HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L Hepadnaviridae, a family of small, non-cytopathic viruses with a relaxed circular DNA (rcDNA) genome. Upon infection, the rcDNA genome is converted into covalently closed circular DNA (cccDNA) in the nucleus of infected cells1. HBV takes human as its only natural host and almost exclusively infects hepatocytes3. HBV infection usually causes either subclinical or symptomatic acute hepatitis, with the latter developing into chronic infection in a majority of infected neonates and young children, and a small percentage of adults4. Chronic HBV infection generally persists for life and has a high risk for progressing onto liver fibrosis, cirrhosis and eventually, hepatocellular carcinoma (HCC)6.Hepatitis B virus (HBV) is the type member of 1. HBV X (HBx) is the smallest (154 amino acid residues) of HBV-encoded proteins and is found in all mammalian hepadnaviruses7. HBx is generally believed to be a non-structural protein and has been shown to play diverse functions in viral life cycle, virus-cell interactions and HBV-related HCC10. For instance, HBx positively contributes towards HBV transcription and replication both in vitro and in vivo13. In hepatoma cells, HBx is apparently involved with various key signaling pathways including Wnt/β-catenin14, NF-κB15, Notch16, RIG-I17, RAR/RXR18, etc., which underlies its association with HBV-related carcinogenesis.HBV genome contains 4 overlapping open reading frames (ORFs) that encode 7 viral proteins through the use of alternative start codons21 and DDB1 (DNA damage-binding protein 1) is one of the earliest HBx-binding host protein identified19. DDB1 is part of the DDB1-containing E3 ubiquitin ligase  complex. Through its interaction with other adaptor proteins, DDB1 recruits substrates to the complex for ubiquitination and eventual proteasomal degradation23. HBx-DDB1 interaction has been shown to subvert this process in the virus’s favor, leading to enhanced cccDNA transcription and viral replication26. The fact that HBx requires interaction with cellular adaptor proteins to be functional suggest the possibility of targeting such interactions for treating liver diseases associated with HBV or HBx.Although multiple studies point to HBx as a regulator of viral and host gene expression, it has not been demonstrated to bind HBV or cellular DNA directly. Instead, mechanistic studies have shown that HBx acts through interacting with cellular adaptor proteins27. In this work, mechanism(s) underlying 2A7’s broad reactivity and its potential usefulness in HBx-targeting applications are explored. We demonstrated that 2A7 recognizes a linear epitope encompassing amino acids 89 to 96 of HBx, which is highly conserved across genotypes and coincidentally overlaps with HBx’s DDB1-interacting segment. In vitro, binding between HBx and DDB1 was inhibited in the presence of 2A7. Complete nucleic acid and amino acid sequences of 2A7 were then obtained and used for the construction of recombinant antibody and single chain variable fragments (scFv). We show that 2A7-derived recombinant antibody and scFv recapitulates 2A7’s HBx-binding capacity and epitope specificity. Furthermore, we explored the possibility of delivering 2A7 into intracellular space for potential HBx-targeting applications.Previously, we described a broadly reactive mouse monoclonal antibody against HBx (anti-HBx 2A7) that detects HBx encoded by representative strains from 7 of 8 known HBV genotypes in Western blot and immunofluorescence assays27. In contrast, another monoclonal anti-HBx obtained in parallel to 2A7, designated 2A2, only reacted strongly with immunogen BPS HBx in Western blot, with minimal reactivity against B200 HBx and no reactivity against B6 or WT HBx  and one genotype C (WT) HBV isolates were used. BPS is the isolate from which the HBx immunogen used for preparing 2A7 was derived. HBx from these isolates display varying differences at protein sequence level  peptides that encompass full-length BPS HBx. Only two peptides, harboring a.a. 81–96 and 89–104 respectively, were recognized by 2A7 in ELISA , K91T (4.96%), K91R/V92Q/H94Y (1.10%), V92I (0.52%), V92L (0.44%) and K95Q (0.42%) were introduced into BPS HBx and tested for 2A7 recognition in Western blot, K91T and V92I mutants retained full and nearly half 2A7 reactivity, respectively, while the other mutants were not efficiently detected by 2A7  concatenated in reciprocal order and fused at C-terminal to human IgG1 Fc, with or without N-terminal mouse Igκ secretion signal sequences Fig. . Upon trDespite its nearly identical epitope-specificity compared to 2A7, scFv VHVL(2A7) is only expressed at fairly low levels in a secretion-dependent manner Fig. . Since HTo test this hypothesis, TR16-Tat was first allowed to incubate with purified 2A7 antibody before being added to culture media of Huh-7 cells. After incubation in the presence of antibody and peptide mixture, cells were washed and treated with trypsin to remove surface-attached 2A7. Western blot analysis of trypsin-treated cells showed that pre-incubation with TR16-Tat enables 2A7, but not 2A2, to enter cells Fig. . The amoin vitro and in vivo7. HBx’s association with and contribution to HBV-related HCC are also supported by numerous studies, although the details of which still require further elaboration and confirmation9. HBx is thus an obvious target for developing drugs against HBV infection or HBV-related HCC. In this work, we report detailed characterizations of a mouse monoclonal HBx antibody (2A7) that is capable of recognizing a predominant majority of known HBx variants  and is involved in regulating liver-specific HBV transcription33. It is likely that variation at 2A7 epitope site on HBx is to some extent constrained by viral dependence on the presence, at nucleic acid level, of functional transcription factor binding site(s) for optimal transcription and subsequent replication. Regardless of the underlying mechanism(s), conservation of this site enables 2A7 to recognize more than 86% of reported HBx protein sequences and such a high level of broad reactivity no doubt establishes 2A7 and its derivatives as powerful tools for HBx studies.Limited information is available regarding possible differences in functions between naturally occurring HBx variants encoded by different HBV genotypes and strains. Lack of usable antibody tools is a possible factor in this regard, especially for studies using whole HBV genomes, because overlapping of HBx ORF with other virus ORFs and regulatory elements prohibits fusing HBx with tags to facilitate detection. It is not surprising that the epitope recognized by the broadly-reactive 2A7 was mapped to a highly conserved region of HBx Figs. , which f19, it was not until recent years that its functional significance in viral life cycle has been demonstrated26. Hijacking of the cellular CUL4-DDB1 E3 ligase by HBx caused abnormal ubiquitination and degradation of proteins such as Smc5/626, which in turn resulted in enhanced transcription from episomal HBV cccDNA and viral replication10. The binding site for DDB1 on HBx coincidentally overlaps with 2A7 epitope and our results show that in the presence of 2A7, binding between HBx and DDB1 is largely inhibited . If achievable, such an intrabody would enable HBx and HBx-DDB1 targeting applications upon liver-specific protein or gene delivery.Since HBx is an intracellular protein, any potential HBx-targeting agents must first gain access to the intracellular space. Compared to IgG, recombinant scFv has much smaller gene and protein sizes, allowing easier manipulation and administration using both nucleic acid- and protein-based delivery methods. Among 2A7-derived scFv constructs that we tested, only those with leading secretion signal sequences were efficiently expressed (and secreted) in transfected cells Fig. , making In addition to intrabody, delivery of protein across plasma membrane with the help of CPP is another possible method for targeting intracellular protein with specific antibody and derivatives. Our results using a fusion peptide combining HIV Tat CPP and 2A7 epitope showed that such a dual-function peptide could indeed bind 2A7 through the epitope and deliver the peptide-antibody complex into cells through the CPP Fig. . FurtherIn summary, results presented here provide 2A7 as a broadly reactive HBx monoclonal antibody capable of interfering with its interaction with a key cellular adapter protein DDB1, which is crucial for HBV life cycle. This suggests a high potential for 2A7 in HBx-targeting diagnostic and therapeutic applications. Availability of the full sequences of 2A7 and demonstration of the functional equivalence of recombinant 2A7 and 2A7-derived scFv compared to original 2A7 also paved way for further engineering of 2A7 for such applications.27. A different anti-HBx mAb designated 2A2 was obtained in parallel with 2A7. 