Aspergillus nidulans. Many strains of A. nidulans have therefore been generated with different proteins endogenously tagged with GFP. To further enhance experimental approaches based upon GFP-tagging, we have adapted the GFP Binding Protein (GBP) system for A. nidulans. GBP is a genetically encoded Llama single chain antibody against GFP which binds GFP with high affinity. Using gene replacement approaches, it is therefore possible to link GBP to anchor proteins, which will then retarget GFP-tagged proteins away from their normal location to the location of the anchor-GBP protein. To facilitate this approach in A. nidulans, we made four base plasmid cassettes that can be used to generate gene replacement GBP-tagging constructs by utilizing fusion PCR. Using these base cassettes, fusion PCR, and gene targeting approaches, we generated strains with SPA10-GBP and Tom20-GBP gene replacements. These strains enabled test targeting of GFP-tagged proteins to septa or to the surface of mitochondria respectively. SPA10-GBP is shown to effectively target GFP-tagged proteins to both forming and mature septa. Tom20-GBP has a higher capacity to retarget GFP-tagged proteins being able to relocate all Nup49-GFP from its location within nuclear pore complexes (NPCs) to the cytoplasm in association with mitochondria. Notably, removal of Nup49-GFP from NPCs causes cold sensitivity as does deletion of the nup49 gene. The cassette constructs described facilitate experimental approaches to generate precise protein-protein linkages in fungi. The A. nidulans SPA10-GBP and Tom20-GBP strains can be utilized to modulate other GFP-tagged proteins of interest.Endogenously tagging proteins with green fluorescent protein (GFP) enables the visualization of the tagged protein using live cell microscopy. GFP-tagging is widely utilized to study biological processes in model experimental organisms including filamentous fungi such as  Aspergillus nidulans [Having the ability to experimentally link proteins to each other within cells provides several experimental avenues to interrogate protein function. For example, it enables experiments to test the effects of preventing proteins from locating to their normal subcellular locations by artificially re-targeting them to another cellular location. Additionally, if the linked target proteins locate stably to specific structures it is possible to experimentally bridge the structures to which the two proteins belong. For example, using this approach we recently provided linkage between nuclear pore complexes (NPCs) and nuclear chromatin in the model fungus nidulans . This wanidulans . To genenidulans . The GFPnidulans –6. This nidulans , 3. GBP nidulans . The disnidulans .pyrG or the pyroA selective nutritional transformation markers to select for Aspergillus transformants. One set of cassettes encodes mRFP linked to the GBP sequence to enable visualization of the generated target protein-mRFP-GBP chimeras (5 linker) [Aspergillus and other fungal cells.In the current work, we report the generation of strains ( linker) . We furt linker) . These c linker) , 11, 12.A. nidulans. In this system, the protein of interest and an anchoring protein are tagged with GFP or GBP . The GBP-mRFP constructs enables visualization of the anchor protein in addition to the GFP-tagged protein that is to be retargeted. The GBP alone version allows a red-tagged experimental protein to also be utilized; when investigating if a GFP retargeted protein is able to attract a red-tagged interacting protein for example. We amplified the GBP or the GBP-mRFP sequence by PCR and added a 15-nucleotide sequence encoding the GA5 linker sequence at their 5’ ends. The GA5 linker between the anchor protein and GBP protein provides a flexible linker [We have adapted the GFP-GBP system for protein retargeting in P or GBP . Becausee linker . The fine linker , universe linker , 11, 12.A. nidulans cells. Because of the defined and distinctive location of septa within cells we first chose to try and retarget GFP-tagged proteins to septa. A. nidulans vegetative cells are syncytial and contain multiple nuclei in a common cytoplasm. Cells grow in a highly polarized manner from a single growing tip and, when first growing from spores, undergo 2–3 rounds of synchronous mitosis without undergoing septation [A. nidulans so that septa contain a central ~50 nm diameter pore which enables direct cell-cell communication [Neurospora crassa [A. nidulans including SPA10 which locates to forming septa, remains stably associated at mature septa [gpdA promoter is transported into nuclei via its NLS  could be retargeted to septa via SPA10-GBP. In this case we utilized a GBP-mRFP cassette to generate the SPA10-GBP-mRFP gene replacement construct. SPA10-GBP-mRFP located at all mature septa . For theWe next asked if a higher abundance protein tagged with GBP at a non-septal location might retarget more Nup49-GFP away from its normal location at NPCs. GBP was therefore fused with Tom20, a transmembrane mitochondrial translocase protein of the outer mitochondrial membrane with a cytosol-exposed C-terminus . TaggingWhile the NLS-DsRed signal helps define the location of nuclei, because of its relative brightness, it also hinders somewhat the visualization of Tom20-GBP-mRFP . We thernup49 and Tom20-GBP-mRFP + Nup49-GFP strains  we successfully targeted cytoplasmic GFP-S-Tag and nuclear GFP-NLS proteins to septa. Endogenously tagged Nup49-GFP was also retargeted to SPA10-GBP at both forming and mature septa. We further used Tom20, a component of the mitochondrial translocase of the outer membrane complex, tagged with GBP (Tom20-GBP) to successfully relocate the majority of Nup49-GFP away from NPCs to the surface of mitochondria causing marked cold sensitivity similar to the effects of the null ΔA. nidulans were carried out as described previously [The techniques for classical genetics, strain construction, media preparation, culture and transformation of eviously , 24, 25.5 linker sequence (GGAGCTGGTGCAGGCGCTGGAGCCGGTGCC) as a 5’ overhang. Genes encoding the nutritional marker genes pyrG or pyroA were amplified from Aspergillus fumigatus DNA to reduce the chance of integration of the construct into the native A. nidulans pyrG or pyroA loci [5-GBP or GA5-GBPmRFP and either the pyrG or pyroA genes as selection markers [pyrG or pyroA and the GBP-mRFP with either pyrG or pyroA base cassettes can now be amplified from different plasmids (GGAGCTGGTGCAGGCGCTGGAGCC) and FN01 (CTGTCTGAGAGGAGGCACTGATGC) when generating GBP gene replacements constructs [Plasmids encoding the GBP cassettes were produced as briefly described earlier . The GBProA loci , 15. The markers . Fusion plasmids  using thnstructs , 12.A. nidulans transformants expressing the GBP-fused anchoring proteins were generated after selection for either the pyrG or pyroA nutritional markers. Cells carrying GBP-fused proteins were confirmed by diagnostic PCR at the endogenous locus of the anchoring proteins [For generation of proteins fused with GBP, the GBP/marker cassettes were amplified from plasmids created above using the universal primers. Replacement constructs were generated using fusion PCR to fuse GBP to the 3’ end of SPA10 or Tom20 coding sequence before their stop codon. This results in the generation of a chimeric protein with GBP fused endogenously to the C-terminus of the anchoring proteins coding for SPA10-GBP or SPA10-GBP-mRFP and Tom20-GBP or Tom20-GBP-mRFP. Positive proteins .A. nidulans expressing fluorescently-tagged proteins was carried out using two different spinning disk confocal systems as described previously [A. nidulans cells for imaging have also been described earlier [Imaging of eviously . The gro earlier . For imaS1 Fig(A) Localization of Tom20-GBP-mRFP in relation to the nuclear marker NLS-dsRed. (B) Localization of the NPC protein Nup49-GFP in relation to nuclear NLS-dsRed. (C) Retargeting of Nup49-GFP from the nuclear periphery to the cytoplasm in cells expressing Tom20-GBP-mRFP with no apparent Nup49-GFP accumulation around the nuclear periphery. Scale bar, ~ 5μm.(PDF)Click here for additional data file.S1 VideoCells expressing both Nup49-GFP and SPA10-GBP were imaged every 2 minutes 23 times and the playback rate is 2 frames per second. Imaging was started just after completion of mitosis. The arrow marks the location of where the septum forms.(AVI)Click here for additional data file.


