Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms. Drosophila melanogaster, and again in 2002 to John Sulston, Robert Horvitz and Sydney Brenner for their discoveries of ‘Genetic regulation of development and programmed cell death’ using the nematode worm Caenorhabditis elegans. These fly and worm systems remain powerful and popular models for invertebrate development studies, while zebrafish (Danio rerio), the dual frog species Xenopus laevis and Xenopus tropicalis, rat (Rattus norvegicus), and particularly mouse (Mus musculus) represent the most commonly used vertebrate model systems. To date, random or semi-random mutagenesis (‘forward genetic’) approaches have been extraordinarily successful at advancing the use of these model organisms in developmental studies. With the advent of reference genomic data, however, sequence-specific genomic engineering tools (‘reverse genetics’) enable targeted manipulation of the genome and thus allow previously untestable hypotheses of gene function to be addressed.Modern developmental biology was born out of the fruitful marriage between traditional embryology and genetics. Genetic tools, together with advanced microscopy techniques, serve as the most fundamental means for developmental biologists to elucidate the logistics and the molecular control of growth, differentiation and morphogenesis. For this reason, model organisms with sophisticated and comprehensive genetic tools have been highly favored for developmental studies. Advances made in developmental biology using these genetically amenable models have been well recognized. The Nobel prize in Physiology or Medicine was awarded in 1995 to Edward B. Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus for their discoveries on the ‘Genetic control of early structural development’ using Box 1.NHEJ (non-homologous end joining) specifies the DNA repair process where double-strand break (DSB) ends are directly ligated without the need for a homologous template. DNA ends processed via the canonical NHEJ pathway are usually protected from significant 5′-end resection by heterodimeric Ku proteins. Many, but not all, NHEJ-based DNA changes result in small insertions or deletions near the DSB cut site.HDR (homology-directed repair) is the general category of DNA repair pathways using sequence homology to repair DNA lesions. One common feature of different HDR pathways is the initial 5′-end processing of one or both DNA DSBs. Single-stranded ends generated in this manner are used to search for homologous sequences either from another site in the genome or from a foreign (donor) DNA. HDR may require multiple cellular steps, including DNA replication and other processes. Unlike classical homologous recombination (HR), HDR can use short DNA templates as donors, including single-stranded oligonucleotides. HDR is widely used to replace an endogenous locus with foreign DNA in genome engineering applications. HR is the most well studied mode of homology-directed repair, whereas HDR also includes HR-independent pathways. Canonical HR involves nucleotide sequence exchanges between two similar or identical molecules of DNA. The initiation of efficient homologous recombination requires long stretches of sequence homology between recombining DNAs. HR-independent HDR mechanisms include single-strand annealing (SSA), a process whereby two 5′-processed single-stranded ends are jointed through base-pair complementation, and break-induced replication (BIR), where one 5′-processed DSB uses its homology sequence as template to initiate DNA replication.Homology-directed repair (HDR) is the general approach of using sequence homology for genomic targeting to replace an endogenous locus with foreign DNA and encompasses homologous recombination (HR)-based as well as HR-independent mechanisms see . BesidesTraditional recombinant DNA technology enables molecular biologists to ‘cut and paste’ simpler prokaryotic DNA plasmids with great precision and superb efficiency. However, equivalent applications for studying development in multicellular eukaryotes require novel tools with more stringent sequence specificity and increased versatility, due to higher genomic complexity, as well as a critical efficiency threshold to enable routine applications. Through decades of innovation, promising designer target-specific endonucleases fulfill this important need. In this Review, we will discuss the properties and limitations of different designer endonuclease platforms for the developmental biologist, each of which can be adapted to introduce molecularly distinct site-specific modifications in eukaryotic genomes. We then explore several successful examples of their use. The principles and applications of these designer targeted endonucleases are largely applicable to nearly all genomes tested so far, although with varying efficiencies and limitations. Thus, we will end with a perspective on how these tools can bring a new era of developmental biology: greatly expanding both the depth and scope of potential research avenues, and making this a very stimulating time for the design and execution of diverse experiments to test both long-standing and new hypotheses in the field.Designer endonuclease-based genome engineering approaches involve introducing a lesion at an intended site of the genome, resulting in a deletion, insertion or replacement of genomic sequence using cellular repair pathways. The key cellular property for genome engineering is the robust recruitment of DNA repair machinery at a desired locus after a double-strand break. The goal of genome engineering is thus to produce reagents, specifically designer endonucleases, that achieve predictable, high-specificity sequence recognition with excellent efficacy. The main inspiration to overcome this hurdle came from understanding how naturally occurring sequence-specific DNA-binding proteins achieve their specificity in reading double-stranded DNA and then using these principles to generate custom enzymes. At present, three major families of designer endonucleases are commonly used: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases.The Cys2-His2 zinc-finger domain, consisting of ∼30 amino acids in a stereotypical ββα configuration, is one of the most frequently used DNA-binding motifs found in eukaryotic sequence-specific transcriptional factors. Upon binding of a zinc-finger domain to its target site, three base pairs in the major groove of the DNA are in close contact with a few amino acids on the surface of the α-helix. As these contacts mediate the sequence recognition specificity of zinc fingers, modifying the key amino acids on the ‘fingers’ can render a degree of selectivity toward a given three base-pair DNA sequence. Proteins constructed with tandem repeats of engineered, sequence-specific DNA-binding zinc fingers constituted the first successful, custom-designed platform to target DNA sequences . Fusing Xanthomonas that contain a DNA-binding domain consisting of 33-35 amino-acid modular repeats that each recognize a single DNA base pair (Transcription activator-like effector (TALE) proteins represent a molecularly unique class of transcription factors from the plant pathogenic bacteria ase pair . A simplase pair . Buildinase pair . By the ase pair .Streptococcus pyogenes are the most widely used CRISPR/Cas9 genomic engineering platform to date. Target site recognition relies on the Cas9-mediated Watson-Crick base pairing between a short stretch of CRISPR repeat RNA  and one strand of target DNA (known as the ‘protospacer’ sequence)  . This prequence) ; the preDrosophila  or can be supplied in the form of a DNA expression vector, transcribed under the control of a RNA polymerase III promoter , meiotic recombination in the germline also helps to dilute off-target lesions, thus only loci near the target sequence are likely to be refractory to genetic dilution. Modern designer nucleases have proven particularly useful in model systems in which homozygosity cannot be rapidly achieved through breeding. In these systems, efficient biallelic chromosome conversion is desirable, and both CRISPRs and TALENs have been shown to accelerate the production of homozygosity in human pluripotent stem cells , while limited reports suggest the Cas9 system is less sensitive to DNA methylation status  . As we have little manipulative capability to guide the cellular DNA repair process to achieve a given outcome, current best practice is to screen through the pool of targeted cells or organisms for a specific modification at the targeted locus. Indeed, the diversity of outcomes can be exploited in model systems, as a range of results can be clonally amplified by subsequent germline propagation. Given the high degree of conservation of DSB repair pathways in eukaryotes, the diverse genomic engineering applications are translatable between different model organisms, although it should be noted that different DNA repair pathways can be differentially favored between different cell types, even within the same organism. In the following sections, we present an overview of the main types of genome editing that are commonly being achieved via different mechanisms of DSB repair induced by custom endonucleases  mutations generated by designer nucleases. The local DNA sequence change may be reflected by RFLP (restriction fragment length polymorphism) if a restriction enzyme site is included around the target sequence. A more general approach takes advantage of local DNA sequence changes that lead to alterations in melting temperature of a PCR-based DNA fragment. Thus, HRMA   or WAVE Drosophila is so inefficient that the donor DNA cannot be directly provided as plasmids for embryonic injection due to the limitation in numbers of injections