INSTIs, in particular bictegravir, are more potent in blocking HTLV-1 transmission than tenofovir disproxil fumarate (TDF), an RT inhibitor. Our data suggest that HIV-1 INSTIs could present a good clinical strategy in HTLV-1 management and justifies the inclusion of INSTIs in clinical trials.More than 10 million people worldwide are infected with the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1). Infection phenotypes can range from asymptomatic to severe adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy. HTLV-1, like human immunodeficiency virus type 1 (HIV-1), is a blood-borne pathogen and viral infection happens in a similar fashion, with the major mode of transmission through breastfeeding. There is a strong correlation between time of infection and disease development, with a higher incidence of ATLL in patients infected during childhood. There is no successful therapeutic or preventative regimen for HTLV-1. It is therefore essential to develop therapies to inhibit transmission or block the onset/development of HTLV-1 associated diseases. Recently, we have seen the overwhelming success of integrase strand transfer inhibitors (INSTIs) in the treatment of HIV-1. Previously, raltegravir was shown to inhibit HTLV-1 infection. Here, we tested FDA-approved and two Phase II HIV-1 INSTIs  Human T-cell lymphotropic virus type 1 (HTLV-1) belongs to the delta-retrovirus genus and is the second most clinically relevant retrovirus after human immunodeficiency virus type 1 (HIV-1). Like HIV-1, HTLV-1 is a blood-borne pathogen and is transmitted horizontally by sexual intercourse, contact with infected blood, and vertically from mother to child during breastfeeding and labor. HTLV-1 is a highly potent oncogenic virus, despite the fact that pathology develops only in about 10% of carriers. The two main pathologies associated with infection are an aggressive form of blood cancer – adult T-cell leukaemia/lymphoma (ATLL)  and a spHuman T-cell lymphotropic virus type 1 infection is of significant epidemiological importance. It is estimated that between 10 and 20 million people are infected worldwide . This fi2+). The irreversible insertion of both vDNA ends into host DNA, termed “concerted integration,” results in a stably integrated provirus which is copied with every cell division. The proviral expression products further stimulate cell proliferation and lead to the smoldering, mitotic spread of HTLV-1  enzyme – a nucleotidyl transferase responsible for insertion of the DNA copy of the viral RNA genome into host cell DNA. Following reverse transcription, IN engages with the viral long terminal repeat (LTR) ends of the DNA thereby forming a synaptic complex also called the intasome. The integration reaction takes place in two steps: (i) in the 3′ processing reaction, IN removes two  nucleotides following the invariant CA-dinucleotide thereby generating 3′-OH groups necessary for the next step in the integration reaction; (ii) following nuclear entry, the intasome engages with host chromatin and the reactive 3′-OH groups attack the scissile phosphodiester backbone on opposing strands of the host DNA stably inserting the vDNA into the host genome . The retf HTLV-1 . Due to f HTLV-1 . These dive site ,b. Succein vitro and in cell-to-cell infection. We describe an optimized in vitro assay that allows for easy screening of INSTI efficacy and IC50 determination. Ex vivo experiments in Jurkat cells infected with HTLV-1 by cell-to-cell transmission indicate that the INSTI inhibition profiles are very similar to those of HIV-1. Taken together, we present data that support the use of INSTIs as prophylactic treatment, and may justify their use in clinical trials.In Human T-cell lymphotropic virus type 1 IN (GenBank ID: AHX00119.1) was expressed and purified from pET28a-SUMO-HTLV-1 IN as described previously  with min2, 5.8 μM ZnCl2, 12.8 mM DTT, 0.53 μM donor DNA, and 0.8 μM HTLV-1 integrase. Following a 30 min room temperature incubation in the presence or absence of INSTIs in the final concentration of 5% DMSO, the reaction was initiated by addition of 300 ng of pGEM-9Zf(−) supercoiled plasmid DNA. The total volume of each reaction was 150 μL. Reactions were carried out at 37°C for 30 min and were processed as described previously  T cell lines (ATCC) were maintained in RPMI supplemented with 10% foetal bovine serum (FBS), 100 U penicillin 100 μg/mL streptomycin and 0.25 μg/mL fungizone. Cells were cultured at 37°C with 5% CO6 cells/mL and exposed to a sub-lethal dose of gamma-irradiation . Cells were resuspended in serum-free RPMI to a concentration of 2 × 106 cells/mL in the presence of drug, and 0.5 × 106 of each cell type were co-cultured for 18 h. Following co-culture, cells were washed in PBS and resuspended in 1 mL of depletion buffer  before gentle tumbling  with 25 μL of anti-CD25 + magnetic beads  to deplete MT-2 cells. Following depletion, the unbound fraction, i.e., Jurkat cells were maintained in RPMI with the drug present for 16 days before genomic DNA harvest . All experiments were done in triplicate. Depletion of MT-2 cells was verified by FACS. For cellular toxicity measurements, Jurkat cells were grown in the presence of the INSTIs as described above. Viability was measured by live dead stain (Hoechst 33258) and analyzed by FACS.Jurkat cells were pre-treated with increasing amounts of INSTI (0.2 pM–2 μM in DMSO), or the carrier (DMSO) for 24 h. MT-2 cells were resuspended to a concentration of 2 × 106 cells were washed extensively in PBS + 3% FBS before incubation with 0.05 μg Alexa 647-conjugated anti-CD3 +  and 0.1 μg Alexa488 conjugated anti-CD25 +  antibodies in the dark for 30 min. Isotype control antibodies were used to determine specificity of the antibodies and staining protocol. Following extensive washing, samples were fixed in 2% formaldehyde for FACS analysis. Single cells were gated, CD3 and CD25 were identified by emission/excitation at 640/670 nm and 488/530 nm, respectively. Data were analyzed on FlowJo software version 10.7.Samples of Jurkat and MT-2 cultures pre- and post-depletion were immunostained for CD3 and CD25 to determine the efficiency of anti-CD25 magnetic depletion. 1 × 10tax gene and the human GAPDH gene  was estimated as previously described . GenomicPDH gene . Gene coted cell . Alu-PCRted cell , with thted cell . Integra1.Human T-cell lymphotropic virus type 1 amino acid sequence  was submitted for automatic homology model building to the on-line software Phyre2 . The seain vitro activity on radiolabeled vDNA donor mimics  double-stranded oligonucleotides mimicking the 3′ processed viral LTR end  and a supercoiled plasmid functioning as the target (tDNA) for integration. The biologically relevant concerted integration where two vDNA mimics are inserted in the tDNA, results in linearization of the plasmid, whilst products of aberrant half-site integration co-migrate with nicked plasmid DNA . These pin vitro strand transfer activity  but was clearly discriminative against the U3-derived donors . Certainactivity . PIPES pactivity , under oactivity .A panel of six INSTIs was selected , to encoin vitro strand transfer at nanomolar concentrations  of Jurkat cells are infected under these experimental conditions. Infections were done in the presence of DMSO (vehicle of the drugs) or a range of drug concentrations (0.2 pM to 2 μM). The resulting dose-response curves show a strong inhibition profile of tested INSTIs, active even at picomolar concentrations , inhibiting HTLV-1 transmission only two- to threefold less efficient than raltegravir or elvitegravir, respectively  is not well structured in our HTLV-1 homology model  in reducing PVL thereby reducing chances of transmission between partners, but also in cases of organ donor transplantation, and possibly preventing the evolution of asymptomatic carrier to HTLV-1 associated disease. Treating pregnant women with INSTIs might warrant some caution; given the reduced exposure of elvitegravir during pregnancy it increases the risk of virological failure and mother-to-child transmission . The WHO defects . Data ob defects . The limen found .Currently, the most devastating pathology caused by HTLV-1 is ATLL. Prognosis is very poor – most patients die within several months after presentation . At the All datasets generated for this study are included in the manuscript and/or the MB designed, executed, and analyzed the experiments, and wrote the manuscript. JM designed, executed, and analyzed the experiments, and edited the manuscript. GM designed and analyzed the experiments, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This report describes the results of experiments examining the pathway by which the human retroviral Gag protein is recruited to sites along the inner leaflet of the plasma membrane where Gag punctum biogenesis occurs. In particular, clever and sensitive experimental methods were devised to image in living cells fluorescently labeled Gag protein derivatives from human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) at the plasma membrane. The photoconvertible fluorescent protein mEos2 was strategically utilized, as the fluorescence emission of Gag at the plasma membrane could be differentiated from that of cytosolic Gag. This experimental strategy allowed for the determination of the Gag recruitment pathway into Gag puncta. For HTLV-1 Gag, puncta recruited Gag primarily from the plasma membrane, while HIV-1 Gag was recruited from the cytoplasm. These observations represent the first report of HTLV-1 particle biogenesis and its contrast to that of HIV-1. The observed differences in the Gag recruitment pathways used by HTLV-1 and HIV-1 Gag provide key information that is useful for informing the discovery of novel targets for antiretroviral therapies directed at eliminating virus infectivity and spread. The assembly of virus particles is a crucial aspect of virus spread. For retroviruses, the Gag polyprotein is the key driver for virus particle assembly. In order to produce progeny virus, once Gag is translated, it must translocate from the location in the cytoplasm where it is synthesized to the plasma membrane and form an oligomeric lattice that results in Gag puncta. The biogenesis of mature Gag puncta can trigger the budding process, resulting in virus particle production. While some aspects of the dynamics of Gag oligomerization and particle biogenesis have been observed with human immunodeficiency virus type 1 (HIV-1), the process of Gag punctum biogenesis remains poorly understood, particularly for other retroviruses. Here, we have conducted the most detailed studies thus far on Gag punctum biogenesis for human T-cell leukemia virus type 1 (HTLV-1). Using mEos2 photoconvertible fluorescent proteins and total internal reflection fluorescence microscopy (TIRF), we have found that HTLV-1 Gag was recruited to Gag puncta primarily from the plasma membrane. This was in stark contrast to HIV-1 Gag, which was recruited from the cytoplasm. These observations imply fundamental differences among retroviruses regarding the orchestration of Gag punctum biogenesis, which has important general implications for enveloped virus particle assembly. A common feature among enveloped viruses that assemble at the plasma membrane is the requirement for membrane accumulation of viral structural proteins and nucleic acid in order for virus particle budding and release to occur. The general strategies used by enveloped viruses to accomplish this task are poorly understood. For retroviruses, such as human immunodeficiency virus type 1 (HIV-1), the Gag polyprotein is known to be the primary driver for virus particle assembly . HTLV-1 infects about 15 million to 20 million individuals worldwide and is the etiological agent of an adult T-cell leukemia/lymphoma (ATLL) –5. Early–Though retroviral Gag proteins have relatively limited amino acid sequence homology, they do share structural similarities among the domains of Gag –10. Gag –It is well known that HTLV-1 is efficiently transmitted via cell-to-cell contacts, i.e., the virological synapse (VS)  cytoplasmic concentrations, and that there is a critical threshold concentration (approaching micromolar) prior to the observation of HIV-1 Gag associated with the plasma membrane . AdditioIn this study, we report new observations that point to novel features of HTLV-1 assembly not seen with HIV-1 or other retroviruses . In partIn order to examine the recruitment of Gag to Gag puncta , the Gag-mEos2 (Gag protein labeled with the photoconvertible fluorescent protein mEos2) expressed in live cells was monitored by changing the color of their fluorescence emission from green to red exclusively at the inner leaflet of the plasma membrane. The photoconversion of Gag-mEos2 at the basal plasma membrane was conducted with incident light at 405 nm delivered by TIRF illumination. The evanescent field of TIRF restricts the activating light to the immediate vicinity of the glass/cell interface, resulting in efficient photoconversion at the basal plasma membrane, while cytosolic Gag-mEos2 remains in the green-emitting form . It has TIRF images of live cells transfected with HIV-1 Gag-mEos2 collected prior to photoconversion revealed puncta that were excited at 488 nm, but not at 561 nm  of the same cell collected before the photoconversion step revealed in addition to the punctate fluorescence a diffuse background, which is caused by green-emitting, cytosolic HIV-1 Gag-mEos2 see . By subt10.1128/mBio.00758-18.1FIG S1 FIG S1, TIF file, 1.6 MB.Epifluorescence images of cells expressing HIV-1 Gag-mEos2. (A) Prior to photoconversion (PC), the epifluorescence image displays diffuse cytosolic fluorescence and puncta upon excitation at 488 nm (left panel). Epifluorescence image of the same cell excited at 561 nm before PC (right panel) shows only autofluorescence near the nucleus. (B) After PC, bright puncta as well as the autofluorescence appear in the epifluorescence image excited by 561-nm light. (C) The images in the right panel of panel A and the left panel of panel B were subtracted to create this difference image. The lack of diffuse fluorescence between the puncta confirms the absence of cytosolic activation. Because of the large intensity range present in these images, the color scale of the images was computationally saturated to visualize both dim and bright features. