing to anatomical or technical problems, despite high reported success rates. Endoscopic ultrasound-guided biliary drainage (EUS-BD) has recently emerged as an effective alternative biliary drainage method after unsuccessful ERCP. EUS-BD can be essentially divided into 3 different techniques—(1) EUS-guided transluminal biliary drainage including choledocoduodenostomy and hepaticogastrostomy, (2) EUS-rendezvous technique, and (3) EUS-antegrade approach. Here, we focus on the current status of EUS-BD in light of these 3 different techniques. Endoscopic retrograde cholangiopancreatography (ERCP) is widely used in current medical practice as a biliary drainage method for biliary obstruction. ERCP occasionally fails owing to anatomical or technical problems such as upper intestinal obstruction, surgically altered anatomy, periampullary diverticulum, or periampullary tumor infiltration, despite a >90 % success rate in most reports. Percutaneous transhepatic biliary drainage (PTBD) or surgical interventions are conventionally performed as alternative biliary drainage methods after unsuccessful ERCP. However, both PTBD and surgical interventions are associated with considerable morbidity and mortality –3.Endoscopic ultrasound-guided biliary drainage (EUS-BD) was first reported in 2001 by Giovannini et al. . FollowiIn EUS-transluminal biliary drainage, the biliary duct is accessed under EUS guidance followed by guidewire placement and fistula dilation. A stent is then deployed between the biliary duct and intestine to create a permanent fistula for biliary drainage. This procedure can be performed in patients with either endoscopically accessible or inaccessible papilla; however, its indication should be limited in cases of unresectable malignant biliary obstruction, given the feature of permanent fistula creation.For EUS-transluminal biliary drainage, the biliary duct is punctured from the upper intestine under EUS guidance followed by cholangiography to ensure proper puncture of the biliary duct and delineate its configuration. A guidewire is then placed into the biliary system and dilation of the needle tract is performed. In this step, a fine needle aspiration (FNA) needle, needle knife, or fistulotome can be used for bile duct puncture with fistula creation performed using a bougie dilator, needle knife, balloon, stent retriever, or diathermic sheath over the guidewire. Finally, a stent is deployed at the fistula between the biliary duct and the intestine for biliary drainage. In terms of access route, this technique is divided further into HGS, in which the fistula is made between the stomach and intrahepatic bile duct (IHBD) of the left lobe, and CDS, in which the fistula is created between the duodenal bulb D1) and extrahepatic bile duct (EHBD)  FNA needle can be used for EUS-RV. A 19-G needle allows a guidewire of up to 0.035 inches to pass through the needle, whereas a 22-G needle only allows a 0.018-inch guidewire to pass through. The guidewire is then manipulated into the duodenum via the obstruction and the ampulla. After the needle is withdrawn inside the outer sheath, the EUS scope and needle are removed while maintaining the guidewire in place. The duodenoscope is reinserted along with the EUS-placed guidewire to the ampulla, where the EUS-placed guidewire exits from the biliary orifice. Biliary cannulation is reattempted along with the guidewire. As another means of obtaining biliary cannulation, the distal end of the guidewire is grasped with forceps or a snare and the guidewire is pulled out through the mouth with the scope or through its accessory channel. An ERCP cannula inserted over the guidewire or the duodenoscope is back-loaded over the guidewire and re-advanced to the ampulla if the guidewire is pulled out through the mouth. Finally, endoscopic biliary stenting is accomplished as planned.Published EUS-RV data are shown in Table In EUS-AG, the IHBD is accessed from the upper intestine with creation of a temporary fistula between the intestine and IHBD. After dilation of the fistula, stent placement or balloon dilation are performed for biliary obstruction through the fistula without the endoscope reaching the ampulla. This technique is suitable for biliary obstruction in patients with surgically altered anatomy or upper intestinal obstruction, in which reaching the biliary orifice endoscopically is impossible or cumbersome.After careful examination of the left lobe of the liver using color Doppler to detect any interposing vessels, the IHBD is punctured from the intestine using an FNA needle primed with contrast agent. Correct biliary puncture is confirmed with bile aspiration and cholangiography through the needle. A guidewire is then inserted into the biliary system through the needle followed by removal of the needle and dilation of the fistula using a bougie dilator over the guidewire. The guidewire is then manipulated into the intestine through the ampulla or anastomosis, with coordinated movements of the guidewire and dilator inside the biliary system. A self-expandable metallic stent is deployed to the malignant biliary obstruction or balloon dilation is performed for a benign biliary stricture in an antegrade fashion. Finally, all devices are removed after confirmation that bile flows well through the stent or stricture.Only several reports exist regarding one-step EUS-AG. The overall success and complication rates of EUS-AG are 77 and 5 %, respectively , 53–56 (All 3 EUS-BD techniques require an experienced endoscopist in both the EUS and ERCP procedures as well as the capability of EUS and fluoroscopy. Availability of alternative biliary decompression, such as PTBD or surgery, is also critical to minimize the risk of bile leakage into the peritoneal cavity in preparation for unsuccessful EUS-BD. The indication for EUS-BD should be decided carefully by taking into account the condition and needs of the patient as well as the endoscopist and facilities available.Once the decision to perform EUS-BD is made, selection of situation-specific EUS-BD techniques should be made, although each EUS-BD technique has overlapping indications and there is no guideline for selection of techniques. Khashab et al.  comparedGiven the results of previous studies regarding EUS-BD, our proposed treatment algorithm using EUS-BD after unsuccessful ERCP is shown in Fig. EUS-BD is a feasible salvage technique for unsuccessful ERCP, although further studies are needed to compare the efficacy and safety between EUS-BD and PTBD and to examine a treatment procedure using EUS-BD techniques.


