BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure ( Cardiovascular diseases are the world's leading cause of morbidity and mortality, responsible for approximately 17 million deaths per annum 2+ concentration ([Ca2+]i) and calcium signalling within vascular cells have long been known to be important for arterial function 2+ pump plasma membrane calcium ATPase 4 (PMCA4) has a role in arterial calcium handling Increased peripheral vascular resistance has been reported as the principal driver for chronically elevated BP PMCA4 is present in vascular smooth muscle (VSM) of larger arteries via physical interaction with nNOS in vivo in our model.PMCAs are classically described as playing a role in calcium extrusion from cells, although it is now evident that they, and specifically PMCA4, also have important roles as scaffolding proteins and in signal transduction In this study, we investigated, for the first time, the effects of PMCA4 ablation on BP and resistance arterial contractile function and, furthermore, examined the acute effects of a recently identified and validated inhibitor of PMCA4 ad libitum access to normal chow diet and water. All experiments were approved by the University of Manchester Ethics Committee and were in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. All animals were humanely killed by cervical dislocation. This study conforms to ARRIVE guidelines on use of experimental animals The effect of global ablation of PMCA4 was assessed using 3‐month‐old PMCA4 germline‐null mutant adult male mice  which we have previously generated In vivo conscious BP of mice was monitored using a CODA™ occlusion tail cuff volume–pressure sensor monitoring system . Mice were acclimatized to the animal holder and the system for three consecutive days prior to experimental recording. For experimental recordings, mice were placed on a pad heated to 37°C and blood flow to the distal tail was occluded with a maximal cuffing pressure of 250 mmHg and then steadily deflated over 15 sec. for a single cycle. Systolic and diastolic blood pressures were automatically recorded during cuff deflation as blood flowed into the tail. Twenty continuous cycles were performed (10‐min experiment), with accepted values  from the latter 10 cycles used for data analysis. Five seconds between each cycle was programmed. Basal BP of PMCA4KO mice was compared to wild‐type littermates (PMCA4WT). In separate experiments, short‐term effects of a recently identified and characterized inhibitor of PMCA4, aurintricarboxylic acid (ATA), were examined in WT and PMCA4KO mice in vitro to completely inhibit PMCA4, and in vivo, this dose reverses cardiac hypertrophy l‐NIO (VLN) at 10 mg/kg bodyweight, a dose comparable to a concentration exceeding the Ki value for inhibition of nNOS 4.7H2O 1.17 mM, NaHCO3 25 mM, KH2PO4 1.17 mM, K2EDTA 0.03 mM, glucose 5.5 mM and CaCl2.2H2O 1.6 mM . Fat and adherent tissue was removed from third‐order mesenteric arteries and aorta. Vascular smooth muscle cells were immediately isolated from mesenteric arteries, or arteries were used for pressure myography studies. Aortic tissue was fixed for immunohistochemical analysis or frozen in liquid nitrogen before being stored at −80°C until proteins were extracted from the tissue.The entire mesenteric bed and thoracic aorta were removed and placed separately into ice‐cold physiological salt solution (PSS) of the following composition: NaCl 119 mM, KCl 4.7 mM, MgSOn = 3 animals as separate experiments) were cleared of attached fat and incubated in HEPES‐buffered physiological solution  supplemented with 1,4‐dithioerythritol  at 2 mg/ml and papain  at 1 mg/ml in a water bath at 37°C for 22 min. The solution was then replaced with HEPES buffer supplemented with type F collagenase from Clostridium histolyticum  at 1 mg/ml for 7 min. The cell suspension was briefly stood on ice before being gently titrated and small drops of cell suspension pipetted onto poly‐l‐lysine‐coated slides . Slides were left at room temperature for 50 min. before ice‐cold methanol was added to the cell suspension for 10 min. Slides were carefully washed in HEPES buffer before being stored under saline solution at 4°C before being probed for PMCA4 as described below.Third‐order mesenteric arteries  and dried overnight at 37°C. Sections were incubated in 3% hydrogen peroxide and washed in double‐distilled water followed by phosphate buffered saline (PBS) before incubation in proteinase K in PBS (0.1 mg/ml). Sections were permeabilized with Triton X‐100  in PBS (0.1%) for 10 min. and washed in PBS. To block non‐specific binding, sections were incubated with 3% bovine serum albumin (BSA) in PBS for an hour and washed in PBS. Isolated VSMCs were incubated under 10% donkey serum in PBS (v/v) for 2 hrs at laboratory temperature. Sections or cells were probed with primary antibody for α‐smooth muscle actin (αSMA) , the endothelial cell marker CD34  or PMCA4  in blocking solution and incubated overnight at 4°C. Slides were washed three times with PBS and then incubated with secondary antibody conjugated with Texas Red (red) or FITC (green) fluorescent labels  for 2 hrs at room temperature. Slides were washed three times in PBS before being stained with 3 μM of 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI)  for 60 sec., washed in PBS and mounted under coverslips using mounting medium . Sections were visualized using an upright confocal fluorescence microscope  with Texas Red, FITC green and blue filters using the EZ‐C1 3.90 FreeViewer software . VSMCs were imaged with a Leica DM5000B microscope and Leica DFC 3000 g camera, and using Leica 10X capture software .Aortas were segmented to approximately 1.5 cm in length, immersed in excess 4% paraformaldehyde (in 0.1 M PBS) for 6 hrs, serially dehydrated in increasing concentrations of ethanol and paraffin‐embedded. Five‐micrometre‐thick cross sections were cut, mounted onto poly‐Aortic tissue was homogenized and proteins isolated and transferred as previously described 2) and left to equilibrate for 30 min. Contractile responses to 100 mM potassium solution (KPSS)  and to cumulative doses of norepinephrine (NE) (1 nM–30 μM) were determined. To assess the effects of acute PMCA4 inhibition on contractile responses, experiments were performed in the presence of 1 μM or 10 μM ATA or 100 μM caloxin 1b1 following an initial 30‐min perfusion of the inhibitor. Such concentrations were chosen based on our previous reports that 1 μM ATA completely blocked PMCA4 activity in membrane microsomes derived from HEK293 cells overexpressing PMCA4 whilst having no effect on Na+/K+ ATPase activity and little effect on PMCA1 activity l‐arginine (LNNA)  l‐NIO (VLN)  2+ chelated PSS solution (Ca2+‐free) with graded increases in intravascular pressure from 5 to 140 mmHg, as previously described Small mesenteric arterial segments of 2–3 mm length were mounted on a pressure myograph  as previously described 2+]i) and arterial contractility, isolated segments of mesenteric arteries were incubated with 20 μM Indo‐1‐AM  in HEPES‐buffered physiological solution for 2 hrs. Arteries were mounted in the bath of a pressure myograph placed atop an inverted microscope, excited at 340 nm and emissions measured via photomultipliers at 400 and 500 nm. The 400 nm:500 nm emission ratio (F400/F500) was determined (following correction for autofluorescence) and used as an indicator of [Ca 2+]i, as previously described For simultaneous measurement of intracellular free calcium (i . Ablation of PMCA4 had no significant effect on the increase in F400/F500 in response to either 100 mM KPSS . However, in response to 100 mM KPSS and 30 μM NE, the rise in