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TIRF images of HTLV-1 Gag-mEos2 before photoconversion observe puncta only with 488-nm excitation (αG = I488/(I488 + I561) where I488 and I561 represent the intensities of a punctum excited at 488 nm and 561 nm, respectively. The limiting values of αG before and after photoconversion were determined experimentally. A histogram of αG from puncta containing unconverted HIV-1 Gag-mEos2 peaked around a value of 0.9  Cells transfected with HIV-1 mEos2-Gag and unlabeled HIV-1 Gag were imaged in the TIRF mode with 488-nm excitation before photoconversion (PC). This image is displayed using a computational color scale so that both the dim and bright puncta are simultaneously visible. (B) The green intensity fraction of the puncta from panel A was displayed as a histogram with a peak at ~0.9 before PC. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the αG corresponding to the maximum extent of photoconversion, an incremental number of pulses of 405-nm light were incident on a cell containing HIV-1 Gag-mEos2 proteins  Cells expressing HIV-1 Gag-mEos2 were exposed to 20-ms pulses of 405-nm light in the TIRF mode. Images of the cell taken in the TIRF mode with 488-nm excitation (A) and 561-nm excitation (B) after applying a specific number of pulses. (C) The green intensity fraction of Gag-mEos2 puncta was calculated from the TIRF images. The mean and standard deviation (error bar) of the green intensity fraction are plotted as a function of the number of 20-ms pulses of 405-nm light. All images are displayed using a linear color scale. The image series of each panel uses a fixed intensity scale to facilitate visual comparison. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the These results establish the experimentally accessible range of the green intensity fraction which spans from ~0.3, representing the full extent of photoconversion of mEos2, to values near 0.9, reflecting entirely unconverted mEos2 proteins. All experiments in this study used conditions that resulted in maximal photoconversion of Gag-mEos2 at the basal plasma membrane.It should be noted that the incident violet light used to photoconvert mEos2-Gag could impact cell viability. To address this, cells were examined with a blue nuclear dye (NucBlue) after photoconversion. Nonviable cells become permeable to this dye. Cells transfected with both HIV-1 Gag-mEos2 and HTLV-1 Gag-mEos2 were examined after photoconversion by using NucBlue. Analysis of cells 1 h after photoconversion revealed no apparent NucBlue staining, which was interpreted as exposure to the incident violet light having no significant impact on cell viability. In particular, no cell nuclei that possessed violet fluorescence 25 min following photoconversion were observed. For a control, ethanol was added to cells, which allowed NucBlue to be readily observed in cell nuclei , confirm10.1128/mBio.00758-18.4FIG S4 FIG S4, TIF file, 1.1 MB.Analysis of cell viability. (A) The HTLV-1 Gag-mEos2 proteins present at the plasma membrane were terminally photoconverted and imaged with TIRF using 488-nm excitation. The outline of the cell is plotted in white on this image. (B and C) Following photoconversion, NucBlue was added to cell culture medium, and the cell was monitored with epifluorescence and 405-nm excitation for approximately 25 min. (D) Efficient visualization of cell death was confirmed by the addition of ethanol to cell culture medium, which resulted in nuclei with prominent violet fluorescence. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We conducted measurements to determine whether the color of the nonpunctate Gag-mEos2 present at the plasma membrane changes over a period of 25 min after photoconversion. In these experiments, HTLV-1 Gag-mEos2 proteins present at the cell’s plasma membrane were terminally photoconverted with a series of short pulses of 405-nm light in TIRF mode. Following the photoconversion step, the intensity of the nonpunctate (diffuse) fluorescence signal was detected with TIRF-based imaging and quantified at selected time points . Because10.1128/mBio.00758-18.5FIG S5 FIG S5, TIF file, 1.1 MB.Intensity of nonpunctate, photoconverted HTLV-1 Gag-mEos2 at the plasma membrane. (A) TIRF images of a HeLa cell with HTLV-1 Gag-mEos2 were collected at the indicated time points before and after photoconversion with 488-nm excitation. (B) The same cell in panel A was imaged with TIRF using 561-nm excitation at the indicated time points before and after photoconversion. (C) The average intensity from the regions of interest outlined in white in images are plotted over time. No green intensity fraction was calculated, because the low intensity from nonpunctate regions contains contributions from background and cytoplasmic TIRF signal, which would result in a biased intensity fraction. The intensity emitted from the nonpunctate HTLV-1 Gag-mEos2 remained approximately the same over a period of 25 min following photoconversion, which indicates that nonpunctate Gag at the membrane is not rapidly recruited into puncta and exists as a stable population over the time scale of our observation period. Because of the large intensity range present in these images, the color scale of the images in A and B was computationally saturated. The same color scale was applied to all images depicted in panels A and B. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −4 µm2/s. Repeated measurements (n = 10) of the mobility of growing HTLV-1 puncta recovered a diffusion coefficient of 1.6 × 10−4 µm2/s (n = 10)  . For com(n = 10) , which wParticle tracking not only identifies the mobility of puncta, but it can also be used to study the recruitment of HIV-1 Gag to puncta . HoweverTo eliminate this problem, we reduced the number of light exposures to only two, one observation right after photoconversion to document the initial state and a second observation after a wait period of 15 min to identify newly formed puncta . A newly formed punctum was defined as a punctum observed in the TIRF image of the cell collected 15 min after photoconversion which was at least 10 pixels (1.5 µm) away from any existing punctum located in the TIRF image of the same cell collected immediately after photoconversion. Because the displacement of existing puncta during the wait period is expected to be ~2.5 pixels, which is based on the measured diffusional mobility of existing puncta discussed in the previous section , the dis10.1128/mBio.00758-18.6FIG S6 FIG S6, TIF file, 0.2 MB.Displacement of Gag puncta. The displacement of existing puncta during a wait period of 15 min was determined by comparing their imaged location immediately after photoconversion and after the wait period. Only sparse regions that contained a single punctum were considered in order to clearly isolate the movement of an individual punctum. We defined a sparse area as a region with a single punctum at least 17 pixels away from its nearest neighbor. The analysis was performed on a subset of the previously imaged cells. Histograms of the measured displacements are shown for HTLV-1 Gag-mEos2 (A), HTLV-1 Gag with unlabeled HTLV-1 Gag (B), HIV-1 Gag-mEos2 (C), and HIV-1 Gag-mEos2 with unlabeled HIV-1 Gag (D). Displacements of 10 pixels or more were rare and comprised at most 11% of imaged puncta. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the αG corresponding to puncta located in images of cells transfected with HIV-1 Gag-mEos2 was determined  Images of a HeLa cell expressing HIV-1 Gag-mEos2 collected before and after PC are scaled linearly according to the absolute maximum and minimum present in the images for each channel (Gr. Exc. and Red Exc.). (E to H) Images of the same cell in panels A to D are presented with a computationally saturated color scale in order to make features in the images more apparent. The white boxes show the locations of the new punctum depicted in Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the G that describe the color of photoconverted and newly formed puncta are essentially identical to the results obtained for the fully labeled Gag . As shown previously in the literature, transfecting cells with this ratio of labeled and unlabeled HIV-1 Gag will result in the production of virus-like particles that contain lattice structures similar to those produced by purely unlabeled HIV-1 Gag . With theled Gag  and D.G of >0.65. Furthermore, there was a minority population of new Gag puncta that were red in color (αG < 0.45). Therefore, the observations using mEos3.2 are comparable to those observed with mEos2  The green intensity fraction of puncta produced by HIV-1 Gag-mEos3.2 and present immediately after photoconversion are plotted in this histogram. (B) After a period of 15 min, the distribution of the green color fraction of new puncta containing HIV-1 Gag-mEos3.2 is similar to the corresponding histogram found for HIV-1 Gag-mEos2. (C) The histogram depicts the color of puncta present in cells transfected with HTLV-1 Gag-mEos3.2 immediately after photoconversion. (D) Following a period of 15 min, the distribution of the green color fraction of new puncta containing HTLV-1 Gag-mEos3.2 is similar to the corresponding histogram found for HTLV-1 Gag-mEos2. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the αG of puncta immediately after photoconversion was distributed in the red with a peak at ~0.2 (αG of newly appearing puncta was identified and their distribution was examined (αG < 0.5) for both data sets. An example of a newly appearing red punctum detected during these experiments is shown in G also display a small shoulder near a value of 0.7  Images of a HeLa cell expressing HTLV-1 Gag-mEos2 collected before and after PC are scaled according to the absolute maximum and minimum present in the images for each channel (Gr. Exc. and Red Exc.). (E to H) Images of the same cell in panels A to D are presented with a computationally saturated color scale in order to make features in the images more apparent. The white boxes correspond to the locations of the new punctum formed depicted in Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the G < 0.45) with a minority population of puncta that were green in color (αG > 0.65). The observed histograms using mEos3.2 are qualitatively similar to those observed with mEos2  was identified by comparison with earlier and later images. Puncta from a total of 10 cells were analyzed to examine the dwell times of HIV-1 Gag-mEos2’s puncta (5 cells transfected with HIV-1 Gag-mEos2 and 5 cells transfected with HIV-1 Gag-mEos2 with unlabeled HIV-1 Gag at 1:3). In parallel, puncta from eight cells were analyzed to determine the dwell times of puncta produced by HTLV-1 Gag-mEos2 (four cells transfected with HTLV-1 Gag-mEos2 and four cells transfected with HTLV-1 Gag-mEos2 with unlabeled HTLV-1 Gag at 1:3).Any new Gag puncta that were identified and tracked in these experiments were labeled short-lived if they disappeared within a minute. Analysis of tracking data revealed that only a small minority of new puncta were short-lived  and B, i10.1128/mBio.00758-18.10FIG S10 FIG S10, TIF file, 0.3 MB.Additional analysis of Gag puncta using particle tracking. (A and B) Particle tracking identified the dwell time of new Gag puncta by measuring the time period between appearance and disappearance of puncta from the image series. Puncta were classified as short-lived if their dwell time was less than 1 min. (A) Number of observed short-lived and total HIV-1 Gag-mEos2 puncta. (B) Number of observed short-lived and total HTLV-1 Gag-mEos2 puncta. (C and F) Recruitment of HIV-1 Gag-mEos2. Images of a cell transfected with labeled and unlabeled HIV-1 Gag (1:3 ratio) and collected with 488-nm excitation were cropped to display a growing