2A7- and 2A2-producing SP2/0 hybridoma cell lines were maintained in RPMI 1640 medium  supplemented with 15% fetal bovine serum at 37 °C with 5% CO2.Mouse anti-HBx mAb 2A7 prepared using bacterially expressed HBx has been described2. Expi293FTM cells were grown in serum-free Expi293TM Expression Medium  at 37 °C with 8% CO2 on an orbital shaker platform  rotating at 125 rpm.Human embryonic kidney cell line HEK293T and human hepatocellular carcinoma cell lines HepG2 and Huh7 were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum at 37 °C with 5% COTM, and Huh7 and HepG2 cells, respectively, according to manufacturers’ instructions.Polyethylenimine , ExpiFectamine™ 293 Reagent  and TurboFect transfection reagent  were used for transfecting HEK293T, Expi293F34) and one genotype C strain (designated WT35) using PCR and inserted downstream of FLAG tag in pcDNA3.0 vector . Expression plasmids for HBx mutants were generated from wild type expression plasmids using KOD-plus mutagenesis kit . Sequences of primers used from generating these HBx constructs are listed in Supplementary Table To construct FLAG-tagged HBx expression plasmids, X ORFs were amplified from HBV replicon plasmids of three genotype B strains . HBx fragment (a.a. 81–96) harboring 2A7 epitope with or without a.a. 47–58 of HIV-1 Tat protein at the C-terminal joined through an miniPEG3 linker, and control HBx peptides  were similarly synthesized.Bacterially expressed BPS-encoded HBx protein purified under denaturing conditions and used as immunogen for mAb generation has been described30 as listed in Supplementary Table S4–6. Amplicons with expected length were recovered from agarose gels and cloned into TA-vector (TaKaRa). Multiple randomly selected clones were then sequenced to obtain candidate IgG cDNA sequences. CDR within heavy and light chain variable regions was predicted using Vbase2 (www.vbase2.org).Total RNA was extracted from mAb-producing hybridoma cells and subjected to RT-PCR using primer sets designed for amplifying mouse IgG constant and variable regions36. LC-MS/MS and raw data collection was conducted by the core facility of Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University on a hybrid quadrupole Orbitrap (Q-Exactive) mass spectrometer  coupled to a Nano Aquity UPLC system . Machine-generated raw MS data were fed into MaxQuant37 software for protein identification using candidate IgG cDNA sequences as queries with default settings, along with commonly occurring protein contaminants.Purified mAb proteins were analyzed by 12% SDS-PAGE under reducing conditions, followed by Coomassie Brilliant Blue G-250 (Sigma) staining. Bands corresponding to heavy and light chains of IgG were identified by apparent molecular weights and excised using clean scalpels. In-gel trypsin digestion, extraction of tryptic peptides and LC-MS/MS analyses were performed essentially as described previouslyTM cells were transfected with the obtained recombinant 2A7 heavy and light chain expression plasmids at 1:2 ratio, and culture supernatant was collected at 6 days post transfection, followed by centrifugation at 5000 g for 30 min and filtration through 0.22 μm filter. Filtrate was loaded onto RoboColumn holding Protein A affinity chromatography resin . After washing with PBS, bound recombinant antibody was eluted with 0.1 mol/L glycine (pH 2.6) into 1 mol/L Tris-HCl (pH 8.8).Human-codon-optimized 2A7 heavy and light chain variable region coding sequences with N-terminal human IgG2 heavy chain secretion signal sequence and C-terminal mouse IgG2a heavy chain or κ light chain constant fragments, respectively, were synthesized and cloned into pcDNA3.4 vector . To produce recombinant 2A7 antibody, Expi293F3 linker through overlap extension PCR with primers listed in Supplementary Table 2A7-derived scFv expression plasmids Igκ-VLVH-Fc and Igκ-VHVL-Fc were constructed by joining 2A7 heavy chain and light chain variable regions in reciprocal order with an intervening (GlyGlyGlyGlySer)450) was measured after the addition of 0.1 mol/L HCl using a microplate reader . Immunofluorescence and Western blot analyses were performed according to standard procedures as previously described38. Densitometry scanning was performed using ImageJ software.Recombinant HBx protein was used for coating 96-well microplates at 100 ng/well in bicarbonate/carbonate coating buffer . For epitope mapping, biotinylated HBx peptides were added to streptavidin coated StreptaWell microplate  at 500 ng/well in PBS. Coating was performed at room temperature for 30 min and plates were then washed with 0.05% Tween-80 in PBS (PBST) and blocked with 3% bovine serum albumin (BSA) in PBS. Antibodies, cell supernatants or lysates, diluted in blocking buffer if necessary, were then added and incubated at 37 °C for 1 h, followed by washing and reaction with horseradish peroxidase (HRP)-conjugated anti-mouse pAb  or anti-human Fc pAb . HRP substrates were then added and optical density at 450 nm  and Protein A/G agarose  were used for capturing FLAG-tagged and Fc-containing proteins respectively in co-immunoprecipitation and pull-down assay. Cell lysates were prepared using IP lysis buffer  containing protease inhibitor cocktail  and mixed with beads. After incubation with rotation at 4 °C for 2 hours, beads were washed 4 times with PBST and then mixed with 1/3 volume of 4 × SDS sample buffer (0.2 mol/L Tris-HCl (pH 6.8), 8% SDS, 0.4 mol/L dithiothreitol, 40% glycerol, and 0.4% bromophenol blue) and heated at 95 °C for 3 minutes to elute the proteins. For pull-down assay with antibody blocking, cell lysates containing HA-tagged DDB1 and HA-tagged Cullin4A were first mixed with or without 2A7 or 2A2 (2 μg/ml), and then mixed with cell lysates containing FLAG-tagged HBx or HBx mutants and incubated with rotation at 4 °C for 2 hours before addition of anti-FLAG beads.2A7 mAb was mixed with different concentration of HBx peptide harboring 2A7 epitope fused with cell-penetrating peptide from HIV-1 Tat protein, incubated at 37 °C for 30 minutes and added to cell culture media. Cells were further cultured for 6 hours, washed 3 times with PBS, harvested following 0.25% trypsin/EDTA digestion and then washed twice with PBS. Harvested cells were lysed in SDS-PAGE loading buffer and analyzed for intracellular 2A7 mAb using Western blot, or lysed in RIPA buffer and analyzed in ELISA. As control, cells were also collected without trypsinization by washing 3 times with PBS and lysing in SDS-PAGE loading buffer. In order to demonstrate specificity of cellular entry enabled by the fusion peptide, HBx peptide harboring 2A7 epitope or neighboring fragment not encompassing 2A7 epitope was added during incubation, or mAb 2A2 was used in place of 2A7.A total of 13950 HBx protein sequences were retrieved from GenBank in December, 2016, from which sequences with insertion or deletion, or not beginning with methionine were excluded, and the remaining 7098 full-length (154 a.a.) HBx sequences were obtained for analysis.Supplementary Figures and Tables 1–7.Supplementary Table 8.