F400/F500 was significantly attenuated in arteries with ATA compared with WT arteries alone  as a measure of [Ca2+]i are shown in Figure Basal Fvia a nNOS‐dependent mechanism and that these effects were independent of PMCA4's role as a calcium pump, as ATA had no significant effect on global [Ca2+]i when nNOS was inhibited.We here show, for the first time, that genetic ablation of PMCA4 in mice has no effect on either conscious peripheral BP or resistance arterial contractility to either KPSS or NE. In contrast, acute inhibition of PMCA4 with ATA, a recently characterized inhibitor of PMCA4 2+ handling proteins in the heart 2+ pumps and channels appear to not compensate for the loss of PMCA4. Our analysis of intact aortic segments from WT mice by immunohistochemistry showed that PMCA4 is principally localized to VSMCs with little being detected in endothelial cells, observations consistent with previous reports The PMCA4KO mouse model used in this study has previously been shown to exhibit a complete lack of PMCA4 protein in testes, sperm +) or NE. Hence, the functional consequence of global PMCA4 ablation is not directly opposite to reported effects when PMCA4 is overexpressed in VSM Previous studies have shown unconscious BP is elevated in mice overexpressing PMCA4 in arterial VSMCs in vitro assay and at 10 μM fully inhibits PMCA4 activity in cardiomyocytes in vitro functional studies reported here. We also selected a comparable dose in vivo of 5 mg/kg, and at this same dose, ATA was shown to significantly attenuate pressure overload‐induced cardiac hypertrophy and it reversed established cardiac hypertrophy in vivo has not been precisely defined; however, at 35 mg/kg it had a significant biological effect for up to 2 hrs in rats As the reason for this remains unclear, we sought to clarify the role of PMCA4 in BP regulation by acutely inhibiting PMCA4 with ATA, a recently identified and validated specific inhibitor of PMCA4 We here present that a single dose of ATA (5 mg/kg) significantly reduced systolic and diastolic conscious BP as measured 90 min. after i.p. injection, an observation consistent with both 1 and 10 μM ATA significantly reducing PMCA4WT arteries contractility to a depolarizing and adrenergic stimulus. Importantly, ATA was found to have no significant effect on BP or on arterial contractility of mesenteric arteries from PMCA4KO mice, thus displaying a specificity of action and supporting our previous findings that ATA has little effect on other calcium handling proteins reduces contractility of mesenteric vessels isolated from WT mice, but not PMCA4KO mice, caloxin 1b1 increased the contractility of mesenteric arteries to both a depolarizing stimulus and to NE. Importantly, caloxin 1b1 increased the contractility of arteries isolated from both PMCA4WT and PMCA4KO mice, suggesting that it can modulate contractility via effects which are independent of the presence of PMCA4 protein. Whilst this may explain, at least in part, the differing effects of ATA and caloxins on arterial contractility, differences in the mechanism of action of the two compounds may clearly contribute. It is worth noting that previous studies reporting that caloxin peptides increased arterial contractility utilized aortic and coronary vessels This study reports, for the first time, the effects of ATA on arterial function and compares these with the effects of caloxins which have previously been the only other available inhibitors of PMCA4 in vitro cellular systems via a nNOS‐dependent mechanism(s). nNOS expression and activity have previously been demonstrated in the vasculature including in mesenteric arteries Further, we sought to understand how inhibition of PMCA4 can reduce arterial contractility and BP. Previous PMCA4 overexpression studies which have reported increased vascular contraction have attributed this observation to the negative regulatory effects of PMCA4 on nNOS via Ca2+‐dependent activation of calmodulin and subsequent Ca2+/calmodulin interaction with nNOS 2+ from a microdomain at the plasma membrane, it may reduce the ability of the associated synthase to be activated 2+]i2+]i. Such a mechanism of action remains to be tested in resistance artery VSMCs; however, although ATA reduced increases in global [Ca2+]i to stimulation in isolated mesenteric arteries, this effect was prevented by LNNA or VLN, suggesting that its effects were due to nitric oxide per se rather than being PMCA‐mediated. Indeed, it is well‐established that nitric oxide, via its second messengers cGMP and PKG, can reduce [Ca2+]i in vascular smooth muscle by mechanisms which include decreased Ca2+ entry and reduced release from the sarcoplasmic reticulum 2+]i and is consistent with the regulative mechanism in the heart, whereby PMCA4 regulates nNOS activity by physical tethering and regulation of subcellular Ca2+. Indeed, ablation of PMCA4 had no effect on global [Ca2+]i. Activation of nNOS is Ca2+‐dependent 2+/calmodulin binding with increases in [Ca2+]i causing Ca2+/calmodulin binding to the autoinhibitory domain of PMCA which, in turn, causes a conformational change and release of the autoinhibitory effect Activation of nNOS has been shown to be The relevance of PMCA4 in the scaffolding of regulators to subcellular domains in recent discussions We propose that arterial PMCA1 or NCX1 does not compensate for ablation of PMCA4 in PMCA4KO mice. However, it remains inconclusive whether nNOS expression and/or function may be up‐regulated in resistance arteries in association with PMCA4 ablation. We have previously shown that global ablation of PMCA4 does not affect the total protein level of nNOS in the heart, but rather that cardiomyocyte nNOS was localized more in the cytoplasm and not at the cells’ plasma membrane via a PMCA4–nNOS‐dependent mechanism. We propose PMCA4 contributes to regulating BP via a nitric oxide‐dependent signalling pathway rather than a direct effect on global [Ca2+]i‐mediated VSMC contraction and highlight the importance of PMCA4 as a scaffolding molecule in resistance vessels . Herein we show specificity of action of ATA for PMCA4, which contrasts with our findings using a commercial PMCA4 inhibitor, caloxin 1b1. Further characterization of ATA is required, and whilst we cannot propose ATA per se as a therapeutic agent, we do propose its use as an important experimental tool to further define the relationship between PMCA4 and BP. Understanding the molecular role of PMCA4 remains important for future development of novel BP lower strategies, of which there is increasing clinical need in an ageing population and with increasing intolerance to current interventions.In summary, we have shown using a novel specific inhibitor against PMCA4, ATA, that inhibition of PMCA4 reduces conscious peripheral BP and isolated mesenteric arterial contractility S Lewis, R Little, S Prehar, F Baudoin performed the research; S Lewis and R Little analysed the data; C Austin, EJ Cartwright and L Neyses designed the research study; L Neyses, EJ Cartwright and C Austin contributed to acquiring funding; EJ Cartwright contributed essential tools; R Little, S Lewis, EJ Cartwright and C Austin contributed to writing the manuscript.The authors declare no conflict of interest.Figure S1 Differential effect of ablation and inhibition of PMCA4 on basal conscious blood pressure.Click here for additional data file.Figure S2 Passive properties of mesenteric arteries from PMCA4WT and PMCA4KO mice are not significantly different.Click here for additional data file.Figure S3 Examples of experimental trace recordings for each mouse genotype and pharmacologically treated group.Click here for additional data file.