punctum located within the white box as a function of imaging time for 488-nm excitation (C) and 561-nm excitation (D). (E) The integrated intensity of the growing puncta is shown for 488-nm excitation (green symbols) and 561-nm excitation (red symbols) as a function of time. The solid lines describe exponential charging curves with a time constant of ~6 min. (F and G) Recruitment of HTLV-1 Gag-mEos2. Images of a cell transfected with labeled and unlabeled HTLV-1 Gag (1:3 ratio) and collected with 488-nm excitation were cropped to display a growing punctum located within the white box as a function of imaging time for 488-nm excitation (F) and 561-nm excitation (G). (H) The integrated intensity of the growing puncta is shown for 488-nm excitation (green symbols) and 561-nm excitation (red symbols) as a function of time. The solid lines describe exponential charging curves with a time constant of ~5 min. Download Copyright © 2018 Eichorst et al.2018Eichorst et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, a particle tracking algorithm extracted from the image stacks the locations and intensities of puncta that progressively increased in intensity. In other words, the puncta must increase in its brightness over a period of minutes to be considered a new punctum. Likewise, any punctum described as new must be well isolated from neighboring puncta during its growth so that accurate fitting and calculations of intensity can be carried out. In addition, the frames preceding the initial detection of the punctum were examined to ensure the absence of any previously existing puncta in the vicinity of the detection area. This filtering of data removed spurious and marginal events, but it also lowers the detection efficiency of new puncta. Similarly, while the excitation power was reduced to decrease photobleaching, it limited our ability to detect dimmer puncta. Thus, particle tracking experiments identified only a fraction of the newly forming puncta.αG calculated from αG = 0.8).An example of a growing punctum found within a cell transfected with both HIV-1 Gag-mEos2 and unlabeled HIV-1 Gag (1:3 plasmid ratio) is highlighted in the images shown in G, particle tracking nevertheless provides a reasonable measure to identify the pathway of Gag recruitment. We classify growing puncta with αG of ≥0.65 as predominantly recruiting Gag from the cytoplasm, while αG of ≤0.45 represents predominate Gag recruitment from the plasma membrane. Any growing puncta with αG between 0.45 and 0.65 are considered intermediate, and no preferred recruitment pathway is assigned to these puncta.While photobleaching will bias the value of the green intensity fraction αAnalysis of approximately 60 HeLa cells transfected with HIV-1 Gag-mEos2 and HIV-1 Gag-mEos2 with unlabeled HIV-1 Gag identified 24 intensity traces of puncta that increased in intensity over a period of several minutes. Twelve of the traces describe puncta produced by cells transfected with HIV-1 Gag-mEos2, and the other 12 traces describe puncta produced by cells transfected with a mixture of HIV-1 Gag-mEos2 and unlabeled HIV-1 Gag.G of ≤0.45, indicating a significant recruitment of membrane-bound Gag. An example with αG = 0.2 is shown in n = 6) were classified as intermediate, and no recruitment pathway was assigned to them.Fourteen of the intensity traces  increased only in the channel excited by 488-nm light, similar to that seen in The rates of increase in the punctum fluorescence intensity were estimated by comparing the intensity values to exponential charging functions e.g., . With thαG = 0.2 is shown in The same experiments were also conducted in HeLa cells transfected with HTLV-1 Gag-mEos2 as well as HeLa cells transfected with HTLV-1 Gag-mEos2 and unlabeled HTLV-1 Gag. The particle tracking algorithm identified 23 intensity traces describing puncta that increased in intensity over time from a set of ~110 HeLa cells transfected with HTLV-1 Gag-mEos2 (11 traces) or HTLV-1 Gag-mEos2 with unlabeled HTLV-1 Gag (12 traces). Ten of the traces exhibited a time-dependent intensity increase over a few minutes with 488-nm and 561-nm excitation with green color fractions less than or equal to 0.45. An example of such a punctum with G ≥ 0.65, which indicates that HTLV-1 Gag is likely being recruited into these puncta from the cytosol (n = 8) had values of αG that were classified as intermediate, and no recruitment pathway was assigned.Five of the collected traces resulted in α cytosol  to H. ThG values obtained from the two sets of traces (23 for HTLV-1 and 24 for HIV-1) by performing a two-tailed Mann-Whitney U test  being utilized by a Gag labeled with the photoconvertible fluorescent protein mEos2 is technically challenging. The primary problem for live-cell imaging studies is that the green-emitting form of mEos2 loses its ability to emit fluorescence (photobleach) 5 to 10 times faster than the more commonly used green fluorescent protein (GFP) derivatives . In contThese considerations prompted us to study recruitment based on the acquisition of only two sets of images. This method eliminates most of the artifacts introduced by photobleaching of mEos2 . In addiThis strategy was used to characterize HIV-1 Gag recruitment to a punctum to a degree not previously achieved  and contPreviously published data that examined the recruitment of HIV-1 Gag into budding sites indicate that almost all puncta produced by HIV-1 Gag-mEos2 grow by recruitment from the cytosol . It is pαG without a detailed model of the photophysics of mEos2 in a punctum. For example, fluorescence resonance energy transfer (FRET) between the two states of mEos2 within a punctum reduces the fluorescence from the green state while increasing the fluorescence of the red state of mEos2, which leads to a lowering of αG compared to αG without FRET. Because no general modeling of the fluorescence intensities in the presence of FRET involving multiple acceptors and donors exists, no quantitative mapping of αG was attempted. Instead, we used αG as a convenient parameter to classify puncta. Specifically, αG < 0.45 and αG > 0.65 were used to mark puncta with predominant recruitment of Gag from the plasma membrane and cytosol, respectively, and were chosen based on reference measurements of mEos2 in the unconverted and fully photoconverted state  were essentially identical . UnfortuIn these studies, we acknowledge that the use of a codon-optimized Gag as a surrogate may not fully recapitulate the expression of authentic Gag in the context of integrated proviral DNA. However, many aspects of our results obtained using codon-optimized HIV-1 Gag  match well with the results of other investigators, which provides a degree of independent validation of our findings. Although a fluorescently labeled full-length expression vector of HTLV-1 Gag has not been published, several groups have examined the distribution of HTLV-1 Gag in cells using fluorescently labeled antibodies , 55, 56.Finally, it is possible that the biogenesis of HTLV-1 Gag puncta by recruitment of Gag along the plasma membrane is a more efficient mode of particle production during cell-cell contacts. Cell-free particle infectivity for HTLV-1 is low, and recent observations have indicated that only a portion of mature HTLV-1 particles have intact cores . InfectiThese results support the conclusion that although HTLV-1 Gag recruitment is primarily from the plasma membrane  and HIV-All experiments were conducted using a Zeiss Axio Observer Z1 microscope equipped with a 100× 1.45-numerical-aperture (NA) oil immersion objective . Images were collected using an Andor electron-multiplying charge-coupled-device (EMCCD) model DV887 camera . A 405-nm laser , a 488-nm laser (Coherent), and a 561-nm laser  were used for illumination in the total internal reflection fluorescence microscopy (TIRF) mode. The color and intensity of light incident on the sample were controlled with an acousto-optic tunable filter (AOTF) . The excitation light and emission light were separated with a multicolor dichroic cube . HeLa cells transfected with HIV-1 Gag-mEos2 or HTLV-1 Gag-mEos2 were imaged with each of the three lasers  using the multicolor dichroic cube to separate excitation from emission.2-independent medium  supplemented with 10% fetal calf serum. The temperature on the microscope stage was maintained at 37°C with a stage heater . HeLa cells were measured 6 to 20 h after transfection. The HIV-1 Gag-EYFP plasmid  were transfected with GenJet following the manufacturer’s protocol. For measurements, cells were maintained in L15 CO plasmid  was modi plasmid , 61 was The initial locations of all puncta were determined from images collected by TIRF using the feature-locating function (mpretack) developed by the Kilfoil group . Once thI(t) of growing puncta was fit to an exponential charging curve defined by I (t) = A[1 − exp(−t/τ)], where A is the amplitude and τ is the time constant. Because reduction of the excitation power reduced the signal-to-background ratio of particle tracking, the green intensity fraction was calculated after subtraction of the background signal determined from the image frames preceding punctum detection.The positions of growing puncta were determined by MatLab implementation of a previously developed algorithm . The out


Vaccination-induced optic neuritis is not common. The development of optic neuritis following various vaccinations have been reported, suggesting a possible association between optic neuritis and vaccination. Of those reported cases, influenza vaccines have been the most common. Although rare, those patients who developed optic neuritis following HPV vaccination also presented with other central nervous system (CNS) demyelinating syndromes, especially following a booster dose. We present a rare case of simultaneous isolated bilateral optic neuritis following the first dose of an HPV vaccination in a young child. She received treatment with a systemic corticosteroid that resulted in a good clinical outcome without developing any demyelinating disease. Optic neuritis is an inflammation of the optic nerve which can be caused by demyelination, infection, post-immunization, or autoimmune diseases. The most common cause of optic neuritis in children is a viral infection. Hence, it seems possible that vaccines can also cause a similar condition by stimulating the host's immune system. The exact mechanism is unknown, but it is believed that the activation of T-cells may cross the blood-brain barrier, resulting in a type IV hypersensitivity reaction that damages the myelin sheath of optic nerve . PresentA 13-year-old healthy female student with no medical illness presented with sudden onset loss of vision in both eyes for three days. It was associated with pain on eye movements. She had her first dose of an HPV vaccination three weeks prior to the presentation and reported having flu-like symptoms after the vaccination. There was no history of limb numbness, weakness, or walking difficulty. The bowel opening and micturition were normal. On examination, visual acuity in the right eye was counting fingers and left eye was hand movement. Both pupils were sluggish and a relative afferent pupillary defect (RAPD) was not apparent. Both anterior segments were unremarkable. Funduscopy showed a diffuse hyperemic swollen disc with dilated vessels in both eyes. The left eye was found to be more profound than the right eye Figure -3. The bNumerous case reports have suggested an association between CNS demyelinating syndrome and several different vaccines -4. Of thThe mechanism of this pathology is still unknown but is presumably immune-mediated. Activation of T-cells secondary to an inflammatory process may cross the blood-brain barrier and induce a type IV hypersensitivity reaction resulting in the destruction of the myelin sheath of the nerves . In vaccPediatric optic neuritis is commonly bilateral and occurs after a viral infection or immunization. It is a clinical diagnosis, according to findings, that includes visual loss, pain on ocular movement, RAPD, dyschromatopsia, and the appearance of the optic disc . HoweverNo clinical trials have been done for optic neuritis in children. Conservative management is the treatment of choice for unilateral or mild bilateral optic neuritis . For bilTo date, there is no conclusive evidence of HPV vaccination in the pathogenesis of demyelinating diseases. For this patient, she refused the booster dose of the HPV vaccination. The decision for the continuation of the HPV vaccination is a personal decision and is not compulsory. Hence, all the patients who are scheduled for HPV vaccination should be informed of optic neuritis as one of the possible adverse events. However, reviews over the safety of HPV vaccines has shown that it is generally safe and well-tolerated -12.Isolated optic neuritis following the first dose of an HPV vaccination is very rare. Response to corticosteroids treatment is promising. However, infective causes need to be ruled out. Longer follow-up is required to detect early demyelinating disease.


