Regulatory cells are important for maintaining immunological homeostasis and tolerance to antigens, including self-antigens. Even though in the majority of autoimmune diseases and inflammatory processes, B cells generally play an important role due to their capacity to secrete antibodies, it has emerged that different B cell subsets may be able to down regulate immune responses, especially in experimental autoimmune disease models. Moreover, it has been demonstrated that B cells are able to drive T cell differentiation in favor of a regulatory phenotype  both in mice and humans .+B-1a cells, CD19hiCD1dhiCD5+ B cells, or marginal zone (MZ) B cells [hiCD38hi and CD24hiCD27+ B cells, which have been identified by their capacity to produce IL10 after appropriate stimulation [In several studies the presence of Breg cells is often related to the development of chronic inflammation or autoimmune disease, but their pathogenic involvement is not completely understood. Researchers have described many B cell subsets with regulatory functions in mice, which are characterized by different surface markers, such as CD5. The development of the allergic environment, for example in asthma, could increase germinal center B cell numbers and MHC II and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen and affect the maturation of regulatory B cell subsets [However, Breg cells represent an heterogeneous group of immunosuppressive cells subset with distinct phenotypic and functional properties, that could play a role also in the induction of immune tolerance to allergens. Breg-derived IL10 is involved not only in inhibition of Th1 polarization, but also in preventing Th2 responses and its expression in mucosal environment is important for the generation of immunological tolerance. Moreover, allergic disorders are characterized by an imbalance of immune response, typically defined by type 2 response and by a functional defect of Treg 3). In particular, allergic inflammation is mediated and regulated by allergen-specific Th2 cells that induce B cells to produce IgG1 and IgE by secreting IL-4. Moreover, Th2 cells also recruit eosinophils via IL-5 production and directly act on epithelial cells and smooth muscle cells through IL-13 production. In partIt is not clear if the alteration in Bregs differentiation in germinal centers is a consequence of the establishment of an allergic environment or vice versa contributes to allergic pathogenesis. Anyway, there is evidence that Breg cells are involved in allergic diseases .A recent review by Palomares and colleagues highlights the importance of IL10- and TFGβ- producing Breg cells because of their immunosuppressive properties during allergic reaction. These cell subsets are increased both in mice and humans in response to high-dose allergen exposure . Bregs aThe aim of the current study was to evaluate the presence of IL-10 competent B regulatory cells in the adenoids of pediatric patients undergoing adenoidectomy for adenoidal hypertrophy and recurrent upper airway infections. The study was also designed to identify possible surface markers of B10 cells.Globally, 111 paediatric patients  were examined for the detection of adenoidal B10 cells, according to the number of adenoidal mononuclear cells (AMCs) recovered after processing the surgical samples. Then, children were subdivided into two groups: 37 atopic and 74 non-atopic children. Atopic children were defined as those with a genetic tendency to develop allergic diseases such as asthma, allergic rhinitis and atopic dermatitis (eczema) according to the American Academy of Allergy, Asthma and Immunology (AAAAI) definition [AMCs from both groups of patients were “in vitro” stimulated with CpG 2006 and CD40L following the protocol of Iwata et al. , but afthi, CD5, CD24hi, CD38bright) and intracellular staining (IL10) and the percentage of each marker was analyzed in flow cytometry.AMCs were then labelled with monoclonal antibodies specific for some surface markers  or median and quartiles if continuous.Statistical analyses were performed using Stata 14.2 . A 2-sided p = 0.212)  for CD24hi CD19/IL10+ cells  Fig. . In addills Fig. .Fig. 1a So far, the presence of this cell subset was described only in murine models of autoimmune diseases . In humaLittle is known about the involvement of B10 cells in the pathogenesis of chronic inflammatory processes or allergic diseases. Moreover, the presence of B10 was never described in literature in children in secondary lymphoid organs, such as adenoids, constitutively exposed to environmental antigens and allergens.The persistence of an inflammatory microenvironment, due to recurrent upper airway infections or related to the atopic condition, could affect the susceptibility of AMCs to in vitro antigen stimulation, as we previously described .To date, no clear phenotype, transcription factor, or lineage markers have been described for B10 cells.A reduction in Breg percentages has been demonstrated only in peripheral blood of allergic patients and no evidence of their presence in secondary lymphoid organs such as tonsils and adenoids, particularly when exposed to environmental antigens has been reported .Our study demonstrated that B10 cells are detectable in the adenoids of children with adenoidal hypertrophy and recurrent respiratory infections. Moreover, the percentage of CD19/IL10+ B cells was higher in atopic patients, probably due to the fact that their regulatory activity on other immunocompetent cells in lymphoid organs may be different than their regulatory activity in peripheral blood. Their suppressive role could be associated with IL10’s ability to inhibit chemokine and pro-inflammatory cytokine production and IL10’s ability to down regulate co-stimulatory molecule expression in APCs .hi CD19/IL10+ cells. CD24 controls T and B cell maturation and function and it could both improve and inhibit immune tolerance, regulating the efficiency of clonal deletion [We also observed greater percentages of all investigated surface markers in allergic patients, significantly for CD24bright subpopulation percentage, but no detectable percentages in CD1dhi/CD5+ and CD24hi/CD38bright double positive cells were observed in both groups (data not shown).It is reasonable to think that the percentage of CD24high cells is related to the IL-10-producing B cells in theCD24high CD38This could explain the association between this marker and higher incidence of autoimmunity, even thought it has not been demonstrated its involvement in allergy development. Moreover, in our study adenoids were removed as very hypertrophic as consequence of recurrent and severe local infections.Recently, Komlósi and colleagues  detectedTherefore, our findings seem to disagree with that obtained by Komlósi and colleagues who reported lower Bregs frequency in atopic subjects. This conflicting finding could depend on different localization, structure, and function of adenoids and tonsils respectively. Palatine tonsils are localized near the entrances to the trachea and the esophagus, marked by a series of crypts, and exposed to both inhaled and alimentary antigens. Adenoids are situated posterior to the nasal cavity, do not possess crypts, have a lymphoepithelial structure, and are predominantly in contact with inhaled antigens and allergens. It is unclear if these characteristics could influence the functionality of immune B cells in tonsils and adenoids, but it has been already demonstrated that T follicular helper cells and B cells from adenoids have a more efficient functional response to bacterial antigens, than tonsillar cells .Even though our current study is preliminary, it provides a first step in the characterization of the B10 cell subset in a secondary lymphoid organ, the adenoids, which are primarily involved in the immune response against microbes and environmental agents in the upper airways.+ cells was not possible; therefore we were unable to perform analyses of other surface markers, the detection of in vitro IL10 production or the evaluation of IL10 mRNA expression. In the future, we propose to characterize B10 cells in human adenoids, not only from the phenotypic point of view, using different surface markers, but also from a functional one, analysing the effect of this cell subset on TCD4+ functionality, proliferation and cytokine production in response to in vitro antigens, with a possible comparison of the results obtained in adenoids with those obtained in peripheral blood of the same patients.Although the data collected in this exploratory study point to important information in the characterization of adenoidal inflammation, we are aware of our study’s limitations. First of all, it was not possible to clearly identify a surface marker distinguishing this B cell subset, nor was it possible to define their functional properties. Due to the low number of cells recovered, purification of CD19/IL10However, the current study has some limitations: the lack of investigation concerning the kind of respiratory infections, the documentation of true allergy, and Bregs were evaluated only at adenoid tissue level.Therefore, further studies should be addressed to respond to these unmet needs, in particular, to investigate possible relationships between the various cell populations located in different organs and in the blood.The current study provides the first evidence that B10 cells are present in adenoid tissue in children with adenoid hypertrophy probably as consequence of infectious inflammation. Atopy might amplify this phenomenon but, unfortunately, we couldn’t give an exhaustive explanation for the observed higher percentage, even though not statistically significant, of B10 regulatory cells in atopic patients, that seems to have an opposite trend compared to what was expected considering B10 role in the modulation of allergic responses. This could be probably due to the structural characteristics of adenoids, as suggested before, or to the different types of antigens and allergens to which the adenoids are exposed. We might speculate that the comprehension of B regulatory mechanisms of action could be useful in order to define B10 cells as a possible cellular marker for monitoring of allergic inflammation and a new therapeutic target for allergic diseases.


Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the DOI:http://dx.doi.org/10.7554/eLife.26886.001 A synapse is a specialized structure where signals are transmitted from a neuron to another neuron or other target cells such as muscles. Proper synapse formation is prerequisite to building functional synapses and constructing neuronal circuits. Synapse abnormalities are suggested to induce neurological and psychological disorders such as autism spectrum disorders and fragile X syndrome . FormatiDrosophila NMJ is a model to study synapse formation and activity-dependent synapse remodeling (Drosophila p21-activating kinase (dPak) localized at the PSD, regulate SSR formation  branched out extensively from initial targeting sites; synaptic boutons revealed by postsynaptic Dlg staining spread evenly along axonal tracks, displaying the beads-on-a-string pattern , large increases were found in null 19-2/19-2 and close-to-null 1/19-2leon . In addition, smearing signals at higher molecular weights representing ubiquitinated substrates were also increased in 19-2/19-2 and 1/19-2leon . The higher molecular-weight smears, however, were increased slightly in 2/19-2leon (1.29 ± 0.78 folds). In contrast, the level of monoubiquitin was strongly increased in hypomorphic 2/19-2leon (1.9 ± 0.77 folds) but the increases were not prominent in 19-2/19-2 and 1/19-2leon . Therefore, the severity in defective ubiquitin homeostasis, represented by the increased levels of free ubiquitin chains and ubiquitinated substrates, correlates with increasing leon mutant strengths.To examine whether ubiquitin homeostasis is disrupted in the  mutants . Whereasleon mutants, the FK2 antibody that recognizes mono- and poly-ubiquitinated substrates, and free ubiquitin chains was used to immunostain dissected larval body walls. In wild-type controls, FK2 signals were detected at NMJs and in nuclei and Z-bands of muscles  and partially suppressed elevated ubiquitinated substrates (0.91 ± 0.04 folds). The partial suppression could be attributed to the limited muscle expression in the mutant. Interestingly, the expression of UAS-ED-Leon induced higher levels of free ubiquitin chains (2.17 ± 0.16 folds) and ubiquitinated substrates (2.19 ± 0.43 folds). It is possible that the enzymatic activity-deficient ED-Leon can associate with ubiquitin chains but cannot deubiquitinate them, and binding to ED-Leon sequesters ubiquitinated substrates from degradation. Taken together, these results indicate that Leon deubiquitinating activity is required for maintaining ubiquitin homeostasis in postsynapses.We then examined the requirement of postsynaptic Leon and its deubiquitinating activity for ubiquitin homeostasis. In  signals . Westernleon mutants prompted us to examine constituents of synaptic organization. Several pre- and post-synaptic proteins were analyzed for their expression patterns and levels in leon mutants. Immunostaining for Dlg that is enriched in the SSR revealed a striking phenotype. In controls, SSR-localized Dlg exhibited thin circular rings in bouton sections. In leon mutants, Dlg-localized rings were expanded, showing much greater thickness and higher protein levels , and postsynaptic dPak . Well-mad normal . Strikinon1/19-2 . Quantifsynapses . The norleon mutants, GluRIIA and GluRIIB clusters were enlarged and overlapped, filling most of the space in bouton sections  that display a pattern of small spreading puncta in boutons . Another independent mEJC amplitudes recording also suggested 1/19-2leon had slightly larger mEJC amplitudes than +/19–2 and 1118w controls . With the 50% or more reduction in the EJC amplitude and the quantal content, other processes are also likely defective in the leon mutants.We then examined whether Leon is required in pre- or post-synapses for restoring the EJC and quantal content in  mutants . Postsynon1/19-2 . The mEJkgrounds . As postn number , and aboleon mutants might cause some aspects of the mutant phenotypes. To test this idea, we examined whether reductions of the gene dosage for these postsynaptic proteins would alleviate leon mutant phenotypes. We first examined SSR-localized Dlg that is required for SSR formation , which were not significantly altered in 2/19-2leon . Thus, the increases in the protein levels, as well as other mechanisms, could contribute to postsynaptic accumulations of Dlg, dPak and Cact in leon mutants.We then addressed whether the accumulations of postsynaptic proteins at the SSR or PSD are accompanied with increases in the total protein levels. Western blots for examining Dlg, dPak and Cact protein levels were performed in isolated body-wall muscles of wild-type and leon mutants, ubiquitinated substrates accumulate . Immunostaining for Ubqn revealed ubiquitous expression at NMJs and in muscles, with enrichment in nuclei  and only weakly in other regions (1.2 folds in muscle). These results are consistent with that the increase in the Ubqn level in leon mutants could mediate expansions of Dlg-positive zones, dPak patches and GluRIIA clusters in leon mutants.Given the reduction in n nuclei . In leonFlag-Ubqn transgene expressed by 24B-GAL4 in postsynapses caused expansions of Dlg-positive zones and GluRIIA clusters , D42-GAL4 (RRID:BDSC_8816), da-GAL4 (RRID:BDSC_55851), UAS-mCD8GFP (RRID:BDSC_5137) and 6dpak (RRID:BDSC_8809) were obtained from Bloomington Drosophila Stock Center, Bloomington, IN. UAS-leonRNAi (ID 17567), UAS-UbqnRNAi#1 (ID 47447), UAS-Rad23RNAi (ID 30497), UAS-Ddi1RNAi (ID 40512), UAS-pllRNAi (ID 103774), UAS-dlgRNAi (ID 41136) and UAS-dpakRNAi (ID 108937) were from Vienna Drosophila Resource Center, Austria. UAS-cactRNAi (5848 R-3) was from NIG-FLY, Japan. UAS-dl-BRNAi was from Steven A. Wasserman  and larval body fillets were fixed in 4% paraformaldehyde for 20 min and washed in PBT (0.01% triton-X-100) for 15 min three times. Fixed fillets were incubated with primary antibodies overnight at 4°C, rinsed in PBT three times, and incubated with secondary antibodies for 2 hr at room temperature. Primary antibodies: mouse anti-Dlg , mouse anti-Brp , mouse anti-Synapsin , mouse anti-CSP , mouse anti-Futsch , rabbit anti-Cact , anti-dPak  chicken anti-GFP , mouse anti-UBQLN2 , goat anti-HRP conjugated FITC, rabbit anti-HRP conjugated TRITC or Cy5  and mouse anti-Leon (1:100) generated against GST-Leon fusion protein . Muscles were visualized by FITC-conjugated phalloidin , which are not shown in figures. For GluRIIA  immunocytochemistry, larval fillets were fixed in Bouins fixative (Sigma-Aldrich.) for 5 min, followed by the protocol described above. Larval fillets were mounted onto slides with PBS containing 87.5% glycerol and 0.22M 1, 4-diaza-byciclo (2.2.2) octane . NMJ images were acquired by confocal Z-stack scanning  using 40x water and 100x oil objectives and processed by LSM 5 image examiner and Adobe Photoshop.NMJ 6/7 phenotypes were analyzed at the A3 segment of late third instar larvae as previously described , 2012b. 2314056; ), rabbit2567913; ), rabbit2568753; ), rabbit2568754; ), mouse 3VO4 and separated in Nupage 4% ~ 12% Bis-Tris gel  for Western blots. For immunoprecipitation, larvae were lysed in lysis buffer or lysis buffer with 0.5% SDS. Lysates with 0.5% SDS buffer were further diluted to final 0.2% SDS for immunoprecipitation. Lysates after incubation with Flag M2 beads  overnight were washed by lysis buffer three times and separated in Nupage 4% ~ 12% Bis-Tris gel for Western blots. Drosophila S2 cell line was purchased from Invitrogen  and Drosophila S2 cells were cultured at 25°C in Schneider’s Drosophila medium with 10% heat-inactivated fetal bovine serum . S2 cells were transfected with plasmids by Effectene Transfection Reagent . Plasmids include Ub-GAL4, Myc-dPak, Myc-Dlg, Myc-Cact, Myc-GFP, Flag-Ubqn, and HA-Ub. After 72 hr transfection, S2 cells were lysed in lysis buffer for immunoprecipitation by Myc agarose beads . Immunoprecipitated lysates were washed by lysis buffer three times and separated in 8% acrylamide gels. Antibodies against Dlg (1:1000), dPak (1:5000), Cact (1:5000), ubiquitin , mouse anti-UBQLN2 , α-Tubulin , anti-Myc  and HA-HRP  were used in Western blots.Third instar larva fillets were homogenized in lysis buffer  supplemented with protease inhibitor cocktails , 1 mM PMSF, 1 mM DTT and 2 mM NaProcessing and analysis of ultrastructures of synaptic boutons by electron microscope were as described previously , with soThe thickness of Dlg-, Cact-, or mCD8GFP-positive zone was analyzed in the original confocal Z-stack images and averaged for lengths across the zone defined by eight straight lines radiating from the bouton center. GluRIIA cluster diameters were analyzed in the original confocal Z-stack images, which were scanned with 0.5 μm intervals and covered overall boutons. Isolated GluRIIA cluster with circular shapes in different Z sections were selected to avoid choosing super-imposed ones for scoring their diameters. When elliptic cluster were chosen, the long and short diameters were averaged to reach the average diameters. Brp number and HRP-labeled NMJ areas were analyzed in the original confocal Z-stack images and Brp density was the ratio of Brp number to HRP-labeled NMJ area. Branch length was calculated from HRP labeling. GluRIIA/IIB/IIC and dPak intensities were the summation from all pixels of NMJs, which was normalized to the total HRP intensity at NMJs by Image J. Synaptic FK2 and Ubqn intensities were calculated by mean pixels in the synaptic area normalized to mean HRP pixels in the synaptic area by Image J. Muscular FK2 and Ubqn intensities were averaged from mean pixels of four muscle areas and normalized to mean HRP pixels in the postsynaptic sites by Image J. The intensities were normalized again to the control genotype for presentation in bar graphs.To estimate expression levels from Western blots, each set of experiments was performed three times independently, and intensities for bands or areas of interest were processed by Image J with normalization to the internal control α-Tub. The areas of interest for mono-ubiquitin, free ubiquitin chains and ubiquitinated substrates are indicated in UAS-UbqnRNAi (#1 and #2), UAS-Rad23RNAi, UAS-Ddi1RNAi, UAS-SyndRNAi and UAS-pllRNAi driven by da-GAL4 were reared at 25°C. mRNA of indicated genotypes was extracted from third-instar larvae by RNAzol RT , followed by the reverse transcription of cDNAs by ImProm-II Reverse Transcription System through oligo-dT . The mRNA expressions were amplified by PCR using Ubqn, Rad23, Ddi1, pll and Rpl19 specific primers.Larvae for knockdowns of 2+-free HL3.1 saline . Preparations were then incubated in modified HL3.1 saline containing 0.6 mM CaCl2 for stimulation, and recordings were taken at room temperature. The two-electrode voltage-clamp was filled with 3 M KCl and impaled in muscle 6 of the A3 segment. One microelectrode (15 ~ 20 MΩ) monitored the muscle membrane potential while the other (5 ~ 8 MΩ) passed electric currents. The muscle membrane potential was clamped at a command value of −60 mV. mEJCs occurring in the background within 100 s were obtained without any stimulation on the segmental nerve. To record an EJC, the segmental nerve was stimulated every 10 s from the cut end that was connected by a suction electrode with 0.1 msec of pulse duration at the voltage two times that of the threshold. Signals were digitized at 50 kHz by a DigiData 1440 interface (Molecular Devices), low-pass filtered at 10 kHz, and saved on an IBM-compatible PC for analysis. For failure analysis, EJC was evoked in 0.2 mM [Ca2+], and the failure rate was calculated by ln(n/N), with n the number of failure events, and N the total number of stimuli.For sample preparation, larvae were dissected with the segmental nerve cut near the ventral ganglion in cold modified Ca In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by K VijayRaghavan as Senior editor and three reviewers, one of whom, Hugo Bellen, is a member of the Board of Reviewing Editors. Although the work is of interest, we regret to inform you that