Odocoileus virginianus) were detected compared to one- and two-camera survey methods. We also compared occupancy rates estimated by survey methods; as the number of cameras deployed per camera-site increased, occupancy estimates were closer to naïve estimates, detection probabilities increased, and standard errors of detection probabilities decreased. Additionally, each survey method resulted in differing top-ranked, species-specific occupancy models when habitat covariates were included. Underestimates of occurrence and misrepresented community metrics can have significant impacts on species of conservation concern, particularly in areas where habitat manipulation is likely. Having multiple camera traps per site revealed significant shortcomings with the common one-camera trap survey method. While we realize survey design is often constrained logistically, we suggest increasing effort to at least two camera traps facing opposite directions per camera-site in habitat association studies, and to utilize camera-trap arrays when restricted by equipment availability.The use of camera traps as a tool for studying wildlife populations is commonplace. However, few have considered how the number of detections of wildlife differ depending upon the number of camera traps placed at cameras-sites, and how this impacts estimates of occupancy and community composition. During December 2015–February 2016, we deployed four camera traps per camera-site, separated into treatment groups of one, two, and four camera traps, in southern Illinois to compare whether estimates of wildlife community metrics and occupancy probabilities differed among survey methods. The overall number of species detected per camera-site was greatest with the four-camera survey method (P<0.0184). The four-camera survey method detected 1.25 additional species per camera-site than the one-camera survey method, and was the only survey method to completely detect the ground-dwelling silvicolous community. The four-camera survey method recorded individual species at 3.57 additional camera-sites (P = 0.003) and nearly doubled the number of camera-sites where white-tailed deer ( The use of remotely-triggered cameras  as a tool for studying wildlife populations is commonplace , 2. ReceCamera trap surveys are also constrained logistically due to costs associated with field equipment, often limiting the total number of cameras available to one per site to maximize the number of sites sampled . In the Across wildlife survey techniques, and particularly in presence/absence surveys utilizing camera traps, researchers experience false-negative measurement errors when a species is recorded as absent despite the species’ true presence –19. AddiThe goal of our study was to investigate how detection rates represented by one, two, and four camera traps per camera-site would affect estimates of occupancy and other wildlife community metrics. We hypothesized that the deployment of one camera trap per camera-site would incompletely record the terrestrial mammal species utilizing the camera-site. We predicted that by increasing the number of camera traps per camera-site we would significantly increase the number of species detected. This increase will result in different occupancy modeling results and potentially reveal habitat relationships not detected by encounter histories generated from one-camera surveys.Data collection used non-invasive, remotely-triggered camera traps and hence did not involve direct contact or interaction with the animals. No bait or lure was used to further limit interference with animals. Fieldwork was done under research permit number SS15-42 to EJH, issued by the Illinois Department of Natural Resources (IDNR).Quercus spp.–Carya spp.) with midstory and understory components of Sugar Maple (Acer saccharium) and American Beech (Fagus grandifolia) on upland sites, while mature mixed hardwoods exist at lower elevations including a significant component of Yellow Poplar (Liriodendron tulipifera)  under one-, two-, and four-camera detection histories to develop a working baseline. From there, we identified the best approximating model given the data for all survey methods. We used the compliment of the estimated detection probability from the null models to estimate the species-specific measurement error under each camera survey method.Odocoileus virginianus), 27% raccoons, 4% coyotes (Canis latrans), 3% Virginia opossums (Didelphis Virginiana), 2% bobcats (Lynx rufus), and 2% eastern wild turkeys. The two-camera method resulted in 369 photographs from 1190 camera-days of effort, and the one-camera survey method yielded 200 photographs with an effort of 592 camera-days , with significant differences among only one- and four-camera survey methods (P<0.05). The four-camera survey method detected 1.25 (53%) additional species than the one-camera survey method and 0.75 (26%) additional species than the two-camera survey method  while four-camera surveys resulted in detections at 10.3 sites  occupancy and detection estimates , where low detection rates across all sites resulted in occupancy estimates of nearly 1. Again, this issue arises when the number of detections across all sites are always low, resulting in maximum likelihood estimates of a very high (and unlikely) occupancy rate with an extremely low detection probability . This card order [Camera trap survey design and efforts are ultimately constrained logistically, whether that is by limited resources or difficult field conditions. Thus, researchers will likely be faced with the trade-off of decreasing the per-unit sampling effort but increasing the number of sites surveyed, or increasing survey intensity over a smaller area with fewer camera-sites. Several authors have provided survey design suggestions , 19, 43,rd order  or smallS1 Dataset(CSV)Click here for additional data file.


DNA methylation have crucial roles in regulating the expression of developmental genes during mammalian pre-implantation embryonic development (PED). However, the DNA methylation dynamic pattern of long noncoding RNA (lncRNA) genes, one type of epigenetic regulators, in human PED have not yet been demonstrated. Here, we performed a comprehensive analysis of lncRNA genes in human PED based on public reduced representation bisulphite sequencing (RRBS) data. We observed that both lncRNA and protein-coding genes complete the major demethylation wave at the 2-cell stage, whereas the promoters of lncRNA genes show higher methylation level than protein-coding genes during PED. Similar methylation distribution was observed across the transcription start sites (TSS) of lncRNA and protein-coding genes, contrary to previous observations in tissues. Besides, not only the gamete-specific differentially methylated regions (G-DMRs) but also the embryonic developmental-specific DMRs (D-DMRs) showed more paternal bias, especially in promoter regions in lncRNA genes. Moreover, coding-non-coding gene co-expression network analysis of genes containing D-DMRs suggested that lncRNA genes involved in PED are associated with gene expression regulation through several means, such as mRNA splicing, translational regulation and mRNA catabolic. This firstly provides study provides the methylation profiles of lncRNA genes in human PED and improves the understanding of lncRNA genes involvement in human PED. Long noncoding RNA (lncRNA) are a class of transcripts that are longer than 200 nucleotides without protein coding capacity. LncRNA genes can be classified into intergenic and intragenic, according to their genome localization. Though the molecular basis of the function of many lncRNA genes is just emerging, the recent work indicates their intricate roles in various biological processes, such as X chromosome inactivation , imprintpancIl17d playing key roles in mouse PED [lncGET, which is essential for mouse PED beyond the two-cell stage via regulating the transcription and RNA alternative splicing at major zygotic genome activation (ZGA) stage [In recent years, several studies have implicated lncRNA genes in mammalian pre-implantation embryonic development (PED). Recently, one study reports a bidirectional promoter-associated lncRNA named ouse PED . In a diIt is well known that DNA methylation is a key regulator of gene expression . DNA metIn this study we performed genome-wide analysis of the DNA methylation of lncRNA genes in human pre-implantation embryos. Comparison of the methylation patterns observed in lncRNA and protein-coding genes identified several distinctive methylation characteristics that differ between these classes of genes. We also analyzed gamete-specific DMRs (G-DMRs) and developmental-specific DMRs (D-DMRs) in lncRNA genes. To investigate the potential roles of lncRNA genes related to the promoter D-DMRs, we performed coding–non-coding gene co-expression (CNC) network analysis, and revealed they display strong association with gene expression regulation. We believe our comprehensive methylation analysis of lncRNA genes would help on a better understanding of molecular regulations that occur in human PED.Previous studies have shown that lncRNA genes have several characteristics which differ from those of protein-coding genes such as length, number of exons and level of expression , 33. Heret al. found the methylation density around TSS was markedly different between lncRNA and protein-coding genes in human H1 cell line and brain tissue, with a V-shaped curve in protein-coding genes and a sharp peak immediately downstream of the transcription start site (TSS) in lncRNA genes [P-value < 2.2 × 10−16). The different observation with the previous study may be due to the special cell stage, pre-implantation embryo, where carrying out the most dramatic genome-wide changes of the methylome. To rule out the influence due to lncRNA genes that fall within protein-coding genes, only lncRNA gene that lie at least 1 kb away from a protein-coding gene were analyzed.The methylation architecture in and around the protein-coding genes is important for gene expression and cell identity . In an eNA genes . HoweverP-value < 2.2 × 10−16).Several studies have shown that there is a strong correlation between CpG density and transcription initiation . We thusWe incorporated recently published single-cell transcriptome data to investigate the relationship between DNA methylation and lncRNA genes expression . As expeP-value < 2.2×10−16). We clustered the G-DMRs using k-means into 6 dynamic patterns separately, and found similar DNA methylation dynamics between lncRNA and protein-coding genes , play indispensable roles during human PED . We thuss Figure . Sperm-ss Figure . In addis Figure . TherefoP-value < 2.2×10−16; Figure P-value < 2.2×10−16; Figure DMRs among multiple samples , are regarded as potential functional regions involved in gene transcriptional regulation . In thisPrevious studies found that promoter D-DMRs are associated with genes that are thought to function in a specific manner . Here, wDNA methylation is an important form of epigenetic modification and serves multiple critical functions, including repression of gene transcription, maintaining genomic integrity, establishing parent-specific imprinting patterns, and repression of transposable elements , 19. DesResearch on PED is important for both reproductive biology and regenerative medicine. Besides, understanding the nature of reprogramming and totipotency of early embryos will enlighten the research on and utilization of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, PED is a special developmental stage, where a series of important distinctive developmental events happened, such as maternal-zygotic transition , ZGA 4242, and sBoth sperm and oocytes contain gamete-specific methylation patterns –22. TherThe identification of DMRs across multiple samples is important in genomic function analysis . Here, wIn summary, we provide the first methylation profiles of lncRNA genes during human PED. Several similarities between lncRNA and protein-coding genes were identified, including the methylation dynamics, TSS methylation distrbution, relationship between promoter and gene expression. The differences in promoter suggested the differential methylation regulation mechanisms between lncRNA and protein-coding genes. G-DMRs and D-DMRs analysis indicated that lncRNA genes contributed more to paternal methylation regulation than protein-coding genes. Our CNC network provide a large-scale network including epigenetically regulated lncRNA and their target genes for further biological research. Our results will help to understand the functions of lncRNA genes and their roles in human PED.The reduced representation bisulphite sequencing (RRBS) dataset of human pre-implantation embryos (GSE49828) was downloaded from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information. The dataset consists of 32 samples from each crucial stages of embryo development. The single-cell RNA-seq dataset of human pre-implantation embryos version is GSE36552, which includes 124 samples ranging from the oocyte to blastocyst stages.The annotations of lncRNA and protein-coding genes were obtained from the hg19 Refseq and NONCODE2016, respectively. Promoter were defined as 1 kb up- and downstream of the transcription start sites (TSSs). The annotated regions, such as CGIs, exons and introns were downloaded from UCSC tables with hg19 track.For RRBS data, sequencing reads were aligned to the hg19 using Bismark tools (version 0.14.5) with default parameters. For RNA-seq data, reads were aligned to hg19 using TopHat version 2.0.9. FPKM (fragments per kilobase of exon per million fragments mapped) was computed with Cuffquant and Cuffnorm.The methylation level of each CpG site was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Single CpG methylation levels were limited to those CpGs that had at least fivefold coverage. For 100-bp tiles, reads for all the CpGs that were covered more than fivefold within the tile were pooled and used to estimate the methylation level as described for single CpGs. The DNA methylation level of each sample is the average of the 100-bp tiles, while the DNA methylation level of each stage is the arithmetic average value of all biological replicates across each stage. The CpG density of every CpG site was calculated as the total numbers of all CpG dinucleotides located within 50 bp up- and downstream of that CpG site. The CpG density for a 100-bp tile is the average of the CpG density for all single CpGs usedto estimate methylation level in the tile.P < 0.05 given by multiple Student's t-test and a Benjamini-Hochberg false discovery rate (FDR) < 0.05.After quantifying the 100-bp tile DNA methylation levels using 100-bp-tile-based methylation calling algorithm, we systematically compared the DNA methylation levels of 100-bp tiles which were covered in both MII oocytes and sperm. We assigned these 100-bp tiles as G-DMRs only if the methylation level of these tiles is in one type of gametes greater than 75%, while in the other type ofgametes less than 25%, with a significant We applied QDMR (version 1.0) to analyze D-DMRs with default parameters. Each region was assigned an entropy value by QDMR based on the methylation levels for all the samples. The regions whose entropy is less than the thrshold were identified as D-DMRs.P-values for each gene pair was estimated through Fisher's asymptotic test implemented in the WGCNA library of R; (4) Keep only gens with the absolute value of Pcc > 0.8 and P-values < 0.05; (5) extract these gene pairs including the genes related to the promoter D-DMRs. The gene networks were visualized using Cytoscape 3.2.0.The single-cell RNA-seq dataset of human pre-implantation embryos were used to construct the coding-non-coding gene co-expression network, including following five steps: (1) we only keep the genes with maximal expression during PED more than 5 and expressional variance ranked in the top 75 percentile; (2) pearson correlation coefficient (Pcc) was computed using R ; (3) Pcc The Database for Annotation, Visualization and Integrated Discovery (DAVID) was a frequently-used bioinformatics resources for GO functional annotation. First, we upload gene lists to DAVID. And then, after selecting identifier for thes genes . Biological process, molecular fuction and cellular component terms was seleted as background gene sets respectively. Hypergeometric Exact test was used to measure gene-enrichment in background annotation terms.