Women’s childbirth experience can have immediate as well as long-term positive or negative effects on their life, well-being and health. When evaluating and drawing conclusions from research results, women’s experiences of childbirth should be one aspect to consider. Researchers and clinicians need help in finding and selecting the most suitable instrument for their purpose. The aim of this study was therefore to systematically identify and present validated instruments measuring women’s childbirth experience.A systematic review was conducted in January 2016 with a comprehensive search in the bibliographic databases PubMed, CINAHL, Scopus, The Cochrane Library and PsycINFO. Included instruments measured women’s childbirth experiences. Papers were assessed independently by two reviewers for inclusion, and quality assessment of included instruments was made by two reviewers independently and in pairs using Terwee et al’s criteria for evaluation of psychometric properties.In total 5189 citations were screened, of which 5106 were excluded by title and abstract. Eighty-three full-text papers were reviewed, and 37 papers were excluded, resulting in 46 included papers representing 36 instruments. These instruments demonstrated a wide range in purpose and content as well as in the quality of psychometric properties.This systematic review provides an overview of existing instruments measuring women’s childbirth experiences and can support researchers to identify appropriate instruments to be used, and maybe adapted, in their specific contexts and research purpose.The online version of this article (doi:10.1186/s12884-017-1356-y) contains supplementary material, which is available to authorized users. Childbirth experiences can have immediate as well as long-term positive or negative effects on life, well-being and health . A positStudies on women’s childbirth experiences have been using different surrogate terms and related concepts such as ‘childbirth satisfaction’, ‘satisfaction with care’, ‘experiences of control’ or ‘of support’, ‘experience of relationship with caregivers’ and ‘experience of pain’ . Women’sFor an instrument to receive good levels of reliability and validity, extensive development and testing of psychometric properties is needed . WithoutNo review specifically focusing on instruments measuring women’s childbirth experiences has been found, but there are two reviews evaluating instruments measuring ‘maternal childbirth satisfaction’ , 20. PerA systematic review is a rigorous method of research that follows a systematic procedure to enable a summary of all findings from multiple studies on a specific topic. The start point is a rigorous search process for capturing the entire body of scientific studies . As resePapers representing instruments measuring women’s childbirth experience.Papers should describe the development or test psychometric properties of an instrument.Instruments assessing both pregnancy, childbirth and the postpartum period are included if one or more dimensions are related to women’s childbirth experiences, and this could be assessed as a separate scale.Papers reporting original research, published in peer-reviewed journal.Reviews were included to enable us to find original papers.Papers published in English or French were included as the researchers could understand these languages.First a review protocol was developed . For the initial screening all the search results were imported into reference management software (EndNote) and duplicates were removed, leaving 5106 titles and abstract to be screened for inclusion. First, papers clearly irrelevant to our topic, such as papers assessing childhood development, contraceptives etc., were removed by one of us (HN). The remaining 809 titles and abstract were assessed independently by two researchers . This identified 266 residual papers which were assessed independently by two of the reviewers (HN and MB) to include papers for more in-depth full text assessment. Sixty-nine papers were retrieved in full text and assessed for eligibility criteria by two reviewers independently . Any potential conflicts were solved by the third reviewer. Fourteen additional studies were found through search of reference lists of included papers and were assessed in full-text by two independent reviewers for eligibility criteria (HN and MB). Three of these papers were included after assessment in full text. In total 83 papers were thus assessed in full text of which 37 did not fulfil the inclusion criteria and were excluded with reason . When ratings differed between the pairs, it was discussed and, when conflict remained, the third reviewer was included in the discussion to reach consensus. An overview of the results of the quality rating of psychometric properties of included instruments is displayed in Table In conducting this review, our focus and aim was on identifying measures and conducting a broad assessment of their psychometric properties. Given the large number of instruments found, and their very different foci, it was not possible to make clear recommendations as to one particular instrument that would suit all purposes. Instead, some general suggestions are made as to the instruments that appear to be emerging as the top ranking tools in terms of the quality measurement performed, and the overall mark given.The following data were extracted for each instrument: Name of instrument/acronym, authors (year), country of origin, aim/motive of instrument, number of items, dimensions/subscales, response scale, timeframe to answer the questionnaire, whether or not the questionnaire was available and a short narrative summary of included instruments. The data extraction was made by the first author (HN) and then checked by the other authors for accuracy.One of the individual papers was conducted by one of the authors (MB). To avoid conflict of interest this paper was assessed for eligibility criteria, and quality assessment was made, by the two other authors (HN and CB).Forty-six articles presenting 36 instruments –59 measuThe Childbirth Trauma Index for adolescents [The Perception of Birth Scale [Support and Control in Birth [The Childbirth Experience Perception Questionnaire [The Birth satisfaction scale and the Birth satisfaction scale - revised [The Birth Memories and Recall Questionnaire [The labour and delivery satisfaction index [Women’s delivery experience measures [Childbirth schema scale [A few of the tools gained a low quality rating, which would indicate the need for further development and evaluation of their psychometric properties. These included: lescents   had marks of 6 or 6.5, which probably indicates a suitable tool, unless there is a higher quality one in the same area. We suggest that the seven instruments with marks of 7 to 9 Additional file 2:Search strategy. (DOCX 15 kb)


Pedigrees contain information about the genealogical relationships among individuals and are of fundamental importance in many areas of genetic studies. However, pedigrees are often unknown and must be inferred from genetic data. Despite the importance of pedigree inference, existing methods are limited to inferring only close relationships or analyzing a small number of individuals or loci. We present a simulated annealing method for estimating pedigrees in large samples of otherwise seemingly unrelated individuals using genome-wide SNP data. The method supports complex pedigree structures such as polygamous families, multi-generational families, and pedigrees in which many of the member individuals are missing. Computational speed is greatly enhanced by the use of a composite likelihood function which approximates the full likelihood. We validate our method on simulated data and show that it can infer distant relatives more accurately than existing methods. Furthermore, we illustrate the utility of the method on a sample of Greenlandic Inuit. Pedigrees contain information about the genealogical relationships among individuals. This information can be used in many areas of genetic studies such as disease association studies, conservation efforts, and for inferences about the demographic history and social structure of a population. Despite their importance, pedigrees are often unknown and must be estimated from genetic information. However, pedigree inference remains a difficult problem due to the high cost of likelihood computation and the enormous number of possible pedigrees that must be considered. These difficulties limit existing methods in their ability to infer pedigrees when the sample size or the number of markers is large, or when the sample contains only distant relatives. In this report, we present a method that circumvents these computational challenges in order to infer pedigrees of complex structure for a large number of individuals. Using simulations, we find that the method can infer distant relatives much more accurately than existing methods. Furthermore, we show that even pairwise inferences of relatedness can be improved substantially by consideration of the pedigree structure with other related individuals in the sample. N generations, where N is the effective population size. However, standard coalescence models, such as Kingman’s coalescent , where t is the annealing temperature.If the new configuration is an invalid pedigree, reject and go back to step 1. If it is a valid pedigree, compute the composite likelihood C times.Repeat steps 1-2 t/f, where f > 1 and go to step 1.Decrease the temperature to Termination: Terminate after I iterations or when the change in composite likelihood is less than e.C, f, I, and e were optimized to achieve a balance between convergence and computational efficiency using a number of trial runs on different simulated data sets. The tuning parameters q is the number of local pedigrees and β > 0. We chose a Poisson distribution with mean n, the sample size, as the distribution of Q. This regularization is conservative in the sense that it favors every individual to remain a singleton unless there is strong evidence otherwise. Our choice to use the Poisson distribution was made, in part, for computational convenience but, as we will discuss in the Results section, resulted in good statistical properties of the method.Since the composite likelihood function is based on pairwise likelihood values, any inference based on it is limited by the quality of the pairwise likelihoods. One important factor that confounds the likelihood computation is linkage disequilibrium (LD), which often causes relationships to be overestimated . UnrelatWe tested the performance of our method on simulated pedigrees. We generated human autosomal haplotypes using msprime  with effSimulation A consisted of 10 singletons and a 45-person family that spanned 5 generations. Of the 45 family members, 10 were sampled and 35 were missing. The kinship coefficients of the sampled relative pairs ranged from 1/4 (e.g. full siblings) to 1/256 (e.g. third cousins). Simulation B was designed to study the performance of our method on smaller family clusters. It consisted of 4 family clusters and 4 singletons. Each family cluster contained 15 to 18 members, of which only 4 of them were sampled. The sampled individuals spanned multiple generations and formed pairwise relationships with kinship coefficients ranging from 1/4 to 1/256. Simulation C was designed the test the method on pedigree structures in which every sampled individual, excluding singletons, has at least one close relative in the data. It consisted of 9 singletons and a 16-person pedigree that spanned 5 generations. The 16-person pedigree contained 7 missing individuals and 9 sampled individuals, where each sampled individual formed a parent-offspring relationship with at least one other sample. Finally, simulation D was designed to test the method on a pedigree that is relatively easy yet more difficult to infer than simulation C. Whereas every sample was connected by parent-offspring relationships in simulation C, some samples in simulation D were connected only by an avuncular relationship.r2 = .05, resulting in about 10,000 markers. The sex of each sample was assumed known, whereas the age was assumed unknown.Each simulation scenario was replicated 100 times. For each sampled individual, we simulated genotyping error by switching each allele to the alternate allele with probability 0.01. To reduce the level of LD among markers, we used PLINK  to pruner2 = 0.05. Due to the unusually high level of LD in the Greenlandic population, we were left with 2173 SNPs after LD-pruning.We applied our method to reconstruct the previously unreported pedigrees of 100 individuals in Tasiilaq villages in Greenland which had been genotyped [We compared the performance of our method on simulated data to PRIMUS (v1.9.0), arguably the state-of-the-art pedigree reconstruction