the findings at this stage are too preliminary for further consideration at eLife. However, we are willing to entertain a resubmission that addresses the reviewers’ comments.Thank you for choosing to send your work, "USP5/Leon deubiquitinase confines postsynaptic differentiation by maintaining ubiquitin homeostasis through Ubiquilin", for consideration at eLife. We would be happy to re-review such a thoroughly revised version.We think that the mechanistic angle including the interpretation of the electrophysiological phenotype will be hard to fix within two months. However, we believe that you should be able to revise the manuscript in a satisfying manner and it should then hopefully be a candidate for Reviewer #1:Drosophila neuromuscular junction (NMJ) development and document that it controls proper postsynaptic differentiation. Leon is the homolog of USP5, a deubiquitinase that functions specifically to disassemble free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin. Previously, the same group described that loss of Drosophila leon is lethal and leads to the accumulation of both free poly-ubiquitin chains, as well as polyubiquitinated proteins. In the current study, the authors describe a role for leon in regulating ubiquitin homeostasis and restricting the growth and development of the postsynaptic density (PSD) and subsynaptic reticulum (SSR): whereas presynaptic components are unaffected, the levels of numerous postsynaptic components  are strongly increased. Surprisingly, the observed phenotypes can be significantly rescued by reducing the levels of Ubiquilin, a protein that transports ubiquitinated substrates to the proteasome. In addition, leon mutants display increased Ubiquilin levels in leon mutants and overexpressing ubiquilin in a wildtype background mimics the phenotypes found in leon mutants, albeit to a lesser extent. These data argue for a role of Ubiquilin in mediating PSD and SSR growth. However, free polyubiquitin chains seem to also play a role as overexpressing these chains also shows a milder version of the phenotypes observed in leon mutants. Finally, combined overexpression of Ubiquilin and free ubiquitin chains in a wildtype background completely mimics leon mutants. Hence, based on the presented data, Leon appears to restrict postsynaptic development by regulating the levels of both Ubiquilin and free polyubiquitin chains.In the current manuscript, Wang et al. assess the function of the deubiquitinase Leon during leon mutants. The figures are nicely organized and in most cases very convincingly bring the message across. It is counterintuitive that reducing the levels of a protein that removes polyubiquitinated proteins in a background in which the levels of polyubiquitinated substrates are elevated. However, the genetic experiments that were performed are clean and clearly show that free polyubiquitin chains, in combination with elevated Ubiquilin levels can affect postsynaptic development. How this occurs remains elusive and is not well discussed by the authors. The experiments to tease out the details of the actual mechanism underlying NMJ overgrowth in leon mutants are not trivial and may represent a continuation / expansion on this paper. I enjoyed reading this paper and would like to recommend it for publication in eLife, if the authors can expand / hypothesize on the consequences of free ubiquitin chains / elevated Ubiquilin levels in the muscle.This is a carefully executed, well-controlled study that consists of an extensive and detailed phenotypic characterization of Major comment:– My major comment is a lack of interpretation of the results in the Discussion. At the moment, the Discussion mostly recapitulates the data. How do the authors think Ubiquilin and free ubiquitin chains affect NMJ development?leon mutants or (b) that the individual proteins that are found at the PSD and SSR are directly regulated by Ubiquilin and/or free ubiquitin chains. This is not discussed at the moment, but is essential for the reader to help interpret the consequences of these findings.As the levels of Dlg, Cac, etc are elevated at the earliest stages of NMJ development, it appears that either (a) the activity and/or levels of the master regulator of postsynaptic density formation, which remains unknown, are increased in How could free ubiquitin chains alter protein levels / activity? Does overexpressing free ubiquitin chains lead to a similar increase in Ubiquilin levels or are these two events independent?leon mutants, can this rescue the increase in Ubiquilin levels? Does this affect NMJ development?In addition, is the increase in Ubiquilin due to a lack of free ubiquitin monomers? For instance, if the authors overexpress ubiquitin in Reviewer #2:The manuscript reports a very pronounced synaptic phenotype for mutations in Leon, the fly homolog of USP5. Studying the neuromuscular junction, they provide strong evidence that there is an extreme expansion of the folds of the subsynaptic reticulum and a concomittent increase in the postsynaptic presence of proteins associated with that structure. In addition, they show that some proteins found in the postsynaptic density of the NMJ, most notably the receptor subunit GluRIIA, the scaffold protein dlg, and the signaling molecule dPAK are also expanded as is the density by EM. These postsynaptic changes are accompanied by a large change in the gross morpology of the synapse, including a reduction in bouton number. The changes in the nerve structure, however, seem to be an indirect consequence of the role of Leon in the muscle.The strength of the paper is in the basic description of these phenotypes. The weaknesses of the paper are in the characterization of the electrophysiology and, above all, in the attempt to formulate a mechanistic explanation of the phenotype. There are small technical issues throughout, such as the failure to quantify Western blots and imprecisions in the description (such as equating Cactus or dlg staining with SSR), but where the manuscript really falls apart is from Major Criticisms:w1118 controls, recorded on the same days, alongside all the measurements for genotypes presented so that one can distinguish effects of the genetic background from those of the mutations. They should use the method of failures to determine quantal content independently since there is good reason to suspect they are misled at present. The increase in quantal size cannot alone explain the decrease in EJC amplitude but might explain a homeostatic decrease in probability of release. In any case, in its current state, the electrophysiology is not interpretable and the final line of that section, which says something vague about correlating with the expansion phenotype, is not really an interpretation and it isn't clear what "correlating" refers to.Nevertheless, the obvious explanation for the decreased EJC amplitude is that there are far fewer boutons present and therefore fewer release sites and less release. I don't understand why they do not make this point. That miniEJC frequency is normal may reflect a homeostatic compensation, but it seems inadequate to compensate for the sharp drop in bouton #.leon mutant NMJs” paragraph two: do the authors intend mEJC amplitude or EJC amplitude. Did they mean the driver by itself or the driver driving the transgene? This sentence was unclear.Subsection “Impaired electrophysiological properties at The same paragraph overstates the result. EJC amplitude are not restored to wt levels . What they mean to say is "partially restores" of "improves relative to the mutant" or something like that.Mechanism: the manuscript does not describe the ubiquitination or a change in ubiquitination for any of the proteins that they claim are misregulated as a result of the leon mutations. They have only overexposed Western blots that are likely to be saturated and no accurate quantification of the levels of the postsynaptic proteins in Re: The authors say that knockdown of syndapin has no effect on cactus or GluRIIA expansion but offer no evidence that their RNAi altered syndapin expression. Similarly, they base all the arguments in leon mutants and the ability of Ubqn loss of function to prevent the expansion of the postsynaptic markers does not really prove that it was directly in the path of the changes. Presumably any protein that was required for the SSR or the PSD would be necessary regardless of whether it was regulated by Leon. Other specific issues for leon mutants. The change in band size is too subtle to tell, especially with the saturation of both the Ubqn and loading control bands. Either a quantification with several replicates or a shorter exposure blot would be necessary. From leon mutants, "highly elevated at SSR rather than other regions"; the magnification is too low to tell, and a quantification is necessary to demonstrate the robustness of the phenotype. If quantification cannot be done, please at least show multiple examples. Figure 7—figure supplement 2C: no loading control shown on the blots.Reviewer #3:Ubiquitin-dependent proteolysis is a complex pathway of protein metabolism implicated in such diverse cellular functions. A late step of the process involves disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. USP5 disassembles branched polyubiquitin chains by a sequential exo mechanism, starting at the proximal end of the chain. Particularly, the enzyme is meant to disassemble free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin, and thus maintain cellular ubiquitin homeostasis.Drosophila, showing that Leon is essential for viability, and that leon mutants accumulate both free and substrate-conjugated polyubiquitin chains Proteasomal enzymatic activities were elevated in leon mutants, while proteasomal degradation of ubiquitinated substrates was impaired.The Chien lab recently published a first genetic analysis for USP5 in Here, the Chien lab executed an analysis of Leon function at the larval NMJ system. Core findings/experiments they present are 1) an expansion of the SSR muscle membrane infoldings and the size of postsynaptic glutamate receptor fields/PSD area due to a lack of muscle Leon function; 2) Western blots and immunostainings of dissected body wall muscles pointing towards increased levels of free and substrate-conjugated polyubiquitin chains; 3) based on TEVC recordings, an impairment of evoked release EJC amplitude and quantal content due to lack of postsynaptic muscle Leon; 4) heterozygous suppression of SSR outgrowth/GluRIIA receptor field outgrowth via dlg /dpak, creating arguments for accumulation of postsynaptic proteins to promote SSR expansion; 5) muscle knockdown of UBA-UBL ubiquitin receptor Ubqn suppressed expansions of Dlg rings and GluRIIA.eLife. My major concerns are the following:Per se, I am impressed by the severity of the phenotypes  they report, and I think if appropriately revised this manuscript might become a strong candidate for leon mutants undergoing pupa formation timely? They should compare precisely timed cohorts to make sure that really the pace of SSR expansion is increased here.1) Can they exclude that they are looking at a "heterochronic" phenotype? In the case of dlg mutants, SSR formation is impaired throughout larval development when comparing precisely timed animals. However, as pupa formation is impaired or completely suppressed in dlg mutants, SSR ultimately growths to over a value found in wild type controls. Are their leon mutant background. How does the phenotype look like in wt background? Are they maybe just seeing the addition of two phenotypes here without any specific functional interaction? Anyway, I must confess that I cannot fully follow the mechanistic connection in between Leon and Ubqn.2) Ubqn knockdown they apparently only tested within 3) They should try to directly monitor a poly-U species of some relevant postsynaptic protein. This would make their case more convincing.4) Concerning the diameter of the GluRIIA fields they report: values appear very high. Can they exclude that they are imaging two or more super-imposed receptor fields as one?5) Somewhat surprisingly but not without example, they find mini amplitudes to be unchanged despite a severe increase of particularly GluRIIA. However, I was surprised to see a down-regulation of evoked release, means that quantal content is severely down here. Concerning the mechanistic basis of their electrophysiological phenotypes, they say: "which correlates with its requirement in postsynaptic differentiation" I am afraid this is insufficient.leon mutants. Is the availability of monomeric Ubiquitin determining their phenotype? Could they test for this?6) They state that both free polyubiquitin chains and ubiquitinated substrate proteins were changed in 7) Why in General comment: the manuscript clearly needs language editing. Moreover, the format of the figures is very diverse, and could be improved by e.g. showing blow ups of the respective GluRIIA phenotypes.