one can practically handle (Homologous recombination (HR) was the preferred methodology for generating site-specific modifications before the widespread use of designer nucleases. A problem with traditional HR has been low efficiency, thus limiting the model systems where it is feasible. For example, classical HR-mediated gene targeting in y handle . Howevery handle B. Pioneey handle . Recent y handle . Thus, iy handle . The funy handle .in vivo . Thus, small DNA elements can be introduced at a particular position in the selected locus. This could result in, for example, the insertion of a short defined stretch of amino acids into a particular site of a protein, an epitope tag fusing in frame with the targeted protein for endogenous tagging, or a recombination site enabling further site-specific engineering. Owing to the ease of making donor DNA through chemical synthesis of short oligonucleotides, such methods for creating local sequence modifications have been successfully demonstrated in many organisms . A highly efficient site-specific transgenesis method without such limitations would be greatly beneficial to developmental biologists.Two recent reports on high-efficiency site-specific gene insertions indicate that such an approach can be routinely adopted with the assistance of designer endonucleases . Both rein vivo study using zebrafish confirmed the specificity and effectiveness of designer nuclease mediated site-specific transgenesis   to generXenopus; Ciona; The availability of a wide range of designer nuclease tools with successful applications in model organisms is revolutionizing the way basic developmental processes can be studied. For example, making static knockout alleles using NHEJ is now standard practice in most developmental biology systems, something either difficult or impossible to do just a few years ago. Moreover, the arrival of versatile, robust and simple genomic engineering tools means that genetic analysis of developmental processes should no longer be confined to traditional model organisms, but can be extended to less well-established organisms, where genetic tools have been lagging behind. Beyond the ready generation of static alleles, these tools offer new options for precise and sophisticated molecular genetic manipulations. Engineering the genomic locus of interest at its endogenous site has many advantages. For example, with single base resolution of genomic editing, it is feasible to mutate specifically a crucial amino acid or to influence the choice of multiple alternative splicing events in order to study the function of a particular protein isoform, all conducted within the context of a complete genomic complex with regulatory and other control elements intact. Introducing an epitope tag at a chosen position in a protein should also be straighforward, thus circumventing the need to generate a protein-specific antibody for immunolocalization or immunoprecipitation. It would even be conceivable to epitope tag every transcription factor within the genomes of key model organisms for systematic chromatin immunoprecipitation approaches. In addition to manipulations of protein-coding regions in the genome, developmental biologists are now equipped with a feasible means with which to probe the vast regulatory genomic landscape  and to study the gene regulation network within an intact genomic context. Many developmentally regulated genes possess multiple regulatory elements, which work together to produce complex spatial and temporal expression patterns during development. Approaches using genomic engineering to examine their roles within their normal genomic context will be crucial to understand the interplay between distinct regulatory elements in controlling gene expression.Furthermore, the ease of targeted manipulation of any genomic region enables the next era beyond genetics in developmental studies, by offering avenues to monitor and manipulate the transcription or epigenetic status of individual loci  with sin


It was demonstrated that second- and third-trimester therapeutic termination of pregnancy (TOP) is feasible in cases with placenta previa. We report a 34-year-old woman with complex fetal malformations associated with placenta previa. An ultrasound examination at 21 weeks of gestation revealed fetal growth restriction (FGR) and complex fetal malformations associated with a placenta previa. After extensive information, the parents opted for careful observation. Thereafter, FGR gradually progressed and we observed arrest of end-diastolic velocity of the umbilical artery. Finally, intrauterine fetal death (IUFD) was confirmed at 33 weeks of gestation. Two days after IUFD, the patient experienced labor pain. The placenta and dead fetus weighing 961 g were vaginally delivered, and total bleeding was 270 mL. Although further studies to confirm the dynamic change of the uteroplacental blood flow are necessary to avoid the risk of maternal hemorrhage, vaginal TOP with placenta previa after feticide or IUFD would be feasible.  While attempting therapeutic termination of pregnancy (TOP), inducing labor at second or third trimester is associated with an increased risk of maternal hemorrhage in cases with placenta previa . RecentlHere we report a case with complex fetal malformations associated with placenta previa vaginally delivered at 33 weeks of gestation 2 days after intrauterine fetal death (IUFD).A 34-year-old woman  was referred to our hospital at 15 weeks of gestation for a suspicious cystic hygroma because of edema around a fetal head. We did not observe the edema or nuchal translucency on ultrasound examination. The patient was carefully followed up. However, an ultrasound examination at 21 weeks of gestation revealed complex fetal malformations  associated with a placenta previa covering the internal cervical os. Trisomy 13 syndrome was highly suspected because of holoprosencephaly, cleft lip, and so on. The oligohydramnios was caused by the trisomy 13 and also by uteroplacental insufficiency.After being provided with extensive information on the severity of the neonatal prognosis, the parents opted for careful observation but not TOP. Thereafter, FGR  gradually progressed and we observed absence of end-diastolic velocity (AEDV) of the umbilical artery. For placenta previa in a patient requiring second- and third-trimester TOP, a cesarean delivery was initially considered as the safest mode of delivery. However, several reports are available on successful vaginal uterine evacuation despite an increased risk of maternal bleeding. No apparent increase in abortion-related infections, postoperative transfusion requirements, hysterectomy, or other complications has been reported for second-trimester pregnancy (at 13–24 weeks of gestation) terminations in the presence of placenta previa . FurtherIt has been demonstrated that feticide before induction may reduce uteroplacental blood flow and thus reduce the risk of maternal hemorrhage associated with this procedure. A French group reported that of the 15 women in their study with complete placenta previa at an average of 22.4 weeks  of gestation who underwent labor induction 9 underwent labor induction without previous feticide. Of these 9 women, 4 required blood transfusions and 1 had a hemostat hysterectomy. Only 1 of the remaining 6 patients with preinduction feticide required a transfusion. They suggested that feticide before inducing labor may reduce maternal blood loss; however, they could not substantiate this with objective evidence [Thus, the Doppler ultrasound examination in cases with placenta previa feticide performed a few days before labor induction demonstrated that although a reduction in placental blood supply could be observed indirectly by an increase in resistance of the uterine arteries, it could be quantified directly using the placental vascularization index (VI) of a 3-dimensional power Doppler ultrasound. Analysis of the variation in VI after feticide indicated that a considerable reduction in placental blood supply may occur 2 days after feticide [Furthermore, contrast-enhanced ultrasound was used to quantify the dynamic changes in uteroplacental blood flow before and after the interruption of fetal villus circulation resulting from feticide during a second-trimester pregnancy termination in a patient with complete placenta previa. The results suggested that placental blood flow reduction after interruption of fetal circulation was a progressive and delayed mechanism. Their interpretation is that, initially, the interruption of the fetal circulation may lead to villous edema, resulting in a subsequent decrease in spiral artery blood flow with a possible delayed progressive evolution . This maIn our patient, FGR gradually progressed and we observed AEDV of the umbilical artery at 31 weeks of gestation. The placenta was studded with irregular high-echo regions which represented calcifications. The presence of preterm placental calcification is reported to be a predictor of poor uteroplacental flow and adverse pregnancy outcome by Viero et al. . FurtherBecause we did not perform contrast-enhanced MRI, the reduction of placental blood supply could not be clearly shown. However, infarction with hemorrhage is depicted as diffuse/circumscribed hypointense lesion and ischemic infarction without hemorrhage is demonstrated as diffuse/circumscribed hyperintense lesion in T2-weighted image . In our Thus, total maternal blood loss was estimated to be only 270 mL. Although further studies to confirm the dynamic change of the uteroplacental blood flow are necessary to achieve a well-organized delivery and to avoid the risk of maternal hemorrhage, vaginal TOP with complete placenta previa after feticide or IUFD would be feasible.


Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection  compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR. Autoantibodies directed against heart antigens are often present in patients with heart failure . StudiesIn transplantation, there is evidence that pre-transplant autoimmunity in the form of autoantibodies is associated with more rejection episodes post-transplant. Studies in humans have shown that pre-transplant autoantibodies to cardiac myosin are associated with an increased risk of cellular rejection following heart transplantation . A direcAfter transplant, both immune cells and antibodies can damage allografts, leading to rejection. In cell-mediated rejection, immune cells infiltrate and damage the allograft. Cell-mediated rejection is diagnosed by endomyocardial biopsy and is typically reversed by increasing immunosuppression. If a heart transplant recipient shows evidence of a decline in heart function, but the endomyocardial biopsy is negative for immune cell infiltration, more specialized immunohistochemical stains are performed, including detection of the complement degradation product C4d , 8. If cDetection of autoantibodies can be laborious as each autoantibody is typically measured by performing an ELISA. Since the autoantibodies may differ from patient to patient, many ELISAs need to be performed to capture the breadth of these reactivities, thus consuming a large volume of patient sample. In order to further understand the role of autoantibodies in heart failure and heart transplantation, a more comprehensive method for profiling these antibodies is needed.Antigen microarrays are a novel, high-throughput technology used for the simultaneous detection of multiple antigen-antibody interactions. In this technique, multiple antigens are spotted onto coated microscope slides using a robotic microarrayer and are probed with antibodies in patient sera, which are then detected using fluorophore-conjugated secondary antibodies . CompareHere we describe the use of a custom-made antigen microarray for the profiling of antibodies in patients both pre- and post-heart transplantation. We also describe the generation of two-color array technique that allows for the discrimination of IgG and IgM reactivities. In two small retrospective studies, we demonstrate the utility of microarrays for antibody detection in the heart transplant population and how profiling antibody reactivities using this technology leads to new insights in both the pre- and post- transplant settings.Ethics approval (08-0732-T) for this project was obtained from the research ethics board at the University Health Network. Serum samples from patients who underwent heart transplantation from June 2003 to January 2011 were obtained from the UHN histocompatibility laboratory. For the study examining pre-transplant autoantibodies, there were 8 patients in the rejector group and 16 patients in the non-rejector group. Patients in the rejector group had at least two episodes of grade 2R rejection as defined by the International Society of Heart and Lung Transplantation (ISHLT)  in the fAntigens were diluted to 0.2 mg/ml in PBS and stored in aliquots at -80°C. A complete list of all antigens arrayed is provided in the Supporting Information  were coated with ribosomal phosphoprotein P0  at a concentration of 10 μg/ml overnight at 4°C. Wells were incubated with human positive control serum with known reactivity to ribosomal phosphoprotein P0  at various dilutions in PBS containing 3% FBS and 0.05% Tween-20 followed by incubation with HRP-conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:5000. Tetramethylbenzidine substrate (Pierce) was added, and OD values were determined at 450 nm.http://robinsonlab.stanford.edu/microarrays/). Fluorescent intensities of features were quantified using an Axon 4200A microarray scanner  and Genepix 6.1 software (Molecular Devices). For each feature, median fluorescent intensity minus local background (MFI-B) on the Cy3 (532nm) and Cy5 (635nm) channels was determined. The single averaged intensity for each antigen was calculated from the features arrayed in triplicate. Significance Analysis of Microarrays (SAM) [Analysis of antigen microarrays was performed as described in online protocols (ys (SAM)  was usedys (SAM)  and heatys (SAM) .Step-wise discriminant analyses were performed to identify clinical variables  and pre-transplant autoantibody levels  that could be used in a function to classify patients as rejectors or non-rejectors. To minimize spurious entry of variables and to accommodate the small sample size , the maximum number of variables allowed to enter the functions was set at 3. Because some of the autoantibody variables displayed significant functional skewness, square root transformations were performed to reduce this property. Analyses were performed using SPSS 16.0 software .Comparison of patient characteristics between groups was performed using a two-tailed unpaired T-test  or alternatively, a non-parametric Mann-Whitney U test. Proportions of individuals with certain characteristics were compared between groups using a chi-square statistic. Correlation studies were performed by calculating Pearson correlation coefficients. A p-value of less than 0.05 was considered to be statistically significant. Analysis was performed using GraphPad Prism 6 software .For our first generation antigen microarrays, we spotted 58 antigens in triplicate. These arrays included systemic lupus erythematosus (SLE) autoantigens, which have been previously validated, along with antigens that have been implicated in cardiovascular disease. We started by conducting proof of principle studies to determine whether our antigen microarray technique would be able to detect autoantibody reactivities known to be enriched in sera of patients with SLE. We therefore probed our arrays with a positive control serum with known reactivity to the SLE antigen ribosomal phosphoprotein P0 and then probed with a single Cy3-labeled secondary antibody that recognized both human IgG and IgM as previously described , 19. FigOnce we were confident of the specificity of the arrays, we further refined the method by using two separate secondary antibodies that carried different fluorescent labels that could distinguish between human IgG and IgM. To demonstrate that the single reactivity detected by the anti-IgG/IgM secondary could be resolved into separate IgG and IgM reactivities using this secondary antibody pair, we probed the microarrays with serum that had known reactivities to the autoantigens ribosomal phosphoprotein P0 and double-stranded DNA (dsDNA) . Slides To compare the sensitivity of our technique to the current gold standard of ELISA, we measured how much serum was required to detect ribosomal phosphoprotein P0 antibodies in this patient’s sera using both the ELISA and array methods. Similar to previous reports , we founThe ability to identify heart transplant recipients at higher risk of rejection is important in developing personalized immunosuppression and biopsy monitoring protocols. Since past studies have recognized that autoantibodies are correlated with rejection episodes, we decided to first pilot our antigen microarrays in the setting of heart transplantation. We investigated whether there was a correlation between specific pre-transplant autoantibodies and rejection following heart transplantation. Patients were designated as rejectors if they had ≥ 2 episodes of ISHLT grade 2R cellular rejection in the first year after transplantation, whereas non-rejectors had no episodes of 2R rejection. All heart transplant recipients received induction therapy as part of an institutional protocol. Clinical characteristics of rejector and non-rejector heart transplant recipients are shown in Antigen microarrays were performed on pre-transplant sera from patients who went on to develop rejection (rejectors) or were free from rejection (non-rejectors) using a single secondary antibody that detects both IgG and IgM. The arrays, which had 58 antigens spotted in triplicate, detected a wide range of reactivity to multiple antigens including cardiac antigens such as cardiac myosin, troponin I, and the beta 1 adrenergic receptor . In ordeAntigen microarrays were also performed to compare antibody profile between patients in the rejector group and healthy controls in order to determine if the identified significant reactivities in the rejector group were also higher than in healthy controls. Healthy controls were sex and age matched to the patients in the rejector group. Not surprisingly antibody reactivities to heart-associated proteins myosin, troponin I, and aldolase were significantly upregulated in the rejector group as compared with the healthy controls by SAM analysis  and classified 100% of the cases . When thTo identify reactivities that distinguished heart-transplant patients with AMR in a retrospective study, we probed these arrays with serum samples taken from 12 heart transplant patients who were diagnosed with AMR and 19 non-AMR heart transplant patients. AMR patient serum was sampled at the time of AMR diagnosis, whereas the serum from non-AMR patients was taken at a similar span of time post-transplant. The clinical characteristics of these patients are shown in Microarrays were probed with patient serum to determine antibody reactivity to all 64 antigens on the arrays. The pair of Cy3-anti-IgG and Cy5-anti-IgM secondary antibodies was used to distinguish non-HLA antibody isotype. A heatmap with all patient reactivities is shown in To investigate the chronological relationship between non-HLA antibody appearance in serum and overt graft dysfunction, we profiled antibody reactivities in two patients over time. We chose the two patients based on sample availability and because they had remained negative for DSA throughout the period of observation. The second patient profiled, AMR6, had one episode of 2R cellular rejection in the early post-transplant period followed by multiple episodes of 1R rejection. During this period, the patient had preserved LVEF and low rheumatoid factor reactivity. At the time of diagnosis of AMR, the patient experienced a significant decrease in LVEF and heart biopsies demonstrated positive IgG staining but negative C4d staining. Clinical graft dysfunction was accompanied by a three-fold increase in the levels of rheumatoid factor . There wAutoantibodies are increasingly being recognized as important components of the host immune response in patients with heart failure and in heart transplant recipients. Here, we describe the generation of custom antigen microarrays to profile autoantibodies in the pre- and post-heart transplant settings. We have further refined previously described technologies to simultaneously quantify IgG and IgM reactivities for a variety of antigens including protein, peptides, and cell lysates. In a retrospective study on pre-transplant serum, we found that several antibody reactivities correlated with increased rates of cellular rejection following heart transplantation. In another study, we identified seven IgM reactivities that were higher in the heart transplant recipients with AMR compared with control recipients. Time course studies in two patients with AMR demonstrated that specific IgM non-HLA antibodies preceded the diagnosis of AMR and thus may signal early graft damage prior to overt graft dysfunction.Antigen microarrays offer several advantages over traditional ELISA assays in detecting and quantifying autoantibodies. First, antigen reactivities are profiled in multiplex fashion on the arrays, allowing for many reactivities to be defined simultaneously . Second,In the first set of experiments, we retrospectively profiled sera from heart failure patients who underwent heart transplantation and were subsequently divided into rejectors and non-rejectors. We identified eight antigen reactivities in pre-transplant sera that were upregulated in rejectors compared with the non-rejectors. Antibodies to cardiac myosin, which have been previously identified as a risk factor for rejection , were foThe other antibodies that were upregulated in pre-transplant serum from the rejectors were against ribosomal phosphoprotein P0, single-stranded DNA, Hsp27, Hsp60, collagen I, and collagen V. It is unclear if these antibodies are pathologic or if they are formed secondary to myocyte damage in the setting of heart failure. During cellular stress, heat shock proteins have been shown to be upregulated and may appear in the systemic circulation . AntibodOnce we identified which autoantibodies were elevated in the rejector group, we performed stepwise discriminant analyses to determine if these autoantibodies added information to current clinical measures for predicting rejection outcomes. First, we examined the predictive power of currently used clinical measures and found that cPRA class I followed by age were important clinical variables in identifying patients who are likely to develop rejection following heart transplantation. Previous studies have demonstrated that a high PRA correlates with increased rates of rejection following heart transplantation . FurtherMeasuring autoantibodies is not only important for predicting rates of rejection post-transplant, but also in identifying heart failure patients who may be candidates for treatment that would delay or obviate the need for transplantation. For example, therapies are being currently developed to neutralize pathogenic autoantibodies that bind to and activate the beta 1 adrenergic receptor in heart failure patients . There aWe also applied antigen microarrays to profile non-HLA antibodies in heart transplant recipients with AMR. In this study, we utilized two-color arrays to identify IgG and IgM reactivities separately. One of the important findings of this study was that heart transplant recipients with AMR developed a distinct profile of IgM antibodies against non-HLA antigens compared with recipients who were AMR-free. The finding that these antibodies were of the IgM isotype suggest that they are likely related to damage within the graft early during the rejection process. Indeed, IgM or natural antibodies have been described in the literature to play a key role in the clearance of apoptotic cell debris . These aThough the seven significant antigens were able to cluster patients into AMR and non-AMR groups, some antibody reactivities were more predominant in some AMR patients than in others . Such a high degree of variability amongst the AMR patients suggests that AMR is characterized by a broad autoantibody “profile” and that multi-antigen panels, rather than single antibody testing, may be required for monitoring non-HLA antibodies. This individuality in autoantibody profile has been seen before among kidney allograft recipients with chronic humoral rejection . AntigenIn conclusion, we have developed custom antigen microarrays to profile autoantibodies in heart failure and heart transplantation. In retrospective pre- and post-transplant studies, we were able to highlight the value of the arrays in detecting new antibody reactivities in these patients. We are in the process of validating these studies using patient samples from additional transplant centers. Given the increasing understanding of the immune system in cardiovascular disease, we expect that these arrays will be useful in detecting autoantibodies in many cardiovascular disorders.S1 FileTable A, Antigen List for First Study (rejector vs. non-rejector). Table B, Antigen List for Second Study (AMR vs. non-AMR). Table C, Sequence of G-protein Coupled Receptor Peptides. Table D, Fold change and q-value for autoantibodies upregulated in pre-transplant sera from rejectors compared with non-rejectors. Table E, Fold change and q-value for autoantibodies upregulated in pre-transplant sera of rejectors compared with sera of healthy controls calculated using SAM analysis. Table F, Fold change and q-value for non-HLA antibodies upregulated in post-transplant sera of AMR patients compared to non-AMR as calculated using SAM analysis. Table G, Fold change and q-value for non-HLA antibodies upregulated in post-transplant sera of AMR patients compared to non-AMR as calculated using SAM analysis (cohort divided into two groups).(PDF)Click here for additional data file.