Transposable elements (TEs) are a class of mobile genetic elements (MGEs) that were long regarded as junk DNA, which make up approximately 45% of the genome. Although most of these elements are rendered inactive by mutations and other gene silencing mechanisms, TEs such as long interspersed nuclear elements (LINEs) are still active and translocate within the genome. During transposition, they may create lesions in the genome, thereby acting as epigenetic modifiers. Approximately 65 disease-causing LINE insertion events have been reported thus far; however, any possible role of TEs in complex disorders is not well established. Chronic obstructive pulmonary disease (COPD) is one such complex disease that is primarily caused by cigarette smoking. Although the exact molecular mechanism underlying COPD remains unclear, oxidative stress is thought to be the main factor in the pathogenesis of COPD. In this review, we explore the potential role of oxidative stress in epigenetic activation of TEs such as LINEs and the subsequent cascade of molecular damage. Recent advancements in sequencing and computation have eased the identification of mobile elements. Therefore, a comparative study on the activity of these elements and markers for genome instability would give more insight on the relationship between MGEs and complex disorder such as COPD. Transposable elements (TEs) account for nearly half (approximately 45%) of the human genome, which is in contrast to the functional genes that constitute a smaller proportion (approximately 5%) of the human genome. Based ocis or trans preference[cis preference (ORF1p and ORF2p) act on other L1 RNAs to aid nuclear import and integration into the genome   are non-LTR elements that lack LTRs at their ends. Most LINEs belong to the LINE-1 (or L1) family and are the only TEs capable of transposing autonomously, which constitute approximately 17% of the human genome. Althougeference. ProteinGenome integrity is a crucial determinant in passing down genetic information from one generation to another. TE-associated genetic alterations such as aberrant mRNA splicing, introduction of premature stop codon and transcriptional disruptions threaten this integrity. Double-stranded breaks (DSBs) generated by TEs produce Microsatellites exhibit high mutation rate compared to point mutations, which makes them a potent regulator of gene expression. MSI, a COPD is a complex lung disorder and is the leading cause of morbidity and mortality. The 2011 WHO estimates indicate that 64 million people have COPD; moreover, COPD is reported to cause 3 million deaths worldwide, making it the fifth leading cause of death worldwide. COPD maTEs can be acquired as somatic mutations over a lifetime; presence of L1 activity in tumour cells but not in the surrounding healthy cells supports this hypothesis. PropagaThe study of heritable non-coding variations is a hot topic, particularly in cancer biology. DNA methylation is one such epigenetic regulator that plays a decisive role in developmental biology and pathobiology by processes such as X-chromosome inactivation and retrotranscription silencing. ApproxiIn recent years, there has been an interest in studying the effects of oxidative stress on epigenetic gene regulation by DNA methylation. Oxidative stress caused by oxidant/anti-oxidant imbalance plays a central role in the pathogenesis of COPD. OxidantUnder oxidative conditions, GC-rich sites are highly susceptible, and guanine with the lowest redox potential oxidizesMarked variability in the distribution of active TEs between individuals is a direct consequence of their activity in somatic tissues and low selection pressure encountered by these elements. It enables them to evolve rapidly at different sites that make their identification in the genome arduous. Over the last 2 decades, new approaches have been applied for identifying mobile elements. Earlier studies mostly used previous knowledge of mutant genes in characterizing the mobile elements by cloning and sequencing,57, whicde novo discovery and homology-based methods are briefly discussed. The algorithm for detecting inserts in de novo method usually involves reading shotgun sequence reads and matching the repeat sequences, followed by clustering the matched pairs to give a consensus sequence of a TE family[de novo sequencing method, homology-based approach uses previous knowledge of TE sequences, such as sequence similarity, in identifying similar class TEs with a low copy number. Figure A few of these methods such as E family. Unlike Research interest in SVs has increased exponentially over the past decade, and with the advent of screening technologies, approximately 5000 insertions have been reported thus far. BecauseAt least 4 principal mechanisms, inflammation, protease-anti-protease imbalance, oxidative stress and apoptosis, have been identified in the pathogenesis of COPD. Of these, the oxidative stress plays a pivotal role in COPD pathogenesis because it directly injures the respiratory tract and regulates other mechanisms.Oxidative stress elicits inflammatory response and inhibits the DNA repair system in a dose-dependent manner that may be altered at the microsatellite level, resulting in genome instability. The vast distribution and complexity of mobile genetic elements in the genome makes another strong argument in genomic instability. In addition to acting as insertional