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by K VijayRaghavan (Senior editor), a Reviewing editor, and one reviewer.Thank you for resubmitting your work entitled "USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below. Please make the textual changes suggested by his reviewer..Reviewer #1:The manuscript is stronger, the electrophysiology more rigorous, and I also think it should be published. The phenotype is big, clear, and the genetics of rescue and RNAi are all very robust. The supplemental table with full statistics is very valuable, as is the inclusion of data from the cd8GFP reporter of the ssr. Some issues can (and should) be improved in the text. My chief concern is that the mechanism behind the phenotypes is not nearly as clear as the authors state. It is fine with me that some things remain poorly explained; but the authors need not try to make it appear as if the very modest changes in certain protein levels are sufficient to explain the phenoptyes or that the changes in bouton number can adequately explain what is happening to quantal content. Ubiquitin homeostasis and protein turn over are very complicated and broadly acting processes and not easily pinned down to the few proteins one looks at. By the end of the Results, a very strong case has been made for the importance of ubiquitination and Ubqn in regulating the development of the postsynapse. The remarks below are to help the authors avoid certain overstatements and premature conclusions in what is otherwise a very interesting and complete manuscript.leon mutants” and in the Discussion, it is a little awkward to say that each bouton houses tens of synapses  because the term synapse is used sometimes to refer to the whole junction, sometimes to a bouton, and sometimes to each release site. If they say each bouton contains multiple release sites that face discrete receptor clusters the sentence will be less awkward.1. In the subsection “Increases of postsynaptic proteins in leon mutant NMJs”: the change in bouton number (which this reviewer suggested as an explanation) doesn't really completely account for the change in the ejc amplitude and quantal content. It may account for some of it, but the ephys parameters are decreased by 50% or more  and bouton # is only decreased by 20-25%. If the density of release sites per bouton and size of boutons is really unchanged, there remains a mismatch that should still be acknowledged. It is tedious, but it would also help if the authors counted brp puncta in an entire NMJ to get a real value for release sites per NMJ. There may be changes in density, for example, that don't reach statistical significance on their own but, coupled with changes in bouton diameter and number might add up to a larger difference in release sites per NMJ. The bottom line is that it is ok if some of the ephys remains mysterious to explain - but this reviewer did not mean to imply that bouton number alone can actually explain the whole phenotype.2. At the end of the subsection “Impaired electrophysiological properties at 3. There is obviously a disconnect between the very large changes in the SSR and dlg and gluR distributions, and the very small  changes in the levels of Pak, dlg, and cact. I think it is fair to guess that many other proteins are increased in order to produce the large changes in the structure of the synapse. In addition, the fact that mutations in those proteins and others can prevent to increase in SSR or GluR markers does not in any way indicate that those proteins are part of the pathway through which leon is acting – it only confirms those proteins are important for normal SSR and GluR development, i.e. they could as easily be part of parallel pathways regulating development as part of a linear pathway.leon mutant phenotypes by reducing Ubqn levels”: the conclusion regarding the Ubqn increases seen in leon mutants is also overstated at this point in the paper. It is certainly likely that this contributes to the other phenotypes and the finding of a large increase in Ubqn in leon mutants and the finding that reducing Ubqn can prevent some or all of the phenotypes is important. But to really prove that the change in Ubqn is causative and not merely part of the phenotype, it needs the data in leon loss of function.  The text for 4. At the end of the subsection “Suppression of  [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:[…] Major comment:– My major comment is a lack of interpretation of the results in the Discussion. At the moment, the Discussion mostly recapitulates the data. How do the authors think Ubiquilin and free ubiquitin chains affect NMJ development?As the levels of Dlg, Cac, etc are elevated at the earliest stages of NMJ development, it appears that either (a) the activity and/or levels of the master regulator of postsynaptic density formation, which remains unknown, are increased in leon mutants or (b) that the individual proteins that are found at the PSD and SSR are directly regulated by Ubiquilin and/or free ubiquitin chains. This is not discussed at the moment, but is essential for the reader to help interpret the consequences of these findings.leon mutants . In addition, we have performed two additional experiments to address the relationship between free ubiquitin chains, Ubqn and individual SSR or PSD-localized proteins. First, overexpressing free ubiquitin chains also induced Ubqn accumulation  could regulate Ubqn levels. The result is that only overexpression of UbAA could upregulate Ubqn, indicating that excessive free ubiquitin chains induce Ubqn accumulation . In contand Cact . The eleIn addition, is the increase in Ubiquilin due to a lack of free ubiquitin monomers? For instance, if the authors overexpress ubiquitin in leon mutants, can this rescue the increase in Ubiquilin levels? Does this affect NMJ development?leon mutants, while Ubqn was increased, the level of ubiquitin monomers was also increased by Western blot analysis . The mEJCs of w1118 and +/19-2 show little difference (689 v.s. 709 pA). The mEJC of 1/19-2leon showed some increases to w1118 and +/19-2, although they are not statistical significant  (1/19-2leon is likely slightly larger than controls.1)The reason to perform electrophysiology recording with the heterozygous mutant  t test) . Taken tw1118 and +/19-2 (2) However, whether the difference in mEJCs is statistical significant or not depends on the genetic background. When performed with GAL4 driver backgrounds , the incw1118, +/19-2 and 1/19-2leon and showed that the quantal content was dramatically decreased in 1/19-2leon  The slight increase in mEJCs, whether it is statistically significance or not, could not account for the large reduction in the quantal content, which is mainly due to the reduction in EJCs. As suggested by the reviewer, we also preformed failure analysis in on1/19-2 . Thus, tThe increase in quantal size cannot alone explain the decrease in EJC amplitude but might explain a homeostatic decrease in probability of release. In any case, in its current state, the electrophysiology is not interpretable and the final line of that section, which says something vague about correlating with the expansion phenotype, is not really an interpretation and it isn't clear what "correlating" refers to.Nevertheless, the obvious explanation for the decreased EJC amplitude is that there are far fewer boutons present and therefore fewer release sites and less release. I don't understand why they do not make this point. That miniEJC frequency is normal may reflect a homeostatic compensation, but it seems inadequate to compensate for the sharp drop in bouton #.leon mutant synapses, we agree that the electrophysiological results are difficult to reconcile in this manuscript. We thank the reviewer for pointing out one better explanation. We rewrote the sentence in the manuscript to describe that fewer boutons might be correlative to lower EJCs and quantal contents detected in leon mutants .Due to severe developmental defects in Subsection “Impaired electrophysiological properties at leon mutant NMJs” paragraph two: do the authors intend mEJC amplitude or EJC amplitude. Did they mean the driver by itself or the driver driving the transgene? This sentence was unclear.leon mutants carrying D42-GAL4 or 24B-GAL4 was significantly larger than that in respective GAL4 driver control, suggesting the mEJC increase in leon mutants might be sensitive to the variation in genetic backgrounds . What they mean to say is "partially restores" of "improves relative to the mutant" or something like that.24B-GAL4 partially restored the EJC amplitude and the quantal content in 1/19-2leon .The alleles we used for pansions . These Ris study . The res2/19-2leon and null 1/19-2leon.We address that Ubqn is in the Leon/USP5 pathway with following data: (1) The Ubqn level increased in leon mutants by both Western blots and immunostaining assays  Musdiameter ; (4) Ubq mutants . Taken tOther specific issues for Ubqn knockdown. Therefore, we did not treat the muscle signals as background. We now show quantifications of FK2 intensities for synapses as well as muscles  from three independent experiments (From leon mutants as suggested (We showed higher magnification figures in Ubqn single channel and used dashed lines to outline the Cact-positive regions (not shown in the figure) for quantification. We also quantified the mean intensity of Ubqn in muscles and Cact-positive zones in wild type and uggested . The resFigure 7—figure supplement 2C: no loading control shown on the blots.We have added loading controls .Reviewer #3:[…] 1) Can they exclude that they are looking at a "heterochronic" phenotype? In the case of dlg mutants, SSR formation is impaired throughout larval development when comparing precisely timed animals. However, as pupa formation is impaired or completely suppressed in dlg mutants, SSR ultimately growths to over a value found in wild type controls. Are their leon mutants undergoing pupa formation timely? They should compare precisely timed cohorts to make sure that really the pace of SSR expansion is increased here.1/19-2leon mutant was one day longer than wild type. The expansion of SSR and PSD in the 1/19-2leon mutants examined in the late third instar stage may be a consequence of extra growth. We have performed the experiment as suggested by the reviewer to compare SSR and PSD phenotypes between control and leon mutants at 72h, 96h, and 120h after egg laying (AEL). Apparently, leon mutants presented enlargement of PSD and SSR as early as 72h AEL, as the Cact-positive zone and GluRIIA clusters are significantly larger than controls, and the differences are further enhanced at later stages  Ubqn knockdown they apparently only tested within leon mutant background. How does the phenotype look like in wt background? Are they maybe just seeing the addition of two phenotypes here without any specific functional interaction? Anyway, I must confess that I cannot fully follow the mechanistic connection in between Leon and Ubqn.Ubqn knockdown in the wildtype background had no effect on SSR-localized Dlg and PSD-localized