Apis mellifera), with non-reproductive tasks being performed by workers while a single queen normally monopolizes reproduction. The decentralized organization of a honey bee colony is maintained in large part by a bouquet of queen-produced pheromones, the distribution of which is facilitated by contact among workers throughout the hive. Previous studies have shown that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures of reproductive potential compared to queens raised from older worker larvae. We investigated differences in the chemical composition of the mandibular glands and attractiveness to workers of “high-quality” queens  and “low-quality” queens . We characterized the chemical profiles of the mandibular glands of high-quality queens and low-quality queens using GC-MS and used the worker retinue response as a measure of the attractiveness to workers of high-quality queens vs. low-quality queens. We found that queen quality affected the chemical profiles of mandibular gland contents differently across years, showing significant differences in the production of the queen mandibular pheromone (“QMP”) components HVA and 9-HDA in 2010, but no significant differences of any glandular compound in 2012. We also found that workers were significantly more attracted to high-quality queens than to low-quality queens in 2012, possibly because of increased attractiveness of their mandibular gland chemical profiles. Our results indicate that the age at which honey bee larvae enter the “queen-specific” developmental pathway influences the chemical composition of queen mandibular glands and worker behavior. However, these changes are not consistent across years, suggesting that other external factors may play important roles in modulating queen quality.Reproductive division of labor is one of the defining traits of honey bees ( Apis mellifera) exhibit extreme reproductive division of labor, with tens of thousands of sterile female workers performing all non-reproductive tasks for a colony. In contrast, one queen monopolizes reproduction, mating with several males (range = 10 to 28 mates depending on the subspecies) during one to several mating flights ). A). A46]).n queens .t-ratio = 11.03, d.f. = 168, P < 0.0001, Retinue response bioassays were conducted in 2012. The number of workers licking, antennating, and grooming queens introduced in observation hives was significantly higher when queens were raised from first instar worker larvae (average retinue size = 17) than when they were raised from third instar worker larvae (average retinue size = 12). High-quality queens attracted a larger number of workers in their retinue compared to low-quality queens  altered the chemical composition of the resulting queen’s mandibular glands, and whether queens raised from younger worker larvae elicited a stronger worker retinue response compared to queens raised from older worker larvae.For the 22 queen mandibular gland compounds analyzed in this study, the mandibular gland extracts from queens raised in 2010 differed from those raised in 2012. Notably for the latter set of queens , there was a difference in the relative concentration of only one mandibular gland component, with low-quality queens showing higher relative amounts of 10-HDA compared to high-quality queens. Perhaps the inconsistent differences in relative concentrations of mandibular gland components between the two years can be attributed to environmental factors such as the year and season when we performed the queen-rearing protocols, the time after mating when the queens were collected for analysis, or the mating frequency of low- vs. high-quality queens across both years (not measured in this study). Furthermore, principal component analysis showed that queen grafting age affected the overall chemical profile of queen mandibular glands, irrespective of the individual chemical profile differences observed between years.Our results are consistent with those of Plettner et al. , who aftPrevious studies have also shown that the amounts of QMP and other mandibular gland compounds increase within weeks after queen emergence, with older queens typically exhibiting more complex and abundant mandibular gland chemical bouquets ,56–59, s. LikewisThe differences we observed in the chemical composition of queen mandibular glands based on grafting age need to be interpreted with caution for several reasons. First, the total amounts of volatiles obtained from experimental queens (96 μg in 2010 and 223 μg in 2012) were relatively low compared to previous studies, which have reported considerably higher values ,58. FurtAnother source of discrepancy between ours and other studies could have been the way in which queens were reared. The necessary use of two different cell-building colonies for the production of the experimental queens could have biased the phenotype of the queens to some degree, since we cannot eliminate the possibility that nurse bees in one cell builder were less attentive in their queen-rearing duties, thus producing queens that were fed or tended to less than the other cell builder. Given that several physiological and external factors seem to affect queen mandibular gland composition in ways that we do not fully understand, queen reproductive quality is not necessarily always reflected in a quantitative or qualitative change of all compounds in the mandibular glands. Rather, reproductive quality, as measured by grafting age (this study), time after mating , and matThis study also found that queens raised from first-instar worker larvae elicited larger worker retinues compared to queens raised from third-instar worker larvae, indicating that queen grafting age significantly affects queen attractiveness to workers. Other studies have also shown that QMP and other queen mandibular gland components regulate physiological and behavioral responses in workers –47,60–65Nonetheless, the queen retinue sizes found in our study  are up to three times higher than those reported by others ,66–67. PFurthermore, while our large sample size for retinue behavior makes it highly likely that our observed differences in gland composition may influence the differences in worker response, we cannot exclude that the increased attractiveness of workers toward high quality queens could be due to other factors, including cuticular lipids , tergal Regardless of these possibilities, any observed differences in queen attractiveness to workers due to grafting age could have an adaptive value. Following queen-rearing protocols similar to our study, a previous investigation on the variation of queen grafting age showed that queens raised from younger worker larvae were larger in size, stored larger numbers of spermatozoa in their spermathecae, and mated with significantly more males than queens that were raised from older worker larvae . A similLow queen attractiveness might be one of the factors influencing the collective decision by workers to either keep their queen or to replace her with a new one (queen supersedure). Rangel et al.  did not S1 AppendixN-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) derivatives obtained from the GC retention times. The column titled "Total" indicates the total amount of compounds (in μg) detected from the glands of each queen. The column titled "Total id" indicates the total amount of compounds that were positively identified using GC-MS, while the column titled "Total UN" indicates the total amount of compounds that were not identified. C) Raw data for the retinue size observed during each 5-min point sampling bout from observation colonies headed by either queens raised from first instar worker larvae  or by queens raised from third instar worker larvae . The total number of instantaneous sampling points for each queen type (n) is denoted within each bar.A) Raw data for the absolute amounts (in μg) and B) Raw data for the relative amounts  of each of 22 compounds identified using GC-MS from mandibular gland extracts of honey bee queens that were raised from either first-instar worker larvae  or third-instar worker larvae  in 2010 and 2012. Kovats index values were calculated for the (XLSX)Click here for additional data file.S1 TableN-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) derivatives obtained from the GC retention times. Differences in absolute amounts of compounds between high-quality queens and low-quality queens were analyzed with two-tailed non-parametric Wilcoxon tests because of unequal variances. Tests were performed separately for queens raised in 2010 and queens raised in 2012  or third-instar worker larvae  in 2010 and 2012. The absolute amount (in μg) of each compound is given for each treatment group (mean ± s.e.m.). Kovats index values were calculated for the (XLSX)Click here for additional data file.