mutagens, alterations such as deletions, inversion and duplication can be attributed to the translocation of these active mobile elements. Studies on lung barrier epithelial cells have proven the effect of airway inflammation and oxidative stress on genome instability. Upon exposure to cigarette smoke, barrier epithelial cells undergo epigenetic alterations that can trigger mobile elements such as L1s, thereby influencing multiple molecular pathways that enhance inflammatory signals. Novel L1 sites can be identified by performing whole genome analysis of epithelial cell DNA from smokers (COPD), ex-smokers (no COPD) and healthy controls against a reference genome. Such new L1 insertions can be compared against the profiles of microsatellite markers in patient samples to study the relationship between mobile genetic elements and genome instability and their potential role in a complex disorder such as COPD.TEs: Transposable elements; MGEs: Mobile genetic elements; LINEs: Long interspersed nuclear elements; DSBs: Double strand breaks; HRR: Homologous recombination repair; NAHR: Nonallelic homologous recombination; MSI: Microsatellite instability; BPD: Bronchopulmonary dysplasia; COPD: Chronic obstructive pulmonary disease; FEV1: Forced expiratory volume in 1 second; FVC: Forced vital capacity; RIP: Retrotransposon insertion polymorphism; CNVs: Copy number variants.The authors declare that they have no competing interests.MW is responsible for suggestions and final revision of the draft. MS is responsible for the concept and design of the paper, preparation of the draft and take full responsibility for the final version of this manuscript. Both authors read and approved the final manuscript.


Philomachus pugnax). Ruff males show enormous variation in the coloration of ornamental feathers, used for individual recognition. This polymorphism is linked to reproductive strategies, with dark males (Independents) defending territories on leks against other Independents, whereas white morphs (Satellites) co-occupy Independent's courts without agonistic interactions. Previous work found a strong genetic component for mating strategy, but the genes involved were not identified. We present feather transcriptome data of more than 6,000 de-novo sequenced ruff genes . None of the identified genes showed significant expression divergence between males, but many genetic markers showed nucleotide differentiation between different color morphs and mating strategies. These include several feather keratin genes, splicing factors, and the Xg blood-group gene. Many of the genes with significant genetic structure between mating strategies have not yet been annotated and their functions remain to be elucidated. We also conducted in-depth investigations of 28 pre-identified coloration candidate genes. Two of these (EDNRB and TYR) were specifically expressed in black- and rust-colored males, respectively. We have demonstrated the utility of next generation transcriptome sequencing for identifying and genotyping large number of genetic markers in a non-model species without previous genomic resources, and highlight the potential of this approach for addressing the genetic basis of ecologically important variation.By next generation transcriptome sequencing, it is possible to obtain data on both nucleotide sequence variation and gene expression. We have used this approach (RNA-Seq) to investigate the genetic basis for differences in plumage coloration and mating strategies in a non-model bird species, the ruff ( Onthophagus spp.) found that patterns of gene expression in growing ornaments between two male morphs  were as divergent as they were between males and females  , for example, a mutation in the MC1R gene causes color pattern variation , a mutation in a beta-defensin gene is involved , which is also linked to dominance and mating strategies, is genetically determined through a dominant (for white) chromosomal polymorphism on autosome 2 , there is a clear genetic linkage between the gene responsible for coat color polymorphism (TYRP) and size  co-determines predominantly light versus dark coloration of the elaborate breeding plumage and male mating behavior . Like other scolopacidae, feather colorations are produced by combinations of eumelanin and phaeomelanin. Only a few genes that affect plumage pigmentation or patterning in birds have so far been identified  and microsatellites from the ruff transcriptome, markers that can be used in follow-up studies of this intriguing study species.Actively growing ornamental feathers were plucked from 11 males in a captive breeding population of ruffs maintained by DBL, and immediately placed into RNAlater , Fig. 2.Total RNA was extracted from the feather samples using the miRvana kit . The RNA was treated with TURBO DNase (Ambion) to eliminate any contamination of gDNA. Synthesis of cDNA was performed using the SMART kit , following the manufacturers' protocol and using 22 cycles for the final amplification step. Each cDNA library was individually tagged and sequenced on half a plate of a 454 Genome Sequences FLX system  at the sequencing facility in Liverpool . All raw 454-sequencing reads are available through the GenBank/SRA database under accession number SRA049313.1.A total of 350,648 sequence reads were produced from the 11 males. After trimming away bad quality, SMART primer and poly A sequence, using NGen 2.0 , 274,325 