GluRIIA . Briefly, elevated free ubiquitin chains in leon mutants induced elevation of Ubqn levels. Accumulated Ubqn stabilizes ubiquitinated postsynaptic proteins to cause NMJ defects.Muscle  GluRIIA . Thus, t3) They should try to directly monitor a poly-U species of some relevant postsynaptic protein. This would make their case more convincing.leon mutants, even with overexpression of Ub transgene. However, in S2 cells, expression of Ubqn stabilizes ubiquitinated dPak, Dlg and Cact, as compared to those without Ubqn overexpression. These results were shown in leon mutants, stabilizes ubiquitinated dPak, Dlg and Cact. Also see response to reviewer#2, point 5.It has been difficult to detect ubiquitinated dPak, Dlg and Cact in body wall muscle preparation of wildtype or 4) Concerning the diameter of the GluRIIA fields they report: values appear very high. Can they exclude that they are imaging two or more super-imposed receptor fields as one?In our analysis,the diameter of GluRIIA clusters is about 0.63 µm in wild type third instar larvae, which is comparable to GluR cluster diameters in a previous report from Graeme Davis’s group, showing that the GluRIIC diameter is about 0.78 µm in third instar larvae and the GluRIIA diameter is about 0.52 µm in second instar larvae . In our study, we were very cautious to select “isolated” GluRIIA puncta in both mutants and wild type. The methodology is described in Materials and methods (“Quantifications”). Briefly,Z stacks of GluRIIA images were scanned with 0.5 µm intervals. Isolated puncta in different Z sections were selected for scoring, thus avoiding super-imposed ones.5) Somewhat surprisingly but not without example, they find mini amplitudes to be unchanged despite a severe increase of particularly GluRIIA. However, I was surprised to see a down-regulation of evoked release, means that quantal content is severely down here. Concerning the mechanistic basis of their electrophysiological phenotypes, they say: "which correlates with its requirement in postsynaptic differentiation" I am afraid this is insufficient.leon mutants, leading to the decreased evoked response (as suggested by reviewer 2). Thus, the description is rewritten as “As postsynaptic expression of Leon also suppressed bouton reduction in the leon mutant  They state that both free polyubiquitin chains and ubiquitinated substrate proteins were changed in leon mutants. Is the availability of monomeric Ubiquitin determining their phenotype? Could they test for this?leon mutants  Why in We think that ED-Leon could associate with polyubiquitin chains but fail to disassemble polyubiquitin chains due to inactivation of the enzymatic activity. It is possible that binding to ED-Leon sequesters ubiquitinated substrates from degradation and leads to their accumulation.General comment: the manuscript clearly needs language editing. Moreover, the format of the figures is very diverse, and could be improved by e.g. showing blow ups of the respective GluRIIA phenotypes.The revised manuscript has been reviewed by an in-house English editor. Also, in the revised manuscript, GluRIIA images are enlarged, and representative enlarged images are shown in [Editors’ note: the author responses to the second round of peer review follow.]The manuscript is stronger, the electrophysiology more rigorous, and I also think it should be published. The phenotype is big, clear, and the genetics of rescue and RNAi are all very robust. The supplemental table with full statistics is very valuable, as is the inclusion of data from the cd8GFP reporter of the ssr. Some issues can (and should) be improved in the text. My chief concern is that the mechanism behind the phenotypes is not nearly as clear as the authors state. It is fine with me that some things remain poorly explained; but the authors need not try to make it appear as if the very modest changes in certain protein levels are sufficient to explain the phenoptyes or that the changes in bouton number can adequately explain what is happening to quantal content. Ubiquitin homeostasis and protein turn over are very complicated and broadly acting processes and not easily pinned down to the few proteins one looks at. By the end of the Results, a very strong case has been made for the importance of ubiquitination and Ubqn in regulating the development of the postsynapse. The remarks below are to help the authors avoid certain overstatements and premature conclusions in what is otherwise a very interesting and complete manuscript.1. In the subsection “Increases of postsynaptic proteins in leon mutants” and in the Discussion, it is a little awkward to say that each bouton houses tens of synapses  because the term synapse is used sometimes to refer to the whole junction, sometimes to a bouton, and sometimes to each release site. If they say each bouton contains multiple release sites that face discrete receptor clusters the sentence will be less awkward.Following the reviewer’s suggestion, we modified the sentences as “At NMJs, each bouton contains multiple release sites paired with discrete receptor clusters that…” and “Each bouton contains multiple release sites where neurotransmitters…”.2. At the end of the subsection “Impaired electrophysiological properties at leon mutant NMJs”: the change in bouton number (which this reviewer suggested as an explanation) doesn't really completely account for the change in the ejc amplitude and quantal content. It may account for some of it, but the ephys parameters are decreased by 50% or more  and bouton # is only decreased by 20-25%. If the density of release sites per bouton and size of boutons is really unchanged, there remains a mismatch that should still be acknowledged. It is tedious, but it would also help if the authors counted brp puncta in an entire NMJ to get a real value for release sites per NMJ. There may be changes in density, for example, that don't reach statistical significance on their own but, coupled with changes in bouton diameter and number might add up to a larger difference in release sites per NMJ. The bottom line is that it is ok if some of the ephys remains mysterious to explain - but this reviewer did not mean to imply that bouton number alone can actually explain the whole phenotype.1118w, 731.6 ± 31.6; 2/19-2leon, 631.9 ± 28.1; 1/19-2leon, 619.5 ± 31.1; n = 10 for all genotypes; Statistics by Student’s t test showed that both leon mutants were p < 0.05 in comparison to 1118w. These reductions of Brp numbers are about 15%, and are close to the 20% reduction in the bouton numbers in leon mutants. As EJC and quantal content were decreased by 50%, we think reduction of bouton and the releasing sites contribute partly to the reduction of EJC and quantal content. We present this quantification and modified the description accordingly.We quantified the Brp number per NMJ: 3. There is obviously a disconnect between the very large changes in the SSR and dlg and gluR distributions, and the very small  changes in the levels of Pak, dlg, and cact. I think it is fair to guess that many other proteins are increased in order to produce the large changes in the structure of the synapse. In addition, the fact that mutations in those proteins and others can prevent to increase in SSR or GluR markers does not in any way indicate that those proteins are part of the pathway through which leon is acting – it only confirms those proteins are important for normal SSR and GluR development, i.e. they could as easily be part of parallel pathways regulating development as part of a linear pathway.leon mutants, and we are only able to examine a handful of them. The slight but not dramatic increases in protein levels of dPak, Dlg and Cact as shown by Western blots could explain at least partly the protein accumulation in postsynaptic specializations. Other mechanisms such as protein recruitment could be also involved. We modified the texts to describe this idea in the Results and Discussion. Regarding whether these proteins are part of the Leon pathway or not, we have not implied that Leon is in direct regulation of these proteins. Instead, we have included Ubqn in the model to explain how these proteins might be stabilized and contribute to postsynaptic structural expansions (Discussion, and We agree that many proteins are altered in 4. At the end of the subsection “Suppression of leon mutant phenotypes by reducing Ubqn levels”: the conclusion regarding the Ubqn increases seen in leon mutants is also overstated at this point in the paper. It is certainly likely that this contributes to the other phenotypes and the finding of a large increase in Ubqn in leon mutants and the finding that reducing Ubqn can prevent some or all of the phenotypes is important. But to really prove that the change in Ubqn is causative and not merely part of the phenotype, it needs the data in leon mutants could mediate expansions of Dlg-positive zones, dPak patches and GluRIIA clusters in leon mutants.”We modified the conclusion for Fig. 7. This modified text was shown in Lines 431-433, as “These results are consistent with that the increase in the Ubqn level in


The most critical step in the initiation of apoptosis is the activation of the Bcl-2 family of proteins to oligomerize and permeabilize the outer-mitochondrial membrane (OMM). As this step results in the irreversible release of factors that enhance cellular degradation, it is the point of no return in programmed cell death and would be an ideal therapeutic target. However, the arrangement of the Bcl-2 proteins in the OMM during permeabilization still remains unknown. It is also unclear whether the Bcl-2 protein, Bid, directly participates in the formation of the oligomers in live cells, even though it is cleaved and translocates to the OMM at the initiation of apoptosis. Therefore, we utilized confocal microscopy to measure Förster resonance energy transfer (FRET) efficiencies in live cells to determine the conformation(s) and intermolecular contacts of Bid within these Bcl-2 oligomers. We found that Bid adopts an extended conformation, which appears to be critical for its association with the mitochondrial membrane. This conformation is also important for intermolecular contacts within the Bid oligomer. More importantly for the first time, direct intermolecular contacts between Bid and Bax were observed, thereby, confirming Bid as a key component of these oligomers. Furthermore, the observed FRET efficiencies allowed us to propose an oligomeric arrangement of Bid, Bax, and possibly other members of the Bcl-2 family of proteins that form a self-propagating network that permeabilizes the OMM. The culmination of interactions of the pro-apoptotic members results in the permeabilization of the outer-mitochondrial membrane (OMM). Caspases are then activated and other proteins are released which enhance apoptosis. Concurrent with these cellular events, there is a third class of member proteins of the Bcl-2 family known as the 'initiators',3 which undergo marked changes in cellular location. There still remain unanswered questions about what role initiators play in the regulation of apoptosis. This study focuses on identifying the conformational changes and intermolecular contacts of the initiator, Bid, in order to gain a more detailed picture of the architecture of the Bcl-2 proteins during the permeabilization of the OMM.The commitment of a cell to undergo apoptosis involves the activation and suppression of certain members of the Bcl-2 family of proteins.