We report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan. Retroviridae, genus Deltaretrovirus, and an etiological agent for adult T-cell leukemia (ATL) (Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus belonging to the family ia (ATL) . Currentia (ATL) . HTLV-1 ia (ATL) . The expia (ATL)  or epigeia (ATL)  of the pia (ATL) .Here, we report the complete HTLV-1 provirus genome sequence of OATL9, a novel HTLV-1/EBV-infected B-cell line isolated from the acute type ATL patient . The stuIt remains to be elucidated how Tax and HBZ contribute to the viral tumorigenesis or how the integration site of the virus affects its gene expression property. We are conducting molecular biological analysis on this cell line to elucidate its mechanistic properties for hematologic immortalization ability.LC183873  and LC378575 .The genome sequences of these HTLV-1 proviral genomes have been deposited in GenBank under accession numbers


The skin functions as a barrier between the organism and the surrounding environment. Direct exposure to external stimuli and the accumulation of genetic mutations may lead to abnormal cell growth, irreversible tissue damage and potentially favor skin malignancy. Skin homeostasis is coordinated by an intricate signaling network, and its dysregulation has been implicated in the development of skin cancers. Wnt signaling is one such regulatory pathway orchestrating skin development, homeostasis, and stem cell activation. Aberrant regulation of Wnt signaling cascades not only gives rise to tumor initiation, progression and invasion, but also maintains cancer stem cells which contribute to tumor recurrence. In this review, we summarize recent studies highlighting functional evidence of Wnt-related oncology in keratinocyte carcinomas, as well as discussing preclinical and clinical approaches that target oncogenic Wnt signaling to treat cancers. Our review provides valuable insight into the significance of Wnt signaling for future interventions against keratinocyte carcinomas. The skin is the largest organ and functions as a protective barrier for the host to prevent fluid loss and regulate body temperature. It also harbors vasculature and sensory organs that transduce changes in temperature and pressure. The skin is composed of two major layers, the epidermis and dermis, forming the outer and inner layers, respectively. The epidermis includes the stratified epithelium, referred to as the interfollicular epidermis (IFE), and skin appendages, including the hair follicle, sebaceous gland, and sweat gland. The IFE and the hair follicle undergo regeneration throughout life. Constant exposure to mutagens, i.e., ultraviolet (UV) light and chemicals, can induce genetic mutations and hyperproliferation of epidermal cells, both of which eventually contribute to the formation of skin cancer. Two major types of non-melanoma skin cancer (NMSC) commonly found in patients are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which together account for more than 95% of total NMSC . As BCC Several signaling pathways that have been shown to play vital roles in development of the skin epithelium are also implicated in the progression of keratinocyte carcinoma, including hedgehog (Hh), transforming growth factor β (TGFβ), mitogen-activated protein kinase (MAPK/ERK) and Wnt signaling ,5,6,7. AWnt signaling pathways govern a multitude of cellular functions including cell proliferation, differentiation, fate specification, migration and polarity ,12,13. AWnt signal transduction is initiated upon the binding of Wnt ligands to cell surface receptors of the Frizzled (Fzd) family. Upon binding, Fzd can associate with other co-receptors, such as low-density lipoprotein-related protein (LRP5/6) or tyrosine kinase receptors , to activate Dishevelled (Dvl) and then trigger diverse signaling cascades ,16,17,18Axin2, c-Myc, and Ccnd1 [The activation of canonical Wnt signaling is characterized by β-catenin-dependent transcriptional activity. In the absence of Wnt ligand, cytosolic β-catenin is captured and phosphorylated by the destruction complex, composed of Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β) ,20,21. Cnd Ccnd1 ,23. 2+) and Wnt/planar cell polarity (PCP) pathways [2+ pathway, Wnt-Fzd interaction leads to the activation of phospholipase C and increases the concentrations of inositol 1,4,5-triphosphate (IP3) and 1,2 diacylglycerol (DAG). IP3 interacts with intracellular calcium channels to release Ca2+ ions, leading to the activation of calcium-dependent kinases, such as protein kinase C (PKC), Ca2+-calmodulin dependent kinase II (CAMKII), or Ca2+-dependent phosphatase calcineurin (CaN) [2+ pathway triggers a wide-range of cellular processes, including actin cytoskeleton remodeling and cell motility [Non-canonical Wnt signaling transduces signals independent of β-catenin, and can be divided into Wnt/Calcium  ,28,29. Pin (CaN)  while CAin (CaN) . In parain (CaN) . Activatmotility . For themotility ,35. Amonmotility . As AP-1motility ,38, the The transduction of Wnt signals depends not only on which ligand is present, but also on which receptor(s) and cognate receptor(s) are expressed in the cell. As such, a single Wnt protein can trigger a combination of multiple signaling cascades that might work together as a dynamic signaling network .The adult skin epidermis is composed of the IFE, hair follicles, sebaceous glands and eccrine sweat glands. Cellular processes including homeostatic maintenance and post-damage regeneration are ensured by the multipotent epidermal SC populations located in both the basal layer of IFE and in the hair follicle .The IFE is continuously being regenerated by cells within the basal layer, which proliferate and give rise to cells that migrate outwards and differentiate into suprabasal keratinocytes, and then terminally differentiate into cornified envelope cells. The control of basal cell proliferation within the IFE is tightly regulated by Wnt/β-catenin signaling ,42. AbseIn mammalian skin, mature HFs undergo regeneration by progressing through cyclical phases of growth (anagen), degeneration (catagen), and rest (telogen). This long-lasting regeneration is fueled by hair follicle stem cells (HFSCs). The activation of HFSCs is tuned by a balance of bone morphogenetic proteins (BMP) and Wnt signals coming from their niche cells . During Beyond the role of Wnt signaling in the normal regeneration cycle of the hair follicle, Wnt signaling also plays a role in the acute response to injury. Upon injury, the adult skin epidermis undergoes a wound healing process which occurs in four overlapping phases: disruption of homeostasis, inflammation, re-epithelialization, and tissue remodeling . Gene exIn summary, epidermal SCs undergo self-renewal and differentiation to regenerate the skin epidermis during homeostasis and upon injury. A growing body of evidence shows that tumor growth is driven by a population of tumor cells presenting SC-like characteristics, such as self-renewal and slow-cycling properties . Thus, SWhile genetic mutations are major factors for the development of keratinocyte carcinoma, disrupted signaling pathways have emerged as necessary cofactors. Disruption of HF homeostasis may lead to hair loss disorders, such as alopecia universalis, or uncontrolled HF proliferation, which may cause follicle-based tumors . NotablyBCC is the most common skin tumor in humans. The main etiological factors provoking the development of BCCs are UV radiation, ionizing radiation, arsenic exposure, as well as traumatic injury or burn ,72. OtheBCCs can be clinically and histologically categorized into several encompassing nodular, micronodular, superficial, infiltrative, morpheiform, and mixture variants. Thus, BCC is generally characterized based on the structure of tumor cells similar to the basal cells of the normal epidermis, however, their molecular characteristics are more related to embryonic hair follicle progenitors . RecentlPTCH1 and gain of SMO were described as causative mutant Hh pathway genes, accounting for 90% of human BCCs [There is extensive evidence that the origin of BCC pathogenesis is predominantly triggered by dysregulation of the Hh pathway ,85. Thisman BCCs ,90. Mousman BCCs , whereasman BCCs . Thus, aman BCCs . Apart fman BCCs . CTNNB1 (the gene encoding for β-catenin), particularly at Ser 33, 37 and Thr 41, perturb the phosphorylation sites for GSK3β, which leads to stabilization of β-catenin and in turn elevates Wnt/β-catenin signaling in favor the event of tumorigenesis [The development and progression of tumors are orchestrated by a network of intricate signaling pathways. In addition to Hh signaling, ample scientific evidence indicates that the Wnt/β-catenin signaling pathway participates in tumorigenesis of BCCs. Of note, constitutive expression of Wnt mediators, e.g., Wnt1, 2, 5A, 11, 13, and 16 and β-catenin, facilitates the progression of BCCs ,96. As rigenesis ,103. As igenesis . Moreoveigenesis . Indeed,igenesis ,105, andigenesis . Hence, Xenopus development [Crosstalk between Hh and Wnt signaling pathways have been recently implicated in the pathogenesis of BCCs. Several genomic studies have indicated that genes encoding for components involved in both Hh and Wnt signaling are commonly altered in human BCCs ,106. It elopment . Simultaelopment , reinforelopment . During elopment . Most reelopment . As a suSCC is recognized based on the tumor lesion composed of both proliferative basal cell and differentiated squamous cell layers. Epithelia arising from different parts of the body may develop different types of SCCs, including cutaneous SCC (cSCC), lung SCC (lSCC), and head and neck SCC (HNSCC). Each type of SCC has characteristics that can be distinguished by multidimensional genome-wide analyses . One of The incidence of cSCC is increasing remarkably each year due to a string of causative risk factors, including UV light from sun exposure, human papilloma virus (HPV) infection, chronic injury, arsenic exposure, immunosuppression, and inflammation ,119. AmoTP53, CDKN2A, RAS, PTEN, EGFR, and genes encoding for NOTCH receptors [H-Ras (A182T) [K-Ras(G12D) was widely utilized to study the process of tumorigenesis [K-Ras(G12D) activation under the control of cell type-specific promoters allowed the origin for SCC formation to be defined. For instance, in combination with Tp53 deletion, K-Ras(G12D) activation in IFE basal cells or HFSCs, but not in transient amplifying HF matrix cells, led to the development of SCCs [TP53 or TP63 [The main mutations of human cSCC are found in eceptors ,122,123.eceptors . In addieceptors ,126. Dececeptors ,128. It  (A182T) , and proigenesis ,131. In  of SCCs . Additio or TP63 ,134,135,K-RAS(G12D) and Tp53 depletion [As previously described for BCCs, the cellular origin of SCCs affects the phenotype of the tumor. For instance, SCCs derived from the IFE are well-differentiated, whereas SCCs developing from the HFSCs frequently undergo EMT . In addiepletion . Other tepletion . In mousepletion ,140, is epletion .2+ signaling, emerges as a major cause of cSCC development and progression [WNT and FZD genes, in cSCCs [TP53 (85.2%), CDKN2A (61.5%), and NOTCH1 (42.6%), but also alterations in Wnt-related genes, LRP1B (22.1%) and APC (8.2%) [Dysregulation of Wnt signaling, including Wnt/β-catenin and β-catenin-independent Wnt/Cagression ,122,141.in cSCCs . This fiin cSCCs . RecentlC (8.2%) . The potential role of Wnt antagonists, SFRPs and Dkks, in tumorigenesis has drawn interest. Several lines of studies indicated that SFRP1, which destabilizes β-catenin by interfering with Wnt-Fzd interactions, was downregulated in human SCC samples . AccordiAberrant accumulation of β-catenin protein or its presence in the nucleus in tumor cells is a common characteristic of SCCs ,147,148.Apart from β-catenin, TCF7L1 , a binding partner for β-catenin, is known to contribute to tumor prevalence and progression in the pathogenesis of several cancers ,156. MotIn addition to cSCC maintenance and progression, β-catenin has an impact on tumor metastasis via EMT, in which E-cadherin-bound β-catenin is released from cell membranes and an increased pool of cytoplasmic β-catenin may enter the nucleus to trigger β-catenin-dependent signaling ,170,171.