N = 49) were immersed into a virtual height situation and a neutral control situation (fear manipulation) with either high versus low sensory realism (presence manipulation). Ratings of presence and verbal and physiological  fear responses were recorded. Results revealed an effect of the fear manipulation on presence, i.e., higher presence ratings in the height situation compared to the neutral control situation, but no effect of the presence manipulation on fear responses. However, the presence ratings during the first exposure to the high quality neutral environment were predictive of later fear responses in the height situation. Our findings support the hypothesis that experiencing emotional responses in a virtual environment leads to a stronger feeling of being there, i.e., increase presence. In contrast, the effects of presence on fear seem to be more complex: on the one hand, increased presence due to the quality of the virtual environment did not influence fear; on the other hand, presence variability that likely stemmed from differences in user characteristics did predict later fear responses. These findings underscore the importance of user characteristics in the emergence of presence.Virtual reality plays an increasingly important role in research and therapy of pathological fear. However, the mechanisms how virtual environments elicit and modify fear responses are not yet fully understood. Presence, a psychological construct referring to the ‘sense of being there’ in a virtual environment, is widely assumed to crucially influence the strength of the elicited fear responses, however, causality is still under debate. The present study is the first that experimentally manipulated both variables to unravel the causal link between presence and fear responses. Height-fearful participants ( Psychological treatments using virtual reality (VR) have shown promising results for different psychopathologies , includipresence, the user’s sense of ‘being there’ in the VE, is widely assumed to be crucial for the fear responses in VR , whereby half of the participants experienced high sensory realism VEs  versus low sensory realism VEs  for the other half . Presence as well as verbal and physiological (skin conductance and heart rate) fear responses were registered. Our hypotheses were: (1) higher quality of visual and auditory content of the VE increases presence, and (2) there is a causal relationship between presence and fear responses, i.e., either (2a) increased fear levels  lead to a higher reported sense of presence (fear → presence), or (2b) increased presence  leads to stronger fear responses (presence → fear).M = 26.84, SD = 10.94; 37 female) were included. The experimental procedure was approved by the Ethics Committee of the Institute of Psychology at the University of Würzburg. All participants gave written informed consent in accordance with the Declaration of Helsinki. Participants received either 8 EUR or course credit for participation.Potential participants were recruited via advertisement and the university subject pool, and were screened for fear of heights using a subset of the Acrophobia Questionnaire AQ,  to prediThe virtual environment was rendered in Unreal Engine 4.12  using assets from the Open World Demo Collection and was displayed on a HTC Vive  with a resolution of 1080 pixels × 1200 pixels per eye at 90 Hz, and a 100° field of view. The experiment ran on a Windows 10 64-bit machine with an Intel Core i5-6600k, 16 GB RAM and a Nvidia GTX 970. A Sennheiser HD 439  was used for audio presentation. Physiological signals  were recorded by a Brainproducts V-AMP 16 and the Vision Recorder 1.2 software .presence manipulation by means of sensory realism  and fear manipulation with different situations . Participants were randomly assigned to the between subject factor.A 2 × 3 mixed design was used for the study. Experimental manipulations were For the fear manipulation, two different environments were created: a control situation which exposed participants to a forest environment surrounded by rocks and trees, and a height situation which exposed participants to the edge of a 30 m deep canyon. These VEs were manipulated regarding sensory realism to induce different levels of presence. This was realized by modifying both the visual realism of the VE as well as the auditory content. The low sensory realism condition was derived from the high sensory realism condition by (1) simplifying polygon meshes by scaling down the vertex count of meshes to 5–10% using the Decimate modifier in Blender, (2) reducing texture quality by applying both a Mosaic filter and Surface Blur filter to the textures in Photoshop see , (3) repAfter the arrival in the laboratory, participants read and signed the informed consent. Participants were then equipped with electrodes for heart rate and skin conductance measurement. During a baseline measure of 5 min, participants filled in questionnaires  and read an information letter, which described the concept of presence in VR, and how it would be rated during the experiment. Subsequently, participants were placed in the center of the VR tracking area and helped to put on the head-mounted display and headphones. The actual experiment consisted of the fixed sequential exposure to three situations, which were presented in either their high or low sensory realism version: the control situation (control 1), the height situation (height condition), and again the control situation (control 2). Each trial consisted of a fade-in of the virtual scene, a 1-min exploration phase where participants could look around, and a rating phase where participants were asked to give their fear and presence ratings, followed by a fade-out of the virtual scene. After taking off the head-mounted display, participants filled in another set of questionnaires .Acrophobia Questionnaire  (α = 0.89), Spatial Situation Model  (α = 0.84), Spatial Presence: Self Location  (α = 0.92), Spatial Presence: Possible Actions  (α = 0.89), and Suspension of Disbelief  (α = 0.88). The three remaining subscales Higher Cognitive Involvement, Domain Specific Interest, and Visual Spatial Imagery were not measured because of the length of the full questionnaire and our focus on subscales that measure spatial presence in the narrower sense. The questionnaire therefore comprised 40 items.MEC-SPQ;  is a selFear ratings were assessed by means of Subjective Units of Discomfort Scales (SUDS) ranging from 0 to 100. Presence ratings were assessed using the question “To which extent did you feel present in the virtual environment, as if you were really there?”  