Despite major advances in antiretroviral therapy against HIV-1, an effective HIV vaccine is urgently required to reduce the number of new cases of HIV infections in the world. Vaccines are the ultimate tool in the medical arsenal to control and prevent the spread of infectious diseases such as HIV/AIDS. Several failed phase-IIb to –III clinical vaccine trials against HIV-1 in the past generated a plethora of information that could be used for better designing of an effective HIV vaccine in the future. Most of the tested vaccine candidates produced strong humoral responses against the HIV proteins; however, failed to protect due to: 1) the low levels and the narrow breadth of the HIV-1 neutralizing antibodies and the HIV-specific antibody-dependent Fc-mediated effector activities, 2) the low levels and the poor quality of the anti-HIV T-cell responses, and 3) the excessive responses to immunodominant non-protective HIV epitopes, which in some cases blocked the protective immunity and/or enhanced HIV infection. The B-cell epitopes on HIV for producing broadly neutralizing antibodies (bNAbs) against HIV have been extensively characterized, and the next step is to develop bNAb epitope immunogen for HIV vaccine. The bNAb epitopes are often conformational epitopes and therefore more difficult to construct as vaccine immunogen and likely to include immunodominant non-protective HIV epitopes. In comparison, T-cell epitopes are short linear peptides which are easier to construct into vaccine immunogen free of immunodominant non-protective epitopes. However, its difficulty lies in identifying the T-cell epitopes conserved among HIV subtypes and induce long-lasting, potent polyfunctional T-cell and cytotoxic T lymphocyte (CTL) activities against HIV. In addition, these protective T-cell epitopes must be recognized by the HLA prevalent in the country(s) targeted for the vaccine trial. In conclusion, extending from the findings from previous vaccine trials, future vaccines should combine both T- and B-cell epitopes as vaccine immunogen to induce multitude of broad and potent immune effector activities required for sterilizing protection against global HIV subtypes. In an USAID global survey, approximately two million new HIV-1 cases were reported in 2015 alone, despite the increased use of the antiretroviral therapies (ART) to control the global spread of the infection . To furtThe current review will describe the findings important to the development of an effective HIV vaccine with emphasis on B-cell and T-cell vaccine epitopes through the coverage of the following topics: 1) Past and present understanding of HIV vaccination, 2) Conserved B-cell and T-cell epitopes on HIV, 3) B-cell epitopes generating broadly neutralizing antibodies (bNAbs), 4) Non–neutralizing antibodies (nAbs) during HIV vaccination and infection, 5) Conserved T-cell epitopes associated with anti-HIV activity(s), 6) Conserved T-cell epitopes associated with protective HLA allotypes prevanlent in HIV endemic countries, 7) Antibodies and T-cell responses enhancing HIV infection, and 8) Conserved T-cell and B-cell epitopes with potent anti-HIV activity for HIV vaccine.Vaccination changed the conventional disease management; an effective vaccine can reduce or even eradicate the infectious disease, which made vaccines as an essential tool against infectious agents. Several unsuccessful attempts made in the past in the development of such vaccines provided extensive information that can be used in the future for more effective vaccines against the virus. Here we reviewed several vaccination attempts and their cause of failures to protect against HIV-1. Since the initiation of the first phase-III clinical vaccine trial VAX004) in 1998, three independent phase-III clinical vaccine trials ended with marginal to no efficacy  4–6]. A in 1998,. A in 19The RV144 trial was minimally successful by conferring 31.2% protection in the combined low/medium/high-risk group but conferring only 3.7% protection in the high-risk group . Since tPrior to the completion of RV144 trial, two phase-IIb clinical vaccine trials  consisting of adenovirus type-5 (Ad5) vectored HIV gag/pol/nef vaccine were in progress and tested whether vaccines based solely on anti-HIV T-cell immunity could confer protection in vaccinated subjects  12–14].–14.12–14Since none of the vaccines in the Phase-III vaccine trials induced potent bNAbs, scientific efforts were subsequently focused on identifying the B-cell epitopes that induce potent bNAbs against global HIV subtypes –32. The An epitope is defined as the site on a molecule or an antigen recognized by either an antibody or B-cell receptor (BCR), or by T-cell receptor (TCR) recognizing the antigenic peptide bound to the major histocompatibility complex (MHC) –39. HencIn recent studies, a majority of bNAbs reacted to conformational epitopes than to linear epitopes  30,31,3,42 makinIn comparison, T-cell epitopes are short linear peptides processed from a protein antigen, and presented on MHC molecules for their recognition by TCR on T cell to induce its effector function(s). The conserved T-cell epitopes on HIV are identical or similar (homologous) in aa sequences and are conserved among the HIV isolates from the same subtype (type-specific) or from multiple HIV subtypes –46, and The bNAbs are those anti-HIV antibodies that potently neutralize a broad spectrum of heterologous HIV viruses including those among global HIV subtypes ,43. The The antigen binding site on an antibody that recognizes an antigenic epitope is called paratope of the antibody . AntibodAlthough few occluded bNAb-inducing Env epitopes  may remain relatively constant in aa sequence, mutation(s) at other viral Env site  of the viral escape variant(s) may expose the occluded bNAb epitopes ,64. For Many bNAbs were determined to be polyreactive and autoreactive to self-antigen(s) . These oin vitro study, Fc mutated variants of wildtype (wt) ADCC-mediating bNAb (b12) retained potent viral neutralization activity similar to wt bNAb but lost ADCC activity [Some nNAbs and many of the bNAbs have been reported to mediate ADCC and antibody-dependent cellular virus inhibition (ADCVI) of the infected cells ,74–76. Aactivity . Howeveractivity . In compNK cells, macrophages, dendritic cells, and neutrophils are the effector cells with FcRIIIa (CD16a) to mediate IgG-based ADCC activity ,76. ADCCSome nNAbs have been reported to enhance HIV infection by complement-mediated enhancement ,87 or byConserved HIV T-cell epitopes for an effective HIV vaccine should induce broad (multiple subtype specificities) and potent (high magnitude) immunity against HIV. Conserved epitopes are often subdominant epitopes since excessive immunity against them or mutations will affect the fitness of the virus ,93. In aThe conserved T-cell epitopes must be able to induce potent CD8+ CTL ,35, polyIn the modestly successful RV144 trial, the polyfunctional T-cell responses consisted of antigen-specific upregulation of IFNγ and IL-2 followed by TNF and IL-21 . Some ofA recent study demonstrated the importance of a vaccine capable of inducing CD8+ CTLs against a simian AIDS lentivirus, SIV. In this study, all macaques immunized with cytomegalovirus (CMV)-vectored SIV vaccine were positive for SIV infection shortly after the challenge with homologous SIV ,106. HowThe T-cell epitopes bound to HLA class I and II molecules are recognized by the TCRs on CD8+ and CD4+ T cells, respectively to exert their function(s). Certain HLA allotypes  confer resistance to HIV infection, while certain others increase susceptibility to the HIV infection –117. HIVAdditionally, certain HLA allotypes correlate with slow disease progression, while certain others correlate with a rapid progression of the disease as determined by virus load, CD4+ T-cell count, and/or disease status ,99. HLA versus susceptible HLA alleles may help identify HIV epitopes and their corresponding immunity required for prophylaxis. In contrast, studies evaluating the control of the HIV infection in HIV+ subjects in terms of slow or rapid HIV progression may identify HIV epitope recognition and immunity more important for immunotherapy. In any event, the T-cell epitopes on HIV identified by these approaches should be tested for their ability to induce potent and broad anti-HIV T-cell activities.The HIV transmission studies evaluating resistant via NF-κB pathway [Non-protective T-cell epitopes can induce either neutral or enhancing effect on HIV infection. For instance, stimulation of CD4+ T cells can cause enhancement of HIV infection –133. Aut pathway –137. Act pathway , whereas pathway ,140. In  pathway –14,21. F pathway . However pathway ,143. In  pathway ,8. There pathway .As discussed above, significant progress has been made to identify the protective B-cell and T-cell epitopes for a highly effective HIV vaccine. Such HIV vaccine should consist predominantly of anti-HIV conserved T-cell epitopes  and seleUntil minimally constructed, highly potent bNAb conformational epitopes become available, the use of bNAb linear epitopes  and most potent NAb linear epitopes from multiple subtypes  should be combined as mosaic vaccine and tested in SHIV/macaque model. Finally, besides bNAbs and type-specific NAbs, every effort should be made to include other conserved B-cell epitopes such as those that induce potent and broad ADCC/ADCVI antibodies as well as conserved T-cell epitopes that induce potent and broad anti-HIV polyfunctional T-cell responses and anti-HIV CD8+ and CD4+ CTLs. T cell-based conserved-mosaic vaccines were shown to enhance the breadth and potency of epitope recognition . In anot