method. Although many pedigree inference methods exist, we chose to use PRIMUS as a benchmark since it is the most flexible of the existing methods in the types of pedigrees it can infer. More specifically, PRIMUS supports the inference of multi-generational, polygamous pedigrees and allows for missing individuals. PRIMUS reconstructs pedigrees that are consistent with pairwise IBD estimates and reports high-scoring configurations.To estimate the pairwise IBD coefficients for the simulated data, we used two different methods: PLINK and RELATE . To use We used PADRE  for simuRecall that CLAPPER maximizes a statistic that incorporates both the likelihood score and the number of family networks c > 0, in order to provide comparable results between methods. The pairwise inference method produces only the best relationship for each pair, which may not result in a valid pedigree when all pairwise relationships are pieced together. Still, it serves as a useful benchmark to evaluate the accuracy of pairwise predictions by our method.We also compared our method to the pairwise inference method. In this method, we used RELATE to compute the pairwise likelihood under each possible relationship  for all We measured the performance of our method in two ways: the frequency of estimating the true pedigree; and the distance between the estimated pedigree and the true pedigree in terms of pairwise relationships. We note that since CLAPPER does not consider inbred pedigrees whereas PP and RP do, we pre-processed the output of PP and RP before measuring the error rate to make a fair comparison. More specifically, we removed all inbred pedigrees from the output of PP and RP and measured the error rate using just the remaining pedigrees.We say that the estimated pedigree is correct if there is a one-to-one mapping between the nodes of the estimated pedigree and the nodes of the true pedigree such that each edge in the estimated pedigree has a corresponding edge in the true pedigree. Note that for PP and RP, which potentially report multiple highest-scoring pedigrees, we say that the estimated pedigree is correct if the true pedigree is in the set of highest-scoring pedigrees.wi is the probability that two individuals share i pairs of alleles IBD at a random locus under the true relationship; and To measure the error rate of the pairwise method, which estimates pairwise relationships directly, we compared the true relationships to the estimated relationships. Therefore, we define the error rate for each pair asficients  are exacFurthermore, to measure the distance between the estimated relationship and the true relationship for each pair, we computed the kinship coefficient distancee and d to measure the pairwise error rate of CLAPPER, where the inferred pairwise relationships are those induced by the estimated pedigree, and the true pairwise relationships are those induced by the true pedigree. For PP and RP, which report all pedigrees with high likelihood scores, we computed the error rate by taking the average across all highest-scoring pedigrees. For PPP and RPP, which report a single best degree of relationship for each pair, we measured the error rate by e and d as defined above.We also used To examine the behavior of the composite likelihood, we simulated a nuclear family with two parents and their four children at 3,000 independent loci. We then computed the likelihood of the data under various pedigree configurations, ranging from the pedigree in which no one is related to the true pedigree. For each pedigree configuration, we computed the likelihood value with three different formulas: the full likelihood using MERLIN , composiThe comparison of the three likelihood formulas are shown in r2 = .025) showed the best relationship prediction accuracy. For third cousin relationships, however, pruning the markers too severely caused too much information loss, leading to a decrease in prediction accuracy. A similar pattern is observed for the second cousin relationships. For our simulated and empirical data, we prune the markers at r2 = .05, which according to our simulations, retained enough information to estimate second and third cousins while keeping the false positive rate (i.e. estimating unrelated pairs as related) relatively low. We note that finding optimal strategies for dealing with background LD when inferring relatedness is an important topic that merits further research.As mentioned in the Methods section, we examined different thresholds for LD pruning. The appropriate level of pruning depends both on the genome length and the types of relationships we want to infer accurately. As shown in For simulation D, all methods had a lower accuracy rate for estimating the true pedigree compared to simulation C. Some of the samples in this scenario were connected only through an avuncular relationship, which made the inference more difficult than the pedigree given in simulation C. Nonetheless, CLAPPER showed a higher accuracy rate than both PP and RP even though we counted the estimated pedigree as correct if the true pedigree was found in any of the best reported pedigrees for RP and PP. Simulations A and B were omitted from our analysis since they contained samples that were not connected by third degree relationships or closer, which made PP and RP inappropriate to use to estimate the full pedigree.ϕ. For simulation A, PPP did not finish successfully in 19 out of 100 experiments due to errors encountered in PRIMUS (e.g. too many likely pedigrees to process). Similarly, PPP did not finish successfully in 24 experiments for simulation B. Furthermore, PP and RP encountered errors or did not produce any outbred pedigrees in some experiments , and relatively low error rates for estimating close relationships (ϕ ≤ 1/32), however, CLAPPER was able to estimate the relationships more accurately than both PPP and RPP. For simulation C, all methods had zero error rates for all relationship categories except PP, which showed a nonzero false positive rate  caused the error rate for estimating distant relatives (ϕ < 1/32) to be very high; but using no regularization at all led to a high false positive rate (Poi(n/2) as our regularization, which still produced a lower false positive rate, yet performed better in inferring distant relatives on simulated data.To demonstrate our method’s ability to infer pedigrees in practical applications, we estimated the previously unreported pedigrees of 100 individuals from Tasiilaq villages in Greenland. Because the Greenlandic Inuit population has high levels of LD, only 1868 SNPs remained after pruning the markers at ive rate . So we cWe ran our algorithm 5 times with different random number seeds, resulting in 5 pedigrees estimates. The top three estimates with the highest composite likelihood scores were within 1.2 likelihood units of each other. The other two estimates were both about 20 likelihood units away from the top three. The inconsistency of the multiple runs was likely caused by the existence of multiple local peaks on the likelihood surface, which makes finding the global optimum difficult in our heuristic optimization. Each run, which consisted of 80 million iterations, finished in about 24 minutes on 2.5 GHz Intel Core i5 processor. In this report, we have shown that the use of composite likelihood allows us to analyze pedigrees containing many individuals at many loci, where computing the full likelihood would be prohibitively slow. Our method can estimate pedigrees when the number of possible pedigrees is too large to enumerate, which is true even for tens of individuals in a multi-generational pedigree. Our method is also one of the very few methods that can support complex pedigree structures such as polygamy, multigenerational pedigrees (up to 5 generations), and missing individuals. In addition, we can incorporate information about sex, age, and the number of generations spanned by the sample to better estimate the pedigree.We have shown that our method has a significant advantage over the pairwise inference method. It can better estimate relationships beyond first cousins  and is aOur method also showed an improvement over PRIMUS (PP and RP) and PADRE (PPP and RPP). PRIMUS’s reconstruction algorithm relies on accurate pairwise relationship assignments based on IBD estimates. If the sample consists mostly of distant relatives, however, relationship assignment becomes uncertain due to high variance in IBD sharing, which often leads to incorrect pedigree reconstruction. Although our method also relies on pairwise information, we showed that working directly with pairwise likelihood values rather than IBD-based relationships assignments improved the power significantly. Furthermore, PRIMUS’s enumeration of possible pedigrees becomes computationally cumbersome as the number of likely pedigrees increases rapidly for a set of distantly related samples. If the data contains many close relationships, however, PRIMUS can reconstruct all likely pedigrees very fast, whereas our method produces a single best pedigree, which may be close but not exactly correct. Thus the performance of each method depends on the sample structure and a suitable method must be chosen accordingly. Similar to PRIMUS, the performance of PADRE depends crucially on accurate estimates of IBD proportions and segments, and poor estimates of either parameter can lead to biases in the relationship inference. We note that IBD estimation is a difficult problem and better estimates of IBD would improve the performance of both PRIMUS and PADRE.We applied our method on the Greenlandic Inuit dataset to demonstrate its ability infer previously unknown pedigrees from genetic data. Although the estimates of distant relationships are uncertain, we can still get a general sense of pedigree structures hidden in the data and take appropriate actions for downstream analyses. For example, the inferred pedigree can be used to filter out close relatives or model relatedness among samples in association studies. Furthermore, we can validate or improve the estimated pedigree with other evidence such as age.Pedigree inference based on our composite likelihood is heavily influenced by how well we can compute the pairwise likelihoods. An important factor that affects the pairwise likelihood computation is LD, which often leads to overestimation of relatedness. Although the HMM by  conditioThere are limitations to our method that require further work. Our method assumes that all individuals are outbred, which may not be true of many systems including some human populations , 51. It Overall, our method provides a computationally efficient way to estimate pedigrees of seemingly unrelated individuals. It improves our ability to validate and discover pedigrees in realistic genetic datasets where we expect a high level of missing data. The ability to estimate pedigrees more accurately opens up possibilities to develop and improve numerous pedigree-based or pedigree-aware studies, from correcting cryptic relatedness in GWAS to estimating demographic parameters of the very recent past. However, as noted in Introduction, the naive use of estimated pedigrees in downstream analyses may not be justified when there is significant statistical uncertainty in the estimation of the pedigree. Such analyses would need to take the statistical uncertainty in pedigree estimation into account, a topic of potential future research.https://github.com/amyko/clapper.Our software is available for download at S1 Table(PDF)Click here for additional data file.S2 TableI for a particular instance of simulation B.Composite likelihood score at various stopping times given by (TIFF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 TextSummarizes a simulation study that shows how CLAPPER performs on inbred pedigrees.(PDF)Click here for additional data file.S1 FigL(unrelated) − L(third cousins), when the true relationship is unrelated. Unrelated pairs often have higher likelihoods for being third cousins when LD is present in the data, as shown by the histogram corresponding to linked markers. The data were simulated with msprime and the likelihoods were computed using RELATE.The figure shows the histogram of the log likelihood difference, (TIF)Click here for additional data file.S2 FigThe x-axis measures how close the test pedigree is to the true pedigree; the test pedigree becomes closer to the truth from left to right. In this simulation, the composite likelihood given by (TIFF)Click here for additional data file.S3 FigAccuracy of simulated annealing method on simulated data at 2000 markers under different levels of regularization.(TIFF)Click here for additional data file.S4 FigShaded nodes indicate sampled individuals; unshaded for unsampled; squares for male; circles female; diamonds for unknown sex.(PDF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.