with a r1. The sequence of interbeat intervals was processed with the R package phyr62. First, the sequence was segmented  and subsequently baseline corrected, using the phase where participants filled in questionnaires as baseline. Second, the mean heart rate change from baseline (ΔHR in bpm) was calculated for each segment.The electrocardiogram (ECG) was derived using three Ag/AgCl electrodes placed under the right collarbone, on the lower left costal arch (reference electrode), and on the lower left back (ground electrode), recorded at a sample rate of 500 Hz. The ECG was filtered offline with a 50 Hz notch filter and a 2.5 Hz high-pass filter. Detection of R waves and correction of interbeat interval artifacts was done in PeakMan 0.3.0The electrodermal activity (EDA) was derived using two 13/7 mm Ag/AgCl electrodes filled with 0.5% NaCl gel. The electrodes were placed on the thenar and hypothenar of the right hand and the signal was recorded at a sample rate of 500 Hz. Segmentation was done analogously to the ECG signal. Before applying the baseline correction, the EDA signal was added to 1 and logarithmized to control for skewness. The change in SCL from baseline [ΔSCL in log(μS + 1)] was then calculated by computing the mean of each segment.post hoc comparisons . In the ANOVA for presence ratings, one participant had to be excluded due to missing data and in the ANOVA for SCL another participant had to be excluded due to technical problems with the electrodes. The cross-lagged panel model was fitted using the lavaan package  = 2.31, p = 0.026, d = 0.66. Participants in the high sensory realism condition  reported significantly higher presence than participants in the low sensory realism condition . For the MEC-SPQ scores, a one-way MANOVA with the five subscales as dependent variables and sensory realism as independent variable revealed no main effect of sensory realism, Wilks’ λ = 0.94, F = 0.56, p = 0.726  as between factor and the fear manipulation (situation) as within factor.F = 5.70, p = 0.021, F = 13.01, p < 0.001, F = 0.07, p = 0.905, post hoc pairwise comparisons  between situations yield a significant difference between control situation 1 and the height situation, t(46) = −4.36, p < 0.001, a significant difference between the height situation and the control situation 2, t(46) = 3.43, p = 0.004, and no difference between control situation 1 and control situation 2, t(46) = −1.69, p = 0.220. Presence ratings in the height situation were higher than in both control situations.For a causal effect of fear → presence, we expected presence ratings to be higher in the height situation compared to the control situations. The ANOVA showed a significant main effect for the presence manipulation, 0.01 see . For theF = 1.02, p = 0.317, F = 161.63, p < 0.001, F = 0.92, p = 0.350, Post hoc pairwise comparisons  between situations yield a significant difference between control situation 1 and the height situation, t(47) = −12.86, p < 0.001, a significant difference between the height situation and the control situation 2, t(47) = 12.98, p < 0.001, and no difference between control situation 1 and control situation 2, t(94) = −1.18, p = 0.469. Fear ratings in the height situation were higher than in both control situations.For a causal effect of presence → fear, we expected fear ratings specifically in the height situation to be higher in the high sensory realism condition. The ANOVA on fear ratings revealed no main effect for the presence manipulation, 0.02 see . Post hoF = 0.13, p = 0.717, F = 81.10, p < 0.001, F = 0.31, p = 0.731, Post hoc pairwise comparisons  between situations yield a significant difference between control situation 1 and the height situation, t(46) = −10.71, p < 0.001, a significant difference between the height situation and the control situation 2, t(46) = 11.06, p < 0.001, and no difference between control situation 1 and control situation 2, t(92) = −1.61, p = 0.251. SCL values in the height situation were higher than in both control situations.Skin conductance and heart rate were analyzed analogously to the ratings. The ANOVA for SCL revealed no main effect for the presence manipulation, 0.01 see . Post hoF = 0.92, p = 0.341, F = 7.97, p = 0.003, F = 0.21, p = 0.713, Post hoc pairwise comparisons  between situations yield a significant difference between control situation 1 and the height situation, t(47) = −3.05, p = 0.010, no difference between the height situation and the control situation 2, t(47) = 1.35, p = 0.377, and a significant difference between control situation 1 and control situation 2, t(47) = −4.07, p < 0.001. Heart rate in the height situation and control situation 2 were higher than in control situation 1.For the heart rate, the ANOVA showed no main effect for the presence manipulation, 0.01 see . Post hopost hoc exploratory analyses were conducted to take a more in-depth look into the associations between presence and fear ratings.Two r(47) = 0.62, p < 0.001. Within the two groups varying in sensory realism (presence manipulation) the correlations were r(22) = 0.80, p < 0.001 for the group with high sensory realism and r(23) = 0.42, p = 0.038 for the group with low sensory realism, with a significant difference between these correlation coefficients, z = 2.13, p = 0.033.First the bivariate correlation between presence and fear ratings in the height situation for the complete sample was, std = 0.82, p < 0.001, and presence in the control situation 2 was predicted by presence in the height situation, βstd = 0.91, p < 0.001; (2) the regression coefficient of presence in the control situation 1 predicting fear in the height situation, βstd = 0.55, p = 0.002; and (3) the correlation between presence and fear in the height situation, r = 0.72, p = 0.005. In the low sensory realism