Prostate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide.Rag2−/−;γc−/− mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR.PCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into PCSD1 xenograft tumor growth capacity was 24 fold greater in the bone  than in the soft tissue  microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < =0.008) and increased AR (p < =0.032) relative to control.PCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche. Prostate cancer is the second leading cause of cancer-related death in men despite advances in prostate cancer management . Since tFor recurrent prostate cancer or metastatic disease on presentation, NCCN guidelines recommend starting androgen deprivation therapy (ADT) with close monitoring . BicalutNext generation ADTs, chemotherapy, and radiopharmaceuticals, along with supportive care, comprises the next stage of care. There are two FDA-approved next generation ADTs, abiraterone acetate and enzalutamide. Abiraterone inhibits the intra-tumoral androgen production by irreversibly binding the CYP17A1 steroidogenic enzyme in an antagonistic fashion, while enzalutamide binds the androgen receptor with strong affinity and diminishes the nuclear translocation of the androgen receptor. Both have been shown to improve survival -11, howeThere is currently no curative treatment for prostate cancer bone metastases . There aet al. ). We tested the anti-androgen, bicalutamide, in PCSD1, our new patient-derived xenograft model of bone metastatic prostate cancer. We transplanted GFP-Luciferase expressing PCSD1 cells intra-femorally (IF) or sub-cutaneously (SC) into immunodeficient mice and provided an androgen deprivation challenge with Bicalutamide. We demonstrated that, as seen in clinical trials of next generation ADT, tumor growth in the bone niche persisted despite a favorable biochemical PSA response. In humans receiving ADT, bone scans have shown persistent growth despite dropping PSA levels [Androgen ablation therapy is standard for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to standard-of-care therapies including androgen deprivation, radiation and chemotherapy. Bone metastases of prostate cancers are difficult to biopsy from patients, therefore, the currently available clinical measures of bone metastatic prostate cancer growth in patients are indirect: PSA in the blood and XRays, CT scans which measure bone density or bone scans which measure bone turnover. Changes in bone density and turnover are used as indicators of metastatic tumor growth. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. In order to study this directly and to have access to the tumor tissue when it is in the bone we established a new xenograft model of bone metastatic prostate cancer growth, PCSD1, from a surgical prostate cancer bone metastasis specimen  resistant growth of human prostate cancer bone metastasis but also can be used to unravel this paradoxical role of AR in castrate-resistant PCa, which has limited the effectiveness of all androgen deprivation therapies. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting androgen deprivation resistant prostate cancer growth in the bone-niche.In this paper we performed the first direct comparison of the effectiveness of bicalutamide in inhibiting PCSD1 prostate cancer growth in the bone to when it is transplanted elsewhere, in this case, just beneath the skin, or, sub-cutaneously. We found that PCSD1 tumors were more resistant to bicalutamide treatment when growing in the bone compared to sub-cutaneously. Importantly, this is the first report to directly demonstrate a striking (24-fold) greater growth of prostate cancer tumors in the bone-niche than the soft tissue, sub-cutaneous niche.The implications of these findings are two-fold. First, the finding that the PCSD1 mouse model closely recapitulates the response in humans suggests that this is a good model for studying castrate resistant bone metastatic prostate cancer. Second, the fact that this phenomenon is unique to the bone-niche and is not seen in the same tumor when injected into the subcutaneous tissue confirms previous findings suggesting a unique interaction between prostate cancer and the bone-niche itself. We are currently exploring possible mechanisms for this. While the Mann–Whitney test showed that the differences in tumor growth rates approached statistically significant p values one limitation of this study is that additional animals in the treatment groups should be used.A small reduction in IF tumor size in the bicalutamide-treated compared to vehicle-treated mice can be seen at the final time point at 18 days of treatment. However, the difference was not statistically significant according to the Mann–Whitney test. More mice are needed to determine whether there is a statistically significant reduction in PCSD1 tumor growth in the intra-femorally transplanted tumors albeit much smaller than the response in the sub-cutaneously transplanted tumors. While longer treatment was not possible due to the tumors having reached the maximal allowed tumor size according to the institutional animal welfare protocol (IACUC), we are currently testing whether earlier treatment may be more effective in suppressing tumor growth in the bone-niche. This may have the clinical implication that bicalutamide treatment in men with any signs of bone metastasis should commence early in the treatment plan. Such a decision would be greatly enhanced with more sensitive, more definitive diagnostic tests for bone metastasis. We are using our PCSD1 model to develop improved imaging of bone metastatic tumors.The PCSD1 bone-niche xenograft model was derived from a bone metastasis from a patient with castrate resistant disease. PCSD1 is both PSA and AR positive by RNA and protein analysis. Bicalutamide challenge of the PCSD1 bone-niche model led to a decrease in tumor PSA at the level of RNA as well as protein as seen in the immunohistochemistry results. AR compensated by increasing RNA expression. We hypothesize that cross-talk from signaling pathways in the bone microenvironment altered AR activity such that it is still functional even in the presence of androgen deprivation therapies such as bicalutamide. Signaling cross-talk as a mechanism of castrate-resistance in prostate cancer that alters steroid hormone receptor activity and transcriptional regulation of gene expression has been demonstrated for AR in as well as other steroid hormone receptors such as GR -23. HereAn alternative hypothesis is that the bone-niche preference and resulting increased tumor growth is due, at least in part, to the physical enclosure of the cells within the endosteal space in the femur compared to the sub-cutaneous injection where the cells may disperse and, thus, may be readily cleared from the body. This was a concern for the IF injected cells as well since the bone marrow is directly connected to the circulation and cells are known to be rapidly cleared unless they have the correct homing signals. To mitigate such a possibility in these experiments, the SC and IF injected cells were resuspended 1:1 in high concentration Matrigel which rapidly solidifies at temperatures higher than 4°C. Once the cells were injected sub-cutaneously, a solidified mass could be palpated at the injection site which was maintained in most mice for several days to weeks so dispersal of the cells was not immediate, however, it was not necessarily precluded. IVIS imaging is being performed at early time points after initial injection in experiments currently underway to monitor the fate of cells just after injection both sub-cutaneously or intra-femorally. In another study from our lab in which we compared bone lesions in mice injected intra-femorally with Matrigel/Media alone compared to PCSD1 plus Matrigel, immunohistochemical analysis revealed solidified Matrigel still present in the endosteal space of the femurs injected with Matrigel alone at 8 weeks post-injection . It is possible that the bone niche enforces and maintains close physical proximity of the tumor cells to each other and to micro-environmental support cells that may be just as important for providing a growth advantage as the signals themselves.In the near future, many urologists anticipate that there may be a rise in the incidence of advanced prostate cancer since the USPSTF recommendations to stop PSA screening were released. There is no curative treatment for bone metastatic prostate cancer. There are currently few mouse models derived from human castrate resistant bone metastases with serially transplantable mouse xenografts into bone other than PCSD1 which was developed and characterized as previously reported . Here, wWe investigated the effect of anti-androgen therapy for prostate cancer in the bone versus soft tissue microenvironments using PCSD1, a new patient-derived xenograft model for bone metastatic prostate cancer. We show for the first time that the bone microenvironment itself significantly increased the tumor growth capacity of the prostate cancer cells, altered AR function and rendered the prostate cancer castrate resistant. We demonstrated that, as seen in a sub-set of patients in clinical trials of next generation ADT, tumor growth in the bone niche persisted despite a paradoxically favorable biochemical PSA response.−/−;γc−/− male mice, and the xenograft tumors are positive for PSA, PSMA, and AR. We have challenged the xenograft in this model with ADT and recapitulated non-concordant favorable biochemical PSA response despite continued growth in the bone niche. We have demonstrated that this phenomenon is unique to the bone niche, while tumor growth responded favorably to ADT in the subcutaneous setting. Bone microenvironment permits castrate-resistant growth in this xenograft model of bone metastatic prostate cancer. One therapeutic implication of our finding is that prostate cancer therapies need to target potent survival signals that bone provides for prostate cancer, and we are currently working to characterize those signals.PCSD1 is a new patient-derived primary prostate cancer bone metastasis xenograft model from a man who developed castrate resistance metastatic disease. The xenograft is serially transplantable via IF or SC transplantation into Rag2