Teamwork, a skill critical for quality patient care, is recognized as a core competency by the Accreditation Council for Graduate Medical Education (ACGME). To date, there is no consensus on how to effectively teach these skills in a forum that engages learners, immerses members in life-like activities, and builds both trust and rapport. Recreational ‘Escape Rooms’ have gained popularity in creating a life-like environment that rewards players for working together, solving puzzles, and completing successions of mind-bending tasks in order to effectively ‘escape the room’ in the time allotted. In this regard, escape rooms share many parallels with the multitasking and teamwork that is essential for a successful emergency department (ED) shift.A pilot group of nine emergency medicine (EM) residents and one senior EM faculty member underwent a commercial escape room as part of a team-building exercise in January 2018. The escape room required participants to practice teamwork, communication, task delegation, and critical thinking to tackle waves of increasingly complex puzzles, ranging from hidden objects, physical object assembly , and symbol matching. Activities required members to recognize and utilize the collective experiences, skills, knowledge base, and physical abilities of the group. After the game, players underwent a structured ‘game-master’ debriefing facilitated by an employee of the commercial escape room; this was followed by a post-event survey facilitated by a faculty member, which focused on participants’ feelings, experiences, and problem-solving techniques.Escape rooms afford learners the opportunity to engage in an activity that rewards teamwork and effective leadership through experiences that directly link to specific ACGME milestones and educational learning theories. EM participants were engaged in the activity and felt that the escape room reproduced an environment analogous to the ED. The debriefing that followed the activity provided a satisfactory conclusion to the experience; but learners preferred a more organized debriefing format that provided them with constructive and specific feedback on their performance. Teamwork is arguably one of the most difficult skills to teach learners in the health professions, especially in chaotic and unpredictable environments, such as the emergency department (ED). Team members must overcome varied levels of training and expertise, conflicting personalities, and diverse skill sets to effectively function as a unit. Studies continue to demonstrate that effective teamwork and collaboration, particularly in high-stakes, high-acuity environments, have the ability to decrease mortality and cost , 2. TrusTraining opportunities in teamwork must be safe, low-stakes, high-impact, and dynamically-engaging if they are to nurture the development of collaborative behaviors. Although teamwork remains a core competency by the Accreditation Council for Graduate Medical Education (ACGME), there is no consensus as to how to effectively teach this skill. Effective team training demands receptive and engaged learners, immersive learning activities and experiences, and participant trust and rapport.http://scottnicholson.com/pubs/erfacwhite.pdf]. Players (or learners) must learn how to quickly acclimate themselves in a foreign environment with other players (possibly strangers) of various skill sets, establish trust, and communicate specific goals and tasks in order to assess the environment, analyze clues, solve complicated problems, reassess the environment, and set-up additional goals and dispositions. To a certain extent, this experience is analogous to caring for real patients in an ED setting. While several effective team-building exercises are described in the healthcare literature [Committed to creating a fun and interactive team training activity, the investigators turned to the growingly popular ‘Escape Room’ as a medium to immerse learners in an environment similar to the ED. Escape Rooms are live-action, team-based games where players must work together, solve clues, and complete a series of mind-bending, fitness-challenging tasks in order to ‘escape the room’ in under 60 minutes [Nicholson S. (2015). Peeking behind the locked door: a survey of escape room facilities. White paper available at terature -7, thereIn this technical report, the investigators describe the use of an escape room as a pilot team-building exercise for providers working in the ED of Thomas Jefferson University Hospital in Philadelphia, Pennsylvania. The investigators contracted with a local escape room agency to allow 10 participants from the ED to participate in a one-hour escape room session, followed by a post-event debriefing.Study settingsThe commercial escape room agency was selected based on convenience of location, given its proximity to the hospital (less than 1 mile). The average rate of successful ‘escapes’ at this location is measured by the agency to be 20%. Four unique escape room formats are available at this particular location, each with a unique theme and unique set of puzzles and challenges. Each escape room also contains multiple sub-rooms to sequentially escape from as the activity unfolds.At the end of the game, players underwent a structured, escape-room debriefing, in which the escape room ‘game-master’ described the puzzles and respective solutions; this allowed players to recount their experiences and problem-solving techniques. The observer (XCZ) did not provide any post-game feedback, nor did he participate in the debriefing.Study participant selectionParticipants were selected on a first-come, first-serve basis, after a flyer that advertised the activity was electronically mailed to ED residents and faculty. The activity was advertised three weeks in advance. The investigation was reviewed and approved by the Institutional Review Board of Thomas Jefferson University.Materials requiredParticipants were not required to bring any additional tools or materials to the escape room.Group structureTen participants were assigned to a single group without any predetermined roles or task designations. Their only assigned group goal was to effectively escape the room within 60 minutes. One of the study investigators (XCZ) also entered the escape room to observe team behaviors and record team interactions; he did not participate in the activity, and participants were asked to ignore his presence.Detailed activity descriptionTo escape the room, participants must practice teamwork, communication, task-delegation, critical thinking, and divergent thinking to tackle a series of increasingly complex puzzles, ranging from hidden objects, physical object assembly , counting, and/or symbol matching. Throughout the activity, the game master watches the participants’ progress through several cameras that are physically mounted throughout the room. Players may receive sporadic visual hints, displayed on a television monitor in the room, which are provided by the game master if participants either fail to progress in solving clues or if participants wish to directly receive a clue.Specific puzzle information and details surrounding the escape room were unknown to the authors and the participants prior to reserving the escape room, as this information is considered proprietary knowledge to the escape room agency.Activity evaluationThe investigators used a validated, post-study survey evaluation adapted from a post-focus group interview guide in order to obtain written feedback about the escape room activity . The refResultsTen total participants  participated in this activity. There were approximately 10 to 12 unique puzzles with topics ranging from, but not limited to, recognizing hidden objects, symbol substitutions, mathematical challenges, searching for objects in images, rearranging objects based on visual aids, light manipulation, assembly of a physical object, riddles, and pattern identification. The team received only three hints throughout the activity  and escaped the room in 46 minutes .A post-activity survey analysis n=10) revealed that eight out of 10 participants had previously participated in escape room activities. Everyone, however, had previously participated in a team-building activity, with ‘sports’ being the most common activity (nine out of 10 participants). All participants reported similarities between the escape room and the ED, with common themes focusing on chaos, communication, strategic thinking, differential diagnoses, teamwork, uncertainty, task delegation, and time constraints. Participants also contrasted the escape room to the ED, highlighting that the escape room felt more linear, clue-based, and low stakes , how to multitask, and how to resolve personal differences. All participants rated the escape room as an effective, fun, teambuilding activity, and even recommended including additional puzzles and additional personnel of varied professions  and varied levels of training for heightened realism.The authors propose that commercial escape rooms can be utilized as effective team building activities by immersing participants in interactive, unpredictable, and chaotic environments that can encourage team members to exercise their unique skill sets to seek out and solve different challenges. Escape rooms share several similarities to the clinical environment, which make them successful learning adjuncts for members of clinical teams.Open-axial qualitative assessment of the survey responses revealed common themes in clinical  and escape-room interactions, such as task switching, role delegation, and working together towards a common goal. Additional common themes included a preference for fewer sequential puzzles  to more closely align with the multiple demands that are expected when working in an ED. Participants would have also preferred a more organized and constructive debriefing.The educational philosophy of this activity is rooted in behaviorism, social learning, and cognitivist and constructivist learning theories. In behaviorism, ‘positive’ behavior is reinforced by providing progression through the escape room process, whereas ‘negative’ behavior either does not help the players (i.e. learners) escape the room or even negatively impacts their ability to escape Table . There iAspects of constructivism are also embedded in this activity: learners construct their own knowledge based on real-time experiences of advancing through several challenges in the escape room. In accordance with the ACGME EM Milestones, escape rooms also afford the learners the opportunity to practice several milestones: Patient Care (PC8 – Task Switching) and Interpersonal Communication Skills  , as well as designing escape rooms with more complex puzzles that require parallel processing to closely approximate the chaotic nature of the ED. The authors found that while the game debriefing provided insight into actions taken during the game, participants would have preferred a more constructive feedback session to help them analyze aspects of communication and problem-solving that can be applied in subsequent scenarios, including the clinical environment.