group, only the autoregressive paths for presence were significant (both p < 0.001). For further visualization of the regression of initial presence ratings predicting later fear ratings in the height situation, the correlation between presence ratings in the first control situation and fear ratings in the height situation were calculated and are displayed in Second, we fitted the presence and fear ratings in cross-lagged panel models, again split by the between-subject factor, to test whether presence and fear ratings would predict ratings in successive trials see , signifiThe present study investigated two research questions: first, whether a manipulation of sensory realism of a VE, i.e., high versus low quality of visual and auditory content, has an influence on experienced presence, and second, whether there is a causal relationship between presence and fear in VR. For this purpose, both presence and fear were manipulated experimentally in VR. Height-fearful participants were immersed into a virtual height situation and a control situation (fear manipulation) with either high or low sensory realism (presence manipulation). During immersion, we assessed ratings of presence and verbal and physiological fear responses.3. However, it remains an open research question, whether VEs that achieved high presence in earlier studies, also induce high presence today . Further research is therefore needed to decide whether costly efforts to increase sensory realism are reasonable to increase presence.The use of highly detailed geometry, increased texture quality, and sound, compared to a low fidelity setup, led to increased presence ratings. This finding is in line with previous research , and theOur study expands previous research on the relationship between presence and fear in VR as our experimental fear manipulation caused an increased sense of presence. Similar effects of fear on presence have been shown for snake phobia by Our study revealed no support of a causal effect of presence on fear as our experimental manipulation of presence did not lead to increased levels of fear in the virtual height situation. Several explanations have to be discussed. First, the strength of the fear response might not be dependent upon presence. If this is the case, putting much effort in creating highly realistic VEs is not necessary for virtual exposure as simpler VEs might be sufficient. Second, effects of presence on fear might only be observable if the manipulation of presence is strong enough. This argument receives support by a comparison of our study’s effect sizes for the manipulation of presence  or quasi-experimental manipulations  of presence have an influence on fear responses.osf.io/8z6gt/.The dataset for this study can be found at DG, MR, IC, and PP contributed to the study concept and design. MR and IC collected the data. DG performed the data analysis and interpretation under the supervision of PP. DG drafted the article and MR, IC, and PP provided critical revisions. All authors approved the final version of the article prior to submission.PP is shareholder of a commercial company that develops virtual environment research systems (VTplus GmbH) for empirical studies in the field of psychology, psychiatry, and psychotherapy. The remaining 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.


Among all antibiotic resistant proteins in the Antibiotic Resistance Genes Database and the Comprehensive Antibiotic Resistance Database, FosB is within 5% of the most number of ARPs identified in Human Microbiome Project reference database but mainly distributed in limited genera, i.e., 122 of total 133 FosB homologues are found from Bacillus and Staphylococcus. Furthermore, these FosB sequences could be divided into three clusters based on their phylogenetic relationship, i.e., two groups of FosB were mainly from Bacillus, and another was mainly from Staphylococcus. Finally, we confirmed that FosB from the group of Staphylococcus presented the highest resistance ability to fosfomycin by in silico and in vitro comparisons. In summary, this study elaborates the specific taxonomic characteristics and resistant abilities of FosB in human microbiota, which might help in developing more promising fosfomycin-like antibiotics.FosB, a Mg Antibiotics are crucial measures in the treatment of infectious diseases. However, the irrational use of drugs and long-term abuse of antibiotics can lead to bacterial resistance. The number of infections caused by multidrug-resistant bacteria has increased during the last decades, and it has been presenting a potential threat to human health . TherefoKnown mechanisms of bacterial resistance to drugs in the human body can be summarized into six major categories: production of inactivated enzymes and modified enzymes , changesfosA  includes most of the publicly available antibiotic resistance proteins (ARPs), which can provide reliable annotations for researchers to investigate the molecular mechanism of bacterial resistance . The ComIn our study, we mainly focused on the unique ARP by comparing the number of homologous genes and taxonomic diversity of ARPs in ARDB and CARD, i.e., FosB exhibited more homologues among all ARPs but was distributed in limited bacteria. The taxonomic distributions of all FosB in human microbiome were investigated systematically, and the representative sequences were selected to compare the structures and fosfomycin resistance. These findings are crucial not only to understand the fundamental principles of bacterial resistance but also to provide more effective strategies to control the proliferation of resistant organisms and even eliminate their resistance.https://www.hmpdacc.org/, contained 1,751 bacterial strains of 1,253 species in September 2014. Basic local alignment search tool (BLASTP)  predicts the genes and related protein sequences of these bacterial genomes. The HMP reference database, (BLASTP)  was usedMaximum likelihood (ML) method in MEGA software (v7.0) was employed in building the phylogenetic tree. It was further trimmed and edited by Dendroscope (v3.4.7).http://bioinf.cs.ucl.ac.uk/psipred/.The alignment among three representative protein sequences was conducted using DNAMAN (v8.0), and colors were modified as needed. The prediction of secondary structures of these proteins were completed by the PSIPRED Protein Sequence Analysis Workbench, https://www.rcsb.org/. The homology modeling was performed using the MOE (v2014.0901). And the molecular docking was completed using Autodock (v4.2.6) to generate 100 docked conformations for each ligand bound to its target (The 14 known 3D structures of FosB homologues and their protein sequences were collected from Protein Data Bank database (PDB), s target . For eacs target . The finfosB genes were cloned into pET28a (+) vector by NcoI/XhoI-digested, respectively. Then the recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells. The recombinant strains were named as FosB-b1, FosB-b2, and FosB-s, respectively.For the expression of these three proteins of FosB, the representative fosB-recombinant bacteria were inoculated into Luria-Bertani (LB) broth containing 50 μg⋅mL-1 kanamycin and incubated at 37°C. The FosB expression of recombinant was induced by isopropyl β-D-thiogalactopyranoside  and then the inoculums were collected for detection of resistant abilities.The three E. coli BL21 (DE3) was used as control strain. The turbidity of all bacterial suspension was adjusted to a bacterial concentration of 0.5 McPherson (about 108 cfu⋅mL-1) by LB broth. A sterile cotton swab was streaked onto the Mueller-Hinton (MH) agar plate and the entire agar surface was drawn. The fosfomycin susceptibility test paper  was applied to the surface of the plate. The diameter of the inhibition zone was measured with vernier caliper after 18 h of incubation at 35°C.Kirby-Bauer susceptibility testing was performed based on the Clinical Laboratory Standards Institute (CLSI) . E. coli600). The reaction system contained 20 μL fosfomycin solutions and 180 μL freshly grown bacteria. The concentration of recombinant bacteria was 106 colony-forming units (cfu)⋅mL-1 . 20 μL of the above cultured solution was used to smear the LB petri dish, and the minimum bactericidal concentrations (MBCs) of fosB-recombinant bacteria were determined by observing colonies growth after culturing at 37°C for 12 h.The resistance to fosfomycin was determined by measuring optical density at 600 nm (ODfu)⋅mL-1 . FosfomyTo identify the distribution of all ARPs, the number of homological sequences of ARPs identified from the HMP reference database was calculated firstly . It was The Shannon Index of ARPs could reflect the diversity of taxonomic distributions of them in ARDB and CARD, i.e., the lower Shannon Index of ARPs indicated the limited distribution of species . Interes133 FosB homologues were identified from the HMP reference database based on the known FosB sequences from ARDB, while only 85 FosB homologues were found in CARD entirely .Firmicutes, and most of them were distributed in two genera. 89 FosB homologues were distributed in Bacillaceae, and 40 homologues were distributed in Staphylococcaceae. Similarly, 61.7% of fosB-containing bacteria belonged to Bacillus, and 30.0% belonged to Staphylococcus  > FosB-b1  > FosB-s   . The unil⋅mol-1) . Thus, tfosB-recombinant strains to express FosB-b1, FosB-b2, and FosB-s protein.To analyse the resistant abilities of the three different FosB to fosfomycin, we formed the E. coli BL21 was used as a control. The inhibition zones of the three fosB-recombinant E. coli was significant smaller than control strain (32.0 mm), and FosB-s (13.7 mm) < FosB-b1 (22.3 mm) < FosB-b2 (23.3 mm)  . Thus, a600 of fosB-recombinant bacteria were measured at different concentrations of fosfomycin. We compared the results of fosfomycin resistance between the concentration of fosB-recombinant bacteria was 107 cfu⋅mL-1  > FosB-b1 (96 μg⋅mL-1) > FosB-b2 (80 μg⋅mL-1)  .-1 fosfomycin, no FosB-b2 colonies were found on the dish with 96 μg⋅mL-1 fosfomycin, and no FosB-s colonies were found on the dish with 144 μg⋅mL-1 fosfomycin, respectively. Together, the FosB-s showed the highest fosfomycin resistance with the MBCs results of FosB-s (144 μg⋅mL-1) > FosB-b1 (112 μg⋅mL-1) > FosB-b2 (96 μg⋅mL-1)  . FurtherSeveral studies have reported that resistance variations are harmful to the environment and human health . In thisBacillus and Staphylococcus. Therefore, it indicated that most of the bacteria in human microbiota might be sensitive to fosfomycin. However, how to control the horizontal gene transfer (HGT) of fosB from these two bacteria to others remains an central issue to fosfomycin resistance.From our results, FosB exhibited one of most homologues among all ARPs in human microbiota, which was consistent with the results from current studies . On the Staphylococcusaureus and Bacillussubtilis (It was reported that FosB had been identified in subtilis , which wsubtilis . BiologifosB might tend to be existing in different genera in other parts of the body, like bone (We also have investigated the prevalence of FosB in the gut microbiota of populations worldwide, based on public metagenome datasets . Howeverike bone , lung (Aike bone .fosB is rarely found in current public gut metagenomic databases, fosB in the environment can still be transferred into human organs. It is worthy to pay more attention to the molecular mechanisms of FosB resistance.Considering the increasing of fosfomycin application  and homoStaphylococcus and Bacillus genera. Moreover, they were divided into three clusters based on their phylogenetic relationship. Overall, the resistant activity comparisons showed that FosB from Staphylococci was more resistant to fosfomycin than from other bacteria, which might be a potential risk to become a pervasive resistance bacteria.In summary, this work presented a macroscopic analysis of ARPs in ARDB and CARD and analyzed the taxonomic distribution of FosB. Our results indicated that although FosB had more homologues, it was mainly distributed in All data generated in this manuscript are included in additional files.JL conceived and designed the study. ZS, XW, XZ, SJ, YL, OA, LQ, PL, and JL collected data and performed the analyses. JL, ZS, and XW interpreted the data and wrote the manuscript. All authors read and approved the final 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.