α-helical fold shared by other members of the Bcl-2 family of proteins.5 Bid, in a similar fashion to the pro-apoptotic member, Bax, translocates from the cytosol to the mitochondria at the initiation of apoptosis.7 In contrast to Bax, which translocates as a full-length protein, the translocation of Bid is believed to be initiated via its cleavage at residue Leu59 by caspase-8 to produce a 7 kDa fragment, p7, and a 15 kDa fragment, p15.7 The p15 fragment, referred to as tBid, associates with mitochondria outer membrane (MOM) and subsequently initiating the process of its permeabilization (MOMP). This fragmentation is necessary for oligomer formation in the presence of Bax or Bak and the eventual release of cytochrome c.8 The cellular and molecular timelines of Bid's cleavage into p7 and tBid as well as its translocation to the mitochondrial outer membrane following apoptosis induction have been studied in the past.11 A more refined breakdown of this timeline would be very useful. A better understanding of this process might also lead to the potential development of better therapeutics that can specifically target different functional aspects of Bid.13Bid is an alpha-helical, 22-kDa protein that adopts a characteristic 3 The initiators, however, contain only one of these domains, BH3. These proteins are known as BH3-only proteins. Other proteins such as p5314 and PUMA15 could also act as initiators. This has led to the hypothesis that the initiators, which include Bid, utilize the BH3 domain to catalyze pro-apoptotic activity by binding to other Bcl-2 proteins. The initiators can accomplish this role by adopting the characteristic α-helical fold of Bcl-2 proteins like Bid or are intrinsically unfolded.16 Indeed, peptides from the BH3 domains of the initiators are known to bind with the pro-apoptotic members16 or pro-survival members17 to enhance pro-apoptotic activity in cell-free assays. Bid can also occupy solution and membrane environments18 to enhance the insertion of Bax into membranes.20 Similar activation has been observed with other initiators in the Bcl-2 family.21 Although the activity of Bid has been well-studied, the possible contributions of other factors that affect its regulation could be overlooked. In a complex biological system, it is important to gather different viewpoints by studying it using various techniques, which individually cannot provide a complete picture. Cell-free assays, which constitute the majority of studies of Bcl-2 proteins, have been very successful in providing accurate descriptions on various states of the proteins. A number of factors but especially the complete components of the OMM, including apoptosis machinery and their physical condition  can modify the states of these proteins within this complex process. Although a wealth of knowledge has been gained from cell-free assays, there still remains the possibility that other features, such as conformational changes that might already be occurring before translocation or exchange equilibrium between membrane bound and cytosolic form of the protein, can be observed when experiments are performed under conditions more closely resembling the live-cell environment. As was shown previously and reproducibly, Bax adopts a different conformation in a test tube in comparison with its native environment within the cell.22 This allows for the equilibration of Bax between the cytosol and mitochondria before the initiation of apoptosis.25 Therefore, we have employed methodologies developed in our lab using live-cell quantitative Förster resonance energy transfer (FRET)-imaging to perform measurements in living cells undergoing apoptosis in order to reach biologically relevant conclusions. From observations of FRET using fluorophores located at positions on Bid as shown in Previous investigations of the initiators have focused on their unique structural features and similarities with the other Bcl-2 proteins. Some member proteins that are pro-apoptotic and pro-survival contain four homologous domains, BH1-4.26We monitored the caspase activation and subsequent translocation of Bid to the mitochondria by monitoring the fluorescence intensity of AlexaFuor546 conjugated to a cysteine at position 35 (p7) relative to the intensity of AlexaFluor633 conjugated to a lysine on tBid (p15). Images of this variant after translocation are shown in 27 and the results are shown in 28 The p15 fragment adopts an extended conformation that can form intermolecular contacts, which is discussed below and in the The conformational changes in the p15 fragment after translocation were probed using our optimized FRET methodology−4 to 1.6 × 10−3 s). The rest of the diffusive behavior (29–33%) exhibited the slow time constant (3.3 × 10−2 to 1.1 × 10−1 s). These relative populations are the result of fitting without correction to differences in quantum yields for the two time constants. As a result, they are only 'apparent populations'. In addition, it is not clear whether to fit the slower time constant to a 3-D or 2-D diffusion model because multiple scenarios could account for the different models of diffusion at a time constant of this magnitude, such as oligomers, association with an organelle or diffusion within a membrane environment. However, these apparent populations are reported to demonstrate the heterogeneity of the diffusion of Bid in a cellular environment.Bid was cross-linked at two different sets of positions shown in After translocation, the intracellular diffusion of these variants differs significantly from that of the uncross-linked variant . The unc2222 In both experiments, the intracellular distribution of Bid is diffuse and punctate before and after translocation to the mitochondria, respectively. In FRET between separate molecules of tBid at the OMM were also measured. These experiments were carried out as described before.22 The FRET efficiencies between positions Bid-118, Bid-140, and Bid-158 are particularly interesting. The observed intermolecular FRET efficiencies between positions Bid-118 and Bid-158 and between Bid-140 and Bid-158 in Intermolecular FRET efficiencies between other positions are shown in Contacts between different regions of tBid and Bax at the OMM were also observed. The translocations of Bid-140 and Bid-88 in the presence of Bax variants are shown in 22 When additional Bid was added in equimolar amounts to the two Bax variants (Bax-78 and Bax-62) and microinjected into cells, an intermolecular FRET efficiency of 0.45±0.11 was calculated from measurements of 16 cells. Based on these results the dimer architecture that was previously observed in Bax is intact when excess Bid is also co-microinjected.Intermolecular FRET efficiencies between tBid and Bax at other positions are shown in 29 before associating with the OMM where it adopts an extended conformation that allows it to form intermolecular contacts. This extended structure is consistent with an NMR study, which showed tBid in a micellar environment retained its helical character in an extended conformation.30 However, in contrast to Bax, tBid does not have any helices that insert into the membrane. This feature would indicate a weaker membrane interaction of Bid relative to Bax. The weaker membrane interaction may allow it to be more fluid in its association with the OMM and could more easily find another Bax molecule to interact and facilitate the insertion of the membrane-spanning helices in Bax. Prior work has shown that tBid is one of the activators that can induce Bax insertion.31 It is also still not fully understood how cleavage at position 59 assists in the translocation of Bid. In fact, Prior to the initiation of apoptosis, Bid adopts a compact conformation similar to the NMR structure of Bax22 equimolar amounts, or excess of Bid relative to endogenous Bax, translocation was observed for both proteins. According to our observations, varying ratios of Bid and Bax also did not alter the rate of translocation to the mitochondria. This is consistent with a previous study6 that showed that Bid interacts in a catalytic nature with Bax to aid in the formation of oligomers. Interestingly, the same study also found that Bid may not be a structural element in the complex forming on the mitochondria membrane. A broad range of ratios of Bid and Bax would allow for permeabilization of the OMM under a variety of conditions. In addition, our observations suggest that a bimolecular interaction between Bid and Bax would not be required for insertion to the OMM. These observations are consistent with a recent study by O'Neill and colleagues.33 Their genome editing experiments show that Bax and Bak can initiate oligomerization in the absence of any other Bcl-2 protein, including Bid. Interactions with the OMM are sufficient to Bax and Bak to be activated at the initiation of apoptosis. Although they propose that Bid and other BH3-only proteins inhibit the activity of pro-survival proteins, our observations provide another aspect of the activity of Bid where Bid is also involved in the interactions that permeabilize the OMM. By having both proteins independently translocating to the mitochondria, the permeabilization of the OMM could occur more efficiently.Bid and Bax translocate to the mitochondria independent of their relative concentrations. Whether their ratio had excess amounts of Bax relative to endogenous Bid,34 as well as synthetic membrane systems,18 whereas in another study, no oligomers were found to be formed.19 These seemingly contradictory results highlight the necessity to be able to observe the protein in a specific spatial and temporal manner. Under conditions of live cells undergoing apoptosis, our study is the first to identify where Bax and tBid make specific molecular contact in these oligomers (tBid:Bax). 522 Some of these interprotein contacts may also be preserved from monomeric interactions. The helices α5, α6, and α7 in tBid may interact with BH3 domain in helix α3 of Bax as the structurally homologous helices α4, α5, and α6 in Bax do. Also, Bak is structurally homologous to Bax and, therefore, might interact with tBid in a similar manner. Bax and Bak mixed dimers have been observed.35 This potential cross interaction of conserved contacts within the Bcl-2 family could allow for the efficient formation of the Bcl-2 oligomers at the initiation of apoptosis.Upon association with the OMM, tBid and Bax form homo- and heteroligomers. Homo-oligomers of tBid (tBid:tBid) have been observed in reconstituted mitochondria36 of oligomerization of Bax in liposomes the presence of tBid was increased by increasing the curvature of the liposomes using Drp1. Therefore, if this network of interactions prefers or even promotes membrane curvature, the Bcl-2 oligomers can become self-propogating until the OMM is fully permeabilized. This scenario is reminiscent of proteins that are involved in membrane sensing and remodeling processes in the cell, such as vesicle budding and fusing. In these processes, a number of different cytosolic proteins arrive at the membrane, concentrate into discrete spots, and deform the membrane surface.40 Some of the proteins that interact with the membrane contain unique functional features, such as the BAR domain or ALPS motif.41 BAR domains exist as monomers in the cytosol.42 However, upon recognition of the membrane surface they oligomerize efficiently.43 The size of the oligomer assembly is defined by the membrane surface topology. The ALPS motif contains a long amphipathic helix that is composed of bulky hydrophobic residues on one side and a polar side consisting of small and uncharged residues.45 ALPS motif can intercalate into lipid-packing defects in a membrane. These defects are the result of the mismatch between membrane curvature and the shape of the lipid molecule.47 In comparison, the C-terminal helix α9 of Bax is often referred to as hydrophobic, yet it consists of bulky hydrophobic residues on one side and a polar side composed of glycine (G179), serine (S184), and threonine . This helix is a crucial component for Bax to target to the mitochondrial membrane. Earlier reports of Bax colocalization with Drp1 and Mfn249 proteins that are involved in mitochondria fission, seem to support that Bcl-2 oligomers can form self-propogating oligomers at discreet regions of the mitochondria.A proposed arrangement of interactions between tBid and Bax is shown in Direct interaction of Bax with tBid as presented in this work, or with possibly other Bcl-2 members in the creation of protein oligomer networks within the OMM, closely parallels