Adherence to combination anti-retroviral therapy for HIV infection is a primary determinant of treatment success, but is often suboptimal. Previous studies have suggested that electronic medication monitoring-informed counseling is among the most effective adherence intervention components. Our objective was to review available evidence about the effectiveness of monitoring-informed counseling and to aggregate findings into quantitative estimates of the effect of such intervention on medication adherence and virologic treatment outcomes.We searched PubMed for papers reporting on randomized controlled trials comparing intervention groups receiving monitoring-informed counseling as one of the intervention components versus control groups not receiving such counseling for their effect on medication adherence and viral load concentrations. The standardized mean difference (SMD) in adherence and the odds ratio (OR) of undetectable HIV RNA in intervention versus control groups were the common effect sizes. Random-effect models with inverse variance weights were used to aggregate findings into pooled effect estimates with 95% confidence limits (CI).A total of 13 studies were included. Adherence was significantly higher in intervention groups than in control groups . Patients in intervention groups were significantly more likely to have undetectable HIV RNA concentrations than patients in control groups . However, in studies in which monitoring-informed counseling was the only intervention component, the difference in adherence and virologic response between intervention and control groups was not statistically significant.Electronic monitoring-informed counseling improved adherence and virologic response compared with control groups not receiving such counseling in studies in which it was one out of multiple intervention components, but not in studies where it was the only intervention component. Adherence to combination anti-retroviral therapy (cART) is a primary determinant of anti-retroviral treatment success. Sufficiently high levels of adherence to cART are necessary to achieve and sustain viral suppression and to prevent disease progression and death –3. Yet, An appreciable number of adherence-enhancing intervention studies have provided patients and/or their health care providers with objective information about the patients’ medication-taking behavior as one of the intervention components. In these studies, medication adherence is typically measured using an electronic medication monitoring device. Electronic medication monitoring devices register the time and date of each opening of the device, which is assumed to represent medication ingestion. The date and time of openings of the device over a long-time period can be shown to patients in the form of a graphical display. Such graphical feedback could make medication-taking behavior and the occurrence of non-adherence more concrete or real to the patient who may be unaware of suboptimal adherence. Personalized feedback based on the pattern of medication use could open discussions between patients and health care providers about adherence barriers and potential solutions to deal with these.Research conducted across medical conditions have suggested that feedback on adherence performance and the accompanying counseling informed by recent adherence performance are among the intervention components that improve adherence most consistently –11. HoweOur objective was to review available evidence about the effectiveness of monitoring-informed counseling among patients who are prescribed cART for a chronic HIV infection and to aggregate findings into quantitative estimates of the effect of such intervention on medication adherence and virologic treatment outcomes. Moreover, we aim to identify study design features that are associated with stronger intervention effects.((“intervention” [tiab]) OR [“intervention” [tw])] AND ) OR ((HIV Infections[MeSH] OR HIV[MeSH] OR hiv[title/abstract] OR hiv-1[title/abstract] OR hiv-2*[title/abstract] OR hiv-1[title/abstract] OR hiv2[title/abstract] OR hiv infect*[title/abstract] OR human immunodeficiency virus[title/abstract] OR human immune deficiency virus[title/abstract] OR human immuno-deficiency virus[title/abstract] OR human immune-deficiency virus[title/abstract] OR [(human immun*) AND (deficiency virus[title/abstract])] OR acquired immunodeficiency syndromes [title/abstract] OR acquired immune deficiency syndrome[title/abstract] OR acquired immuno-deficiency syndrome[title/abstract] OR acquired immune-deficiency syndrome[title/abstract] OR ((acquired immun*) AND (deficiency syndrome [title/abstract])) or “sexually transmitted diseases, viral”[mh]) OR HIV[title/abstract] OR HIV/AIDS [title/abstract] OR HIV-infected[title/abstract] OR HIV[title] OR HIV/AIDS[title] OR HIV-infected [title])) AND (adhere*[tiab] OR complian*[tiab] OR adhere*[tw] OR complian*[tw] OR Patient Compliance[MeSH] OR Medication Adherence [MeSH])) AND (“1996/01/01”[PDat]: “2014/12/31”[PDat])))We searched PubMed for papers published from August 1996 to October 2014 using the following strategy:The reference lists of the papers retrieved were reviewed for additional relevant publications. Additionally, we searched abstracts from the International AIDS conference , the IAS Conference on HIV Pathogenesis, Treatment, and Prevention , the HIV Drug Therapy Glasgow Meeting , and the International Conference on HIV Treatment and Prevention Adherence (years 2010–2014).Eligible studies met the following criteria: (1) randomized controlled (cross-over) trial (2) comparing monitoring-informed counseling as one of the intervention components versus not receiving such counseling. Intervention groups could thus consist of multi-component and single component interventions; (3) outcomes are medication adherence and/or viral load concentrations; 4) participants are HIV-infected persons prescribed cART for a chronic HIV infection. We included English language papers only.We extracted the following information from each study: name of the first author, year of publication, sample size, whether patients were initiating, restarting, or switching a cART regimen or were already on ART, whether the intervention was only administered to patients with low pre-intervention adherence levels (yes/no), or if patients were triaged to different levels of intervention intensity depending on their adherence level (yes/no), the percentage of patients with undetectable viral loads at baseline, duration of the intervention period (weeks), number of intervention sessions, and intervention components. The categorization of intervention components was adapted from two previous systematic reviews of anti-retroviral adherence interventions , 8. Intet-, x2-, or F-statistics, contingency table data, or exact p values (We defined adherence as the percentage of prescribed doses of cART taken. We used the standardized mean difference (SMD) as the common effect size to express the difference in adherence between intervention and control groups. If studies did not provide the SMD, we contacted authors to get additional data. If no additional data were available, we calculated the SMD from correlation coefficients, means and SDs, odds ratios (OR), p values . When stp values .We used the OR as the common effect size to express the difference in the percentage of patients with an undetectable viral load in intervention versus control groups. When studies reported an insignificant effect of the intervention on viral load without data, we assumed an OR of 1.01. Random-effect models with inverse variance weights were used to aggregate individual SMDs and ORs into pooled effect estimates with 95% confidence limits (CI) using Review Manager 5.3.We compared pooled effect estimates of adherence and undetectable viral loads between studies in which monitoring-informed counseling was the only intervention component with studies in which monitoring-informed counseling was one out of multiple intervention components. We conducted a sensitivity analysis to examine potential bias resulting from over-representation of studies with more than one intervention arm. We examined the extent to which results would change when studies with more than one intervention arm were excluded from the analysis or if only a single intervention arm was included.We examined whether variation in effect sizes of adherence and viral load were significantly associated with study design features. We investigated the effect of the following study design features: whether patients were initiating, restarting, or switching a cART regimen (yes/no) or were already on ART, the percentage of patients with undetectable viral loads at baseline, whether the intervention was only administered to patients with low pre-intervention adherence levels (yes/no), or if patients were triaged to different levels of intervention intensity based on their adherence level (yes/no), duration of the intervention period (weeks), number of intervention contacts, whether intervention components administered to the intervention group included the following: didactic provision of information about HIV, cART, and adherence (yes/no), behavioral, cognitive behavioral, or motivational counseling (yes/no), provision of reminder devices (yes/no), social support enlistment (yes/no), depression screening/treatment/referral (yes/no), financial incentives for good adherence (yes/no), and substance use screening/treatment/referral (yes/no).Q statistic (Q-between) within a mixed effects model using the method of moments estimation. These analyses were conducted using the SPSS macro’s MetaF and MetaReg from Lipsey and Wilson  Figure . This reWe identified three studies in which monitoring-informed counseling was the single intervention component and was compared with a control group not receiving such counseling , 14, 22.A total of 12 out 13 studies included compared a single intervention arm with a control group. In one study, two intervention arms were compared with the same control group . One of Q = 6.87, p = 0.009). Studies providing financial incentives for good adherence . Both effects remained statistically significant in a multivariate model.Variation in effect sizes of adherence were significantly associated with the study design features; percentage of patients with undetectable viral load at baseline and providing financial incentives for good adherence. Studies with a lower percentage of patients with undetectable viral loads at baseline (dichotomized at the median of 35%) yielded larger effect sizes than studies with a higher percentage of patients with undetectable viral loads at baseline . Studies in which the intervention components included behavioral, cognitive behavioral, or motivational counseling yielded higher effect sizes than studies not including this component . We were unable to include both variables in a multivariate model due to high collinearity.Variation in effect sizes for undetectable viral loads were significantly associated with whether the intervention components included didactic provision of information about HIV, cART, and adherence, or behavioral, cognitive behavioral, or motivational counseling. Studies in which the intervention components included didactic provision of information about HIV, cART, and adherence yielded higher effect sizes than studies not including this intervention component , with an absence of small studies yielding negative effects   Figure .Our meta-analysis of randomized controlled trials (RCTs) investigating the effect of monitoring-informed counseling on treatment adherence and