E. coli by introducing foreign phophoketolase enzymes. The results showed that the carbon yield improved from 0.19 to 0.24 for xylose and from 0.30 to 0.43 for glycerol. This further proved that the introduction of NOG pathway can be used as a general strategy to improve P3HB production.Poly-(3-hydroxybutyrate) (P3HB) is a polyester with biodegradable and biocompatible characteristics suitable for bio-plastics and bio-medical use. In order to reduce the raw material cost, cheaper carbon sources such as xylose and glycerol were evaluated for P3HB production. We first conducted genome-scale metabolic network analysis to find the optimal pathways for P3HB production using xylose or glycerol respectively as the sole carbon sources. The results indicated that the non-oxidative glycolysis (NOG) pathway is important to improve the product yields. We then engineered this pathway into  Escherichia coli greatly improved P3HB product yield [In our recent work, based on genome-scale metabolic network analysis, we designed an optimal pathway for P3HB production from glucose using the non-oxidative glycolysis (NOG) pathway and the construction of this pathway in ct yield . Considect yield , it is dRalstonia eutropha using xylose as a sole carbon source can produce 2.31 g/L of P3HB with a content of 30.95 wt% [Hemicellulose is the third most abundant polymer in nature and can be easily hydrolyzed into fermentable sugars . Xylose 0.95 wt% .E. coli containing P3HB synthesis pathway could produce 8 g/L P3HB (65% content) in a 7.5 L bioreactor using crude glycerol as the sole carbon source [Glycerol is the main by-product of the biodiesel production. During the process of transesterification of vegetable oil with methanol or ethanol, 1 ton of glycerol is inevitably produced for every 10 tons of biodiesel . BiodiesE. coli strains to produce P3HB from xylose or glycerol and the higher P3HB yield in the strains with NOG pathway validated the theoretical prediction.In this paper, we first theoretically evaluate the effect of NOG pathway on P3HB production from xylose and glycerol through genome-scale metabolic network analysis. Based on the computational analysis results, we engineered E. coli could increase the theoretical carbon yield of P3HB to glucose from 66.7% to 88.9% by calculating the optimal pathways for P3HB production using flux balance analysis of an extended E. coli metabolic network model [−1 h−1, the maximal P3HB monomer production rate was 8.33 mmol gDCW−1 h−1 and 5 mmol gDCW−1 h−1 respectively, equaling to a 66.7% carbon yield considering the different numbers of carbon element in the two substrates (5 in xylose and 3 in glycerol). The carbon loss is due to the release of CO2 in the pyruvate decarboxylation step. In contrast, the P3HB carbon yields of the optimal P3HB production pathways calculated from the extended metabolic network with NOG pathway was 88.9% for xylose (11.1 mmol gDCW−1 h−1 P3HB from 10 mmol gDCW−1 h−1 xylose) and 100% for glycerol (7.5 mmol gDCW−1 h−1 P3HB from 10 mmol gDCW−1 h−1 glycerol) as shown in 1 Computational simulation indicates improved P3HB yield through the NOG pathway In our previous study, we showed that the introduction of NOG pathway in E.coli2 Improved PHB/xylose yield through the introduction of NOG pathway into E.coli by the over-expression of phosphoketolase (encoded by fxpk) and fructose-1,6-bisphosphatase (encoded by fbp) [E. coli and many other bacterial due to the carbohydrate catabolite repression (CCR). Quite different CCR metabolisms have involved in gram-negative and gram-positive organisms [xylA), xylulokinase (encoded by xylB) and arabinose transporter (encoded by araE) from Bacillus subtilis were introduced into E.coli to simultaneously utilize glucose and xylose due to different mechanisms of CCR between these two kind of bacteria (unpublished data). To reduce glucose repression and increase xylose assimilation, plasmid pZABE harboring genes xylA, xylB and araE from B. subtilis was constructed and transformed into the control and engineered strains. P3HB production and yield were also detected using 10 g/L glucose and 5 g/L xylose as the carbon source. The P3HB production was improved from 2.0 g/L to 3.0 g/L, and yield was improved from 0.21 to 0.31(To verify the effect of NOG pathway on the P3HB synthesis using xylose as sole carbon source in MM medium , we intrrganisms . The xyl1 to 0.31 and 2D. E.coli3 Improved PHB/glycerol yield through the introduction of NOG pathway into 2HPO4 and MgSO4,, pH 7.2 [Experiments with shaken flasks were performed with 10 g/L glycerol as carbon source in basal medium that contained yeast extract, peptone, NaE.coli, the carbon yield of NOG strain was increased up to 0.24, about a 26.3% increase compared with the control strain using xylose as sole carbon source. Similar phenomenon was observed using glycerol as sole carbon source, the carbon yield of NOG strain reached 0.43, about a 43.3% increase compared with the control strain. Thus, low cost carbon sources have the potential to produce P3HB, and introducing NOG pathway into E. coli might be a universal strategy using xylose and glycerol as the sole carbon source.To reduce the production cost of PHA, the use of low cost carbon sources is urgently required. In this work, the simulation results showed that with NOG pathway introduce, both xylose-input and glycerol-input were displayed high carbon yield compared with the classical pathway. Experiments were also conducted and verified the results. Through engineering of NOG pathway into


Taken together, our data suggest that BafA1 may offer potential as an effective therapy for HCC.Hepatocellular carcinoma (HCC) is refractory to chemotherapies, necessitating novel effective agents. The lysosome inhibitor Bafilomycin A1 (BafA1) at high concentrations displays cytotoxicity in a variety of cancers. Here we show that BafA1 at nanomolar concentrations suppresses HCC cell growth in both 2 dimensional (2D) and 3D cultures. BafA1 induced cell cycle arrest in the G1 phase and triggered Cyclin D1 turnover in HCC cells in a dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) dependent manner. Notably, BafA1 induced caspase-independent cell death in HCC cells by impairing autophagy flux as demonstrated by elevated LC3 conversion and p62/SQSTM1 levels. Moreover, genetic ablation of LC3 significantly attenuated BafA1-induced cytotoxicity of HCC cells. We further demonstrate that pharmacological down-regulation or genetic depletion of p38 MAPK decreased BafA1-induced cell death via abolishment of BafA1-induced upregulation of Puma. Notably, knockdown of Puma impaired BafA1-induced HCC cell death, and overexpression of Puma enhanced BafA1-mediated HCC cell death, suggesting a role for Puma in BafA1-mediated cytotoxicity. Interestingly, pharmacological inhibition of JNK with SP600125 enhanced BafA1-mediated cytotoxicity both  Bafilomycin A1 (BafA1), a specific vacuolar H + ATPase (V-ATPase) inhibitor, is frequently used at high concentrations to block the fusion between autophagosomes and lysosomes and as an inhibitor of lysosomal degradation134In this investigation, we show that BafA1 at nanomolar concentrations substantially inhibits the growth of HCC cells in both 2D and 3D cultures and in mouse models. We further demonstrate that BafA1 induces caspase-independent HCC cell death via targeting of autophagy and MAPK pathways. Our data supports further exploration of BafA1 as a drug candidate in the treatment of HCC.in vitro.To investigate the effect of BafA1 on HCC cells, BEL7402, HepG2, Huh7 and SMMC-7721 cells were treated with BafA1 and a number of cell-based analyses were performed. The non-transformed human liver cell line, LO2 was also included. MTT assays were carried out to determine the growth kinetics of the cell lines in response to BafA1 at increasing concentrations for 24–72 h. As shown in To dissect the mechanism by which BafA1 suppresses HCC cell growth, we first examined whether BafA1 perturbs the cell cycle distribution. BafA1 (5 nM) increased the percentage of BEL7402 and HepG2 cells in the G1 phase of the cell cycle, with a concomitant decrease in cells in G2/M phases, indicative of a G1 phase arrest . ConsistCyclin D1 