the interaction between proteins that sense and remodel membranes. This analogy also fits well with the concentration independent formation of the Bid-Bax oligomers in the mitochondrial membrane. This feature of the general network of protein interactions on the mitochondrial membrane lends itself to our understanding of the irreversibility of this step in the initiation of apoptosis.30 This was then followed by the replacement of various residues at desired locations with a cysteine that was used for the conjugation of molecular probes. Bid variants had either one or two cysteines. All mutations were generated using the pET15b-Bid plasmid as a template and the QuickChange site-directed mutagenesis kit . BL21(DE3) Escherichia Coli cells were transformed with the modified plasmids and grown according to previously published conditions,30 except Luria Bertani broth was used instead of minimal media with isotopic enrichment. After harvesting and resuspending the cells in lysis buffer , the cells were passed twice through an emulsifier before centrifugation at 34 000 × g for 30 min at 4 °C. The cell lysate was then passed through a 5-ml HisTrap HP column  equilibrated with lysis buffer. The His6-tagged Bid variant was eluted using an increasing gradient of elution buffer . Bid fractions were then pooled and buffer exchanged into Buffer A  using a PD10 column . Further purification was performed by anion-exchange chromatography using a 1-ml HiTrap HP Q column  equilibrated with Buffer A. Bid was eluted using a linear salt gradient with Buffer B . Removal of the Histidine tag was not necessary because His6-tagged Bid exhibits wild-type (WT) behavior in live cells . All Bid and Bax mutants, that are not modified by cross-linking, translocate from the cytosol to the mitochondria at roughly the same rate as the WT proteins.Bid mutants with an N-terminal 6X-Histidine tag were produced by replacing all native cysteines with a serine in the original pET15b-Bid plasmid carrying human Bid cDNA.°C. The protein concentration was 100 μM. To react the FRET donors to Bid, the ratio of protein to dye was 2:1 for proteins with two cysteines and 1:1 for single-cysteine variants. To conjugate the FRET acceptor, Dabcyl, to Bid a 7:1 excess of dye to protein was used. Unreacted and reacted species with one fluorescent dye molecule were separated with a mono-Q column as described previously.27 The internal reference, AlexaFluor633, was conjugated to the above dye-labeled Bid proteins using recently published protocol at Lys144 or Lys146 .27 Liquid chromatography mass spectrometry (LC-MS) of the labeled protein variants and their fragments, following pepsin digestion, confirmed the stoichiometry and location of dyes, respectively, as previously described.27Bid was site-specifically labeled at engineered cysteines using maleimide chemistry. AlexaFluor546 and AlexaFluor488 (Invitrogen) were used as FRET donors and Dabcyl (Anaspec) was used as the FRET acceptor. For the conjugation reaction of each probe to Bid, the conditions were 20 mM Tris-HCl at pH 8.0 with 1 mM TCEP, in the dark for 1 h at 25 27 All imaging experiments were carried out using serially immortalized mouse embryonic fibroblast (MEF), which was generously provided by Richard Youle, .48 Additional experiments were performed on a Zeiss 510 system but with the same excitation and emission filter sets that were used in the previous studies. The total concentration of conjugated protein that was microinjected for FRET measurements ranged from 200 to 400 nM. Equimolar amounts of 200 nM of different Bid variants were co-injected for intermolecular FRET measurements, whereas equimolar amounts of 200 nM of Bid and Bax variants were co-injected for interprotein FRET. To ensure that Bax:Bax contacts were intact for Bid:Bax experiments, an additional 200 nM of unlabeled Bid was added and co-injected with 200 nM of fluorescent Bax labeled at position 78 and 200 nM of Bax labeled at position 62. Previously we estimated that there was a dilution factor of 1:5–1:10 of the injected protein concentration relative to the final concentration in the cell.The confocal microscopy and microinjection setup to study Bid was described earlier.0), which corresponds to a distance between the fluorophores with 50% FRET efficiency. To measure intramolecular FRET between two cysteines, the F' sample consisted of a Bid variant with two cysteines with only AlexaFluor546 or AlexaFluor488 labeled on one of the cysteines and the internal reference, AlexaFluor633. Then, a FQ' sample was prepared for this same variant that was labeled with the either AlexaFluor546 or AlexaFluor488 as well as Dabcyl on the other cysteine in addition to the internal reference, AlexaFluor633. FRET efficiency, E' was calculated as described previously.27 To measure intermolecular FRET between two cysteines, the F' sample was labeled with AlexaFluor546 and the internal reference, AlexaFluor633. The FQ' sample consisted of co-microinjecting the F' sample with another single-cysteine variant of Bid labeled with Dabcyl. FRET efficiency, E' was calculated as described previously.27 The locations of the cysteines used in these experiments are indicated in the Tables.FRET efficiency depends on a distance separating two fluorophores. The choice of pair of fluorophores depends on their characteristic Föster radius (RμM staurosporin (STS) into the cell culture medium. This method of initiation was chosen to ensure consistency with our previous studies of Bax.27 To verify that the translocation of Bid using STS was consistent with inducing translocation of Bid with TNFα,51 a colocalization assay with Mitotracker Green FM was performed using 100 μg/ml CHX and 1 μg/ml TNFα with cells in PBS. Colocalization of Bid and Mitotracker Green FM, as well as similar Bid translocation times, was observed using both methods of apoptosis initiation 3, and 1 mM TCEP in 100 mM Tris-HCl at pH 7.5 for 4 h in the dark at 37 °C. Completeness of the reaction was verified by LC-MS via the addition of 352.13 Da to the mass of the Bid variant . Fragments produced by pepsin that are linked by the cross-linker were also detected . Subsequent to cross-linking, these variants were labeled with AlexaFluor633 and microinjected into MEF cells for analysis.To verify whether the observed conformational changes had an effect on translocation, two variants of Bid were cross-linked with BM(PEG)3 . One variant was cross-linked between Arg88Cys and Lys158Cys, whereas the other variant was cross-linked between Gln136Cys and Gln180Cys. The conditions for the cross-linking were 35–60


We report here the discovery and development of boron-assisted oxime formation as a powerful connective reaction for chemical biology. 4 M–1 s–1, the largest to date for any oxime condensation. Boron's dynamic coordination chemistry confers an adaptability that seems to aid a number of elementary steps in the oxime condensation. In addition to applications in bioconjugation, the emerging importance of boronic acids in chemical biology as carbohydrate receptors or peroxide probes, and the growing list of drugs and drug candidates containing boronic acids suggest many potential applications.We report here the discovery and development of boron-assisted oxime formation as a powerful connective reaction for chemical biology. Oximes proximal to boronic acids form in neutral aqueous buffer with rate constants of more than 10 Herein we describe that O-alkylAIBs form with remarkable speed and selectivity in neutral aqueous buffer. The reaction is unaffected by proteins, carbohydrates, biothiols, and human serum, and has an equilibrium constant of >108 M–1.We selected the 2-formylphenylboronic acid , or HPLC injection . More complex hydroxylamines, such as the pentapeptide shown in entry 4, were also excellent substrates, delivering complete conversion after the first injection. Condensations with ketones were slower than with 2-FPBA (entry 7), but still faster than normal ketoxime condensations. Pinacol boronate esters also gave complete conversion after one minute (compares entries 2 & 8) but the hydrolysed boronic acid 2a was the main product with only 10% of pinacol ester oxime 2f observed; after ninety minutes only 2a was present. The ketoximine pinacol product (entry 9), on the other hand, was more stable and comprised 97% of the product after the first injection (the remainder being 2e); after thirty minutes 2g had also completely hydrolysed to 2e. The ability to use pinacol esters is valuable since these are readily obtained from Pd-catalysed routes to boronic esters, and their deprotections in organic solvents are often tricky.As shown in panel b of –1 s–1 – several orders of magnitude faster than the fastest neutral pH oxime condensations.34 Although fluorescence quenching is an indirect measurement technique, HPLC injections of aliquots from the assay mixture showed only product and starting materials (indicating a clean reaction) and gave conversions qualitatively in agreement with the fluorescence measurements. In addition, the fact that 10 and 1 µM reactions monitored by 1H NMR showed >90% conversion at the earliest possible measurements . The asymmetry in the pH dependence curve is inverted in comparison to a typical oxime condensation (acetone with hydroxylamine),35 which shows a more pronounced drop-off in rate at higher pH than at lower pH. Based on the build-up of a tetrahedral intermediate at higher pH, Jencks attributed this pH dependence to the need for a proton in the dehydration step.35 Although we have thus far not been able to detect intermediates, we speculate that the near-neutral pKa of amine-substituted aqueous boronates36 provides the ideal environment for acceleration of the normally rate-limiting dehydration.The reaction is fast and displays second order kinetics. We established the rate in three independent ways: HPLC, NMR, and fluorescence quenching. While the time resolution and sensitivity of HPLC and NMR could provide only a lower bound for the rate constant, fluorescence quenching gave a more quantitative assessment. A hydroxylamine bearing a lissamine fluorophore and a 2-FPBA connected to a quencher were prepared see  and gave2a . From this value and the k1 value obtained from fluorescence quenching . The additives led to no detectable reduction in reaction efficiency see  with theO-alkylhydroxylamines are also available. A shortcoming of the present method in comparison to the classical oxime condensation is the size of required 2-FBPA motif. Although for most applications this should present no difficulties, examples where the compactness of the oxime is critical 37 would not be possible. The ability to run conjugations at 1 : 1 ratios of partners at biological pH means that either or both components can be complex, precious materials. Although we have focused on oxime condensation for proof-of-concept the coordinating properties of boron in aqueous media could potentially be exploited to accelerate other important reactions whose rates are limited by the kinetics of Schiff base formation.11A great advantage of the present method over many coupling reactions is the simplicity and ready availability of the starting materials. There are commercial libraries of phenylboronic acid and boronic ester compounds, many of which contain an aldehyde or can be trivially elaborated to incorporate one. Furthermore, the widespread use of oxime conjugations for connective processes at high concentration means that a variety of