+ cells and revealed that targeting Lgr5+ cells by inhibition of Wnt signaling  in combination with Vismodegib treatment resulted in regression of BCCs [Targeting the Wnt signaling pathway is recognized as a potential therapeutic approach to treat various cancers. Currently, several preclinical and clinical trials on compounds targeting Wnt signaling are in progress. The conventional treatments for localized keratinocyte carcinomas include surgical excision, Mohs surgery, cryotherapy, curettage and electrodessication, topical treatment, photodynamic therapy, radiation therapy and chemotherapy individually or in combination ,176,177. of BCCs . This opFor cSCCs patients, the primary treatments are surgical excision, Mohs surgery and/or adjuvant radiation therapy. Radiation therapy followed by adjuvant chemotherapy is recommended for metastatic cSCCs ,185. TheDespite of the advanced treatments available for cSCCs, these treatments may gradually lose durable responses or cause a number of side effects, and tumors may relapse or become resistant to treatment . Also, tOwing to the involvement of Wnt signaling in diverse cellular functions across different normal tissues, treatments targeting Wnt signaling might affect healthy tissues or organs. For instance, porcupine inhibitors, e.g., LGK974 and Wnt-c59, might negatively impact on bone volume and strength . It was In this review, we emphasize the crucial roles of Wnt signaling in different aspects of keratinocyte carcinoma development, including their participation in tumor initiation, progression, invasion, and metastasis. Canonical Wnt signaling is essential for keratinocyte carcinoma initiation and progression by enhancing tumor cell proliferation and regulating the maintenance of CSCs. On the other hand, non-canonical Wnt signaling is necessary for regulating the process of tumor invasion and metastasis. Despite the availability of various analyses to screen the mutations or biomarkers for these cancers, it is plausible that due to heterogeneity some tumors might escape detection due to factors from the tumor microenvironment and existing quiescent CSC populations. Future investigations should focus not only on the intricate molecular communications within tumors, but also on extrinsic factors from stromal cells, including fibroblasts and immune cells, as well as identification of CSCs. In view of the complexity of multiple signaling coordinating skin tumorigenesis, it will be of utmost importance to better understand and dissect the cross-interactions of Wnt signaling with other oncogenic pathways. This would allow the development of new effective therapeutic interventions to eradicate Wnt signaling-related malignancies.


The CAI patients generally had flatter talar joint surfaces and a flattened calcaneal ground‐contact surface. These findings suggest that specific bone shapes may increase the risk of developing CAI after sustaining a LAS. © 2019 The Authors. ®Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1892–1902, 2019Bone shapes, particularly those defining the subtalar joint (STJ), have not received much attention yet as a risk factor for developing chronic ankle instability (CAI) after sustaining a lateral ankle sprain (LAS). This study aimed to compare three‐dimensional (3D) shape variations in the STJ bones within individuals with CAI and healthy controls. 3D statistical shape models (SSMs) of the STJ bones were built to describe the bone shape variations observed within a population consisting of 26 individuals with unilateral CAI and 26 healthy controls. Using the SSMs and analysis of covariance test, age‐ and gender‐adjusted shape variations in the bones were compared within individuals with CAI and healthy controls. The mean age of the CAI patients  and healthy controls  was 29 (standard deviation [SD] = 11) and 36 years (SD = 11), respectively. Tali and calcanei did not significantly vary between ipsilateral CAI and their contralateral ankle. Two shape modes, one for the talus ( Lateral ankle sprain (LAS) comprises approximately 80% of all ankle sprains,Most patients experiencing a LAS can be successfully treated and regain functional ankle stability with conservative treatment.Limited evidence is available suggesting that surgical treatment is more effective in decreasing the frequency of recurrent ankle sprains.A great deal of effort has been put into identifying the factors associated with the development of CAI  are presented in Figure n = 40) were the individuals who had voluntarily undergone CT scanning either as a part of a study investigating the in‐vivo measured range of motion of the STJThe computed tomography (CT) scans of a number of patients with confirmed CAI and healthy controls were retrieved from the database of Amsterdam UMC . All CAI patients visited Amsterdam UMC for persistent complaints of ankle instability in combination with recurrent lateral sprains, with or without pain and had undergone non‐surgical treatments at least for 6 months without success. Healthy controls . The acquisition parameters  and the tomographic reconstructions showed certain levels of variability within the data set. In particular, the voxel sizes varied between 0.3 × 0.3 × 0.3 mm and 0.7 × 0.5 × 0.5 mm.n = 98) and tali (n = 98) were segmented following the same segmentation protocol as used in our previous study.All calcanei  were retrieved from our previously published studies for the tali σ = 3 mm, nm = 2,000, λ = 5 × 10−4).The differences between the bone instances due to their relative translations, different orientations, and different scaling factors were minimized by aligning all the tali  using an unbiased registration algorithm.n = 8,667) and calcanei  was established using the coordinates of all points and surface normals as described in a study performed by van de Giessen et al.x, y, z) of the corresponding points established across all tali  of the CAI patients as well as those of the healthy controls. Then, a principal component analysis was performed on the covariance matrix. As a result, the modes of shape variation  with a descending order of variance  were obtained. The modes of shape variation describe the directions of shape changes, while the variance describes how much variation is present in the corresponding direction.Following the registration process, a dense correspondence Fig. , step 4 x) can be represented, using the mean shape of the talus  (x®) and a weighted sum of the modes of shape variation for the talus  (Φ), asb values  describe the contributions of the first c modes of shape variation to the mean bone shape. In other words, a shape parameter  for a given mode of shape variation  describes how far a shape x is away from the mean shape x® in the specified direction  .A new or existing talar  shape  regardless of its group , the shape parameters  are different.b values) for the first c modes of shape variation were compared between the three groups using an analysis of variance test. For pairwise comparisons of the groups , the Bonferroni post‐hoc analysis was performed. Only the shape modes that represented more than 5% of the total shape variation were included.While the mean talus  shape, b values) across all tali and calcanei for each mode of shape variation conform to a normal distribution, a Kolmogorov–Smirnoff test was applied prior to our statistical analyses. All statistical analyses were carried out using SPSS .To evaluate whether the observed variations in the bone shape were affected by age and gender, an analysis of covariance (ANCOVA) test was performed considering the age and gender as covariate factors. To assess whether the shape parameters .The CAI patients were almost equally distributed in terms of their gender . The mean age of the patients was 29 years (standard deviation [SD] = 11). There were 12 males and 14 females in the healthy control group with a mean age of 36 years (SD = 11). In 62% of the patients, the right ankle was affected by CAI , respectively described 49% and 45% of the total shape variance in the talus and calcaneus . For both bone types, the distributions of the shape parameters for the first c modes did not significantly differ from a Gaussian distribution (p > 0.05).The number of the modes of shape variation retained for statistical analyses, The descriptions of the bone shape variations are only provided for the first three shape modes of the talus Fig.  and calcTalus Mode 1: the lateral rotation and the size of the talar head.Talus Mode 2: the curvature of the talar lateral process and the inclination angle of the talar neck relative to the body.Talus Mode 3: the lateral projection of the talar process and the length of the talus.Calcaneus Mode 1: the length of the calcaneus.Calcaneus Mode 2: the inclination of the sustentaculum tali.Calcaneus Mode 3: the medial and lateral tuberosity and the contour of the anterior articular surface.The first three modes of shape variation of the talus Fig.  and calcp > 0.05) differ between the ipsilateral and contralateral sides of the subjects who had a unilateral CAI .The shapes of the talus and calcaneus did not significantly (p = 0.015) as well as between the CAI contralateral controls and healthy controls (p = 0.035).The shape variations of the talus described by Mode 2  and CAI contralateral controls vs. healthy controls (p = 0.001).Shape variations represented by the Mode 3 of the calcaneus  caused no changes in the statistical significance of the reported results.In this study, a 3D SSM technique was used to compare the shapes of the talus and calcaneus between both sides of the patients with unilateral CAI as well as between CAI patients and healthy controls. The results indicate specific shape differences in the bones. These make individuals with CAI distinguishable from healthy individuals with no known history of ankle joint pathology.D) between both axes will increase. In turn, the pronatory external moment will be higher and will cause extra strain on the medial muscles and ligaments. A patient may minimize the lateral shift of the GRF axis and avoid losing balance