CIViC has seen increased adoption by, and also undertaken collaboration with, a wide range of users and organizations involved in research. To enhance CIViC’s clinical value, regular submission to the ClinVar database and pursuit of other regulatory approvals is necessary. For this reason, a formal peer reviewed curation guideline and discussion of the underlying principles of curation is needed. We present here the CIViC knowledge model, standard operating procedures (SOP) for variant curation, and detailed examples to support community-driven curation of cancer variants.Manually curated variant knowledgebases and their associated knowledge models are serving an increasingly important role in distributing and interpreting variants in cancer. These knowledgebases vary in their level of public accessibility, and the complexity of the models used to capture clinical knowledge. CIViC (Clinical Interpretation of Variants in Cancer -  ExamplVariant Aliases are alternative names, descriptions, or identifiers that differ from the primary CIViC Variant Name. These terms are manually curated and are incorporated into the search fields within the CIViC interface. Curators should include one or more aliases such as protein changes on alternative transcripts , dbSNP IDs [bSNP IDs , COSMIC bSNP IDs  or otherVariant Summary is a user-defined summary of the clinical relevance of the specific CIViC Variant. The Variant Summary should be a synthesis of the existing Evidence Statements for the CIViC Variant. When curating a Variant Summary, basic information on recurrence rates and biological/functional impact of the variant may be included, but the focus should be on the clinical impact. For Predisposing Variants, any appropriate American College of Medical Genetics (ACMG) evidence codes (ACMG-AMP 2015 criteria [The criteria ) that arVariant Type(s) are used to classify variants by Sequence Ontology terms [http://www.sequenceontology.org/browser/obob.cgi) is recommended to identify appropriate terms. When choosing variant types, selection of multiple terms is supported in order to capture both functional and structural effects of the variant  by the values assigned to Evidence Level . The Variant Evidence Score is a relative measure of the total amount of curation in the database for a specific CIViC Variant and does not take into account conflicting evidence.The Primary and Secondary Coordinates for each CIViC Variant are manually curated and verified. Each Variant is assigned representative genomic coordinates  for the assigned reference assembly . Primary Coordinates are generated for all Variants. Secondary Coordinates are utilized for structural variants involving two loci . Specific guidelines for choosing representative coordinates and transcripts are described below.Allele Registry [Although multiple genomic changes can often lead to functionally equivalent alterations , CIViC uses representative coordinates to provide user-friendly variant context rather than enumerate all possible alterations that could cause the Variant. When choosing a representative CIViC Variant, Curators should use the most common or highly recurrent alteration observed , and Curators should always include the transcript version number . There is rarely only one correct transcript. Representative transcripts must contain the variant but are otherwise chosen based on priority criteria such as: wide use in the literature, having the longest open reading frame or most exons, containing the most common exons between transcripts, or having the widest genomic coordinates  Sequence Variant Nomenclature (HGVS Expressions) to describe sequence variation in genomic, RNA, coding DNA, and protein coordinates [ClinVar IDs for each CIViC Variant. ClinVar IDs and HGVS nomenclature must be entered individually in the Variant editing interface and may capture ClinVar IDs and HGVS entries not described by the representative coordinates. Manual entry is required  to permit entries for complex or Categorical CIViC Variants and to support alternate transcripts and reference build versions . EIDs follow a structured knowledge model with 12 required fields  with additional optional fields . Based on the Evidence Type, additional required or optional fields become available  and Drug Interaction Type if multiple drugs are involved). Figure At the core of the CIViC knowledge model lies the CIViC Gene Name and Variant Name are required for EID submission. The Gene Name field will auto-fill using type-ahead search for genes in the Entrez database or their associated Aliases. The Variant Name will also auto-fill based on existing CIViC Variants. User-defined variants are also permitted if the desired Variant record does not already exist. To prevent redundancy, it is recommended that the Curator browse existing Variant Names for the gene of interest and consider possible variant synonyms before creating a new CIViC Variant.A Source Type and Source ID, which link the EID to the original publication supporting clinical claims. Currently, CIViC accepts publications indexed on PubMed or abstracts published through the American Society of Clinical Oncology (ASCO). If a PubMed Source Type is selected, the Curator can then enter the PubMed ID, which can be verified by comparing the desired source to the abbreviated citation that is automatically generated below the PubMed ID field. If an ASCO Source Type is selected, the ASCO Web ID should be entered into the source ID field. Additionally, Clinical Trial Registry Number(s) are automatically linked via the PubMed database, when available.Each Evidence Item must be associated with a Variant Origin categorizes the variant based on method of acquisition. Options for this field include: Somatic, Rare Germline, Common Germline, Unknown, or N/A. The Variant Origin should be entered as somatic if the variant is only found in tumor cells , and is not expected to be inherited or passed to offspring. The Variant Origin is not applicable (N/A) in some circumstances, particularly in variants that involve differences in expression, methylation, or other post-translational modifications  database [http://disease-ontology.org/faq/).The database . The fieEvidence Level describes the robustness of the study supporting the Evidence Item. Five Evidence Levels are currently available: Validated association (A), Clinical evidence (B), Case study (C), Preclinical evidence (D), and Inferential evidence (E)  association described by the Evidence Item’s clinical summary. Five Evidence Types are currently supported: Predictive , Diagnostic, Prognostic, Predisposing, and Functional. Each Evidence Type describes the clinical or biological effect a variant has on the following: therapeutic response (Predictive), determining a patient’s diagnosis or disease subtype (Diagnostic), predicting disease progression or patient