Background: Nutrition medical education training programs that are focused on home cooking are emerging. Objective: This short communication describes the first synchronous tele-nutrition medical education training program using a novel Culinary Coaching (CC) model. Design: Seven health coaches were trained and each coach delivered CC programs to four patients . Evaluations included:1) two questionnaires before, immediately after, and six months post training program; and 2) one questionnaire after each patient program. Results: CC training significantly improved coaches’ attitudes about and confidence to deliver CC from pre-program means of 3.61 and 3.65 (out of 5), respectively, to post-program means, 3.77 (p<0.01) and 3.86 (p<0.05), respectively, and remained higher 6 months after the training program . Health coaches described a high usage of CC principles and tools through the patient programs. Conclusions: This early evidence suggests that the CC model can be successfully expanded to health coaches, thus improving nutritional care. Nutrition medical education training programs that focus on home cooking as a way to improve nutrition are emerging . This is‘a behavioral intervention that aims to improve nutrition and overall health by facilitating participant’s home cooking through an active learning process that combines culinary training with health and wellness coaching competencies’ [The culinary coaching (CC) was developed to improve participants’ nutrition through a two-pronged approach of combining culinary training and health coaching . Health tencies’ . This reThe CC training program was developed as a remote synchronous medical education training program aimed at educating health coaches to improve patients’ home cooking habits. Competencies were set within each category of the health coaching competencies  with a focus on trainees’ personal behavior as a lever for professional activities . The synSeven (for an effective group coaching) certified health coaches with culinary credentials from seven North American sites were recruited through social media. Inclusion criteria: (1) Health and Wellness Coaching certification, (2) culinary credential (or a proven 5 years’ culinary experience), and (3) active coaching practice where patient programs could be delivered. All seven recruited coaches completed the training program. In addition, each health coach completed a 6-month internship where each implemented the CC in four programs with patients with cardio-metabolic risk factors from her practice ,11 , seven Likert-scale items assessing confidence to deliver home cooking education using coaching principles , and open-ended questions assessing the overall training program, and motivation and expectations from patients’ programs.Implementation of the training content areas was measured by a Program Questionnaire after each of the four patient programs. The questionnaire included nine Likert-scale items assessing the usage of key CC culinary principles and communication strategies that were discussed in the training program, and open-ended questions assessing the patient program challenges and the training program contribution to provider competencies.Program evaluation included engagement in both the training program and the patient coaching program, and a mixed method evaluation through three questionnaires:Items for questionnaires 2 and 3 were revised from questionnaires assessing the feasibility of a web-based professional training , attitudQuantitative responses were tested across time points by linear mixed effects model . Qualitative descriptive analysis  was condp < 0.05) and 4.88 (0.69) (p < 0.01) after the training program, and remained improved 6 months after the training program, 1.11 (0.49) (p < 0.05) and 4.86 (0.74) (p < 0.01). Further, coaches’ attitude about and confidence to use the CC model improved from pre-program means of 3.61 (0.68) and 3.65 (0.57) out of 5 (higher is more confident), respectively, to post-program scores of 3.77 (0.63) (p < 0.01) and 3.86 (0.87) (p < 0.05). Scores remained higher 6 months after the training program (3.93 (0.56) (p < 0.01) and 3.93 (0.81) (p < 0.05)). These results are reflective of, for example, 4/7 participants responding that they were very confident about home cooking activities before training program compared to 6/7 after training program (Cohen’s effect sizes ~0.9–1.1).Health coaches improved negative attitudes about and self-efficacy to perform various home cooking activities from a mean (standard deviation) of 1.22 (0.51)  and 4.64 (0.62) , respectively, before the training program to 1.07 (0.46) (Twenty-two (79% of the 28) patient programs were completed during the program’s internship. ‘The shift from “coach” to “trainer” is the area I want to strengthen. Ideally the transition would be seamless.’ (2) The practice sessions were one of the training program highlights. Participants praised their learning experience from both practicing and observing the program faculty as well as their colleagues. ‘I am now even more aware of my strengths and weaknesses, my skills as a chef and a coach and how that relates to the manner in which I present guidance to clients, and how I see myself as an agent of change.’ (3) Participants reported a beneficial educational experience from using culinary videos for their own education as well as a potential resource for patients. ‘These videos could also be a resource for clients.’Coaches’ perceptions from the training program were positive with notable themes: (1) The CC’s two-pronged approach was praised as a powerful tool. However, graduates perceived navigating between training and coaching as challenging. This pilot demonstrates that a synchronous remote medical education training program in CC is feasible, and was well-received by trainees who reported improved personal culinary behavior and confidence to use CC. These results are consistent with reports of onsite culinary trainings which are shown to improve providers’ personal and professional outcomes . The valThe curricular principles can also be adapted to address a variety of educational needs. For example, The American College of Preventive Medicine developed a short 2-h remote asynchronous training program in CC for their national training . We are The CC tele-nutrition training program has potential limitations that might impact viability such as individuals without access and/or skills to use internet-enabled devices. Also, further research is required to evaluate the impact of CC on culinary behaviors, health outcomes and healthcare costs of patients with various health conditions. This study used a small sample of convenience and larger studies are needed for confirmation. Our goal is to determine whether the remote CC initiative can make an efficient, effective, and scalable impact in supporting clinicians and health coaches in delivering home cooking education, and therefore, providing value-based preventive care.


N-methyl-2-pyrrolidone (NMP) and N-ethyl-2-pyrrolidone (NEP) in Germany. NMP and NEP metaboliteconcentrations were determined in 540 24-h urine samples of the GermanEnvironmental Specimen Bank collected from 1991 to 2014. With these data we wereable to investigate NMP/NEP exposures over time and to evaluate associatedrisks.The aim of this study was to get a first overview of the exposure tothe solvents and reproductive toxicants N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) and NEP metabolites5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP)and 2-hydroxy-N-ethylsuccinimide (2-HESI)were determined by stable isotope dilution analysis using solid phase extractionfollowed by derivatization (silylation) and GC–EI–MS/MS.NMP metabolites 5-hydroxy-We were able to quantify 5-HNMP and 2-HMSI in 98.0 and 99.6% and5-HNEP and 2-HESI in 34.8 and 75.7% of the samples. Metabolite concentrationswere rather steady over the timeframe investigated, even for NEP which has beenintroduced as an NMP substitute only in the last decade. Calculated median dailyintakes in 2014 were 2.7 µg/kg bw/day for NMP and 1.1 µg/kg bw/day for NEP. Forthe combined risk assessment of NMP and NEP exposure, the hazard index based onthe human biomonitoring assessment I values  was less than0.1.Based on the investigated subpopulation of the German population,individual and combined NMP and NEP exposures were within acceptable ranges inthe investigated timeframe. Sources of NEP exposure in the 90s and 00s remainelusive.The online version of this article (10.1007/s00420-018-1347-y) contains supplementary material, which is available to authorizedusers. N-Methyl-2-pyrrolidone  andN-ethyl-2-pyrrolidone are dipolar aprotic solvents with high solvent power and water solubility. In Europe18,000 tons of NMP are produced each year  and agriculture(component in selected pesticide formulations). Different consumer products mayinclude NMP up to an amount of 5%, such as printer inks, toners, coatings, cleaners,and ink ECHA . Due to In 2009 NMP was classified as a reproductive toxicant Cat. 1B (H360),with labeling requirements in mixtures containing ≥ 5% NMP [Commission Regulation(EC) No 790/2009]. The classification is based on studies where dose-relateddecreases have been observed in fetal body weights of rats after oral and inhalationexposure and at levels below maternal toxicity  and the Verband der ChemischenIndustrie e.V.   isformed, which is further oxidized to N-methylsuccinimide (MSI) and 2-hydroxy-N-methylsuccinimide (2-HMSI). 5-HNMP and 2-HMSI are the mainmetabolites which can be detected in urine. In example, 43.8% of NMP are excreted as5-HNMP and 19.7% as 2-HMSI with elimination half-times of ~ 4 h for 5-HNMP and~ 17 h for 2-HMSI. Investigations on NEP metabolism in humans is limited  and 2-hydroxy-N-ethylsuccinimide (2-HESI), have been identified after oraldosages. Here, 28.9% of the dose are excreted as 5-HNEP and 21.6% as 2-HESI. Theelimination half-times are ~ 7 h and ~ 22 h, respectively.Both, NMP and NEP can be absorbed by skin, inhalation or by ingestion.Human NMP metabolism has been previously studied in detail  for NMP andNEP were published by the German Human Biomonitoring Commission (HBM Commission) and the German Environment Agency (UBA) . Samples were from students between 20and 30 years of age. Sampling location was the area of Münster. Demographic data was collected by a standardized self-reportedquestionnaire. Details can be found in Table N-ethyl-2-pyrrolidone (5-HNEP), 2-hydroxy-N-ethylsuccinimide (2-HESI) and the ethyl chaindeuterium-labeled analogues (±)-5-hydroxy-N-ethyl-2-pyrrolidone-d5 (5-HNEP-d5) and (±)-2-hydroxy-N-ethylsuccinimide-d5 (2-HESI-d5) were synthesizedand characterized. The chemical purity of the aforementioned compounds was> 97%. The deuterated internal standards had no detectable impurities ofnon-labeled analogues.All reagents and chemicals were purchased and used as previouslydescribed , 200 µL water, and 200 µL 1% acetic acid were added to600 µL urine. After homogenization, 1000 µL of this solution were loaded ontosolid phase extraction cartridges , preconditioned with 2 × 500 µL ethyl acetate/ethanol(4:1 v/v), 2 × 500 µL methanol, and 4 × 500 µL 1% acetic acid. After washingwith 250 µL 1% acetic acid and 750 µL water, the analytes were eluted with1750 µL ethyl acetate/ethanol (4:1 v/v). Samples were dried under a gentlestream of nitrogen, then dissolved in 200 µL