virologic treatment response, yielded significantly improved adherence and virologic treatment response only when such counseling was part of a multi-component intervention. The improvement in adherence constituted a medium-sized effect. The improvement in medication adherence achieved in the intervention groups was clinically relevant as it was accompanied by an increased likelihood of having an undetectable viral load.We distinguished between studies in which monitoring-informed counseling was the only intervention component and studies in which monitoring-informed counseling was one out of multiple intervention components. In the vast majority of studies included, monitoring-informed counseling was one out of multiple intervention components that patients received. Consequently, the separate contribution of monitoring-informed counseling to the improved levels of adherence and virologic treatment response is difficult to establish. However, this reflects the current state of the art in HIV adherence support research in which most interventions consist of multiple components . MoreoveWe identified only three single component studies with divergent results. Two single component studies found insignificant effects of the intervention on adherence , 14. By By contrast, the single component study that yielded improved levels of adherence in the intervention group mentioned that participating health care providers had received practice and role-playing training sessions during which it had been emphasized that the goal of counseling was to help subjects to improve their medication-taking behavior, not to scold them about poor adherence . The autWe aimed to identify study design features that were associated with larger effect sizes for adherence and virologic treatment response. Lower percentage of patients with undetectable viral loads at baseline and providing financial incentives for good adherence were significantly associated with larger effect sizes for adherence but not for virologic treatment response. The clinical relevance of these findings is therefore uncertain. Studies including the didactic provision of information about HIV, cART, and adherence, or behavioral, cognitive behavioral, or motivational counseling as intervention components yielded larger effect sizes for virologic treatment response than studies that did not include these intervention components. We were unable to assess the independent effect of these two intervention components in a multivariate model, due to the low number of included studies and due to the fact that many studies including one of the two intervention components also included the other component. While the multiple component studies were associated with larger improvements in adherence and virologic treatment response than the single component studies, it remains thus largely unknown which intervention components are responsible for this difference.There is also more a fundamental reason why it could be difficult to determine which adherence intervention components are most responsible for improvements in adherence than others. It is increasingly recognized that multifactorial barriers may influence patient adherence and that these barriers may differ between patients and change within patients over time. There is also increasing recognition that interventions should be targeted to people who are clearly identified as needing that specific intervention. By analogy with a medical condition that can only be adequately treated when an accurate diagnosis is established, the treatment, i.e., adherence intervention, should be matched to the diagnosis, i.e., barriers to adherence that the patient is experiencing . For exaThere has been attention in the field to the content of adherence care that is provided in control groups of adherence intervention studies. It was previously found that the difference in adherence intervention components provided in the intervention and control groups was a significant predictor of the difference in viral load and adherence success rates between intervention and control groups . In the The present study has several limitations. First, because in the vast majority of studies monitoring-informed counseling was one out of multiple intervention components, the separate contribution of such counseling to the improved levels of adherence and virologic treatment response is difficult to establish.Second, we searched a single electronic database only, i.e., PubMed, which may have resulted in publication bias. However, we supplemented this database with searches in abstracts of the most relevant HIV conferences for the subject of medication adherence.Third, if studies reported insignificant differences in adherence or virologic response between intervention and control groups without data, we assumed an effect size of 0.01. This may have been a too conservative estimate, which may have resulted in an underestimate of the effectiveness of the concerning interventions.Fourth, we categorized adherence intervention components in several broad categories, for example, behavioral, cognitive behavioral, or motivational counseling. Within these broad categories, distinct types of intervention components may have had a different impact on adherence and virologic response that will remain undetected in the present global analysis, e.g., cognitive behavioral counseling could have had a different impact on adherence and virologic response than motivational interviewing.Fifth, when more than one type of intervention was tested within a single study, we considered data from each arm of the intervention as separate data points. This may have resulted in an over-representation of a study with more than one intervention arm. However, a sensitivity analysis in which we excluded this study from our analysis or included only one of the intervention arms did not change our overall results.In conclusion, monitoring-informed counseling improved medication adherence and virologic response compared with control groups not receiving such counseling in studies in which it was one out of multiple intervention components, but not in studies where it was the only intervention component.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wiwaxia from the Cambrian Stage 3 Chengjiang Konservat-Lagerstätte show that the Wiwaxia scleritome comprised nine equivalent transverse rows associated with outgrowths of soft tissue, but did not possess a separate zone of anterior sclerites. This serial construction is fundamentally incompatible with the circumferential disposition of sclerites in early molluscs, but does closely resemble the armature of certain annelids. A deep homology with the annelid scleritome must be reconciled with Wiwaxia’s mollusc-like mouthparts and foot; together these point to a deep phylogenetic position, close to the common ancestor of annelids and molluscs.Wiwaxiids are a problematic group of scale-covered lophotrochozoans known from Cambrian Stages 3–5. Their imbricating dorsal scleritome of leaf-like scales has prompted comparison with various annelids and molluscs, and has been used as a template to reconstruct the articulation pattern of isolated Small Shelly Fossils. The first articulated specimens of  Wiwaxia is best known for its stalked carbonaceous sclerites, which together comprise an imbricated dorsal scleritome. Articulated scleritomes have previously been reported from five localities spanning 15 million years12345Wiwaxia scleritome is remarkably conserved, notwithstanding species-level variety in sclerite proportions and orientation. An anterior zone of sclerites is followed by eight transverse rows across the body, with morphologies varying consistently according to location: ventrolateral sclerites are sickle-shaped; lower-lateral sclerites are oval; upper-lateral sclerites are rounded and symmetrical, and dorsal sclerites are asymmetric. In addition, mature specimens – those longer than a centimetre – exhibit twin series of erratically arranged dorsal spines. The anterior body region seemingly corresponds to a distinct zone of rounded scleritesThe distinctive mid-Cambrian organism Wiwaxia. The sclerites bear the distinctive signature of microvillar secretion – an internal microstructural fabric of long, narrow chambers – which assigns Wiwaxia to the lophotrochozoan total group81012Wiwaxia foot  and feeding apparatus12The construction of the sclerites and scleritome represents important, if ambiguous, evidence with respect to the phylogenetic affiliation of Wiwaxia based on articulated specimens from the Cambrian Stage 3 Chengjiang Lagerstätte. Post-mortem enrolment and soft tissue preservation in these fossils, in combination with critical W. corrugata material from the Burgess Shale, allows a timely re-evaluation of the Wiwaxia scleritome, and expounds the scleritome’s implications for the affinity of this confounding taxon.Here we report a new species of Wiwaxia specimens, each comprising part and counterpart, have been collected from Chengjiang by the Early Life Institute working team, and deposited in the Early Life Institute and Department of Geology, Northwest University, Xi’an, China (Prefix: ELI). These complete, articulated specimens represent organisms in various states of enrolment, preserved at various orientations to the plane of splitting. Sclerites and mouthparts are represented by regions with a deep purple to black colouration, and in the best cases correspond to an intact layer of carbon. Regions corresponding to soft tissue are coated with rust-coloured framboids, apparently arising through the oxidation of pyrite.Five new W. corrugata are deposited in the Smithsonian Institution National Museum of Natural History (NMNH), Washington DC, and the Royal Ontario Museum (ROM), Toronto, and represent unweathered carbonaceous compression fossils associated with diagenetic aluminosilicate filmsBurgess Shale specimens of http://zoobank.org/Referencesurn:lsid:zoobank.org:pub:1B5E0AE5-2FB2-4EFF-B35B-8293D919DEE8This published work and the nomenclatural acts it contains have been registered in Zoobank: Family Wiwaxiidae Walcott, 1911 Wiwaxia Walcott, 1911Genus Ovoid body bearing nine transverse rows of ribbed carbonaceous sclerites, arranged in bundles and directed towards the posterior. Anteriormost sclerite row terminal. Sclerites comprising narrow root and wide blade, and incorporating narrow internal longitudinal chambers. Sclerite morphology varying consistently across each transverse row; medial sclerites rounded, ventro-lateral sclerites elongate and curved, usually with pointed tip. Dorsal surface of adults often with elongate spinose sclerites. Ventral surface comprising unarmoured ‘foot’. Toothed feeding apparatus comprising two to three rows of curved carbonaceous teeth arranged on tongue-like supporting apparatus.Wiwaxia papilio sp. nov.LSID. urn:lsid:zoobank.org:act:E5A1A18C-D087-4FA1-AC60-A0CE65603E9BPapilio (Latin), butterfly, reflecting the butterfly-like arrangement of the fans of sclerites.ELI-W001 , an almoELI-W002–ELI-W005 , 2a,b.Lagerstätte at the Jianshan Section in Haikou, Kunming. Other taxa recovered from this site include the early agnathan HaikouichthysSpecimens were collected from the yellowish-green to greyish-green mudstones of the Chengjiang Single order of widely spaced sclerite ribs (4–6 ribs on sclerites 500–1000 μm in length). Non-ventrolateral sclerites long and wide relative to body length .Wiwaxia corrugata in terms of its overall body size, the form of its mouthparts, the relatively large size of dorsal sclerites, the broad yet short ventrolateral sclerites, and the absence of dorsal spines. Adult specimens are conceivably represented by the larger isolated sclerites that have also been reported from ChengjiangWiwaxia species. W. papilio sp. nov. is distinguished from W. corrugata based on the low number ribs on its sclerites; detailed comparison with other species is hampered by the shortage of comparative material1The examined material resembles juvenile specimens of W. papilio sp. nov.  