reads with a mean read length of 297 nucleotides remained see . The seqhttp://www.ensembl.org/info/data/ftp/index.html) using stand alone version 2.2.18 of blastx  that could not be annotated from the chicken protein database, we also blasted sequences against zebra finch gene predictions, human protein database, and the nr (non-redundant) sequence database using the web blast interface of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi).All contigs and singletons were annotated using a blast approach. The contig and singleton sequences were compared with the chicken protein database   from neutral expectations of the relationship between heterozygosity and FST. Positive selection was inferred from the LOSITAN analysis if the given P-value was higher than 0.975 and the estimated FST was above 0.25.Tests for genetic differentiation between groups of individuals with different color and mating strategy were performed using the “population differentiation” option in GenePop (http://www.ensembl.org/biomart) using the ClustalW algorithm  using the program MsatCommander . Note, however, that several contigs may map to different parts of the same gene. A total of 371 genes were present in the transcripts of all 11 sampled individuals, while 2286 genes were present in the transcripts of one individual only.P < 0.01) in black males compared with others (P > 0.05).The differential expression analysis revealed 22 genes that were up-regulated and 20 down-regulated (unadjusted h others a. In rush others b. Six geh others c. HoweveP = 0.0039), but did not remain significant after the false discovery rate correction in the test across all transcripts.Out of the 28 pre-identified coloration candidate genes , 11 had SNPs were identified in 822 of the 8943 contigs  or between different mating strategies . No SNPs were identified in any of the 28 pre-identified coloration candidate genes.There was no correlation between gene-wise nucleotide differentiation and expression divergence between color morphs . They identified a number of genes (mainly related to lipid metabolism and immunity) with differential expression between the two morphs. To the best of our knowledge, the only RNA-Seq study so far published that has investigated variation in sexually selected traits in a non-model organism was performed on antler growth in the Sika deer , gene expression was compared between two sequenced cDNA libraries from skin tissue. One of these was from a black-pigmented spot and the other from an adjacent yellow pigment region. Several genes downstream of the MC1R gene in the melanin synthesis pathway were identified as having higher expression in the black tissue compared with the light one . Our small sample size and the large individual variation in gene expression severely limited our power to detect small differences in gene expression between morphs, especially for lowly transcribed genes. A number of recent studies have showed significant gene expression divergence between individuals differing in ecologically important traits, such as disease resistance , future studies will be able to make better estimations of gene expression levels between and within individuals, as well as obtaining expression information from a larger number of genes. For future studies, it will also be preferable to include comparisons between samples from differentially colored feathers from the same individual to control for inter-individual effects.Several SNP markers showed significant nucleotide divergence between males of different color morphs and mating strategies. Most striking was, perhaps, the large number of hits on feather keratin genes. The keratin gene family has been well characterized in chicken  belong to non-annotated genes or sequenced cDNA clones with unknown function. These genes may be specific to birds or are too rapidly evolving to identify the mammalian homologs. Follow-up studies need to verify if these are truly important for mating strategy decisions, and if so what their molecular functions are.FST outlier analysis performed using the LOSITAN software  gave only partially overlapping results. In particular, the We also used a candidate gene approach to investigate variation in known coloration genes in more depth. Previous studies have investigated variation specifically in the MC1R gene involved in the melanin synthesis pathway , 6460 genes were identified as being expressed in skin tissue using a similar sized RNA-Seq dataset as presented here. Biological functions related to cytoskeletal structures and cell proliferation were overrepresented in genes primarily expressed in zebra finch skin . In this species, 8825 unique genes were identified and functionally annotated , which represent about a quarter of all genes annotated from the very well-studied chicken transcriptome, where data are available from a range of different tissues. Almost ten thousand of the chicken genes have been identified as being expressed in skin tissue (Chan et al. From our ruff transcriptome sequence data, we were also able to identify more than 800 SNPs and almost 200 microsatellites with flanking sequences. These molecular markers, together with the presented inference of genes important in coloration and mating strategy variation, will provide a valuable resource for further studies of ecology and genetics in this extremely interesting and ecologically well-studied species.