acetonitrile, and again dried undernitrogen. After adding 30 µL pyridine and 30 µL N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide for derivatization , thesamples were finally diluted with 50 µL ethyl acetate and analyzed byGC-EI-MS/MS.200 µL internal standard . Helium 6.0 (1 mL/min) was used as carriergas, injection volume was 1 µL. Injection was performed in pulsed splitless modeat an injector temperature of 280 °C. The temperature program was as follows:90 °C (5 min); 10°C/min to 200 °C (5 min); 30°C/min to 280 °C (6 min). Transferline, ion source, and quadrupole temperatures were set to 280, 230, and 150 °C,respectively. Collision gas (nitrogen 5.0) flow rate was 1.5 mL/min and quenchgas flow rate (Helium 6.0) was 2.25 mL/min. Quantification (quantifier) andconfirmation  of the respective analytes were done in EI MRM mode analysis was performed using anRtxhttp://www.g-equas.de) for NMP metabolites.LOQs were determined according to a signal to noise ratio of nine.The LOQ for 2-HMSI and 2-HESI was 2 µg/L and for 5-HNMP and 5-HNEP it was2.5 µg/L. Information about within-series and day-to-day imprecision are givenin Table Statistical analysis was performed using IBM SPSS Statistics 22(2013). Boxplots were generated with OriginPro2015G (OriginLab Corporation).Values below LOQ were set as LOQ/2 , values below LOD (limit of detection) were set as LOQ/4. To investigatepossible associations between creatinine adjusted metabolite levels and gender,the Mann-Whitney U test was applied. Jonckheere–Terpstra test was applied toanalyze time trends.m1 and cm2 (5-HNMP and 2-HMSIor 5-HNEP and 2-HESI) in µg/L and their respective molar masses M(m1) and M(m2)in g/mol, the molar masses of NMP or NEP M(compound) in g/mol, the 24-h urinevolume in L/d, the urinary excretion factors fUE(m1) andfUE(m2) of the respective metabolites was calculated as the sum of hazard quotients (HQ) for NMP and NEP , being a factor of 15 below the HBM-I value for adults(15 mg/L) and a factor of 49 below the respective HBM-II value (50 mg/L). Themaximum concentration of the sum of NEP metabolites was 1,312 µg/L (1.31 mg/L),being a factor of 11 and 30 below the HBM-I (15 mg/L) and HBM-II (40 mg/L) levels.These HBM values apply to the evaluation of a single substance. Due to the verysimilar toxicological profiles of NMP and NEP, a mixed exposure to both substanceshas to be taken into account in risk assessment [Stellungnahme der Kommission“Humanbiomonitoring” des Umweltbundesamtes ]. The hap < 0.001), 5-HNMP (p < 0.001), and 2-HESI (p = 0.012). Due to the lower detection rates, 5-HNEP was excludedfrom this and further statistical analyses. Potential sources of exposure of thegeneral population to NMP and NEP cannot clearly be identified, although it has beenreported that NMP exposure may arise from the use of pharmaceutical or cosmeticproducts . The NEP metabolite 2-HESI was detectedin the samples of all years with median concentrations between 2.5 and 38.4 µg/L(3.4 µg/g creatinine to 46.0 µg/g creatinine). For 5-HNEP median concentrations wereall below LOQ except for 1999 with a median concentration of 9.7 µg/L (10.1 µg/gcreatinine). For 5-HNEP more than 50% of the samples had concentrations < LOQ.Accordingly, 5-HNEP was excluded from time trend analysis. 2-HESI did not show anytrend over the timeframe investigated. Thus, in this study with urine samples from20 to 30 year old students from Münster, we could not observe any immediate effectsof regulatory measures (starting in 2009 for NMP and 2013 for NEP) on generalpopulation exposure to the two alkyl pyrrolidones NMP and NEP. One possibleexplanation could be an increase in production volumes of products containing NMP orNEP, compensating the decrease in alkyl pyrrolidone concentrations. However, data onproductions volumes of such products is not available so this remains speculative.Anyway, as already discussed above, even maximum exposures observed were and stillare well below HBM I values  for single andcombined NMP/NEP exposures.The urinary alkyl pyrrolidone metabolite concentrations for theinvestigated years between 1991 and 2014 are presented in Table N-vinyl-2-pyrrolidone . This would indicate to lower median NEP exposure, compared to NMP,but higher upper bound exposures to NEP, compared to NMP, which is also reflected inthe highest hazard quotient found for NEP. Currently, it is still unclear, whichsources are responsible for this NEP exposure, especially in samples pre 2008. Weare not aware of any significant  NEP uses up to this time. We onlyfound hints that NEP could be a side product in the synthesis of p < 0.001), no trend was observable for NEP (p < 0.080). These results  are in line withthe observations from the creatinine adjusted metabolite concentrations.Finally, we calculated the daily intakes (DIs) for both NMP and NEPaccording to Eq. . As can With this study we were able to show that young adults of the Germanpopulation have been continuously exposed to NMP and NEP at least since 1991, up to2014. While exposure to NMP has been expected due to its wide field of  applications, the ubiquitous exposure to NEP (previously marketed as asubstitution product to NMP) came unexpected. For NEP, the sources of past exposuresneed to be unveiled. We did not observe an effect of regulatory measures enacted inrecent years in the EU (2009–2013) on lowering alkyl pyrrolidone exposures in ourstudy population. However, metabolite concentrations in all ESB samples were clearlybelow the HBM values for NMP and NEP derived by the German HBM Commission. Acumulative risk assessment by calculation of the hazard index (HI) based on the HBMI values of NMP and NEP as acceptable levels revealed a maximum HI of 0.09 which isclearly below 1, the level that would indicate a combined exposure above HBM I.Consequently, the results based on the ESB study population do not raisetoxicological concerns towards the level of combined NMP and NEP exposure.Ongoing measurements of 24-h urine samples of the ESB in the next yearsmay investigate the possible effect of the regulatory measures imposed in themeantime on both alkyl pyrrolidones. In addition, study populations other than theESB (20–30-year-old university students) should be investigated to broaden thedatabase on exposure to alkyl pyrrolidones in the full German population, includingsusceptible subpopulations. Surveys such as GerES


Pan troglodytes) and western lowland gorillas (Gorilla gorilla gorilla) would show greater flexibility when presented with a causally-clear task. We presented six chimpanzees and seven gorillas with a clear tube from which they had to remove straws to release a reward. To first evaluate the apes’ causal understanding, we recorded the efficiency with which the apes solved the task . To further explore how they solved the task, we also recorded the order in which they removed the straws, which allowed us to determine if habitual action sequences emerged. All apes spontaneously solved the task in their first trial and across repeated trials the majority of their solutions were efficient (median = 90.9%), demonstrating their understanding of the puzzle. There was individual variation in the consistency of straw removal patterns exhibited by the apes, but no ape developed an exclusive habit in the order with which they removed the straws, further indicating their causal understanding of the task. Next, we presented the apes with a new configuration of the same task that required the apes to remove fewer straws to obtain the reward. All apes switched to a more efficient straw removal sequence even though their previously-successful, but now less-efficient, solution remained available. We theorize that because the apes understood the causality of the task, they did not form habits and were not conservative.In contrast to reports of wild primates, studies of captive primates’ flexibility often reveal conservatism: individuals are unable to switch to new and more efficient strategies when task demands change. We propose that such conservatism might be a result of task design and hypothesize that conservatism might be linked to primates’ lack of causal understanding in relation to experimental apparatuses. We investigated if chimpanzees ( Gorillas’ tendency for conservatism has not been investigated as extensively as for chimpanzees; two studies have tested three gorillas in different tasks, both revealing some inflexibility when task conditions changed and some success in overcoming a change in conditions  by removing only the straws below the reward. As a second indicator of causal understanding, we also documented the order of straw removal the apes used in each trial to determine if the apes formed action habits when solving the task in the first configuration. Specifically, we propose that habit formation might be indicative of reduced causal understanding because habitual actions are based on antecedent stimuli rather than the causal consequences of each action . We alsoThe subjects were six chimpanzees  and seven western lowland gorillas  . All of This study was approved by the Lincoln Park Zoo Research Committee, which is the governing body for research at Lincoln Park Zoo. This research adhered to legal requirements in the United States of America and to the ASAB/ABS Guidelines for the Use of Animals in Research. The apes were tested in their social group on exhibit, except for two gorillas who were tested individually when voluntarily separated as part of daily husbandry routines. All testing was voluntary for the subjects and no changes were made to their typical husbandry routine for this study. The subjects were never food or water deprived. The food rewards and straws used for the experimental protocol were approved by veterinary staff.The “Ape-plunk” apparatus  was a clear PVC tube  attached to the outside of the cage mesh . Five hoTo begin a trial, we inserted paper straws through the tube from the bottom to the top, but not far enough so the ape could access them, and baited the Ape-plunk with a high-value food reward (either a grape or peanut in-shell depending on the subject’s preference), such that it rested on one of the straws threaded through the tube . Once alWe ran two sequential phases with each subject. For each trial in phase 1, we set up the Ape-plunk with four straws below the reward, and one above it  and each subject completed phases 1 and 2 in an average of 11 sessions (SD = 4). All testing was completed between June 2016 and September 2017.To prevent excessive straw consumption, or the apes from using straws as tools to retrieve rewards in later trials, a keeper asked the apes to trade straws back before the next trial began. The subjects were given lower-value food rewards  than the reward in the Ape-plunk in exchange for trading the straws to ensure that they did not begin to value straws more than solving the task.sensusensusensuAlthough many subjects were tested while in their social group to ensure all testing was voluntary, enhancing validity and welfare sensu, we “staThe sessions were recorded using a Sony Handycam (HDR-CX160) on a tripod that was set up to record each Ape-plunk available and the participating ape. All data were transcribed into Excel and are available in the supplementary materials. A second coder, blind to the experimental condition, recorded the order of straws removed according to straw position for one randomly-selected session of each