survival (Prognostic), disease susceptibility (Predisposing), or biological alterations relevant to a cancer phenotype  , but include sufficient experimental detail to interpret and evaluate the evidence without repeating the original text or using domain-specific acronyms or colloquialisms. Such details include the type of study , controls used, outcomes measured, the number of individuals involved and relevant statistical values . Data constituting protected health information (PHI) should not be entered in the Evidence Statement field.The Drug Names field will become available. Multiple drugs can be added to a single Evidence Item, requiring a Drug Interaction Type  that describes the relationship of these drugs in the study. The Drugs and Drug Interaction Types should be explicitly stated in the source supporting the Evidence Item and not inferred by the Curator. Trade names should not be used for Drugs. When available Drug Names are taken from the NCI Thesaurus  database [When additional phenotypes not captured by the Disease field alone are indicated, database  can be aThe last field in the Evidence Item submission form permits free-form text for additional comments about the Evidence Item. For example, Curators can call an Editor’s attention to a particular comment using macro notation  /Version(s)  used by the Curator to assemble the Assertion. Important specific details from practice guidelines (e.g. NCCN) should be included in the Summary, including disease stage, and in the case of predictive Assertions, treatment line  which practice guidelines recommend.Each Assertion requires a one-sentence Supporting Evidence grid allows users to associate Evidence Items with Assertions. This collection of Evidence Items should cover the important clinically relevant findings for the CIViC Variant in the context of a specific cancer. For Predictive Assertions, the collection of Evidence Items should also consider the Drug(s) and their Drug Interaction Type. Assertions do not require Evidence Items for development; however, complete (revised and accepted) Evidence Items must be added to the Assertion before it can be accepted by CIViC Editors . Citation of CIViC has also been used as supporting evidence in ClinVar submissions (See ClinVar variant 523644). Submission of CIViC Assertions to ClinVar also requires documented curation protocols for variant tiering to obtain 1-star status. Finally, efforts to obtain FDA recognition for the CIViC database and ClinGen Somatic curation process are underway and would benefit from documented curation procedures for CIViC. Therefore, the need for a clear and peer reviewed CIViC SOP is apparent. We hope that this work may build on published SOPs for evaluation of evidence and curation in this space [While an earlier publication has introduced the CIViC database , no publis space , 32 and Implementation of this SOP will promote standardization of data across CIViC, which is intended as a platform for the generation and dissemination of a large volume of structured data. Utility of large data sets is highly dependent on standardization of the manner in which data is encoded into the format , 34. TheMultiple efforts exist to aggregate, formalize and structure cancer variant data, or provide classification and clinical tiering of somatic or germline variation. Publication of SOPs can help clarify interrelation between these different efforts. For instance, considering the guidance described here for Predictive (Therapeutic) annotations in CIViC (Additional file Additional file 1. Supplementary Materials, contains supplemental figures and tables outlining curation SOP guidelines illustrated by workflow figures, tables, and screenshots from the CIViC interface.


Schistosomiasis is one of the most prevalent parasitic diseases in low- and middle-income countries (LMICs), being regarded as a neglected tropical disease in sub-Saharan Africa. Praziquantel is the conventional treatment recommended for schistosomiasis in mainstream healthcare systems. In many poor settings, while many people reportedly use both traditional medicine and public sector mainstream healthcare systems, little is known if those infected with schistosomiasis use both African traditional and prescribed antischistosomal medicines. This review aims to map evidence of the concomitant management of schistosomiasis by traditional health practitioners (THPs) and health care professionals (HCPs) in communities with a high prevalence schistosomiasis infection in LMICs.Guided by Arksey and O’Malley scoping review framework and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), we will map the evidence from relevant studies dating from 2007 to 2019 published in LMICs. An electronic keyword search of the following databases will be conducted: PubMed, Cochrane Library, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and MEDLINE via EBSCOhost, Google Scholar, and WILEY online Library. Peer-reviewed articles, gray literature sources, and reference lists will be included to identify eligible studies. Following title screening, two reviewers will independently screen the abstracts and full texts. Any study that focuses on managing schistosomiasis will be included. The data will be analyzed using thematic analysis with the help of NVIVO software version 12, with the Mixed Method Appraisal Tool (MMAT) being used to assess the quality of the included studies.This review will map the evidence in the literature of the concomitant management of schistosomiasis by THPs and HCPs in communities with a high prevalent infection in LMICs. The review findings will be important for policy makers across the healthcare continuum and be used to inform stakeholders’ consensus process to explore the development of a generic set of patient-centered quality indicators that are applicable to multiple care settings. It will also identify research gaps in schistosomiasis management in LMICs and provide direction for future research. The results will be disseminated through a peer-reviewed publication and presented in relevant conferences.CRD42017078198PROSPERO The online version of this article (10.1186/s13643-019-1088-3) contains supplementary material, which is available to authorized users. Infectious diseases are both a major public health concern and a socioeconomic problem in tropical regions within most low- and middle-income countries (LMICs), where neglected tropical diseases (NTDs), such as schistosomiasis, are cause for concern . These NPraziquantel, the recommended treatment against all forms of schistosomiasis , is inexZea mays with Glossonema boveanum were specific for intestinal schistosomiasis, while Cissus quadrangularis and Stylosanthes erecta were reported for the first time in Mali to be used against urogenital schistosomiasis [Alternatively, traditional medicine has demonstrated