PET studies have shown increased tracer retention in patients clinically diagnosed with dementia of AD type and mild cognitive impairment in regions that are known to contain tau lesions. In vivo uptake has also consistently been observed in midbrain, basal ganglia and choroid plexus in elderly individuals regardless of their clinical diagnosis, including clinically normal whose brains are not expected to harbor tau pathology in those areas. We and others have shown that [F-18]-AV-1451 exhibits off-target binding to neuromelanin, melanin and blood products on postmortem material; and this is important for the correct interpretation of PET images. In the present study, we further investigated [F-18]-AV-1451 off-target binding in the first autopsy-confirmed Parkinson’s disease (PD) subject who underwent antemortem PET imaging. The PET scan showed elevated [F-18]-AV-1451 retention predominantly in inferior temporal cortex, basal ganglia, midbrain and choroid plexus. Neuropathologic examination confirmed the PD diagnosis. Phosphor screen and high resolution autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined with the exception of neuromelanin-containing neurons in the substantia nigra, leptomeningeal melanocytes adjacent to ventricles and midbrain, and microhemorrhages in the occipital cortex , in addition to incidental age-related neurofibrillary tangles in the entorhinal cortex. Additional legacy postmortem brain samples containing basal ganglia, choroid plexus, and parenchymal hemorrhages from 20 subjects with various neuropathologic diagnoses were also included in the autoradiography experiments to better understand what [F-18]-AV-1451 in vivo positivity in those regions means. No detectable [F-18]-AV-1451 autoradiographic binding was present in the basal ganglia of the PD case or any of the other subjects. Off-target binding in postmortem choroid plexus samples was only observed in subjects harboring leptomeningeal melanocytes within the choroidal stroma. Off-target binding to parenchymal hemorrhages was noticed in postmortem material from subjects with cerebral amyloid angiopathy. The imaging-postmortem correlation analysis in this PD case reinforces the notion that [F-18]-AV-1451 has strong affinity for neurofibrillary tau pathology but also exhibits off-target binding to neuromelanin, melanin and blood components. The robust off-target in vivo retention in basal ganglia and choroid plexus, in the absence of tau deposits, meningeal melanocytes or any other identifiable binding substrate by autoradiography in the PD case reported here, also suggests that the PET signal in those regions may be influenced, at least in part, by biological or technical factors that occur in vivo and are not captured by autoradiography. TheLiterature on [F-18]-AV-1451 PET imaging in patients clinically diagnosed with α-synucleinopathies is still scarce , 20, 28.We have had the opportunity to study in detail the [F-18]-AV-1451 imaging-pathologic correlates in an autopsy-confirmed PD case and have used this to investigate the off-target in vivo signal observed in this patient in midbrain, basal ganglia, choroid plexus and some focal areas in the cortex. Additional legacy postmortem material containing basal ganglia, choroid plexus and parenchymal hemorrhages from 20 subjects ) were also studied for comparison purposes to better understand what [F-18]-AV-1451 in vivo positivity in those regions means.The study was approved by the local Institutional Review Board and informed consent for neuroimaging and autopsy was obtained for each subject. Demographic and postmortem data are shown in Table The PD subject underwent [F-18]-AV-1451 PET scan 12 months prior to death. [F-18]-AV-1451 PET  data were acquired using a Siemens/CTI  ECAT HR+ scanner . PET data were then reconstructed and attenuation corrected, and each frame was evaluated to verify adequate count statistics and absence of head motion. T1-weighted MRI images were acquired on a 3 T Tim Trio (Siemens) and segmented using Freesurfer (FS) as previously described [http://amide.sourceforge.net) [[F-18]-AV-1451 PET images were co-registered and fused with 3 T MRI images. A manual method was used to identify and match 31 ROIs defined on postmortem 10 mm-thick coronal brain slabs and aligned visually to the corresponding coronal T1-weighted MRI images with coregistered PET images. To optimize correspondence between ROIs defined on PET images and their pathological substrate, we used the gyral and ventricular morphology on tissue slabs to guide visual identification of matching ROIs on MRI-T1 images, using a dynamic resampling of MRI in planes of view that matched the tissue blocks planes of sectioning. ROIs were then drawn manually on MRI-T1 images using the medical software AMIDE v.1.0.5  , 52, expIn addition to the PD case, legacy postmortem brain material from 20 representative cases with various neuropathologic diagnoses  from the Massachusetts Alzheimer’s Disease Research Center (MADRC) Neuropathology Core were included in this study Table . These aFrozen tissue blocks from the left hemisphere from the index case containing multiple ROIs along with blocks containing basal ganglia, choroid plexus, and parenchymatous hemorrhages from the 20 additional cases Table  were sec[F-18]-AV-1451 phosphor screen and high resolution nuclear emulsion autoradiography were performed following protocols previously described in detail elsewhere . SDD-AGEp < 0.05. Statistical analysis and graphs were generated using GraphPad Prism v6.0 software .Correlations between [F-18]-AV-1451 in vivo uptake (SUVRs) and SDD-AGE total-tau and PHF-tau measurements in different ROIs were performed using a linear regression test. Significance was set at The patient was a Caucasian male who developed progressive stiffness in his left extremities in his early 50s, along with resting tremor and bradykinesia in his left hand. He received a clinical diagnosis of idiopathic PD and had a favorable response to L-DOPA therapy. His symptoms remained stable for many years but he subsequently developed hypophonia and stuttering speech, constipation, postural instability with falls, dysphagia and occasional visual illusions. His cognition was only mildly impaired 1 year before his death , left middle frontal and left occipital cortices  deposition in the SN and entorhinal cortex (EC), consistent with a diagnosis of PD Braak Stage 3 . There wAs expected, given the absence of tau pathology in this case , phosphor screen autoradiography failed to show detectable [F-18]-AV-1451 binding in most ROIs analyzed. The only exceptions, exhibiting both autoradiography signal and elevated in vivo retention, were EC (#10), substantia nigra (#18), thalamus (#12) and occipital cortex (#16) Fig. . High reOf note, putamen and pallidum (#2 and #11) were among the regions showing the highest in vivo tracer retention in this patient  Neuropathology Core, including 4 AD, 4 controls (CTL), 2 PSP, a Pick disease (PiD) case and a DLB case, were also examined  cases containing multiple parenchymal hemorrhages were also included in autoradiography experiments. In agreement with our previously published observations , [F-18]-This is the first imaging-pathological correlation of novel PHF-tau PET tracer F-18]-AV-1451 in an autopsy-confirmed PD case with minimal-to-none AD co-pathology. The study of this single case has been particularly informative to learn new and valuable information about the frequently observed in vivo off-target retention of this tracer in brain regions like midbrain, basal ganglia and choroid plexus, and investigate the underlying substrate/s that may be responsible for such signal in the absence of brain tau pathology. Importantly, this same PET pattern is frequently observed in elderly individuals, including those clinically normal -AV-1451 , 40, 45.Our experiments using [F-18]-AV-1451 phosphor screen and high resolution autoradiography in multiple brain regions from this PD case showed that this tracer bound with strong affinity to age-related NFTs in the EC, neuromelanin-containing neurons in the substantia nigra and leptomeningeal melanocytes adjacent to the lateral ventricles, and to a lesser extent to microhemorrhages in the cortex. All these findings are consistent with our previously published observations , 33. In In our previous studies we observed robust off-target binding of [F-18]-AV-1451 to neuromelanin- and melanin-containing cells and alerted on the importance of carefully taking this finding into account when interpreting [F-18]-AV-1451 in vivo retention patterns , 33. OthAs noted above, elevated in vivo F-18]-AV-1451 retention in basal ganglia has been observed in a significant proportion of elderly individuals with different clinical diagnosis, including AD -AV-1451  and non-, Fig. To further investigate the mismatch between elevated in vivo F-18]-AV-1451 retention in basal ganglia and lack of autoradiography signal in this region, we performed [F-18]-AV-1451 phosphor screen and high resolution autoradiography in basal ganglia sections from 12 cases with various neurodegenerative diseases . Of note, due to the close location of choroid plexus to medial temporal lobe structures, elevated in vivo signal in this area can potentially interfere with assessment of “true” tracer retention in the hippocampus and entorhinal cortex; thus, it is important to understand the underlying substrate of tracer’s uptake in the choroid plexus. Our autoradiography study of postmortem tissue samples, which included choroid plexus from 6 individuals, detected tracer binding in three of them corresponding to the presence in these cases of abundant leptomeningeal melanocytes (see representative cases in Fig. In our PD case we also noted increased in vivo [F-18]-AV-1451 retention in focal areas of frontal and occipital cortices in the left hemisphere. Our autoradiography experiments revealed, in the limited number of sections analyzed, the presence of tracer binding to an occipital microhemorrhage. It is conceivable that our PD case may harbor additional microhemorrhages that would only be revealed by extensive brain sampling. The analysis of additional legacy postmortem material from two CAA cases harboring multiple brain hemorrhages further confirmed tracer binding to those lesions in autoradiography Fig. . This isIn conclusion, the imaging-pathologic correlation analysis of the first autopsy-confirmed PD patient who underwent [F-18]-AV-1451 PET scan prior to death confirms that this tracer not only binds with strong affinity to NFT tau pathology in AD, but also exhibits off-target binding to neuromelanin and melanin-containing cells and, to a lesser extent, to brain hemorrhagic lesions. These substrates likely explain, at least in part, the enhanced PET in vivo signal frequently noticed in midbrain, basal ganglia and choroid plexus regardless of the clinical diagnosis and of the presence or absence of tau-containing lesions in those regions. However, the robust off-target in vivo retention in basal ganglia and choroid plexus, in the absence of tau deposits, meningeal melanocytes or any other identifiable binding substrate by autoradiography in the PD case reported here, suggests that differential uptake and clearance profiles of this compound in these brain regions deserve to be further investigated. All together these data offer new important clues for the accurate interpretation of the patterns of [F-18]-AV-1451 retention observed by in vivo neuroimaging. Additional imaging-pathological studies on postmortem material from individuals studied by imaging methods prior to death will continue to provide insight into the implications of [F-18]-AV-1451 signals.