phase per subject, and had 100% agreement with the primary coder.The apes’ responses in a given trial were either classed as efficient (they removed only relevant straws below the reward) or inefficient (they removed both relevant and irrelevant straws) to obtain the reward. In phase 1, there were a total of 120 different possible orders that the apes could remove all five straws, 24 (20%) of which would be efficient, removing the four straws below the reward first. In phase 2, only 12 (10%) of the 120 possible combinations would be efficient, removing the two straws below the reward first.For all trials, we also recorded the order in which the subject removed the straws using the numbering system in All analyses were conducted in R version 3.2.3 . All graW = 18, N = 13, p = 0.72). Collectively, the apes removed only relevant straws for a median of 90.9% of their trials and four of the 13 apes used an efficient strategy in every trial of phase 1  did so efficiently . Across all their trials, the chimpanzees were efficient for a median of 90.9% of their trials and the gorillas were efficient for a median of 95.2% of trials in phase 1 . There w phase 1 . The apehttps://figshare.com/articles/Chimpanzee_problem_solving_and_flexibility/6654896/2). Collectively, the apes used 38 different action sequences to solve the task (120 were possible)  the apes used when they solved the task in phase 1 varied widely between and within individuals (ossible) . Specifiossible) .W = 8, N = 13, p = 0.07). The apes’ median run length ranged from 2–5 trials .Not only was there variance across individuals as to the way in which they solved the task, there was also variance in the number of different action sequences each individual used across trials, ranging from seven to 14. Indeed, the apes’ individual Shannon H index ranged from 1.11 to 2.36 , where t5 trials  and did W = 169, N = 13, p < 0.01). In contrast to chimpanzees, the gorillas most frequently removed straws from the bottom of the apparatus until they reached the reward  (median= 35.0%), however they did not use this strategy significantly more than the chimpanzees .The action sequence most commonly used by the chimpanzees in phase 1 was repeatedly removing the straw directly below the reward , which occurred for a median of 60.8% of individuals’ trials . In contp < 0.001). For example footage of a chimpanzee switching from an efficient strategy in phase 1 to a different, but efficient, strategy in phase 2, see this video: https://figshare.com/articles/Chimpanzee_problem_solving_and_flexibility/6654896/2. Five of the 13 apes were efficient in every trial in phase 2 .In their first trial of phase 2, when presented with the new configuration of the Ape-plunk, all 13 apes removed fewer than four straws to obtain the reward . Furthermore, 12 of the 13 apes switched to the most efficient strategy on their first trial , which was significantly different than chance , revealing that chimpanzees used it more frequently than gorillas. Gorillas used the alternative efficient action sequence  significantly more often than chimpanzees . Indeed, one gorilla (AZ) used the 1,2 action sequence for all of his trials  and seven that were inefficient. The gorillas used six different action sequences, the two efficient ones and four inefficient ones. The chimpanzees’ modal response was the 2,1 action sequence while the gorillas’ modal response was the 1,2 action sequence . In phass trials .W = 24, N = 13, p = 0.67). Overall the apes’ diversity indices in phase 2 were significantly lower than in phase 1 , another indicator of their flexibility and efficiency since there were only two efficient action patterns in phase 2 . This suggests that there was no relationship between consistency of action sequences used in the first phase and ability to employ efficient action sequences when contingencies changed (flexibility). The apes’ median run length ranged from 3–20 . The apes’ median run length was also not significantly different between phase 1 (median = 2.50) and phase 2 (median = 4.00) .In Phase 2, chimpanzees had a median Shannon H index of 0.80 and the gorillas’ was 0.65 where the index would be 0 if they used only one sequence and if they used all possible solutions 2.37. As in phase 1, their diversity did not differ by species , they also showed exploration within phases in terms of the action sequences they used , indicating that they did not form immutable habits. The apes expressed a wide range of variation within and between subjects in the action sequences they used to solve the task, continuing to explore new sequences throughout phase 1. In phase 1, the most efficient manner in which the apes could solve the task was to remove all of the four straws below the reward, but they could do so in any order  scores and their long median action sequence run lengths. Even though some individuals had these tendencies, they were still able to transition away from their preferred action sequence when the task configuration changed. This is further supported by the reduction in response diversity (H index score) shown by both species in phase 2 compared to phase 1, when fewer combinations of action sequences could be performed while remaining efficient .Although the apes showed exploration of the different action sequences, we also noted that certain individuals persisted in using certain sequences, as highlighted by their low diversity . We theorize that this was because of two mutually-reinforcing factors. The chimpanzees learned the simple association to pull out whichever straw the reward rested on because doing so caused the reward to fall and move closer to the final goal. In this way, the chimpanzees received proximate reinforcement for each action, even though it was only the final one (removing straw 1) that released the reward and so was reinforced with receipt of food. It is likely that the chimpanzees’ attention was initially drawn to the straw with the grape on it due to a form of stimulus enhancement—the straw became a potent stimulus via association with the food reward touching it. Similarly, in phase 2 the chimpanzees’ most commonly-used action sequence  followed this same rule, accounting for 69% of their responses. Given this, it is possible that the chimpanzees did not understand the task mechanism holistically, but following this simple rule (remove straw that reward rests on) is both what allowed them to solve the task efficiently and change their response pattern flexibly when the task configuration changed. It is worth noting, however, that 46% of the chimpanzees’ responses in phase 1, and 31% of their responses in phase 2, were via action sequences other than 4,3,2,1 or 2,1 respectively. Thus, they did not follow this rule exclusively but were consistently efficient.In contrast to the chimpanzees, the gorillas’ most commonly-used action sequence in phase 1 was 1,2,3,4 , which represented 26% of all their responses. They used the 4,3,2,1 action sequence (the chimpanzees’ preferred technique) less frequently—it represented only 13% of their trials. Interestingly, the gorillas’ second most common response was 2,1,3,4 , which might represent a failed attempt at 1,2,3,4 . Within the 1,2,3,4 action sequence, only the last action is rewarded and reinforced. No changes are triggered when straws 1, 2, or 3 are removed prior to 4. It is unclear what motivated the gorillas to prefer this action sequence. Perhaps they were motivated to remove the straws associated with where they obtained the reward . In phase 2, at a species level the majority of the gorillas’ responses followed the pattern of removing straws from the bottom upwards—in 54% of their trials they used the 1,2 action sequence—but at the individual level, each gorilla showed a preference for either the 1,2 or 2,1 sequence. Potentially, the gorillas who preferred removing straws sequentially from the bottom of the apparatus in phase 1  would not need to understand the task to be flexible when the task configuration changed in the second phase. However, due to the variety of methods used by the gorillas across their trials in phase 1, we conclude that this is not the case. In spite of these individual preferences, the gorillas showed greater behavioral diversity in terms of their action sequences than the chimpanzees and so certainly were not bound to a single technique. Greater understanding about how these apes solved the task, and what caused them to explore multiple action sequences, as well as what explains the species differences we observed, is required. For example, is it possible that the species had a different causal understanding of the task , or do tIf the apes did not have causal understanding of the task, or had not been motivated to obtain the reward efficiently, we might have expected them to simply remove all five of the straws, regardless of the reward location. Although this strategy is inefficient, the effort to remove extra straws is relatively low. Despite this, in phase 1, the apes used an inefficient 5-straw action sequence in fewer than 10% of their trials and, in phase 2, only one ape removed all of the five straws, and only in one trial. Thus, the apes were not only efficient in phase 1, but showed flexibility in their responses such that they continued to use the most efficient responses as new newly-efficient action sequences became available, discarding previously-adopted action sequences. The apes’ drive to obtain the reward in the most efficient way possible is reminiscent of previous studies of ape problem solving and social learning in which they have been shown to adopt the most efficient strategy, even ignoring socially-demonstrated actions if they did not pertain directly to reward retrieval e.g., .below the reward to look at the limits of the apes’ flexibility and further explore the interplay between conservatism and causal understanding. Another future direction could be to explore apes’ responses when presented with a task that was opaque. Without causal feedback, apes might persist with the first successful action sequence, which would become an engrained habit, and would be difficult to shift away from when contingencies of the task changed.Although it was a secondary aim of our study to ascertain if habit formation was an indicator of reduced causal understanding, it is likely that our study design did not provide the apes with a long enough exposure for a habitual action sequence to develop in phase 1. In spite of this, we did see runs of action sequences emerging across trials in the apes’ responses. Furthermore, We predicted that if apes have causal understanding of a novel task they would be flexible when task demands changed. The apes’ overall efficiency solving the novel task demonstrated their causal understanding of it, likely supporting their later flexibility. The apes also were flexible in the sequences in which they removed straws to solve the task, and although there was some individual variation, they did not appear to form habits. In conclusion, we theorize that the apes’ causal understanding of the task (perhaps underscored by simple associative learning rules) protected them against habit formation and also against conservatism, but future work is required to understand the species differences we observed in the pattern of the apes’ responses .10.7717/peerj.6195/supp-1Data S1Click here for additional data file.