levels in mammalian cells is mainly controlled by glycogen synthase kinase 3β (GSK3β)11To determine whether BafA1-induced growth inhibition in HCC cells was due to cell death, BafA1-treated BEL7402 and HepG2 cells were analyzed by flow cytometry with FITC-conjugated Annexin-V and PI double staining. As illustrated in Recent studies indicate that BafA1 triggers caspase-independent cell death in cancers of different origins8To confirm that the observed double-membrane vesicles were indeed related to autophagy, GFP-LC3 dot formation was investigated. HCC cells were transfected with GFP-LC3. The number of cells with GFP-LC3 punctate in BafA1-treated cells was significantly higher than in control cells . The effet al. showed that BafA1 at low concentration (1 nM) inhibits the early stage of autophagy by promoting mTOR activation and Beclin1 association with Bcl2 in pediatric B-cell ALL cellsWe next examined the role of autophagy in BafA1-induced HCC cell death. A recent report by Yuan We thus examine whether autophagosome formation is involved in BafA1-mediated inhibitory effects on HCC cell growth. To this end, we established BEL7402 and HepG2 cells in which LC3 was stably depleted with lentiviruses carrying specific shRNA. The efficiency of knockdown was confirmed by immunoblot assay . As showThe three major mitogen-activated protein kinase (MAPK) pathways  are known regulators of cell survival, proliferation and stress response. Consistent with previous observations in breast cancer cellsTo dissect the underlying mechanism of action of p38 MAPK in BafA1-induced cytotoxicity of HCC cells, protein expression of LC3II and p62 in addition to several members of the Bcl2 family of proteins were determined by immunoblot analysis. Both LC3II conversion and p62 accumulation by BafA1 in BEL7402 and HepG2 cells were decreased following pre-treatment with SB202190 , indicatWe further examined the effect of BafA1 on HCC growth in xenografts derived from BEL7402 and HepG2 cells. BafA1 markedly abrogated the progression of established tumors compared with controls . Furtherin vitro and 10 mg/kg BafA1 markedly abrogated the progression of established tumors. Furthermore, the in vivo effect of BafA1 on HCC cells was enhanced in response to combination treatment with BafA1 and the JNK inhibitor, SP600125. Given that HCC is refractory to almost all forms of chemotherapy, our data suggest that BafA1 alone, or in combination with a JNK inhibitor, may represent a promising therapeutic approach in HCC.High concentrations of BafA1 (>0.1 μM) have been widely used in the inhibition of lysosomal activity, blocking autophagy flux and/or inducing cytotoxicity in various cancer types, including HCCSeveral studies have documented that BafA1 induces cell cycle arrest in cancer cells, a process involving deregulation of cell cycle regulators5et al. in BafA1- treated pediatric B-cell ALL cells, we observed that BafA1 treatment inhibits the activation of the class I PI3K/Akt/mTOR/p70S6K signaling pathway, suggesting that BafA1 treatment at low concentration may result in autophagy induction in HCC cells. Therefore, in BafA1-treated HCC cells, the observed LC3II accumulation  in HCC cells, consistent with previous observations in breast cancer cellsWith the increasing incidence of HCC in the Europea and Asia in recent years2 at 37 °C.BEL7402, LO2 and SMMC-7721 cells were maintained in RPMI 1640 cell culture media. Huh7 cells were cultured in DMEM and HepG2 cells were maintained in EMEM. All culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). Cells were cultured in a humidified incubator in 5% COAnti-microtubule-associated protein 1 light chain 3 (LC3), p62 and β-actin were obtained from Sigma. Anti-Cyclin D1, Cyclin E, CDC6 and Rb were obtained from Santa Cruz. The following, antibodies were purchased from Cell Signaling Technology: phospho-ERK1/2, phospho-p38, phospho-JNK, phospho-GSK3α/β, phospho-Rb, p53, Caspase-3, Bcl2, Puma, Beclin-1 and DYRK1B, along with antibodies directed against Erk1/2, p38, JNK and GSK3α/β. The inhibitors PD98059, SB202190, SP600125 and AZ191 were purchased from Selleck. Bafilomycin A1 (BafA1) was purchased from Merck Millipore. Drugs were dissolved in dimethyl sulfoxide (DMSO) as stock solutions and stored at −20 °C.The following lentiviral constructs were purchased from Santa Cruz: MAP LC3β shRNA (sc-43390-V), p38α shRNA (sc-29433-V) and noncoding shRNA (sc-108080). Lentiviral particles were used to directly infect BEL7402 and HepG2 cells. Stable clones were subsequently selected using puromycin (Sigma).RNA interference was used to knock down DYRK1B and Puma. DYRK1B①: 5′-GGUGAAAGCCUAUGAUCAUTT-3′; DYRK1B②; 5′-GCCUGGUAUUUGAGCUGCUGUCCUA-3′. Puma①: 5′-GCCUGUAAGAUACUGUAUATT-3′; Puma②: 5′-GGAGGGUCCUGUACAAUCUTT-3′. A scrambled siRNA was used as a negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′.Tumor cell growth was measured daily using the MTT -2,5-diphenyl tetrazolium bromide) assay as previously describedBEL7402 and HepG2 cells were seeded (300 cells/well) in ultra-low attachment 96-well plates and maintained in serum-free DMEM/F12 medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) and B27 (B27 and medium at a 1:50 volume ratio). Seven days after seeding, the propagated spheroid bodies were collected and digested by StemPro Accutase to single cell suspensions to generate a second generation of spheroidsCells were treated with various agents as indicated in figure legends. After treatment, cells were collected and processed for immunoblotting analysis as previously describedFor cell cycle analyses, cells were treated with vehicle or BafA1 for 24 h and then were collected and fixed in cold 70% ethanol overnight at 4 °C. To ensure that only DNA was stained, cells were treated with PBS  and then were incubated for 10 min at room temperature in the dark. All samples were analyzed by flow cytometry. For analysis of apoptosis, cells were treated with vehicle or BafA1 and then they were subjected to flow cytometric analysis of membrane redistribution of phosphatidylserine using an annexin V and propidium iodide (PI) double-staining techniqueCells were seeded in glass bottom cell culture dishes  and were transfected with mRFP-GFP-tagged LC3 for 24 h. Fluorescent images of live cells were captured directly using an inverted confocal microscope (Leica). For quantification of autophagic cells, fluorescent puncta were determined from samples in triplicateBEL7402 and HepG2 cells were fixed and embedded. Thin sections (90 nm) were examined at 80 kV with a JEOL 1200EX transmission electron microscope. Three fields containing more than 5 randomly selected microscopy-captured images were counted and autophagosomes were defined as double-membrane vacuoles measuring 0.1 or 1.0 μm.6 cells in 100 μL PBS/mouse) to induce tumor development. When tumors reached an average volume of 200 mm3, tumor-bearing mice were intra-tumorally inoculated with BafA1. Mice were randomly divided into four groups (three mice per group): (a) vehicle control, (b) intraperitoneal (i.p.) treatment with JNK inhibitor (SP600125) alone, (c) intratumoral administration with BafA1, (d) BafA1 treatment in combination with SP600125, the inhibitor (SP600125) was administered one day prior to treatment with BafA1. After five weeks treatment, tumor sections (5 μm) were subjected to either hematoxylin-eosin (H&E) staining or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as previously describedSix week old female nude mice were subcutaneously inoculated in the right dorsal flank with BEL7402 or HepG2 cells  × 2/2. The experiment was terminated when tumors reached 1 cm3 in volume and/or symptomatic tumor ulceration occurred. Surviving mice were sacrificed under anesthesia. The animal experiment was conducted at Dalian Medical University , complying with the national guidelines for the care and use of laboratory animal. All animal experiment were approved the experimental animal ethics committee of Dalian Medical University.For the in vivo data, statistical significance between groups was calculated based on the LSD test using SPSS 17.0 software . A p-value of p < 0.05 was considered statistically significant. All experiments were carried out in triplicate as three independent experiments.Comparisons of data were first performed using one-way analysis of variance (ANOVA). Multiple comparisons between treatment groups and controls were evaluated using Dunnett’s least significant difference (LSD) test. For analysis of How to cite this article: Yan, Y. et al. Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways. Sci. Rep.6, 37052; doi: 10.1038/srep37052 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated.The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish  Adamts20 deficient bt/bt mice have defects in melanoblast survival [Adamts9 haplo-insufficient mice on an Adamts20 deficient (bt/bt) background present with a secondary cleft palate [The A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motifs (ADAMTS) metalloproteinases have important functions during developmental morphogenesis and are also implicated in chronic disease. The proteoglycanase subfamily of ADAMTS1, 4, 5, 8, 9, 15 and 20 have broad functions, many attributed to their ability to remodel extracellular matrix (ECM) components, such as the chondroitin sulphate proteoglycans versican and aggrecan. For example, t palate  and ADAMt palate ,7. Adamts5, Adamts9 and Adamts20 in mice prevented generation of bioactive fragments of versican that are necessary for interdigital tissue apoptosis during development [Adamts5 knockout mice also developed myxomatous heart valves [ADAMTS5 has been implicated in classic morphogenesis during development as well as in chronic diseases such as arthritis and atherosclerosis. For example, combinatorial knockout of elopment ,9. Adamtt valves . Furthert valves ,12 and mt valves .Adamts9 as necessary for umbilical cord vascular development due, at least in part, on its facilitation of Shh signaling [ECM remodeling is crucial to many developmental and disease processes, in part due to its role in controlling cell signaling. Heparan sulphate proteoglycans bind fibroblast growth factors (FGFs), thereby regulating their bioavailability to their receptors (FGFRs)  during dignaling . Furtherignaling . Combineadamts5 expression disrupted Shh signaling during somite differentiation and reduced the expression of the myogenic regulator myod. Importantly, somite differentiation was synergistically dependent upon Shh and ADAMTS5. Moreover, these functions of ADAMTS5 were independent of catalytic function. These data indicate that ADAMTS5 plays an important non-enzymatic role in regulating the Shh pathway during embryogenesis that impacts on muscle development. This may be relevant in conditions where ADAMTS proteins interact with the Shh signaling pathway, such as osteoarthritis and umbilical cord vascular complications, as well as disorders where the myogenic program is disrupted, such as muscular dystrophies. This study identifies a role for ADAMTS5 during zebrafish embryogenesis. Abrogation of Adamts5−/− mice [adamts5 gene that is maternally inherited and then dynamically expressed in early-stage embryos [We have previously elucidated a role for ADAMTS5 during myoblast fusion in post-natal skeletal muscle from −/− mice . To inve embryos . To obta embryos , which i embryos ,22. At 8adamts5 function, the gene was targeted using two independent morpholino antisense oligonucleotides (MOs) that were directed to either the AUG translation start site (AUG-MO) or the splice site at the exon 2/3 boundary (2/3-MO)  B, since blotting C. To conanalysis D. This ip codons D. The AU5 2/3-MO . myod expression during myogenesis [no tail (ntl) is an independent marker for axial mesoderm (notochord) [shh and ntl remained unchanged in 12 hpf adamts5 morphants compared to controls [shh  . Expresscontrols . Howeverols [shh A,D and nshh  ADAMTS5 were co-injected, with both able to partially rescue the reduced paraxial mesodermal myod expression , a receptor in the Shh signaling pathway  was usedmesoderm .myod in the paraxial mesoderm and disrupted myotome boundaries.Zebrafish myogenesis is controlled by multiple pathways ,30,31. Sadamts5 morphants were rescued with mRNA encoding both wild-type and catalytically-inactive ADAMTS5. Although unexpected, there is some precedence for ADAMTS family members demonstrating non-catalytic functions as reviewed recently [363A) ADAMTS15 were able to reduce breast cancer cell migration on matrices of fibronectin or laminin [The phenotypes induced by the recently . For exa laminin . ADAMTSL laminin  suggestimyod [adamts5 morphants displayed altered myod expression in the paraxial—but not adaxial—mesoderm despite levels of shh expression in the notochord being unaffected. This might be explained by reduced bioavailability of Shh in the absence of ADAMTS5. Since the adaxial myod-positive cells represent the slow muscle precursors that subsequently move through the fast muscle region where they impact on fast muscle differentiation [adamts5 morphants.Shh is an important regulator of musculoskeletal development, given its role in somite and neural tube patterning. Duplication, and presumed overexpression, of Shh is associated with congenital muscular hypertrophy in humans . Shh alsmyod . The adantiation , it woulThe results obtained using agonists and antagonists of the downstream Smo pathway suggest that ADAMTS may work both upstream, as well as in parallel with Shh signals. Wnt/β-catenin signaling has been shown to act in co-operation with Shh (and BMPs) in embryonic myogenesis . This coadamts5 morphants. In addition, U-shaped myotome boundaries have also been observed in mutants of ECM components, such as fibronectin [Defective notochords have been identified in mutants of ECM components, such as fibrillin , collageronectin  and lamironectin , or the ronectin , providiThis study has identified a new function for the metzincin ADAMTS5. By exploring the role of ADAMTS5 in zebrafish, understanding has been gained of a potential non-catalytic function in the regulation of muscle development and maintenance via interaction with the Sonic Hedgehog signaling pathway. Since both ADAMTS5 and Shh have independent—as well as potential combinatorial—roles during musculoskeletal development, the complex interplay between ADAMTS and Shh could be relevant to the development of musculoskeletal diseases, such as muscular dystrophies and arthritis. Further biochemical and functional characterization of potential interactions between ADAMTS5 and Shh in such diseases may reveal new insights into the development and progression of these diseases. Given that treatment options for these diseases are limited, this knowledge could then be applied to the development of novel therapeutics that specifically modulate this interaction to slow the progression of these debilitating conditions.Tg(acta1:lifeact-GFP)/Tg(acta1:mCherryCaax) [Wild-type and rryCaax)  zebrafisrryCaax) . Embryosadamts5 (GenBank: JF778846.1) were used to ablate the adamts5 gene. MOs were solubilized in 1× Danieau buffer and 1 nL injected at a concentration of 1 mg/mL into one-cell stage embryos, as previously described [adamts5 species were confirmed by Sanger sequencing  of RT-PCR amplicons generated with flanking primers (5′-ggcggatgtaggaactgtgt and 5′-ttacgcacctcacactgctc). Capped RNA encoding full-length wild-type or catalytically-inactive (E411A) ADAMTS5 [Morpholino antisense oligonucleotides  targeting the ATG start codon (5′-atgctgtcgaaattacaggagtttggcgcgtat) and exon 2/3 splice site (5′-ctatcattgaggacgacggcctgcacgctgccttcactgtggctcatgagatc) of zebrafish escribed . Alterna ADAMTS5  were synFor other studies, injected embryos were treated with 5 µM cyclopamine  in DMSO or 10 µM Smoothened agonist SAG (CAS 364590-63-6)  in water, along with the corresponding vehicle control at 5.5 hpf and fixed at 12 hpf in 4% PFA/PBS.shha and myod [no-tail (ntl) [Whole-mount in situ hybridization was performed as described . The foland myod , and no-il (ntl) . Immunofil (ntl) .Cell lysates were extracted from 24 hpf embryos and subjected to Western blotting as described previously , using tt-tests were performed between all treatment groups compared to the respective control groups with scoring performed blind. Significance was achieved at a p-value ≤ 0.05, with Gaussian distribution assumed in all cases.Two-tailed paired