by an inversion movement  as well as a flattened calcaneal ground‐contact surface Fig. B. Variatent Fig. C. This cβ > α). Considering that the STJ axis is oblique, an analogy can be made to a gear mechanism shown in Fig. β > α), less rotation around the horizontal axis of the talus in relation with the calcaneus is observed for a given rotation around the vertical axis  is difficult, as they are subtle and are distributed over bone surfaces. Moreover, not all of these modes of shape variation explain meaningful shape variations, as they may simply describe the noise caused by scanning and point sampling.The main limitation of this study is the retrospective nature of the data collection. Although instability is certainly present in the CAI patients, more concomitant problems  may be the cause of the observed bony shape differences. Moreover, information on the activity level of the patients was not available, which may be a contributing factor as well. The age and gender of the subjects were, however, known. Therefore, the effects of age and gender could be accounted for in our statistical analyses.Investigating the mechanisms that can lead individuals to develop CAI after sustaining a LAS were outside the scope of the current study. Nevertheless, we attempted to explain some mechanisms that can be associated with shape variations in the STJ bones and may play a role in the onset of CAI. With this perspective, bony shape variations described by Mode 2 of the talus and Mode 3 of the calcaneus were considered. Such explanations are, however, limited in the sense that the SSMs of the talus and calcaneus were developed independently from each other, without having a coupling between them to describe simultaneous shape variations. Irrespective of the exact mechanism, the most important finding of the current study is that two specific talar and calcaneal shape variations appear to be significantly different between CAI patients and healthy controls. Determining whether these findings represent post‐traumatic changes or whether recurrent LAS originate from these variations requires further research in a prospective setting. However, the fact that bone shape did not vary within individuals with one unstable ankle suggests that these shape variations play a role in the development of CAI after a first‐time LAS and are not caused by CAI.The bone shapes of the subjects with and without CAI overlap. Some of the healthy subjects exhibit geometrical features that are similar to those identified as being typical for the CAI group. These individuals may have not developed CAI yet or may have not sustained an injury that leads to the onset of CAI, but may nevertheless be at the risk of developing CAI. Inversely, some of the CAI patients do not show the shape features that we found to be typical of the CAI group, but had nevertheless sustained events that had led to the genesis of CAI, potentially due to other risk factors.Within this study, the shape variations in the bones of the TCJ of the CAI patients and healthy controls were not considered. Instead, we focused on the STJ bones and their shape variations. That is due to the fact that the TCJ and its relation with CAI have already been the main focus of researchers, while the STJ and its potential contribution to CAI have been often overlooked. Nevertheless, the methodology presented here is not limited to the STJ bones and could be applied to the TCJ bones as well.This study is a next step in identifying the risk factors that originate from the shape of our bones. Creating SSMs that are based on a prospective data set would help in assessing the prognostic values of the bone shape variations on the development of CAI. Moreover, if the described shape variations could be translated into reliably measurable parameters for conventional radiology, they can be also included in the risk assessment models that are used in clinical settings. This can provide clinicians with more insight into which treatment options may work the best for individual patients who have sustained LAS.The 3D statistical shape models of the talus and calcaneus were built based on the mixed data of patients with unilateral CAI and healthy controls. These enabled us to quantitatively compare shape variations between the ipsilateral and contralateral sides of CAI patients, the ipsilateral sides of CAI patients and healthy controls, and between the contralateral sides of CAI patients and healthy controls. We found two specific statistically significant shape differences between CAI patients and healthy controls. In the case of the talus, the identified shape mode affected the curvature of the talar lateral process as well as the inclination angle of the talar neck relative to the body. As for the calcaneus, the identified shape mode was related to the medial and lateral tuberosities combined with the contour of the anterior articular surface.All the authors contributed to the study design. G.V. and G.K. contributed materials and G.V. checked the data set to discard CT scans satisfying exclusion criteria. Scans of healthy controls were provided by G.V., G.K., and L.B. L.B. contributed materials and SSM generation tools. N.T. post‐processed the data set and analyzed the data together with G.V., L.B., G.T., and A.Z. All authors revised the paper for intellectual content and approved the final version of the manuscript to be published.Supporting information.Click here for additional data file.


Targeting epigenetic reader proteins by small molecule inhibitors represents a new therapeutic concept in autoimmune diseases such as rheumatoid arthritis (RA). Although inhibitors targeting bromodomain protein 1 (BRD1) are in development, the function of BRD1 has hardly been studied. We investigated the therapeutic potential of BRD1 inhibition in joint-resident cells in RA, synovial fibroblasts (SF) and macrophages. The proliferation of SF was decreased upon BRD1 silencing, accompanied by the downregulation of genes involved in cell cycle regulation. Silencing of BRD1 in SF decreased the basal expression of MMP1 but increased TNF-α- and LPS-induced levels of MMP3, IL6 and IL8. In monocyte-derived macrophages (MDM), silencing of BRD1 decreased the LPS-induced expression of TNF-α, but did not significantly affect basal and the TNF-α- and LPS-induced expression of IL6 and IL8. Our data point to a cell type- and a stimulus-specific function of BRD1. Inhibiting BRD1 could have potential beneficial effects in RA via decreasing the proliferation of SF. Anti-inflammatory effects were limited and only observed in MDM. Bromodomain proteins (BRD) are epigenetic readers of acetylated histones. Targeting BRDs by small-molecule inhibitors has emerged as a new potential therapy in inflammation and arthritis2. Inhibitors against the family of bromodomain and extra-terminal (BET) proteins exhibit anti-inflammatory properties3 and show beneficial effects in RA synovial fibroblasts (RASF)4 and experimental arthritis5. First inhibitors targeting members of a distinct BRD family, the bromodomain and plant homeodomain finger-containing (BRPF) family, including BRPF1, BRD1 (BRPF2), and BRPF3 have recently been developed7, however, they have not been tested regarding their anti-inflammatory potential.Histone lysine acetylation, in combination with other post-translational modifications and DNA methylation determines the epigenetic code that regulates gene expression. Enzymes that read and erase histone acetylation marks are involved in regulating pathogenic pathways in rheumatoid arthritis (RA), including inflammation8. BRD1 has been functionally linked to the acetylation of histone 3 lysine 14 (H3K14ac)9, a histone mark that co-occurs together with H3K9ac marks at active promoters. Furthermore, H3K14ac marks prime inactive genes for stimuli-dependent activation in mouse embryonic stem cells10. In anticitrullinated peptide antibody-positive RA patients, an intronic single nucleotide polymorphism  in BRD1 was associated with the progression of joint destruction in stage I of a genome-wide association study11, providing a potential link between RA and BRD1. In this work, we evaluated the function of BRD1 in joint resident cells, specifically synovial fibroblasts (SF) and macrophages, and explored the potential of BRD1 inhibition as a treatment strategy in RA.BRD1 has been identified as a subunit of the MOZ/MORF histone acetyltransferase (HAT) complex12 of these tissues , cyclin A (CCNA2) and cyclin B1 (CCNB1). The mRNA expression of p21 was not affected by BRD1 silencing  after silencing of BRD1. Silencing of BRD1 in RASF and OASF reduced the basal MMP1 mRNA expression and secretion  have recently been developed16. BRD1 plays an important role in T cell development and is indispensable for the activation of the CD8 gene during early thymocyte development15. In SF, silencing of BRD1 exhibited rather pro-inflammatory and pro-destructive functions in presence of inflammatory stimuli. We did not observe any differences in SF from RA and OA patients, in accordance with the comparable expression of BRD1 in RA and OA tissues. Furthermore, there were no differences after BRD1 silencing in SF derived from different joint origin with respect to measured gene products. In MDM the LPS- induced expression of TNF-α was decreased by BRD1 silencing. This anti-inflammatory effect in MDM was stimulus-specific, since TNF-α did not affect the expression of TNF-α mRNA upon BRD1 silencing. This is in line with previous studies that showed a stimulus-specific expression of TNF-α in different cell types in vitro and in vivo19. A cell-type specific regulation of IL6 in SF and CD14+ monocytes has been described also previously by Noss et al. who identified a single nucleotide polymorphism in the IL6 promoter that affected only the expression of IL6 in SF from RA and OA patients but not in monocytes20. We have recently showed a cell-type specific regulation of a larger panel of cytokines, chemokines and MMPs in SF and MDM upon repeated stimulation with LPS. This coincided with a different epigenetic architecture at the promoters of these genes in unstimulated SF and monocytes, leading to a cell-type specific regulation of these genes after stimulation with LPS21. BRD1 is associated with a HAT complex adding H3K14ac marks to active gene promoters9. H3K14ac marks are rapidly induced in promoter regions of IL6, IL8 and TNF-α by inflammatory stimuli in different cell types23. In THP-1 monocytes, H3K14ac marks preceded the recruitment of the p65 in the TNF-α promoter upon stimulation with S100b proteins22, suggesting a role of H3K14ac in the activation of the TNF-α promoter. Unfortunately, there are no public data sets on H3K14ac marks available that would enable a comparison of this histone modification in inflammatory gene promoters in macrophages and SF or other fibroblasts.Beside the anti-proliferative effect in SF, we showed that BRD1 regulates cytokine and MMP gene expression in a cell type- and stimulus-specific manner. Effects of BRD1 inhibition on other arthritis-relevant cell types in addition to SF and MDM are likely since BRD1 is expressed also in T- and B-cells7, making the interpretation of the results difficult. In addition, BRPF inhibitors were mentioned to impair the RANKL-induced differentiation of monocytes into bone resorbing osteoclasts7. These results, together with the reported BRD1 SNP (rs138845) and our findings suggest a potential role of BRD1 in the pathogenesis of arthritis. In contrast to BET protein inhibition, that has a broad range of anti-inflammatory and anti-proliferative effects in diverse cell types in vitro and in vivo24, BRD1 inhibition rather exhibits a cell type and stimulus-specific functions with only limited anti-inflammatory effects with respects to the studied gene products and cell types. Therefore, BRPF inhibitors should be tested in many different cell types and under different stimulatory conditions.In IL-4-stimulated macrophages, pan-BRPF inhibitors modulated the secretion of CCL-22. However, depending on the concentration of these inhibitors, both a decreased and an increased secretion of CCL-22 were observed21. All patients fulfilled the criteria for the classification of RA25 and OA26. The characteristics of patients undergoing joint replacement surgery is shown in Table Synovial fibroblasts of RA and osteoarthritis (OA) patients and monocyte-derived marcophages (MDM) from healthy volunteers and RA patients were cultured as described5 RASF or OASF and 6.5 × 105 MDM were transfected with a pool of two different siRNAs (2 × 0.75 μM) targeting BRD1 or scrambled siRNAs (Qiagen) as a control using the Amaxa Basic Nucleofector Kit for Primary Mammalian Fibroblasts or the Human macrophage nucelofector kit (Lonza). 