Each ventro-lateral sclerite fully overlaps its posterior neighbour  Formation contains disarticulated sclerites that correspond to the size range of sclerites in adult Wiwaxia corrugata, and includes elongate sclerites that conceivably represent spines325Wiwaxia adults16Wiwaxia are present in great abundance at the deeper water localities on Fossil Ridge and Mount Stephen41228In contrast, deep-water settings are replete with adult Wiwaxia juveniles occur in almost all geographic and ecological settings31Wiwaxia’s biological affinity.Since the discovery of the first articulated specimensWiwaxia sclerites are the conspicuous dorsal scales (elytra) of aphroditid and polynoid annelids10Wiwaxia sclerites4Wiwaxia sclerites, chrysopetalid paleae occur in a series of bundles or fans across a transverse rows35Wiwaxia sclerites, comprise a proximal root and a broad distal blade, and on a more superficial level may exhibit ribs, a granular ornament, and a distal prong  bear comparable sclerites. Polyplacophoran sclerites exhibit crystalline cores that are surrounded by a thin cuticular layer with a microvillar texture45Wiwaxia1349Wiwaxia would have to represent a surprisingly early and extremely derived aplacophoran that retained larval features to adulthood and developed a precise sclerite organization unseen in modern representativesWiwaxia’s scleritome.Could the molluscs provide a more reasonable analogue for the Wiwaxia scleritome has no precise equivalent in modern or fossil groups, this is not to say that it does not share homologies at a deeper level. Carbonaceous sclerites are secreted by microvilli in bryozoans, brachiopods, molluscs and annelids4651Wiwaxia sclerites is paralleled by basal annelids, and possibly brachiopods5455Wiwaxia could conceivably foreshadow the neuropodial and notopodial bundles observed in crown-group annelids. Whilst it is conceivable that the iterated nature of the Wiwaxia scleritome arose convergently, we prefer to assume homology in the absence of clear evidence to the contrary.Although the detailed construction of the Wiwaxia scleritome must be balanced against the conspicuously mollusc-like nature of its mouthparts and foot121457Hallucigenia, which bears onychophoran-like claws alongside cycloneuralian-like mouthparts; this is resolved if cycloneuralian-like mouthparts occurred in the common ancestor of onychophorans and cycloneuraliansWiwaxia is less well established, there is more than one way to reconcile its molluscan and annelidan features. Either annelid-like sclerite rows or molluscan mouthparts may have been present in the common ancestor of annelids and molluscs and been retained for some time in the stem lineages of each phylum.The broadly annelid-like serial construction of the Wiwaxia is a stem-group mollusc .


Analysis of complex metatranscriptome datasets of these communities poses a considerable bioinformatic challenge since they are non-restricted with a varying number of participating strains and species. For RNA-Seq data a standard approach is to align the generated reads to a set of closely related reference genomes. This only works well for microbial communities for which a near complete catalogue of reference genomes is available at a small evolutionary distance. In this study, we focus on the design of a validated de novo assembly metatranscriptome pipeline combined rRNA removal, IDBA-UD assembler, functional annotation and taxonomic classification. Different assemblers were tested and validated using RNA-Seq data from an in silico generated mock community and in vivo RNA-Seq data from a restricted microbial community taken from a mouse model colonized with Altered Schaedler Flora (ASF). Precision and recall of resulting gene expression, functional and taxonomic profiles were compared to those obtained with a standard alignment method. The validated pipeline was subsequently used to generate expression profiles from non-restricted cecal communities of four C57BL/6J mice fed on a high-fat high-protein diet spiked with an RNA-Seq data set from a well-characterized human sample. The spike in control was used to estimate precision and recall at assembly, functional and taxonomic level of non-restricted communities.The here developed de novo assembly pipeline for metatranscriptome data analysis was designed for microbial ecosystems, which can be applied for microbial metatranscriptome analysis in any chosen niche.A generic  High throughput metagenomics have revolutionized our knowledge of microbial communities such as those that populate the human gastrointestinal (GI) tract. Complementing 16S ribosomal RNA gene-based compositional analyses, metagenome sequencing of these communities provided a broad description of the genetic content and relative abundance of individual members . Metagende novo assembly method can be used. De novo assembly of RNA-Seq reads into contigs increases the information content and therefore grants a more reliable annotation of the expressed genetic content of an unknown microbial community [de novo assembly of Illumina sequencing data [de novo metagenome assemblies’ will not reliably work for RNA-Seq derived contigs. Consequently additional verification strategies, such as PCR, are necessary as confirmation of the genetic context predicted by assembled contigs.To overcome these problems a ommunity ,17. Subsing data ,19. Mosting data ,17. Sequde novo assembly pipeline for single-end Illumina sequencing using existing assemblers. The method provides better gene assignment results then alignment methods, and we evaluated sensitivity, reliability and validity of the method for the function analysis of complex metatranscriptome data. The generic de novo assembly method developed was validated by PCR and using metatranscriptome datasets of community-restricted samples and a spike-in of a human control sample, and enabled a reliable functional profiling and taxonomic binning of samples obtained from the mouse GI tract.The microbiome of the GI tract of healthy human individuals fulfils a variety of beneficial functions for human health . NumerouA generalized metatranscriptome assembly and analysis pipeline was designed . Brieflyin silico generated mock community composed of eight strains, was used for a primary evaluation of the entire workflow. The second RNA-Seq dataset used to further validate the workflow resulted from an in vivo study of a restricted intestinal microbial community of limited complexity obtained by the colonization of inbred non-obese diabetic (NOD) mice with Altered Schaedler Flora (ASF) [de novo assemblies obtained from this complex non-restricted mouse community, RNA-Seq data from a well-characterized sample from the human small intestine [In order to validate the proposed pipeline, two single-end RNA-Seq metatranscriptome datasets were used. The first RNA-Seq dataset, obtained from an ra (ASF) . The datra (ASF) . To estintestine  was usedin silico mock metatranscriptome was built by merging RNA-Seq data of eight single species transcriptome profiling experiments downloaded from public repositories  for the mock community as a whole as well as for each of the individual members  , and appEven though there were many contaminating reads in these samples that could interfere with the assembly process only a few false positive reads assignments were detected. The precision and recall for functional annotation was 0.97 and 0.58 respectively. The precision was therefore comparable to what was observed for the mock community, whereas the recall is much lower, which is probably due to a much lower sequencing depth of ASF samples .de novo assembled sequences. For a taxonomic classification a two-step procedure was developed. First, from the set of de novo assembled sequences from the ASF microbiome a subset of 103 proteins derived from essential single copy genes (ESCG) [Additionally we explored the performance of a taxonomic classification of s (ESCG)  was idens (ESCG)    . Relativ family) .de novo assembly of 24077 contigs and allowed for the prediction of 36012 partial and full length ORFs within these contigs. Of these ORFs, 25897 were exclusively derived from the mouse datasets, whereas 9707 were assembled exclusively from the human dataset. A total of 407 hybrid ORFs were assembled consisting of reads obtained from both data sources. Virtually identical results were obtained for the mouse-derived RNA-Seq data when the mouse reads were assembled separately (results not shown). This suggests that a substantial increase in microbial complexity essentially did not influence the outcome of the mouse assembly. Of the RNA-Seq mRNA reads derived from the human small intestine sample, 85% was captured in an assembly whereas this fraction was 61–71% for the mRNA reads derived from the four individual mouse cecum samples. The fraction of the reads that was assigned to an ORF was 80% and 45–55% for the human small intestine sample and mouse cecum samples, respectively   was usedAssemblies were performed using the assemblers’ default setting –52. WhenS1 FileAssembler Performance: Testing of various assemblers and output performance; Filtering: Ribosomal RNA, adapter and quality filtering results. Mock_TNF_hybrids: Number of shared assemblies and tetra nucleotide frequency occurrence regression coefficient between mock members. Coverage_Assembly: Distribution of assembled proteins based on transcriptome coverage. Mock_assembly ouput: Precision and recall for functional assignments. NOD_mouse_taxonomy_output: Precision and recall for taxonomy assignments(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 FileTaxonomy profiles for all mouse and human samples using blastx, megablast and assembly strategies.(PNG)Click here for additional data file.S4 File(PNG)Click here for additional data file.S5 File(PDF)Click here for additional data file.