its contribution to managing schistosomiasis . In an esomiasis . A rangesomiasis . A studysomiasis .Due to the unavailability of conventional therapy, or the expensive cost of praziquantel and other antischistosomal medicines, people in rural areas may not have access to modern treatment for schistosomiasis, which results in them using traditional medicine , 19. ColThis review aims to map the evidence of the concomitant management of schistosomiasis by traditional health practitioners and health care professionals in communities with a high prevalence of schistosomiasis infection in LMICs. Thus, the purpose of this review is to provide evidence to enable the implementation of policies and guidelines to manage schistosomiasis with traditional medicine contributing to primary healthcare.This review will be based on the framework originally proposed by Arksey and O’Malley  and furtWhat is the evidence of the healthcare seeking behavior among individuals in communities with high prevalent infections of schistosomiasis?What is the evidence of the use of traditional, complementary, and alternative medicine for managing schistosomiasis?Is there a bidirectional referral of patients between THPs and HCPs for managing schistosomiasis?The following research questions are formulated to guide the review in meeting its aims and objectives. The general research question is: “What is the evidence about the concomitant management of schistosomiasis by THPs and HCPs in communities with a high prevalence schistosomiasis infection?” The specific research questions are as follows:The Population, Intervention, Comparison and Outcomes (PICO) for the research questions has been used to break down the clinical questions into searchable keywords , MEDLINE, Google Scholar, Cochrane Library, WILEY online Library, and grey literature. Reference lists of included studies will also be searched. The keywords search will include the following: schistosomiasis; neglected tropical diseases; collaboration; investigation, traditional health practitioners; health care professionals; access; low- and middle-income countries, schistosomiasis, or collaboration or traditional medicine or neglected tropical diseases (NTDs) or natural products or leads. The Boolean search terms (AND and OR) and MeSH terms  will be included in the search. Gray literature will be identified through website source links in references. Peer review studies and gray literature reporting on the evidence of managing schistosomiasis by THPs and HCPs in LMICs published between 2007 and 2019 will be included. Authors of primary studies or reviews will be contacted for further information or to access missing studies where relevant. If the authors do not respond, then their publications will be excluded.Evidence of the population of interest in this review (THPs and HCPs)Evidence of the intervention (schistosomiasis treatment and co-morbidities)Evidence of the comparison of treatment by THPs and HCPsEvidence on the outcomes of managing schistosomiasisArticles published in English and French will be included.The following criteria provide a guide to clearly understand what is proposed by the reviewers and, more importantly, a guide for the reviewers themselves upon which to base decisions about the sources to be included in this review . As explArticles published before 2007.Articles not addressing the treatment of schistosomiasis and comorbidities.Articles not addressing integration and collaboration between traditional medicine and conventional medicine.Studies that are not reporting evidence from LMICsThe search strategy for this review aims to be comprehensive to identify both published and unpublished (grey literature) primary studies and reviews , 26. A pPublications duplicated in the research results will be treated as a single study for the review. To maintain transparency in the review selection process, a PRISMA Flow Diagram will be followed in each stage of the selection process. In addition, a list of the studies excluded during the full-text review will be documented as an Additional file For the quality appraisal of the included studies, we will use a Mixed Methods Appraisal Tool (MMAT), version 2018 . The MMAWe will conduct data extraction to enable a logical and descriptive summary of the search results , “duration of intervention”, “aims”, “methodology adopted”, “key findings” (evidence established), and “gaps in the research”. For each category reported, a clear explanation will be provided.Throughout this research, we will examine the above-mentioned themes and critically identify their link to the research question. Reviewers will analyze the significance of the findings according to the aim of this research and their implications for future studies, practice, and policy.This review is part of larger studies evaluating the management of schistosomiasis by THPs and HCPs in South Africa. There is little evidence to support the current South African policy to manage schistosomiasis. This systematic scoping review aims to build on the work of the existing Cochrane study  by furthArticles not addressing integration and collaboration between traditional and modern medicine will not be part of this research, as it intends finding a way to manage schistosomiasis via bidirectional referral of patients between THPs and HCPs. Due to the prevalence of schistosomiasis, a collaboration of THPs and HCPs could help to manage the infection, as traditional medicine is available to most people in LMICs, with not less than 80% of people worldwide depending on it .It is anticipated that this review will identify gaps in the current literature on this topic and provide direction for future research in this area of this review. The summary and dissemination of these research findings may be of interest for policy makers and stakeholders (practitioners and consumers) who are involved in the NTDs management, especially those involved in schistosomiasis management using either the mainstream healthcare systems or African traditional medicine.Additional file 1: Results of pilot database search. (DOCX 14 kb)Additional file 2:PRISMA-P  2015 checklist: recommended items to address in a systematic review protocol. (DOCX 22 kb)Additional file 3:Sample extraction form. (DOCX 15 kb)