Schizophrenia is a highly heterogeneous disorder, and around a third of patients are treatment-resistant. The only evidence-based treatment for these patients is clozapine, an atypical antipsychotic with relatively weak dopamine antagonism. It is plausible that varying degrees of response to antipsychotics reflect categorically distinct illness subtypes, which would have significant implications for research and clinical practice. If these subtypes could be distinguished at illness onset, this could represent a first step towards personalised medicine in psychiatry. This systematic review investigates whether current evidence supports conceptualising treatment-resistant and treatment-responsive schizophrenoa as categorically distinct subtypes.A systematic literature search was conducted, using PubMed, EMBASE, PsycInfo, CINAHL and OpenGrey databases, to identify all studies which compared treatment-resistant schizophrenia  to treatment-responsive schizophrenia (defined as known response to non-clozapine antipsychotics).Nineteen studies of moderate quality met inclusion criteria. The most robust findings indicate that treatment-resistant patients show glutamatergic abnormalities, a lack of dopaminergic abnormalities, and significant decreases in grey matter compared to treatment-responsive patients. Treatment-resistant patients were also reported to have higher familial loading; however, no individual gene-association study reported their findings surviving correction for multiple comparisons.Tentative evidence supports conceptualising treatment-resistant schizophrenia as a categorically different illness subtype to treatment-responsive schizophrenia. However, research is limited and confirmation will require replication and rigorously controlled studies with large sample sizes and prospective study designs.The online version of this article (doi:10.1186/s12888-016-1177-y) contains supplementary material, which is available to authorized users. Schizophrenia is highly heterogeneous and better characterisation of this heterogeneity is needed to progress research into aetiology, mechanisms and treatment. While the majority of patients with schizophrenia respond to typical or atypical non-clozapine antipsychotics, roughly a third of patients do not respond well and are considered treatment-resistant . DiffereEvidence suggests that treatment-resistance is a stable trait, as an early lack of response to treatment has been consistently shown to predict poor treatment outcome and diagnosis of treatment-resistance –5. FirstThe dopamine hypothesis  is arguaApproximately a third of patients are treatment-resistant , 8, thouAs Farooq et al. comment , classifOur inclusion criteria were: original data comparing patients with treatment-resistant and treatment-responsive schizophrenia; all patients have a primary diagnosis of schizophrenia or schizoaffective disorder; the definition of treatment-resistance incorporates (as a minimum) two failed antipsychotic trials; the treatment-responsive group comprises of patients with a known response to non-clozapine antipsychotics; publication in a peer-reviewed journal.Studies were excluded if: treatment-resistance was classified based on only one antipsychotic trial; treatment-responsiveness was defined solely as not meeting treatment-resistant criteria; the group labelled “treatment-responsive” included patients on clozapine; if studies of clinical variables were cross-sectional .th January 2015, including Medline, Embase, PsycInfo and PsycArticle), PubMed (on 2nd February 2015) and CINAHL (on 2nd February 2015). The grey literature database OpenGrey was also searched on 2nd February 2015. Search terms included synonyms for schizophrenia and treatment-resistance  10% of titles and abstracts identified by the database search and b) 20% of full-text articles identified by title and abstract screening. Cohen’s kappa was calculated to provide a conservative measure of inter-rater reliability; this produced kappas of 0.8 and 1 respectively, indicating substantial agreement. If the second reviewer identified novel potentially eligible studies, these were re-screened collaboratively.A standard data extraction form was used , electroencephalography (EEG), positron-emission tomography (PET), and magnetic resonance spectroscopy (MRS). All report controlling for basic demographics like age and sex but only three , 18, 27 Two investigated brain volume, measured by MRI. In a study of 37 New Zealand patients , Anderson et al. reported significantly lower grey matter volumes, especially in cortical areas, for treatment-resistant patients, but no differences in whole brain or white matter volume . Ultra-tIn the same study, Molina et al. also investigated P300 parameters - the late component of the event-related potential - with EEG but found no differences between responsive and resistant patients .One study compared striatal dopamine synthesis capacity with PET measurement of [18 F]-DOPA uptake, in a matched-design study of 24 British patients  . No partTwo studies measured regional brain metabolite concentrations using MRS. Demjaha et al.  carried There were nine gene-association studies, with no eligible genome-wide association study (GWAS) papers. All of them used white participants.G169A polymorphism  or the C270T polymorphism [dinucleotide repeat polymorphism than treatment-responsive patients [Two investigated polymorphisms in the brain-derived neurotrophic factor (BDNF) gene. In a study of 94 Finnish patients , no group differences were found for the morphism . In a stpatients .T102C polymorphism. In a study of 102 Canadian patients , a significantly higher frequency of the C/C genotype in treatment-resistant patients was reported [Two studied the serotonin 2A receptor (5-HT2A) gene, specifically the reported  and a streported .The latter also reported a significantly higher frequency of the C/A genotype for the tryptophan hydroxylase enzyme (TPH1) gene in treatment-resistant patients, but no group differences for the guanine-nucleotide-binding protein (GNB3) gene .5’UTR (ccG repeat) polymorphism of the reelin gene, a significantly higher frequency of ccG10 alleles in treatment-resistant patients was found [In a study of 193 French patients  considering the as found .Another study investigated the regulator of g-protein signalling (RGS4) gene in a study of 93 Finnish patients , but found no group differences .Bal I polymorphism, was examined in a study of 89 French patients  which reported significantly less homozygosity for treatment-resistant patients [The dopamine receptor 3 (DRD3) gene, specifically the patients .HLA-A1 allele frequency for treatment-resistant patients [A study of 38 Finnish patients  investigated the human leukocyte antigen (HLA) genotypes and found significantly higher patients . In contUnfortunately, none of these findings are reported to survive multiple-testing correction: four do not report correction , 34, 36 There was one study of familial genetic loading and prevalence in relatives, which studied the prevalence of schizophrenia spectrum disorders, cluster A personality disorders and long-term psychiatric care in relatives, and calculated family loading scores and morbidity risks . In a stTwo studies investigated whether clinical variables were associated with subsequent treatment response or resistance.There was one prospective study  of PositMeltzer et al. studied age of onset . In a stThere were two papers looking at neurocognitive function.Joober et al. examined 75 Canadian patients  . TreatmeDe Bartolomeis et al. investigated verbal memory, working memory, motor speed, verbal fluency, processing speed and executive functions, in a study of 41 Italian patients  . The onlThere was one study measuring demographic characteristics. Meltzer et al.– described above - studied gender and ethnicity . The stuThis is the first systematic review directly addressing whether treatment-resistant schizophrenia is categorically different from treatment-responsive schizophrenia, a question with substantial implications for research and clinical practice. It is somewhat unusual for a systematic review to address a theoretical question like this, but the benefits of comprehensiveness, reduced bias, and replicability remain desirable. To ensure that findings were not biased towards research fields that unduly influenced the conclusions, a broader search strategy than is conventional was necessary. This provides a comprehensive overview allowing the emergence of a pattern across research fields. It also highlights the areas lacking in quantity or quality of research. Another strength of this review is the focused definitions of treatment-resistance and treatment-responsiveness; they relate to clinical practice for diagnosing and treating treatment-resistance and provide a comparison between two groups of patients with distinctly different responses to antipsychotics.However, this study does have limitations. Firstly, while every effort was made to seek out all available research there is a stronger chance of publication bias for non-RCT studies, as there are less protective measures  in place; however, we did identify several studies reporting null findings . Secondly, despite not excluding non-English language papers, papers were largely Western and participants were largely white. Thirdly, significant heterogeneity remained in definitions of treatment-resistance and treatment-responsiveness and in the populations and methods used to recruit participants, making comparison different. Fourthly, in comparing based on treatment outcome, antipsychotic response and resistance are not readily separable from group differences in illness symptomatology or severity. Also, the binary comparison of treatment-resistant patients to confirmed treatment-responders arguably ignores some of the complexity of the disorder. Not only can treatment-response be considered along a spectrum , but schTwo studies identified in this review indicate that treatment-resistant schizophrenia may be differentiated from treatment-responsive schizophrenia by measures of dopaminergic function: specifically, lower levels of striatal dopamine synthesis capacity, equivalent to levels in healthy volunteers , and lesIf these findings reflect that treatment-resistant patients lack the striatal dopaminergic elevations typically detected in schizophrenia, this could explain why treatment-resistant patients show little response to D2 dopamine receptor blockade with conventional antipsychotic treatment . This woThe studies identified report that treatment-resistance may be differentiated from treatment-responsive schizophrenia by predominance of the C allele of the T102C polymorphism of the 5HT2A gene (encoding the main excitatory serotonin receptors) , 33 and Nonetheless, other studies  have found that lower 5-HT plasma levels, as well as a subsequent increase during treatment, predict poorer response to atypical antipsychotics, with responders showing equivalent levels to healthy volunteers . SimilarThe identified studies indicate that treatment-resistance may be differentiated by abnormalities in brain glutamate concentrations not seen in treatment-responsive patients; as with the dopamine findings, this