24 h after transfection cells were stimulated with TNF-α (10 ng/ml) or LPS (100 ng/ml) as indicated for another 24 h.3.5 × 102O2. Slides were permeabilized with 0.1% Triton in PBS. Nonspecific protein binding was blocked with 5% goat serum in antibody diluent (Dako). Polyclonal rabbit anti-BRD1 antibodies (abcam) and rabbit IgG1 were applied over night at 4 °C. Slides were washed in PBS-T (0.05% Tween 20 in PBS) and incubated with biotinylated goat anti-rabbit antibodies (Jackson ImmunoResearch). The signal was amplified with ABC reagent for peroxidase  and detected with 3,3′-diaminobenzidine (Vector Laboratories). Staining intensities (SI) of tissues were scored (1: little staining – 4: strong staining) independently by two researches. For double staining, slides were stained with either the fibroblast marker prolyl 4-hydroxylase beta (Acris), CD68 (DakoCytomation) or IgG the day after staining for BRD1. Slides were washed in PBS-T and incubated with biotinylated goat anti-mouse antibodies. The signal was amplified with ABC reagent and detected with either Vector blue (Vector laboratories) or HistoGreen (Linaris). For immunofluorescence stainings, RASF were cultured in chamber slides , fixed with 4% paraformaldehyde and permeabilized with tris-buffered saline (TBS) containing 0.1% Triton X-100. Nonspecific binding was blocked with 5% goat serum. Slides were incubated with polyclonal rabbit anti-BRD1 antibodies and rabbit IgG1 overnight at 4 °C. After washing, slides were incubated with FITC- conjugated secondary antibodies (Thermo Scientific). Actin filaments were stained with phalloidin (Sigma-Aldrich). Slides were covered with fluorescent mounting medium (Dako) and analysed with a fluorescence microscope (Axio Imager).After deparaffinization, tissue sections of RA and OA patients were pre-treated with citrate buffer . Endogenous peroxidase activity was disrupted with 3% HTotal RNA was isolated from cells using the ReliaPrep RNA Cell Miniprep System (Promega) including on-column DNaseI digest and reversed transcribed. Real-time PCR was performed using SYBR green (Life Technologies). Primer sequences are available on request. Constitutively expressed human ribosomal protein large P0 (RPLPO) was measured for internal standard sample normalization. Relative mRNA expression was calculated by the comparative threshold cycle method (ΔΔCt).4. Membranes were probed with antibodies against BRD1 (abcam) or α-tubulin (abcam). As secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Jackson ImmunoResearch) were used. Signals were detected using the ECL Western blotting detection reagents  and the Alpha Imager Software system (Alpha Innotech).Western blotting was performed as described in detail previouslyMMP1, MMP3, IL6 and IL8 were measured using the human total MMP-1 ELISA kit, the human total MMP-3 ELISA kit, the human IL-6 ELISA Set, and the human IL-8 ELISA Set (BD Biosciences), respectively in cell culture supernatants.Proliferation and adhesion (towards plastic) of transfected RASF was analysed using an impedance-based system for Real-time proliferation and adhesion assays . Cells were seeded into E-plates 24 h after transfection at a densitiy of 2500 cells/well. TNF-α (10 ng/ml) was added simultaneously during seeding. Each condition was analysed in quadruplicates. Impedance changes were recorded every 15 minutes (0–24 hours) and every 30 minutes (24–146 hours). Adhesion was analysed over the first 90 minutes of the experiment. Proliferation was analysed during the exponential phase of the slopes (40–80 hours) and over the entire period of the experiment (0–146 hours).SF were incubated with FITC Annexin V (BD Biosciences) and propidium iodide (Sigma-Aldrich) for 15 minutes at room temperature in the dark, as recommended by manufacturer’s protocol (BD Biosciences), and analysed by flow cytometry .Statistical analysis on data sets was carried out by using the GraphPad Prism program . N numbers in all experiments represent biological samples from different patients. Differences between experimental groups were analyzed by paired t-test or Wilcoxon signed rank test for analysis of basal expression. Differences in proliferation (0–146 h) were analysed by one-way ANOVA. Data are reported as means ± standard deviations. P values < 0.05 were considered significant. Raw data files are available on request.Dataset 1


The caveolae microdomain plays pivotal roles in various cellular processes such as lipid homeostasis, signal transduction, and endocytosis, and also serves as a signaling platform. Although the caveolae microdomain has been postulated to have a major contribution to the development of cardiac pathologies, including cardiac hypertrophy, recent evidence has placed this role into question. Lack of direct evidence and appropriate methods for determining activation of caveolae-specific signaling has thus far limited the ability to obtain a definite answer to the question. In this review, we focus on the potential physiological and pathological roles of the multifunctional kinase Ca This review can help to highlight targets of research focus and specific questions to tackle toward gaining a better understanding of the molecular mechanisms linking caveolae signaling and heart health, toward establishing new therapeutic strategies.Caveolae are unique flask-like membrane invaginations of 50–80 nm in diameter, which are enriched in cholesterol and sphingolipids . CurrentThere are two types of structural proteins in caveolae: caveolins and cavins. Caveolin is comprised of three isoforms, caveolin-1, caveolin-2, and caveolin-3 , with sp2+-dependent signaling molecules such as calcineurin and CaMKII play vital roles in the development of cardiac hypertrophy, and activation of these molecules is associated with LTCC activity  kinase that is activated in a Carylation , oxidatirylation , and glyrylation . CaMKII rylation . Activatrylation . Geneticrylation . Thus, Crylation . In addirylation . Two isorylation , 2003. Mcoupling , whereasnduction . Furthernduction . Collectnduction . HoweverOne of the major obstacles in determining the precise pathophysiological role of CaMKII in the caveolae microdomain is the lack of efficient and simple methodology to assess microdomain-specific activation of the kinase. Conventionally, the activation of a kinase is determined biochemically by detecting the phosphorylation of its specific substrate using a radioisotope or fluorescence from a whole cell lysate. However, these methods are not suitable for the assessment of microdomain-specific signaling, since fraction preparation is complicated and time-consuming. Alternatively, the detection of phosphorylation using a phosphor-specific antibody is a simple, useful, and reliable method. The detection of mitogen-activated protein kinases (MAPKs) such as ERK and p38 MAPK is a representative example of the application of phosphor-specific antibodies for assessing signaling pathway activation.Figure 1) (We recently developed a novel tool to examine the caveolae-specific activation of CaMKII using a fusion protein comprising 22 amino acids of the cytosolic domain of phospholamban (PLN) fused to caveolin-3 (igure 1) . PLN is igure 1) . The cytigure 1) . Notablyigure 1) . Therefoigure 1) .2+ influx for inducing contractions by triggering Ca2+-induced Ca2+ release  or a CaMKII-specific inhibitory peptide (inhibition).Besides cardiomyocytes, several reports suggested the involvement of CaMK in caveolae-related biological effects, such as 1α,25(OH)2D3-dependent signaling or P2X3 receptor-mediated Ca+ influx . In card+ influx . Phospho+ influx . Several+ influx . However+ influx . By cont+ influx . Therefo+ influx . Thus, s+ influx . Howeverertrophy . Howeverin vivo. Third, the methodology for assessing signals other than CaMKII needs to be developed. Finally, the role of the specific activation of these signals in subcellular organelles such as the mitochondrion should be determined. Methods based on a phosphorylatable peptide-tag have great potential to help tackle these questions. Taken together, the evidence accumulated to date indicates that selective inhibition of target molecules involved in caveolae-specific signaling based on their subcellular location could be a promising therapeutic tool to treat cardiac hypertrophy and heart failure in the future.We have here summarized the current knowledge on the effects of caveolae-specific signal activation in relation to the pathogenesis of cardiac hypertrophy. The related molecules such as CaMKII and LTCC have multiple cellular functions that seem to depend on the corresponding subcellular localization of the molecules, including intracellular organelles and microdomains. Thus, regulation of location-dependent signal activation is a potential therapeutic target for heart failure. For instance, inhibition of the specific population of CaMKII or LTCC, those which are involved in the development of cardiac hypertrophy or induction of cell death, could potentially improve the prognosis of patients with heart failure without disturbance of excitation-contraction coupling. However, several questions remain to be answered regarding the role of microdomain-specific signaling in the development of cardiac hypertrophy and heart failure. First, the contribution of other microdomains such as lipid rafts  or couplST and HN wrote the manuscript. YF checked and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Giardia duodenalis is an important zoonotic parasite which can parasitize in the intestines of humans and various animals. However, the information about the