In a few short months, there will be a gathering of the clan to celebrate the 50th anniversary of the first annual meeting of the American Association for Cancer Education (AACE). I would encourage all who read this editorial to make plans to attend. In addition to the regular fare of presentations on innovative cancer education programming, there will be special presentations commemorating our 50th. The AACE, and the annual meeting, has undergone numerous changes since it was formed, but has remained true to its mission “…of bringing together members and invited guests for discussion and demonstrations related to cancer teaching activities and varying pedagogical views…” . In an eThe annual meeting will also offer a number of workshops where attendees can gain new knowledge or hone their skills. As of this writing, confirmed workshops (and their leaders) include: Cancer Survivorship ; Genetics and Cancer Care ; Health Literacy ; Palliative Care ; and Use of a Translator . An expansive collection of workshops that will surely meet the needs of every attendee. As a past participant and presenter at our workshops, I can attest to their value. Dialog begun in these workshops continues through the year and is a positive influence on one’s research and/or program development.The annual meeting proper will include both invited speakers as well as concurrent sessions. Sessions will cover the entire cancer education waterfront from highly competitive abstracts submitted for presentation. Another beneficial aspect of the meeting is the Poster Sessions where we get an opportunity to personally meet and query presenters from around the world. These are always such great learning and networking opportunities. In commemoration of our 50th anniversary, there will be a luncheon panel of the History of Cancer Education. One of the highlights of the annual meeting is the presentation of the Samuel Harvey lecture which will be given this year by Dr. Huerta. We will also hear from Dr. Charles Kelly the President of the EACE on Shared Decision Making as an Educational Tool in Cancer Management. I trust that this preview of the annual meeting has whet your appetite. Please visit the website to register for what will undoubtedly be a dynamic meeting.I would like to reemphasize that the meeting is now known as the “International Cancer Education Conference.” This title more accurately reflects the diversity and applicability of topics and presenters at the meeting. No one individual exemplifies this more than Dr. Amr Soliman (current President) who is internationally known for his efforts in cancer education and cancer epidemiology. He, as well as a number of our members, have given lectures and developed training programs throughout North Africa, the Middle East, and Europe. I have had the honor of traveling with him and witnessing his dedication and proficiencies. I have also benefited from attending meetings of the EACE. The 2015 meeting in Heidelberg, Germany, was particularly insightful as I came to meet Radislaw Tarkowski, MD, PhD and a group of his students from Wroclaw Medical University in Poland. I was very impressed by the number and quality of their presentations such that I had numerous conversations with Dr. Tarkowski at the meetings and continued these upon my return to the States. Our conversations began to focus on the development of a 2-week training program at Wroclaw University focused on the art of scientific publishing and the principles and methods of cancer education research. A noble goal, but where to find funding? Our answer resided with the Fulbright Scholar Program. This program awards grants to US faculty/professionals to engage in short-term collaborative projects. Many of you have likely heard of the Fulbright which provides funding to support long-term exchanges, but they also operate a Fulbright Specialist Program  which provides funding for shorter periods (2 to 4 weeks). This allows, as they state, “greater flexibility to pursue projects that work best with their current academic or professional commitments.” This is a two step process requiring the application/review/approval of the Specialist and a separate application/review/approval of the host institution. We found the application process to be quite straight forward and administered by knowledgeable, responsive professionals. I would recommend that anyone interested review their website and consider applying. This program is named for Senator William J. Fulbright (Arkansas) who was a gifted statesman who saw the benefit of scholarly exchanges as a means of advancing mutual understanding. An impactful program named after a visionary Senator. The Specialist Program allowed us to develop a 2-week program for medical students and even faculty.Wroclaw is the fourth largest city in Poland . Wroclaw Medical is one of the most renowned public medical schools in Poland and offers a 6-year curriculum taught in English. The school curriculum, like so many others, focuses on clinical care and does not offer sufficient time for medical research let alone those areas aligned with cancer education. The program that Dr. Tarkowski and I developed incorporated didactic lectures, workshops, and private/group consultations. Full details of this experience will be presented in a future manuscript in this Journal that Dr. Tarkowski and I will co-author. I found the attendees to be highly motivated, intelligent, and engaged. While we both had initial concerns over how active the students would be, these reservations were laid to rest early in the first session. The students were highly inquisitive and asked highly focused questions which often lead to an ever expansive and detailed conversation. I cannot praise the students enough for their level of participation. Many brought completed or manuscripts in progress for discussion at the workshops. These discussion not only lead to a strengthening of their manuscripts, but the discussions addressed areas of interest to all participants which should lead them to ever better study design. I also cannot thank Dr. Tarkowski enough for his leadership and for his gracious generosity as a host. I came to know the land of my ancestors at a very personal level. My wife (Terry O) and I not only became more keenly aware of the rich history of Polonia but also benefited from its rich culinary experience. I encourage all to explore the many benefits of the Fulbright Program.Be well and Na zdrowie!AMM


InteractiveXRDFit is a custom-made MATLAB program that calculates the X-ray diffracted intensity for heterostructures. InteractiveXRDFit is a custom-made MATLAB program that calculates the X-ray diffracted intensity for oxide thin films and heterostructures based on a library of inorganic materials. These fascinating materials can be studied with a plethora of advanced techniques. Independent of their properties, their characterization usually starts with conventional X-ray diffraction (XRD) to attest their crystalline quality. Indeed, most crystal-growth laboratories are equipped with X-ray diffractometers, standard instruments that are relatively inexpensive, fast and non-invasive.InteractiveXRDFit was developed to allow the user to simulate the (00L) diffractogram of heterostructures combining different materials grown on different substrates . The user can choose the substrate and the different materials composing a heterostructure from a long list of compounds (mainly perovskite oxides), choose between (001) or (111) substrate orientation, and play with the different structural parameters (unit-cell size and number of layers). It is possible to build a superlattice composed of up to three different materials, and to add a top and/or bottom layer . The simulation is quick and allows the user to compare it directly with experimental measurements, so as to give a rapid determination of the crystalline parameters of the sample.Based on these observations, the program This paper describes the program, how to use it and how it is constructed, with an example for illustration.2.InteractiveXRDFit was written using MATLAB R2015b  for Mac. It is compatible with Linux, Unix, Mac OS and Windows. The version available at the date of publication of this paper can be found in the supporting information. However, this program is constantly being adapted to suit users’ needs, and anyone interested in using the latest release can obtain it from GitHub. If the user does not already have an account, he/she should start by creating a personal GitHub account (for free): https://github.com. The user should then request access to InteractiveXRDFit.InteractiveXRDFit.m.The user can then download every file for the latest version of the program, open MATLAB on his/her computer and run 3.InteractiveXRDFit.m, three windows will appear:Once the user runs a),(i) the XRD fitting parameters window Fig. 1a,c display , and(ii) the ay Fig. 1c, andb).