Current diagnostic assessment tools remain suboptimal in demonstrating complex morphology of congenital heart disease (CHD). This limitation has posed several challenges in preoperative planning, communication in medical practice, and medical education. This study aims to investigate the dimensional accuracy and the clinical value of 3D printed model of CHD in the above three areas.Using cardiac computed tomography angiography (CCTA) data, a patient-specific 3D model of a 20-month-old boy with double outlet right ventricle was printed in Tango Plus material. Pearson correlation coefficient was used to evaluate correlation of the quantitative measurements taken at analogous anatomical locations between the CCTA images pre- and post-3D printing. Qualitative analysis was conducted by distributing surveys to six health professionals  and three medical academics to assess the clinical value of the 3D printed model in these three areas.Excellent correlation (r = 0.99) was noted in the measurements between CCTA and 3D printed model, with a mean difference of 0.23 mm. Four out of six health professionals found the model to be useful in facilitating preoperative planning, while all of them thought that the model would be invaluable in enhancing patient-doctor communication. All three medical academics found the model to be helpful in teaching, and thought that the students will be able to learn the pathology quicker with better understanding.The complex cardiac anatomy can be accurately replicated in flexible material using 3D printing technology. 3D printed heart models could serve as an excellent tool in facilitating preoperative planning, communication in medical practice, and medical education, although further studies with inclusion of more clinical cases are needed. Congenital heart disease (CHD) is the most common birth defect among the newborns . DespiteTo date, diagnostic assessment and pre-operative planning of CHD is based on volumetric data such as computed tomography (CT), magnetic resonance imaging (MRI), and echocardiography. In conjunction with the three-dimensional 3D) reconstruction method, 3D virtual model of human’s heart can be generated D reconst, 8. EvenIn order to resolve this shortcoming, there have been several studies on fabrication of 3D printed cardiac models of CHD. These models were reported to facilitate the surgical decision-making and planning, enhance the patient-doctor communication, and improve the knowledge acquisition among the patients, students, and junior doctors , 12–30. However, the evidence to suggest the dimensional accuracy of the 3D printed cardiac models is lacking. Most of the studies in current literature only investigate the clinical usefulness of the models without reporting their dimensional accuracy. Additionally, most of the current studies are case reports or case series focusing on the utility of 3D printed heart models in preoperative planning , 29–31. Thus, this study presents a preliminary experience in creating flexible 3D printed heart model from cardiac CT angiography (CCTA). The aim of this study was twofold: (1) to investigate the dimensional accuracy of the 3D printed heart model, and (2) to evaluate the clinical value and feasibility of flexible 3D printed models of CHD, in particular for preoperative planning, medical education, and communication in clinical practice.Ethical approval was obtained from Curtin Human Research Ethics Committee. The ethics committee waived the requirement for informed consent due to the retrospective nature of the study and use of de-identified images. Five cases of de-identified CCTA images of complex CHD in Digital Imaging and Communications in Medicine (DICOM) format were obtained from a radiology archive. During the cases selection process, only cases with patients aged from 0–15 years old were included.All CCTA was performed at 70 kV on a 256-slice GE Revolution scanner  or a 128-slice Siemens Definition Flash . The contrast medium was administered according to the following protocol:Injection rate: weight (kilograms) x 0.1 mL/sVolume of contrast medium: injection rate x 20 sTo reduce the radiation dose to the paediatric patients, prospective electrocardiogram (ECG) triggering technology and tube current modulation were utilized. The selected cases were also ensured to have slice thickness of less than 0.75 mm to guarantee the resolution of acquired images for 3D printing purpose.Mimics Innovation Suite software  using thresholding and region growing methods [3-Matic software . The digital model was hollowed and smoothed before being exported in Standard Tessellation Language (STL) format [All five cases were segmented using  methods . In the ) format .3-Matic was loaded into Geomagic Studio 12  to separate the digital model into two compartments. This was done so that the intra-cardiac structures can be better demonstrated. The model was cut through a plane transecting right atrium and right ventricle to provide a viewing window for the ventricular septal defect (VSD). Mesh doctor function was also used to optimize the file for 3D printing.The STL file exported from The time to generate the STL model was limited to 2 hours to ensure the practicability of producing 3D printed heart models. Only one digital model that requires least manual edition but with excellent demonstration of cardiac anatomical structures and pathologies was chosen for 3D printing.The chosen digital model was printed with a commercial printer Stratasys (Objet Eden 260VS) using Polyjet printing technology in Tango Plus material, which is a rubber-like material that has more resemblance to human heart tissue when compared to hard plastic , 34. AftHoros (Horos Project), which is an open-source DICOM viewer  was demonstrated between the CCTA and 3D printed model measurements, indicating that the 3D printed model generated in this study is highly accurate in replicating anatomical cardiac structures. Overall, the 3D printed model marginally over-estimated the size of the heart when compared to the CCTA measurements, but only by 0.23 mm in average. Overall, all the participants have at least three years of working experience in the corresponding professional field. Eight out of nine participants had encountered challenges in their profession before due to limitations of current visualization techniques, whereas six out of nine participants reported that they had used 3D printed cardiac models in their practice previously. The average satisfaction score of the model was 8.4 out of 10.Four out of six health professionals indicated that the 3D printed models are useful in planning interventions, while three out of six reported that it could be useful in testing devices for pre-surgical simulation . There wBoth the cardiac surgeons agreed that the 3D printed models would be able to provide additional information of the pathology compared to conventional imaging and computer simulation. They reported that the 3D printed cardiac model is helpful for them to appreciate potential procedural difficulties, and hence will be able to increase the success rate of the surgery.The two cardiac surgeons and two cardiologists were asked whether the 3D printed model is useful in enhancing patient-doctor communication, and all of them reported that the model would be invaluable in improving the consultation experience as they would be able to explain the CHD to the patients more efficiently. They also thought that the patients could understand the condition of their heart better with the use of 3D printed model. However, when being asked whether the consultation time could be reduced, only two of them responded ‘yes’ .Interestingly, when all the health professionals were asked whether they prefer to use 3D printed model or medical images to communicate with the patients or other health professionals, four out of six indicated they prefer to use both of them as a medium in communication .The three medical academic staff members were asked about their opinion of using 3D printed cardiac models in teaching anatomy and pathology. All of them found it invaluable, and indicated that the students will be able to learn the pathology quicker with better understanding. However, two medical academics implied that the 3D printed model generated in this study could not help the students to learn normal heart anatomy . This is3D printing in medicine was initially introduced in maxillofacial and orthopedic specialties, mainly for preoperative simulation. It was only in recent years that this technique has expanded to cardiovascular diseases. Its clinical value in this area is promising, although more research is needed , 26, 29.Only a few existing studies reported on the dimensional accuracy of the 3D printed heart models. Despite so, all of them are in general agreement that the 3D printed heart models are accurate, with the reported mean difference ranges from 0.05 ± 0.17 mm to 0.4 ± 0.9 mm , 27, 30.The preliminary experience in this study demonstrates that: (1) it is feasible to accurately replicate a heart in flexible material; and (2) the 3D printed heart model of CHD is useful in pre-operative planning, medical education, and communication in medical practice. These positive findings are significant as this is the first report to investigate the accuracy and the multi-directional utility of the 3D printed heart model in a holistic approach. To our knowledge, this is also the first time to confirm model accuracy by performing contrast-enhanced CT scan of the 3D printed heart model for measurement of the intra-cardiac structures , since oThe mean difference of 0.23 mm between the measurements of the 3D printed heart model and the original CCTA could be caused by several reasons. First, it is possible that the measured locations on both CT datasets were not perfectly at the same point. Even though the CT scan of the 3D printed model was reconstructed manually to match the heart orientation of the original CCTA, there is a chance to have slight variations between the analogous locations, impeding a perfect comparison. Second, the image resolution and noise of the source data could also introduce errors in defining the boundaries of the anatomical structures . AlthougOverall, the cardiac surgeons and cardiologists responded positively with respect to the use of 3D printed heart model in communication within medical practice. However, not all of them agreed that the duration of consultation could be reduced with the use of 3D printed heart model. In a study carried out by Biglino et al, it was found out that the consultation involving the use of 3D printed model lasted 5 minutes longer than usual . This miThis study has several limitations. First, although comparing the measurements of the original CCTA and 3D printed heart model at ten different anatomical locations was an indicator of the 3D model’s accuracy, it is worthwhile to note that the results may not necessarily reflect the accuracy of the entire model, as the measurements were only taken in axial plane . It is nSecond, there is only one 3D printed heart model generated in this study, therefore, the results lack generalization. Despite this limitation, findings in this study are consistent with those reported in a multi-centre study representing a larger group of patients with 3D printing of CHD . In theiAs 3D printing in cardiovascular specialty is still in its infancy, future studies should include more cases and participants to confirm the dimensional accuracy and clinical value of the 3D printed heart models. It will also be worthwhile to include surveys with the parents or patients with CHD, as they are the stakeholders who would benefit directly from this novel technology. Additionally, future studies should focus on developing standardized method in generating 3D printed heart models. In the current literature, there is a great variation in terms of how 3D printed models were fabricated, including the source data and segmentation method , 38, 39.In conclusion, this study has preliminarily assessed the dimensional accuracy as well as the clinical value of flexible patient-specific 3D printed heart models. The flexible 3D printed heart model can be accurately reproduced from cardiac CT images. It is found to facilitate preoperative planning and improve the surgical outcome; enhance patient-doctor and inter-professional communication; and improve approach in medical teaching. These findings warrant further investigations in 3D printing of congenital heart disease.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.