again suggests a categorical difference rather than one of severity. A study by our group found higher levels of glutamate in the ACC in treatment-resistant but not treatment-responsive patients compared to healthy volunteers . We haveOther reviews of neuroimaging studies of treatment-resistance have differed in their interpretation of this literature, with a shift towards confidence that glutamatergic differences exist. One 2015 review  reportedOne possible mechanism underlying the efficacy of clozapine in treatment-resistant illness may relate to its’ ability to attenuate glutamate release, as demonstrated in several rat studies , 55. TheStudies identified by this review find that treatment-resistant patients have reduced grey matter volumes in comparison to treatment-responsive patients , 29. TheDespite evidence that clozapine induces distinctive structural changes in grey matter , one stuThese findings are consistent with the two other reviews of treatment-resistant vs treatment-responsive patients , 53 whicIn contrast to our discussion above, Mouchlianitis et al. conclude that grey matter abnormalities appears to be a difference of degree along a continuum ; howeverRegarding white matter, Molina et al.  report tStudies in this review indicate that treatment-resistant schizophrenia may be differentiated from treatment-responsive schizophrenia by shorter alleles of the dinucleotide repeat polymorphism , but notThe null result for Val66Met is somewhat surprising, as two larger studies have found robust associations when comparing clozapine users to non-clozapine users  , and in Studies identified also suggest that treatment-resistance may be differentiated from treatment-responsive schizophrenia by greater repetition of the CCG repeat for the 5’UTR polymorphism on the reelin gene . HoweverOne study identified indicates that treatment-resistant schizophrenia may be differentiated from treatment-responsive schizophrenia by higher familial loading scores and greater risk of schizophrenia in relatives; in fact, the study found that treatment-responsive patients did not show the significantly higher risk compared to healthy volunteers that treatment-resistant patients did . An earlOne study identified indicates that treatment-resistance may be differentiated from treatment-responsive schizophrenia by predominance of the HLA-A1 allele  but anotWider literature has indicated the importance of the immune system in treatment-response, with two studies finding that treatment-resistant patients show significantly higher levels of serum IL-6 levels than healthy volunteers (with non-resistant patients presenting with intermediate levels) , 71  create challenges for interpreting the literature .Addressing the inconsistency of treatment-resistant definitions and the comparison groups used would be of great value for future research, especially when attempting to determine if there are categorical differences between these groups of patients. We recommend that the field develops and follows a consensus for more strictly defining and referring to treatment-resistance, incorporating the clinical guidelines as a minimum. This is underway with the Treatment Response and Resistance in Psychosis (TRRIP) Working Group, with guidelines in press . In addiFuture studies should also seek to both further replicate the findings of glutamatergic and grey matter abnormalities, and advance knowledge in areas where there are contradictions or a simple lack of research. Investigating neurochemical differences between treatment-responsive and treatment-resistant patients may be of particular importance, as these could inform different approaches to pharmacological intervention. Neuroimaging studies may also use resting state data to investigate functional connectivity, as a recent study  and a re


Lumateperone (ITI-007) is a first-in-class investigational agent in development for the treatment of schizophrenia. Acting synergistically through serotonergic, dopaminergic and glutamatergic systems, lumateperone represents a new approach to the treatment of schizophrenia and other neuropsychiatric disorders. Lumateperone is a potent antagonist at 5-HT2A receptors and exhibits serotonin reuptake inhibition. Lumateperone also binds to dopamine D1 and D2 receptors acting as a mesolimbic/mesocortical dopamine phosphoprotein modulator (DPPM) with pre-synaptic partial agonism and post-synaptic antagonism at D2 receptors and as an indirect glutamatergic (GluN2B) phosphoprotein modulator with D1-dependent enhancement of both NMDA and AMPA currents via the mTOR protein pathway.Lumateperone was evaluated in 3 controlled clinical trials to evaluate efficacy in patients with acute schizophrenia. In Study ITI-007-005, 335 patients were randomized equally across 4 treatment arms: one of two doses of lumateperone, risperidone (active control) or placebo QAM for 4 weeks. In Study ITI-007-301, 450 patients were randomized equally across 3 treatment arms: one of two doses of lumateperone or placebo QAM for 4 weeks. In Study ITI-007-302, 696 patients were randomized equally across 4 treatment arms: one of two doses of lumateperone, risperidone (active control) or placebo QAM for 6 weeks. In all 3 studies, the primary endpoint was change from baseline on the Positive and Negative Syndrome Scale (PANSS) total score compared to placebo. Also, a 6-week open-label safety switching study was conducted. In this ITI-007-303 study 302 patients with stable schizophrenia were switched from standard-of-care (SOC) antipsychotics and treated for 6 weeks with lumateperone QPM outpatient and then switched back to SOC for 2 weeks.Two of the 3 randomized studies were positive. In Studies ITI-007-005 and ITI-007-301, lumateperone  met the primary endpoint with statistically significant superior efficacy over placebo at Day 28 as measured by the PANSS total score . In Study ITI-007-302, neither dose of lumateperone separated from placebo on the primary endpoint in the intent-to-treat population; a high placebo response was observed in this study. Across all 3 efficacy trials, lumateperone improved symptoms of schizophrenia with the same trajectory and same magnitude of improvement from baseline on the PANSS total score.Lumateperone was well-tolerated with a favorable safety profile in all studies. In the two studies with risperidone included as an active control, lumateperone was statistically significantly better than risperidone on key safety and tolerability measures including prolactin, glucose, lipids and weight. In the open-label safety switching study statistically significant improvements from SOC were observed in body weight, cardiometabolic and endocrine parameters worsened again when switched back to SOC medication. In this open-label study, symptoms of schizophrenia generally remained stable or improved. Greater improvements were observed in subgroups of patients with elevated symptomatology such as those with comorbid symptoms of depression and those with prominent negative symptoms.Lumateperone represents a novel approach to the treatment of schizophrenia with a favorable safety profile in clinical trials. The lack of metabolic, motor and cardiovascular safety issues presents a safety profile differentiated from standard-of-care antipsychotic therapy.


Over the past few years, microRNAs (miRNAs) have not only emerged as integral regulators of gene expression at the post-transcriptional level but also respond to signalling molecules to affect cell function(s). miRNAs crosstalk with a variety of the key cellular signalling networks such as Wnt, transforming growth factor-β and Notch, control stem cell activity in maintaining tissue homeostasis, while if dysregulated contributes to the initiation and progression of cancer. Herein, we overview the molecular mechanism(s) underlying the crosstalk between Wnt-signalling components  and miRNAs, as well as changes in the miRNA/Wnt-signalling components observed in the different forms of cancer. Furthermore, the fundamental understanding of miRNA-mediated regulation of Wnt-signalling pathway and vice versa has been significantly improved by high-throughput genomics and bioinformatics technologies. Whilst, these approaches have identified a number of specific miRNA(s) that function as oncogenes or tumour suppressors, additional analyses will be necessary to fully unravel the links among conserved cellular signalling pathways and miRNAs and their potential associated components in cancer, thereby creating therapeutic avenues against tumours. Hence, we also discuss the current challenges associated with Wnt-signalling/miRNAs complex and the analysis using the biomedical experimental and bioinformatics approaches. The Wnt proteins comprise a highly conserved and diverse family of genes found in humans, mice, Xenopus, Zebrafish and Drosophila  The pathger RNAs . Recent The canonical Wnt-signalling cascade refers to the transduction of series of signals mediated via the interaction of specific Wnt ligands with their target receptor resulting in the accumulation of β-catenin Fig. . AmassmeOFF-state), the membrane receptor complex is inactivated, thereby preventing the clustering of a trimeric complex composed of Wnt ligand/Fzd/LRP receptors and eventually results to β-catenin ubiquitination and degradation , but also plays a significant rate-limiting role in the initial stages of majority of sporadic colorectal cancers [In the absence of Wnt ligand interaction , to Frizzled and LRP 5/6 co-receptors at the cell surface [myc, c-jun, Axin2, EphB/ephrin-B and CyclinD1. These target genes all play crucial roles in the regulation of a plethora of cellular processes including but not restricted to cell proliferation, cell division, stem cell maintenance as well as regulation of various stages of embryogenesis [Conversely, the  surface , 17. Add surface , with st surface . Formati surface , 21, whi surface , 23 to aogenesis , hence aogenesis , 25–27. ogenesis –31.2+, the Wnt/JNK and the Wnt/planar cell polarity (PCP) pathways  are composed of ~22 nucleotide sequences in length and belong to the class of non-coding endogenous small RNAs. The functions of miRNAs in posttranslational gene regulation involves the direct interaction of miRNA with the 3’ un-translated region (UTR) of the target messenger RNAs , 50. In Additionally, apart from the canonical miRNA biogenesis pathways described above, alternative mechanisms, particularly through mRNA splicing , have bein-vitro [Numerous scientific evidences have implicated miRNAs as regulators of the Wnt-signalling pathway in the context of embryonic , osteoblin-vitro . The netin-vitro , colorecin-vitro , 71, brein-vitro , liver cin-vitro  and othein-vitro –78. For in-vitro . This rein-vitro .Another miRNA, miR-200a, has been identified as a potential negative regulator of the Wnt/β-catenin signalling pathway. This miRNA targets mRNAs of Zeb1 and Zeb2 which are repressors of E-cadherin. Therefore, by degrading Zeb1 and Zeb2 mRNA, E-cadherin becomes abundant and available for binding with β-catenin thereby forming E-cadherin-β-catenin complex which promotes cell-cell adhesion . The recThe controversial cancer stem cell (CSC) theory is based on the phenomenon that cancer cells may be derived