(iii) the fit display Fig. 1b.3.1.a), the user has the following options:From the XRD fitting parameters panel Fig. 1a, the u(i) Quit the program..csv file). When loading data for comparison, two vertical green lines are displayed together with the data (in red) and the simulation (in blue). They delimit the region over which the r.m.s. values are evaluated , NdAlO3, NdGaO3, Si, SrTiO3, TbScO3 and YAlO3.Substrates can be chosen from DyScO2, BaO, BaTiO3, BiFeO3, CaCuO2, LaAlO3, La2CuO4, LaFeO3, LaMnO3, LaNiO3, La2NiMnO6, LaO, LSMO (La0.67Sr0.33MnO3), MnO, MnO2, MnTiO3, NiO2, NdNiO3, Nd2NiMnO6, NdO, PbO, PbTiO3, (PbxSrx1−)TiO3, Pb(ZrxTix1−)O3, PrBa2Cu3O7, RuO2, SmNiO3, SrO, SrO2, SrRuO3, SrTiO3, SrVO3, TiO2, VO2, YBa2Cu3O7 and ZrO2.Layer materials can be chosen from AlOc-axis value. The c-axis value either can be a constant (in most cases) or can vary as a function of depth within a layer  to help visualize the evolution of the c axis throughout the heterostructure.The ph Fig. 1c to hel3.3.b) after each modification of a parameter, to follow rapidly any changes in intensity as a function of the different parameters and compare the simulations with the measurement. In this window, one can display the data as a function of either 2θ , the lower one having to cover a longer distance (in red) equal to 2dsinθ. The beams are characterized by their wavelength λ and their wavevectors k = k′ − k = Q, where Q is a Bravais vector of the reciprocal lattice, as shown in Fig. 2b). Further details can be found in the work of Ashcroft & Mermin /λ]. Since this program evaluates the Bragg diffraction along the Q direction as shown in Fig. 2b), only the z value for the different atoms and the distance d between equivalent planes of the corresponding material are needed: The structure factor is then obtained for each unit cell using the Wyckoff positions within this cell: 4.3.1.ABO3, as shown in Fig. 3c. In the (001) orientation .Most of the materials used in this program have a perovskite structure on Fig. 3, top, thF is then The structure factor x, y and z directions, the lattice parameter units are equal to c(2)1/2, c(2)1/2 and c/(3)1/2, respectively, where c is the lattice parameter of the unit cell.In the (111) orientation Fig. 3, bottom,z of the groups of atoms (A2 O4 O5 O6 B2) and (A3 O7 O8 O9 B3) are equivalent to (A1O1 O2 O3 B1). We therefore use the first set of five atoms as our unit cell to calculate the structure factor F:One can easily see that the positions along 4.3.2.4 is a tetragonal material used as a substrate, with a = b = 3.75576 Å and c = 12.6377 Å, as shown in Fig. 4LaSrAlOF is then The structure factor 4.4.g for the whole heterostructure is then obtained by adding the contributions from each layer. However, this neglects the effects associated with the absorption of the beam as it passes through the sample, or the reduction in intensity due to scattering and diffraction. The dynamic theory of diffraction takes these effects into account, but involves a degree of complexity that is beyond the scope of this program  providing point-and-click control of the program, so that the user can run the application with no need to learn the MATLAB language or type commands.Other parts of the program calculate the atomic scattering factors, structure factors, scattering amplitude and scattered intensity of the heterostructure built by the user. In order to save time, the substrate scattering amplitudes have all been calculated beforehand and are uploaded by the program.The program is composed of the following MATLAB scripts and functions:InteractiveXRDFit.m. This is the heart of the program. It initializes all the variables and creates the GUI.(i) Substrate.m. This script loads the correct substrate data and intensity (from Substrates.mat) depending on the user’s choice.(ii) GenerateSubstrates.m. This script generates the XRD intensities for different substrates. It is never called from the other scripts. This is the script that was used to calculate the substrate scattering amplitudes beforehand in order to save time (saved in Substrates.mat). It is made available only for the sake of completeness.(iii) PlotFitAndData.m. This script plots the calculated XRD intensities on the fit display panel. If the user has loaded a measurement for comparison, this script also displays the measured XRD intensities, together with the lines delimiting the region over which the RMS (root mean square) is evaluated. On the c display panel, it plots the x-axis profile of the simulated heterostructure.(iv) ProgcNSimu.m. This script calculates the XRD intensity and c-axis profile of the heterostructure defined by the user. It is used every time the user modifies a parameter in the XRD fitting parameters panel.(v) expzm.m. This function calculates the contribution from each layer material to the diffracted intensity.(vi) FLayer.m. This function calculates the form factors of the different materials depending on their crystallographic structures. ‘z’ are the out-of-place atomic positions in the different unit cells, expressed in relative unit cells.(vii) StructureFactor.m. This function calculates the structure factor for materials with a perovskite structure .(viii) AtomicScatteringFactor.m. This function calculates the different atomic scattering factors used in this program, based on International Tables for Crystallography, Vol. C.(ix) RMS.m. This script is used to calculate the RMS of the pairwise differences of the fit and measurement, xmin and xmax over which the RMS and RMS(log) values are calculated . This is done only if the user loads measured data, to compare them with the fit.(x) 6.As discussed in §4.47.3 samples grown on SrTiO3 substrates, standard simulations  were not giving satisfactory results. This is shown in Fig. 53 with N = 127 unit cells and c = 4.139 Å (in blue) is compared with the measurement (in red). The value of c is determined by the PbTiO3 peak position and the number of unit cells is optimized to fit the finite size oscillations. However, the width of the simulated film peak is narrower than the measured one, and the oscillations to the right of the film peak are less intense than the measured ones.An interesting example is shown in Fig. 5c-axis value described with an exponential function was added to the program. The result obtained for N = 125 and cz = −0.079exp(−z/20) + 4.146 Å  is shown in Fig. 5The option to fit the diffractogram using a depth-varying 8.InteractiveXRDFit was developed to answer the need for users to simulate rapidly the (00L) diffractogram of heterostructures combining different materials grown on different substrates.The program Epitaxy  from Malvern Panalytical allows thorough analysis of X-ray diffraction data with efficient fitting procedures, taking the instrumental details  into account and allowing the user to determine the sample structure properties , but the materials data remain fixed as they are in the materials database. SUPREX  , a versatile program using the differential evolution algorithm for fitting X-ray and neutron reflectivity data as well as surface X-ray diffraction data  Python scripts for assisting X-ray diffraction experiments.Other fitting programs are already available and they are efficient, but they may lack some flexibility with some of the parameters that this program allows to be modified. For example, InteractiveXRDFit is simple to use and has the advantage of allowing the user to carry out rapid simulations of different heterostructures and see how changing the parameters modifies the diffractogram for a better understanding of the measurements.Additionally, it can be easily adapted to the user’s needs, with more materials and functionalities added on demand. Requests for any new requirements are most welcome.Click here for additional data file.10.1107/S1600576718012840/vh5084sup1.zipApp allowing direct installation of the free-standing executable of the program. DOI: Click here for additional data file.10.1107/S1600576718012840/vh5084sup2.zipMATLAB main program InteractiveXRDFit.m, 9 MATLAB sub-programs and file containing the calculated intensities of the different substrates (Substrates.mat). DOI:


Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied. A striking decrease in the level of occupancy at the Asn-565 glycosite was observed in dimeric IgM compared to that in monomeric IgM, and no glycan occupancy of Asn-565 was observed in tetrameric IgM. Glycopeptide analysis with liquid chromatography-electrospray ionization tandem mass spectrometry revealed mainly complex-type glycans with substantial heterogeneity, with neutral; monosialyl-, disialyl- and trisialylated; and fucosyl-and non-fucosyl-oligosaccharides conjugated to grass carp serum IgM. Glycan variation at a single site was greatest at the Asn-262 glycosite. Unlike IgMs in other species, only traces of complex-type and no high-mannose glycans were found at the Asn-565 glycosite. Matrix-assisted laser desorption ionization analysis of released glycans confirmed the overwhelming majority of carbohydrates were of the complex-type. These results indicate that grass carp serum IgM exhibits unique N-glycan features and highly processed oligosaccharides attached to individual glycosites. Glycosylation represents a major post-translational modification of proteins involved in various biological processes, including transcription, differentiation, apoptosis, cell adhesion, receptor-ligand binding, as well as oncogenic transformation and immune response –4. Almos2Man3) backbone but vary with respect to the structures attached to this core  . N-linkehis core .The N-linked oligosaccharides presenting on immunoglobulins have received particular attention because changes in the attached glycans can impact immunoglobulin solubility, structural stability, and biological function . In humaSalmo salar) and 10% for Atlantic cod (Gadusmorhua) IgM (Ginglymostomacirratum), a cartilaginous fish, revealed that the most abundant glycan was high-mannose type. Moreover, the structures of both complex glycans and high-mannose on nurse shark IgM were very similar to the vertebrate species . The heavy chain possesses four constant domains, CH1–CH4, containing the sites for the binding of effector cells , cytotoxhua) IgM , 24. Atl species .Compared to mammalian IgM, teleost IgM displays a significant degree of structural diversity, with different polymerization states, including monomers, dimers, trimers, and tetramers, being revealed –29. StruThus far, however, no data have been reported on the site-specific N-glycan of teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first comprehensive study of the carbohydrates conjugated at each N-glycosylation site, as well as the occupancy at the Asn-565 residue in the conserved C-terminal tailpiece, among grass carp IgM isoforms. These data expand the limited information