prevalence and genetic diversity of G. duodenalis in goats in China is limited. It is yet to be known whether Yunnan black goats, a unique goat breed in subtropical Yunnan province, southwestern China, are infected with G. duodenalis. Thus, a total of 907 fecal samples were collected from Yunnan black goats in five regions in Yunnan province, to estimate the prevalence and genotypes of G. duodenalis using a PCR-based approach. The G. duodenalis prevalence is 4.2% (38/907) in Yunnan black goats by nested amplification of the β-giardin (bg) gene, and the genotypes are identified as assemblage E, with 5 novel subtypes (E11-E15). Multilocus sequence typing revealed that 11, 18, and 38 samples were amplifiable on tpi (triose phosphate isomerase), gdh (glutamate dehydrogenase), and bg locus, respectively, and identified three novel multilocus genotypes (MLGs): MLGE9-MLGE11. To our knowledge, this is the first report of G. duodenalis prevalence and genotypes in Yunnan black goats in China, which extended the host range of G. duodenalis and provided basic data for controlling G. duodenalis infection in Yunnan black goats. Giardia duodenalis  is a common enteric protozoan parasite which can infect humans and a wide range of animal species. G. duodenalis infection can cause a series of diseases which have important effects on human and animal health, such as abdominal cramps, diarrhea, weight loss, and malabsorption [ G. duodenalis infection can be caused by ingesting cysts in contaminated water or food, or through fecal-oral access due to wastewater [ G. duodenalis is approximately 10% in the world population [ G. duodenalis prevalence ranged from 0 to 15.6% in humans [ G. duodenalis has a high prevalence in some low-income areas and developing countries [sorption –3. G. dustewater –6. Accorpulation , and then humans , 9 and 1ountries . G. duodenalis isolates from humans and various animals have been classified into eight different assemblages (A-H) on the basis of molecular characterization [So far,rization , 15. Amorization . Assemblrization , Thailanrization , and Egyrization . G. duodenalis infection in goats in other countries with prevalence ranging from 2.9 to 35.8% [Yunnan province is the fifth largest producer of goats in China , and aboto 35.8% , 21, butto 35.8% –25. G. duodenalis. Thus, the objectives of the present study were to estimate the G. duodenalis prevalence in Yunnan black goats based on characterization of the β-giardin (bg) gene sequences and identify its genotypes using multilocus genotyping (MLG) targeting gdh gene, tpi gene, and bg gene sequences [Yunnan black goat is a unique breed of goat distributed in subtropical Yunnan province, southwestern China. It is yet to be known whether Yunnan black goats are infected withequences , 26.A total of 907 fecal samples were randomly collected from Yunnan black goats in Chuxiong, Lijiang, and Xishuangbanna prefectures, Yunnan province, southwestern China . All of Fecal specimens were washed repeatedly with ultrapure water until all the potassium dichromate was removed, and then genomic DNA was extracted from 200 mg of each fecal sample in a 2 ml centrifuge tube using the commercial E.Z.N.A® Stool DNA kit  by following the manufacturer's instruction. The obtained DNA samples were stored at −20°C for further study. G. duodenalis by PCR-based sequencing of the 511 bp fragment of the bg-gene [Each fecal specimen was examined for the presence and genotype of bg-gene . In addi bg-gene –30. The μl of template from the first PCR product, 2 μL deoxyribonucleotide triphosphate (dNTP) mixture, 2.5 μL of 10×PCR buffer, 3 mM of MgCl2, and 0.2 μM of each primer in a total volume of 25 μL. PCR amplifications were performed as follows: 1 cycle for 5 min at 94°C, followed by 35 cycles of 45 s at 94°C for denaturation, 45 s at 67°C for annealing, and 45 s at 72°C for an extension. All of amplification products were subsequently visualized on 1.5% agarose gels with ethidium bromide. For each PCR amplification, a positive sample (sequenced DNA) and negative (PCR water) control sample were included.The secondary reaction mixture contained 2 http://www.ncbi.nlm.nih.gov/GenBank) using Basic Local Alignment Search Tool (BLAST).All nested-PCR products were sent to Xi'an Qingk Biotechnology Company for two-directional sequencing on an ABI PRISM 3730 XL DNA Analyzer  using relevant internal nested primers for PCR amplification. The sequences obtained were compared with relevant sequences available in GenBank database , and statistically significant differences were considered when P <0.05.The relationships between G. duodenalis-positive based on the amplification of the bg gene. G. duodenalis prevalence was significantly different among the study areas , between different age groups , and between different genders . The G. duodenalis prevalence in Yunnan black goats was higher than that (2.9%) in goats in Heilongjiang province [ G. duodenalis prevalence in Yunnan black goats was markedly lower than in goats in Greece (40.4%) [ G. duodenalis prevalence may be related to feeding conditions, geographical difference, and animal husbandry practices as well as different susceptibility of different breeds of goats.A total of 907 fecal samples were collected from Yunnan black goats in five regions in Yunnan province , and 38 province , but lowprovince , Shaanxiprovince , and Henprovince , China.  (40.4%) , Spain ( (40.4%) , and Bel (40.4%) . The difG. duodenalis prevalence ranged from 0% to 7.03% among the sampled areas. The highest G. duodenalis prevalence was found in Yunnan black goats in Mohan , Xishuangbanna prefecture  and two novel assemblage E subtypes  based on sequence analyses of the bg gene loci  and one novel assemblage E subtype  based on the gdh gene sequences and two novel assemblage E subtypes  based on the tpi gene sequences were also identified among G. duodenalis-positive samples from Yunnan black goats  from other animals and humans  and two known assemblages E subtypes (E5 and E10). MLGs analysis identified three novel MLGs within assemblage E of G. duodenalis. These results not only extended the host range of G. duodenalis distribution, but also enriched the genetic diversity of G. duodenalis in humans and animals, which also have implications for controlling G. duodenalis infection in Yunnan black goats.This is the first report of prevalence and molecular characterization of


On the contrary, inhibition of the Hh signaling pathway by cyclopamine treatment suppressed the expression of osteoblast-related genes and decreased bone mineralization. In agreement with these findings, blocking Hh signaling through knockdown SHH and Gli2 genes led to defective osteoblast differentiation, while promoting Hh signaling by knockdown Ptch1 was beneficial to osteoblast differentiation. Our results thus support that activation of the Hh signaling pathway negatively regulates autophagy and consequentially promotes osteoblast differentiation. On the contrary, induction of autophagy inhibits osteoblast differentiation. Our work reveals the mechanism underlying Hh signaling pathway regulation of bone development.Impaired osteoblast differentiation may result in bone metabolic diseases such as osteoporosis. It was reported recently that hedgehog (Hh) signaling and autophagy are two important regulators of bone differentiation. In order to further dissect their relationship in bone development, we used a zebrafish larvae model to investigate how disruption of one of these signals affects the function of the other and impacts osteoblast differentiation. Our results showed that activation of Hh signaling negatively regulated autophagy. However, suppression of autophagy by knocking down  Summary: Our report of an essential regulation role of hedgehog signaling and autophagy on osteoblast differentiation may contribute to research on bone development biology, hedgehog signaling and the autophagy pathway. Osteoblasts are responsible for bone formation, and impaired osteoblast development leads to serious bone diseases such as osteoporosis . Zinc fiatg5 alleviated the PLY-induced inhibition of differentiation  signaling pathways, mediated by sonic hedgehog (Shh) and Indian hedgehog (Ihh), are recognized as indispensable regulators for osteoblast differentiation and morphological transition. Specifically, the transmembrane receptor Patched (Ptch1) binding to a secreted ligand initiates Hh signaling, whereas unliganded Ptch1, as a negative regulator of Hh signaling, inhibits the activity of the membrane protein Smoothened (Smo), which regulates downstream Gli transcriptional effectors. Among three Gli proteins, Gli2 is a very important activator for the Hh signaling pathway . ActivatInterestingly, accumulating evidence suggests that the Hh signaling pathway cross-talks with autophagy pathway. For example, Hh signaling inhibited formation of autophagosome both in basal level and in autophagy-induced conditions . In pancDanio rerio (zebrafish) has a high similarity with human in bone architecture, bone cell types (osteoblasts and osteoclasts) and matrix proteins  and Gli2MO, respectively. Gli2Mis was used as a negative control. Consistent with results observed under cyA treatment condition, downregulation of SHH and gli2 expression , an Hh signaling pathway inhibitor, for 3 days at 3 dpf downregulated SHH protein expression A, but exn levels B. Furthen levels C. Secondpression D increasand atg5 E and autand atg5 F–H. ThosPtch1, the Hh signaling negative regulator, by injection of Ptch1MO or Ptch1Mis into 1–4 cell stage embryos. As anticipated, knockdown of Ptch1 expression increased SHH protein and autophagy protein p62, but decreased the autophagy proteins ATG5 and LC3BII  wild-type AB strain was originally obtained from the College of Life Sciences and Technology, Tsinghua University. Fish feeding, breeding and maintenance were according to the literature (Zebrafish (terature . Embryoshttp://www.gene-tools.com), 5MO and MisMO sequences were synthesized according to our previous published data  solution in DMSO was added to the embryo media at final concentrations of 10 µM from 3 dpf to 6 dpf, and cyA , were dissolved into DMSO, and then was diluted to 10 µM and 50 µM. 0.1% DMSO treated larvae was used as negative control. 3MA  was dissolved into heated ddHGSE120170.Larvae at 6 dpf were collected into Trizol Reagent (Sigma-Aldrich). Total RNA was extracted following the Trizol Reagent RNA extraction kit manual. First-strand cDNAs were synthesized by reverse transcription using the M-MLV RTase cDNA Synthesis Kit (Promega). qPCR primer pairs are listed in The procedure has been previously described in our previous literature . Briefly2), 50% ethanol and 25% ethanol, bleached with 3% H2O2 containing 1% KOH for 20 min .Larvae at 6 dpf were collected and fixed for 2 h by 4% paraformaldehyde at room temperature, fixed larvae were stained with 0.1% Alizarin Red  in 0.5% KOH overnight. Stained larvae were gradient rehydrated with 75% ethanol (100 mM Tris pH 7.5, 10 mM MgClr 20 min , and rinData in figures represent mean±s.d., derived from at least three experiments. Statistical analyses were performed using one-way ANOVA tests.