from a rare population of cells possessing stem cell properties –87. ExpeRecent studies have established the importance and significant roles played by the canonical Wnt-signalling in stem cell self-renewal activities in various tissues and cell types . For insThe role of miRNA in the regulation of stemness of CSCs is another aspect of miRNA activity currently under investigation. Reports suggest that miR-193a expression inhibits tumourigenicity and invasiveness by directly targeting KRAS and plasminogen activator urokinase (PLAU), respectively, with these two factors highly expressed in human colon adenocarcinomas , 95. AnoAs critical biological modulators, it is no more news that miRNAs acts to suppress or facilitate cancer and tumour development by interacting with targets of the Wnt-signalling pathway. A summarized description showing the alterations of miRNAs/Wnt/β-catenin signalling in cancer has been provided Table . A more Following the initial discovery of the association of miRNA with cancer over more than a decade ago, technological advances that have produced multiple high-throughput bioinformatics methods designed for profiling miRNA expression and identification of miRNA targets involved in a plethora of pathways that regulate normal physiology and various diseases have being established . CompareThe challenging enormity of tumour heterogeneity, both in the primary tumour and metastasis has become explicit. Although, individual interactions between components of the canonical Wnt pathway and miRNAs in normal and cancer state have already been established , 111, weIn the following section we will briefly describe the different bioinformatics approaches that have been utilized in recent advances to identify and characterize Wnt/β-catenin-regulated miRNAs and their associated targets as well as the experimental strategies equally used for the validation of bioinformatics data Fig. .Fig. 3BiUGUAAACAUCCUUGACUGGAAG-3′ in mature miR-30e sequences derived from human, mouse, and rat. Additionally, the binding properties of β-catenin/TCF4 with miR-30e were validated and confirmed by performing EMSA and super-shift assay.Recent advances in next-generation DNA sequencing combined with chromatin immuno-precipitation (ChIP-Seq) have provided methods ideal for identification of transcription factor binding sites (TFBSs) with exceptional sensitivity as well as predicting mRNA targets of miRNA . ChIP haSimilarly, ChIP experiments carried out in mESCs and P19 cells showed that Tcf3 binds directly to the miR-302 promoter regions to repress the transcription of the miR-302 gene , thus suAPC, CTNNB1 and TCF4 genes and other components of the Wnt-signalling pathway as key oncogenic drivers in different forms of cancers [As previous studies have implicated mutations of  cancers , 123, ap cancers . Importa cancers .There have been several recent papers detailing ChIP-Seq limitations, for examples difficulty in transferring and maintaining data due to the large file sizes , and ideThe comprehensive and efficient study of miRNA expression profiling has been made convenient via the evolution of next generation sequencing technologies. Our understanding of the regulatory roles of miRNAs in gene expression, and association with diseases triggering multiple changes in gene expression levels facilitating cancer and tumour progression has been improved upon by advances in miRNA expression profiling research , which cTCF4 was observed to contain three mir-802 binding sites in its 3′ UTR with additional validation experiments revealing a significant correlation with Zeb1 and miR-21. Several studies have also confirmed Zeb1 as a key Wnt target [Identification of Wnt-regulated miRNAs through expression profiling methods can be achieved by enforced expression of the Wnt ligand which consequently results to the subsequent activation of both or either canonical or non-canonical Wnt-signalling pathways depending on the type of Wnt ligand. Specifically, expression levels of individual components that are crucial to the progression of the cascade such as APC, β-catenin and Tcf4 transcription factors can be matched with different miRNAs with the aim of observing patterns of miRNAs regulated by the Wnt pathway. For instance, characterization of non-coding transcriptomes of tissues derived from six normal pancreas and pancreatic cancer (PDAC) tissues using high-throughput NGS-based technology algorithms such as small RNA-sequencing (sRNA-Seq) and Massive Analysis of cDNA Ends (MACE) identified miR-802 as a negative regulator of TCF4 , which ut target , 133 as t target . Some ott target , miRGen t target , miRBaset target , miRExprt target  and CLIPt target .Notwithstanding, traditional platforms for the execution of miRNA expression profiling have also been useful in identifying miRNA targets of the Wnt-signalling pathway and vice-versa. In colorectal cancer, global miRNA expression profiling carried out on 13 cancer and adjacent normal samples showed a significant down-regulation of 61 and up-regulation of 42 miRNAs with enrichment in pathways promoting tumour progression such as the Wnt pathway in addition to TGF-β and MAPK pathways . Also, fA major limitation/complication of miRNA expression profiling includes the recent discovery of isomiRNAs that tend to display sequence variations by shortening or lengthening of the 3’ end , 148. UnCrosslinking and immunoprecipitation sequencing (CLIP-Seq) offers the luxury of analysing RNA/protein interaction. This approach is similar to RIP-Seq, however, stabilization of protein-RNA complexes are achieved by ultra-violet (UV) crosslinking. UV crosslinking is used as an ideal crosslinking agent in RNA studies due to the inability for UV crosslinks to form between proteins. miRNAs are subsequently reverse transcribed to cDNA and analysed with sequencing (RNA-Seq) for mapping of miRNA binding sites on their target mRNAs with high confidence . These sCLIP-Seq and target prediction studies effectively identify individual mRNAs regulated by multiple miRNAs, therefore proving that the transcriptional regulation of a single gene may be dependent on the combined effect of multiple miRNAs. Next-generation sequencing of RNA (RNA-Seq) allows for the generation of tissue-specific gene expression profile data which can be useful in developing a novel pathway analysis methodology for the prediction of miRNA function. Some web applications used for the identification of miRNA-regulated pathways in a tissue specific manner include miTALOS v2 , ToppMirAlthough experimental studies focused specifically on the identification of miRNAs that regulate the Wnt-signalling pathway are scarce, pathway analysis using CLIP-Seq of the miR-200 cluster family (miR-200b/c/miR-429 and miR-200a/miR-141) which has been established to be involved in cell migration and EMT, was able to identify a connection between miR-200b/c/miR-429 and the Wnt-signalling pathway in hepatocellular carcinoma (HCC) and liver fibrosis tissue samples . AdditioA major limitation of CLIP-Seq however is the inability to reduce background noise/signals due to the nature of high-throughput sequencing. For instance, Matthew et al.  reportedFollowing the elucidation of Wnt-mediated regulation of miRNAs or vice versa, potential candidate targets are confirmed and validated by additional experimental analyses that interrogate the pathological and physiological implications of the discovered regulations. These validations can be performed via two conventional strategies: an approach involving the exposure of a tumour tissue/sample to genetically altered oncogenic candidates, or an approach which involves the systematic manipulation of oncogenic candidates into becoming wild-type tissues . The latGain-and-loss-of-function studies are one of the most efficient approaches employed to validate the oncogenic/tumour suppressor potential of a target gene following NGS methodologies. However, in comparison to the relative potency of numerous overexpression strategies, the methodologies developed for miRNA downregulation appear to be less robust , 159. MoFor instance, using HT29 and HCT116 CRC cells transfected CRISPR/cas9 vectors were able to reduce the expression levels of mature miR-200c as well as miR-141 and miR-17 by 96 % , with reDespite the outstanding potential of CRISPR/Cas9 in transcription regulation, genome editing and gene therapy, some important issues, such as off-target mutations , PAM depThe use of genetically engineered mouse models (GEMMs) can also provide an avenue for the validation of miRNAs involved in the regulation of Wnt-signalling pathway following bioinformatics analysis. Although there exist some differences between humans and mice, new models possess the ability to accurately mimic erratic human malignancies and tumour development, thus enabling efficient tracking of both primary and metastatic tumour progression from initial stages than hitherto possible . These min-vivo was confirmed using premalignant mammary tissue of MMTV-Wnt-1transgenic mice [To elucidate the importance of miR-184 in modulating Wnt-signalling in the retina, delivery of formulated nanoparticle-derived miR-184 in the retina of oxygen-induced retinopathy (OIR) mice significantly inhibited Wnt-signalling . OIR is The evolution of tissue engineering have given rise to organoid culture which is a novel and effective tissue stem cell derived three dimensional (3D) model and a useful tool predominantly for functional study , 186. BaAPC-deleted colon-derived organoids overexpressing either miR-483 or Igf2 showed that enforced miR-483 expression promotes high-grade dysplasia [in vivo [in vivo [Contextual modelling in ysplasia . Althoug[in vivo , 196, ot[in vivo . The abi[in vivo . Further[in vivo  in order[in vivo . The rel[in vivo , more ef[in vivo .Apart from the above mentioned techniques and experimental designs utilized in the validation of the gene expression levels and numerous short sequences generated by NGS platforms, other validation techniques widely used include quantitative RT-PCR which is often used for the validation of differentially expressed genes discovered using microarray and RNA-Seq , 202. FuAlthough the application of bioinformatics in the elucidation of the miRNAs-mediated regulation of Wnt-signalling is extremely beneficial Fig. , particu