currently available concerning teleost IgM glycosylation and provide a knowledge base for understanding the biological significance of glycosylation on teleost IgM.The general approach to characterizing IgM glycosylation structures is shown in Figure Grass carp (Ctenopharyngodonidella idella) 350–500 g, were bred and kept in 1,000-L tanks with recirculating and Uv-sterilized water. Fish were anesthetized with 10 ppm Benzocaine  for 3–5 min, blood was collected from the caudal vein from 10 naïve grass carp, allowed to clot for 1 h at room temperature and overnight at 4°C, serum was collected after centrifuging at 4,000 rpm for 10 min, pooled and stored at −80°C. All animal experiments were approved by the Institutional Animal Care and Use Committee of Central China Normal University.IgM was purified from pools of 10 individuals using a combination of polyethylene glycol (PEG) precipitation and ion-exchange chromatography as described previously . BrieflyTo isolate the heavy and light chains of grass carp IgM, 4 ug of purified IgM was mixed with denatured loading buffer and loaded onto 4–10% gradient SDS-polyacrylamide gel, then subjected to electrophoresis at 25 mA till the dye reached the bottom of the gel. The gel was stained with Coomassie Brilliant blue (Sigma) and the heavy and light chains was determined according to molecular weight markers. Previously electrophoretic analysis revealed carp fish serum containing different populations as monomeric, dimeric, and tetrameric forms of IgM , 42. To m = +0.984 Da). Sequence and potential N-glycosylation sites for grass carp serum IgM were obtained from GenBank (accession No ABD76396.1).To determination the glycosylation site on grass carp IgM, coomassie-stained SDS-PAGE gel pieces containing IgM heavy chain were destained with a solution of 50 mM ammonium bicarbonate in 50% acetonitrile  with shaking, and followed by 100% acetonitrile. The sample pieces were reduced by incubating with 64 mM DTT at 56°C for 60 min, followed by alkylation with 130 mM iodoacetamide at R.T. for 45 min in the dark. The samples were washed with 50 mM ammonium bicarbonate and dehydrated in acetonitrile, dried in a SpeedVac centrifuge, rehydrated in 50 mM ammonium bicarbonate. N-glycans were released by incubating the sample with 100 Units/ml of PNGase F for 12 h at 37°C, the N-linked glycans were collected for further MALDI-TOF analysis. Gel piece was dehydrated in acetonitrile, rehydrated in 50 mM ammonium bicarbonate and treated with trypsin (Promega) at an enzyme/substrate ratio of 1:20 (w/w) or with chymotrypsin (Promega) at an enzyme/substrate ratio of 1:13 (w/w) overnight at 37°C. Peptides were extracted from the gel pieces with 50% acetonitrile in 5% formic acid. The resulting peptides were then extracted and desalted with a C18 tip (Millipore) for analysis by LC-ESI-MS/MS. Glycosylation site were determined by mass of deglycopeptide-peptide  coupled on-line to a Q Exactive Plus mass spectrometer  in positive polarity mode. Chromatography column was Acclaim PepMap, C18, 50 um × 150 mm, particle size 2 um, 100Å  with buffer A (95% water and 4.9% acetonitrile with 0.1% FA) and buffer B (90% acetonitrile and 9.9% water with 0.1% FA) at a flow rate of 300 nL/min. The gradient profile was set as follows: 3–5% B for 6 min, 5–25% B for 54 min, 25–40% B for 25 min and hold 5 min, 40–80% B for 5 min, and 80–100% B for 5 min. The electrospray voltage was 2.0 kV. Peptides were analyzed with high-energy collisional dissociation (HCD) fragmentation mode by data-dependent MS/MS acquisition. In MS1, the resolution was 70,000, the AGC target was 3eABD76396.1) using Proteome Discoverer V2.2 (Thermo Fisher Scientific). Carbamidomethylation of Cys was set as a fixed modification, oxidation of Met and converting of Asn to Asp were set as variable modifications. The mass tolerance was set at 10 ppm for precursor ions, and the MS/MS mass tolerance was set at 0.01Da. Trypsin or chymotrypsin was set with up to two missed cleavages. Annotation of the corresponding glycopeptide spectra was performed in using Byonic software v1.09 (Protein Metrics) (+), and at least one of the following oxonium ions as 186.076 (HexNAc-H2O+), 168.066 (HexNAc-2H2O+) and 366.140 (HexNAc-Hex+) within 50 ppm. The existence of peptide or peptide+HexNAc ions in MS/MS spectra was checked manually to determine the glycosite-containing peptide and glycan compositions of the glycopeptides. Spectra were also checked manually about b and y ion data from peptide portion of the corresponding glycopeptides.N-linked-glycosylation sites were determined in de-glycosylated peptides based on converting Asn to Asp by PNGase F treatment, the corresponding glycosylated peptides were confirmed by HCD tandem MS. The obtained de-glycopeptide MS data of were searched against grass carp IgM sequence (GenBank accession No Metrics) . SearcheMetrics) . Additiohttp://web.expasy.org/glycomod/) . N-glycans were dissolve in 10 μl 0.1% TFA followed by spotting 1 μl glycan sample with 1 μl 2,5-dihydroxybenzoic acid matrix  onto a MALDI plate. Positive ion MALDI spectra were obtained with a Bruker ultraflextreme™ Waters-MicromassToF Spec 2E time-of-flight (TOF) mass spectrometer  operated under flexControl 3.3 . Spectra were obtained in positive linear ion mode in the range 1,000–3,200 m/z, using an accelerating voltage of 2.2 kV and a laser energy of 6.5 kV. The positive control in the MALDI-TOF MS analysis was authentic complex-type glycans from mAb rituximab, 2,5-dihydroxybenzoic acid matrix was used as negative control. The average MS spectra  were used for glycan assignment by GlycoMod software (ycomod/) . N-glycaTo determine the glycosylation status of grass carp IgM, IgM was purified by polyethylene glycol (PEG) precipitation and ion-exchange chromatography from serum. Purified IgM migrated two major protein bands with molecular weights of 75 kDa and 23 kDa under reducing conditions, representing the heavy and light chains, respectively Figure . To idenAEH76780.1 and AEH76777.1) also indicated no N-linked carbohydrate attached to the grass carp IgM light chain (data not shown). Analysis indicated that each of the heavy chains contains four potential N-glycosylation sites restricted to the constant regions. Two of these are located at Asn-262 and Asn-303 in the CH2 domain, one at Asn-426 in the CH3 domain, and one at the Asn-565 residue within the conserved C-terminal tailpiece of the CH4 domain  and with the additional oxidation at the methionine residue (2528.2104 Da/2529.2130 Da)  and with methionine oxidation (2643.2383 Da/2644.2287 Da)  Figure , Table 1m = 0.984 Da) could be clearly determined in comparison with non-glycosylated Asn-565 peptides via mass spectrometry  peptide are shown in Figure m/z 204.08 (HexNAc+), 186.07 (HexNAc-H2O+), 168.06 (HexNAc-2H2O+), and 366.14 (HexNAc-Hex+) are readily discernible within the fragmentation pattern. The composition of each glycan was identified by data-dependent MS/MS with an accuracy of 10 ppm or less. On the basis of previous information regarding fish IgM N-glycosylation (m/z 292.09 (NeuAc+) and m/z 657.23 (NeuAc-Hex-HexNAc+), indicated the presence of sialylated glycans, with fragment ions corresponding to core-fucose on the peptide+HexNAc residue suggesting core-fucosylated glycans , rather than N-glycolylneuraminic acid (NeuGc), was the only form of sialic acid observed in grass carp serum IgM.Three representative glycans detected on the sylation , the deo Figures . Glycan  Figures –C. NotabAssignments a set of glycans at each glycosite were achieved by selectively detecting tryptic glycopeptides, as shown in Tables 3Hex6Fuc1 composition on Asn-426 glycosite, for which the identification as a hybrid or complex type glycan was ambiguous. Heterogeneity was greatest at the Asn-262 glycosite, with both neutral and mono/disialylated, as well as small and large glycans, were observed. The Asn-303 and Asn-426 glycosites exhibited the next highest levels of variation. Three novel oligosaccharides, HexNAc9Hex3, HexNAc9Hex4, and HexNAc9Hex4Fuc1, with redundancy in the HexNAc monosaccharide  were identified on grass carp serum IgM with a mass accuracy within 5 ppm. No other glycan composition was possible with respect to the observed m/z values, suggesting the presence of glycans on grass carp serum IgM containing the diacetyllactosamine motif. Unlike IgMs in other higher vertebrates and some reported fish species, which possess major high-mannose glycans at the C-terminal tailpiece glycosite, the Asn-565 site of the grass carp serum IgM possesses exclusively complex-type N-glycans and is devoid of high-mannose structures. Together, these data indicate that the glycans on grass carp serum IgM are highly processed. It should also be pointed out that only a minority of Asn-565 peptides were conjugated with carbohydrates with both tryptic and chymotryptic glycopeptide analysis, further confirming the incomplete glycan occupancy at the Asn-565 glycosite.Of the 38 total glycan compositions observed on grass carp serum IgM by tryptic and chymotryptic glycopeptide analysis, 17 were sialylated and 21 were not. Notably, when the glycans were grouped into high-mannose, hybrid, complex types, the glycans identified on grass carp serum IgM were limited to complex-type carbohydrates, with the exception of a single carbohydrate with a HexNAc3−6Hex4−9Fuc1NeuAc1,3 complex-type glycans , as are common on invertebrate glycans as well as the N-glycans of zebrafish embryos  with access codes: PXD010308.The RAW MS data of grass carp serum IgM analysis, as well as the original search results of pGlyco 2.0 have been uploaded to the PRIDE partner repository (HG, Y-AZ, and BW conceived and designed the experiments. Y-LS, M-DH, Z-WC, and JW performed the experiments. Y-LS, QY, and HB analyzed the data. HG, Y-FC, LX, and C-HW wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.