Doxorubicin (DOX) is an effective chemotherapeutic drug in the treatment of various types of cancers. However, its clinical application has been largely limited by potential development of cardiotoxicity. Previously we have shown that ultra-violet radiation resistance-associated gene (UVRAG), an autophagy-related protein, is essential for the maintenance of autophagic flux in the heart under physiological conditions. Here, we sought to determine the role of UVRAG-mediated autophagy in DOX-induced cardiotoxicity. Mouse models of acute or chronic DOX-induced cardiotoxicity were established. UVRAG deficiency exacerbated DOX-induced mortality and cardiotoxicity manifested by increased cytoplasmic vacuolization, enhanced collagen accumulation, elevated serum activities of lactate dehydrogenase and myocardial muscle creatine kinase, higher ROS levels, aggravated apoptosis and more depressed cardiac function. Autophagic flux was impaired in DOX-induced cardiotoxicity. UVRAG deficiency aggravated impaired autophagic flux in DOX-induced cardiotoxicity. Intermittent fasting restored autophagy and ameliorated pathological alterations of DOX-induced cardiotoxicity. Collectively, our data suggest that UVRAG deficiency exacerbates DOX-induced cardiotoxicity, at least in part, through aggravation of DOX-induced impaired autophagic flux. Intermittent fasting, which restores blunted autophagic flux and ameliorates pathology in the mouse models of DOX-induced cardiotoxicity, may be used as a potential preventive or therapeutic approach for DOX cardiotoxicity. Doxorubicin (DOX) is an effective chemotherapeutic agent in the treatment of a broad range of human solid and hematogenous malignancies. However, clinical application of DOX is largely limited by its cardiotoxicity1456Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process delivering cytoplasmic components to lysosome for degradation and recycling. Autophagy process in mammalian cells consists of several sequential stages-autophagosome formation, maturation, lysosomal degradation and recycling of degradation products. Among them, autophagosome formation can be further divided into four steps: initiation, nucleation, elongation and closure911121415161718Ultraviolet irradiation resistance-associated gene (UVRAG), an autophagy-related protein, has been shown to regulate autophagosome formation23Young adult UVRAG-deficient mice we obtained have normal cardiac morphology and functionNext we examined the effects of UVRAG deficiency on chronic DOX-induced cardiotoxicity. Again, the survival rate of DOX-treated UVRAG-deficient mice was significantly reduced compared with DOX-treated WT controls . CytoplaIt is well established that DOX acts as an electron acceptor to stimulate mitochondria to generate ROS2Apoptosis, which can be triggered d by ROS, also contributes to DOX-induced cardiomyopathy28Growing evidence has shown that inflammation is induced in DOX-induced cardiotoxicityThe ROS production contributes to mitochondrial damage, which in turn, stimulates more ROS production, forming a vicious cycle. In addition, damaged mitochondria are capable of triggering cell death33Next we examined whether UVRAG deficiency affected autophagic flux in DOX-induced cardiotoxicity. LC3 II and p62 protein abundance were significantly increased in LVs from UVRAG-deficient mice compared with corresponding WT controls in acute DOX cardiotoxicity  and at 4Short-term fasting restores autophagy in several pathological conditions373839Interestingly, intermittent fasting reduced lysosomal markers LAMP-1 and LAMP-2 in LVs from WT mice after acute but not chronic DOX treatment . MoreoveOur present results suggest that impaired autophagic flux contributes to the pathogenesis of DOX-induced cardiotoxicity. Given that intermittent fasting is capable of restoring autophagy in this scenario, therefore, we propose that intermittent fasting ameliorates pathology of DOX-induced cardiotoxicity. Intermittent fasting mitigated degenerative vacuoles and fibrosis in LVs from WT and UVRAG-deficient mice after acute  and chroAutophagy has recently been linked to DOX-induced cardiomyopathy. Previously we have shown that UVRAG regulates autophagic flux in the heart at autophagosome maturation step4243ROS has been considered the major mediator in DOX-induced cardiotoxicity. Mitochondria, which are abundant in cardiomyocyte, are the main source of ROS. Turnover of damaged mitochondria through autophagy is essential to maintain cardiomyocyte structure and functionInflammatory cytokines are elevated by UVRAG deficiency after DOX treatment. Previous studies show that ROS is capable of inducing expression of inflammatory cytokines such as TNFα, which in turn promotes ROS generation, leading to a vicious cycleThe key question is what are the potential mechanisms that DOX impairs autophagic flux and UVRAG deficiency aggravates impaired autophagic flux in DOX-induced cardiotoxicity? By means of detecting LC3 II and p62 protein abundance in the heart elicited by CQ-treated WT mice, we showed that autophagic flux was impaired in acute and chronic DOX-induced cardiotoxicity, which is in agreement with several other studies15Fasting exerts a number of beneficial effects on health mediated by remodeling of mitochondria dynamics, enhanced mitochondria biogenesis, changes in energy metabolism, decreased signaling pathway associated with survival such as insulin and insulin-like growth factor-1 signaling, reduction in mitochondria oxidative stress and promotion of autophagyInterestingly, large LAMP-1- and LAMP-2-positive dots examined by immunohistochemistry were present in the hearts following DOX treatment. More importantly, intermittent fasting reduced LAMP-1- and LAMP-2-positive dots in the hearts following acute but not chronic DOX treatment. Thus, chronic DOX treatment may cause more severe damage to lysosomes or autolysosomes. Furthermore, intermittent fasting showed no impact on LAMP-1 or LAMP-2-positive dots in DOX-treated UVRAG-deficient mice, consistent with the fact that UVRAG regulates ALRIn conclusion, UVRAG deficiency leads to exacerbation of DOX-induced cardiotoxicity, at least in part, through aggravation of impaired autophagic flux. Intermittent fasting is capable of rescuing impaired autophagy and ameliorating pathology of DOX-induced cardiotoxicity. Given that intermittent fasting and multiple fasting cycles suppress tumor growth and sensitize a wide range of tumors to chemotherapy50All mice used in this study were of the FVB/NJ background. Animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Directive 2010/63/EU of the European Parliament), with the experimental protocols approved by the Institutional Animal Care and Use Committee of Shanghai, China [SYXK(SH)2011-0112]. Generation of UVRAG-deficient mice was described previously23Acute DOX-induced cardiotoxicity was induced in 2-month-old WT and UVRAG-deficient mice by a single intraperitoneal injection of DOX  dissolved in saline. Chronic DOX-induced cardiotoxicity was induced in eight-week-old WT and UVRAG-deficient mice by intraperitoneally injected 6 doses of DOX  at weekly intervals. Mice were observed for another 2 weeks before assessment of cardiac function.In both acute and chronic DOX cardiotoxicity, mice were anaesthetized by isoflurane inhalation and euthanized by rapid cervical dislocation at the indicated time points and left ventricles were dissected from the excised hearts for western blot and ROS analysis. For histological and immunohistochemical staining, mice were anaesthetized by isoflurane inhalation and the entire hearts were used to prepare paraffin-embedded serial sections in four-chamber view. Generally time-course analysis of vacuolar degeneration and fibrosis in LVs were performed in acute and chronic DOX cardiotoxicity. ROS and apoptosis were assessed 5 days after DOX treatment in acute DOX-induced cardiotoxicity and at 4 weeks of DOX treatment in chronic DOX-induced cardiotoxicity.In the fasting experiments, mice were subjected to intermittent  fasting, starting from one day before the initial DOX treatment. Fasted mice had free access to water. LVs or the perfusion-fixed hearts from the fasted group were dissected during the day of feeding after fasting treatment as indicated. In this study, we dissect left ventricle to make consistent tissue sampling to exclude the possible impacts of different hemodynamics between left ventricle and right ventricle on the experimental results.Mouse anti-LC3 monoclonal antibody (M152-3), Rabbit anti-LC3 polyclonal antibody (PM036) and rabbit anti-Beclin 1 polyclonal antibody (PD017) were from MBL International Corporation , rabbit anti-p62 polyclonal antibody (#5114), rabbit anti-Atg5 monoclonal antibody (#12994) and rabbit anti-UVRAG monoclonal antibody (#13115) were purchased from Cell Signaling Technology , mouse anti-tubulin monoclonal antibody (628802) was obtained from Gene Tex, Inc , rat anti-LAMP-1 monoclonal antibody (sc19992), rat anti-LAMP-2 monoclonal antibody (sc19991), mouse anti-ubiquitin monoclonal antibody (sc8017), rabbit anti-XIAP polyclonal antibody (sc5550) and rabbit anti-GAPDH polyclonal antibody (sc25778) were from Santa Cruz Biotechnology , rabbit anti-α-smooth muscle actin (α-SMA) polyclonal antibody was obtained from Abcam Limited , and mouse anti-β-actin monoclonal antibody (AM1021B) was purchased from ABGENT .® Elite ABC Kit  as previously described® Plus Peroxidase In Situ Apoptosis Detection Kit  according to the manufacturer’s instructions. For quantification of vacuolar degeneration of cardiomyocytes, fibrosis and α-SMA expression, around 20 microscopic fields randomly selected in LVs per section, 3 sections from each of 3–5 mice were assessed. For quantification of apoptotic cells in the heart, 20–25 microscopic fields randomly selected per section, 3 sections from each of 3 mice were evaluated by TUNEL assay.Following anesthesia, mouse hearts were perfused with ice cold PBS followed by fixation by 4% paraformaldehyde in PBS. Fixed hearts were embedded in paraffin and cut into serial sections in four-chamber view at 5 μm in a Leica RM2255 rotary microtome . Heart sections were stained with H&E or picrosirius red for evaluation of routine morphology and collagen accumulation. Immunohistochemistry was performed on the paraffin sections using the VECTASTAINSerum LDH activity was assayed by means of reflectance spectroscopy using LDH detection kit . Serum CK-MB activity was determined using mouse CK-MB ELISA kit . Determination of LDH and CK-MB were performed according to the manufacturer’s instructions.In vivo autophagic flux assessment was evaluated as described previously2O) or chloroquine  and LVs from excised hearts were harvested for analysis of LC3 II and p62 protein levels by western blot. Immunohistochemical staining for LC3 II was performed on paraffin-embedded heart sections.Analysis of ROS was performed as described previouslyTNFα, IL-6 and IL-1β were assessed in heart homogenates using ELISA kits  according to the manufacturer’s instructions.TM Western Chemiluminescent HRP Substrate . All western blot quantifications were performed using the Image J software.Proteins extracted from left ventricular tissues were separated by SDS-PAGE gel and transferred to nitrocellulose membrane. The membranes were then blocked for 1 hour at room temperature with 5% skim milk powder in Tris-buffered saline with Tween 20 (TBST) buffer and incubated with primary antibodies overnight at 4 °C. After washing with TBST buffer, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature and again washed with TBST buffer. The protein bands were detected by using ImmobilonEchocardiography was performed as described previouslyP < 0.05 were considered statistically significant.All data were expressed as mean ± S.E.M. Statistical differences were analyzed by one-way ANOVA followed by Bonferroni’s test for multiple comparisons using GraphPadPrism software. Values of How to cite this article: An, L. et al. UVRAG Deficiency Exacerbates Doxorubicin-Induced Cardiotoxicity. Sci. Rep.7, 43251; doi: 10.1038/srep43251 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.