When students from two or more professions learn about, from, and with one another in joint learning activities, interdisciplinary perceptions and attitudes improve and collaborative knowledge and skills increase.3 Most, more or less, hinge on the development of competencies deemed necessary for effective collaborative practice, such as communication and ethical values.The contribution of these skill sets to the positive and productive function of patient care teams is both apparent and universal. For example, abilities to listen and constructively consult, discuss, and debate are obvious elements for collegial interactions. Respecting patient dignity and maintaining confidentiality reflects a commitment to professional conduct and engenders trusting relationships. Such behaviours and attitudes are further recognized as collective competencies given they have been adopted by many North American health professional training programs as discipline-specific educational outcomes. By both accounts, these principles also apply to the scholar competency role.Different frameworks exist to guide integration of IPE programming into health professional curricula worldwide.4Similarly, the American Association of Medical Colleges outlines an entrustable professional activity milestone whereby trainees ‘form clinical questions and retrieve evidence to advance patient care’.5Albarqouni et al recently enlisted over 200 multidiscipline clinicians and academics from 28 countries to agree upon a set of evidence-based practice competencies for health professional teaching and learning.6 Through Delphi survey and consensus processes,they arrived at a set of 68 core competencies further organized into introductory concepts and five main evidence-based practicesteps including: ask, acquire, appraise and interpret, apply, and evaluate.While the authors acknowledge somecompetencies likely require modification to suit specific needs of any given discipline, most are indeed fundamental skills broadly applicable across professions. Examples include converting clinical questions into structured, answerable formats; constructing and carrying out an appropriate search strategy; distinguishing evidence-based from opinion-based clinical practice guidelines; explaining the evidence to patients and integrating their preferences into decision-making processes; and managing clinical decision-making uncertainty in practice. The consensus statement represents an important step towards unifying expectations of evidence-based practice. The details can guide instructional and assessment design and delivery in health professional curricula and for a number of programs, these may fall within the scholar competency role  .From 50 years agowhen McMaster University launched a novel medical program where students first learned about patient problems concurrent with epidemiology and biostatistics, Canadian physicians-in-training today are meant to ‘identify pertinent evidence, evaluate it using specific criteria, and apply it in their practice’ as part of the scholar competency role.nt care) .7 That is, how are these competencies truly interprofessional in nature and not simply common skills expected by many different professions? First, this set of competencies is an acknowledgement that a majority of professionals employ evidence-based practice in patient care. Although decision-making may occur within the discrete scope of a profession’s practice, we can be assured of a common language when this care is informed by reported literature, such as randomized controlled trials, meta-analyses, or clinical practice guidelines. Like the fundamental aspects of interprofessional communication  these competencies represent an opportunity for standardization of practice principles across patient care providers. Furthermore, when teams of various disciplines convene to develop treatment protocols or care pathways, there is a shared understanding of how the evidence under consideration may be interpreted.Clinicians may enlist similar approaches to navigate incomplete available data or ambiguous study findings to make treatment choices.If as Nandiwada&Kormos further contend, this work substantiates the existence and importance of evidence-based practice competencies across health professions, howdo they expressly promote shared or collaborative care?Interprofessional competencies are meant to promote collaborative care that ultimately improves patient outcomes. Integration of evidence-based competencies into shared decision-making clearly has the potential to do so. Patient adherence may be enhanced when care providers can clearly explain the data underpinning the rationale for the selected treatment and offer realistic estimates of potential risk. Clinicians who can interpret and synthesize a wide array of literature may have greater abilities to individualize care while also incorporating patient preferences. In this regard, optimal evidence-based practice must be enabled by the communication and collaboration skills endorsed by both profession-specific and interprofessional competency frameworks. Just as effective team-based care must draw upon the collective contributions of its diverse members, quality healthcare also relies on this complement of a clinician’s knowledge, skills, and behaviors.


Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues  together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research. Alongside with the exciting advancements in genomic research in the recent years, biomedical research field also witnesses the significant advancement in proteomic research. With increasing accumulation of biological data at both genomic and proteomic levels, using bioinformatic tools to analyze these data has become the focus of biomedical research in the perceivable future. Consequently, the success of bioinformatical research raises demand for more and better quality of data from the biological and biomedical research community, a task can only be achieved through technique advancement at genomic and proteomic levels.Mass spectrometry (MS) and immunoblot analysis are two prevailing techniques of protein analysis presently. MS has dominated the proteomic research in the recent years to enable analysis of thousands of individual proteins simultaneously. The immunoblot-based techniques, including western blot and dot blot, on the other hand, have also played a significant role in the protein research even since its invention1234568We have developed an immunoblot method suitable for population studies with the key characteristics of being convenient, high throughput, quantitative, and suitable for absolute determination of protein content, and named this technique quantitative dot blot analysis (QDB)All animal procedures were carried out in line with Binzhou Medical University Animal Use Directive and approved by the ethical review board of Binzhou Medical University.2, 10 mM Na2P2O7, 1% Triton X-100, 10% glycerol), supplemented with protease and phosphatase inhibitors .Take 50 mg mouse tissue into a 1.5 mL Tube. Add 200 µL lysis buffer  and take out 1 µL for protein concentration assay with BCA kit. Note: IgG free BSA is used as a negative control and the standard protein is used as a positive control. The specificity of the antibody is demonstrated by showing one band of right size using the western blot analysis.Prepare sample lysates (20 µg) in section 1.4, IgG free BSA (20 µg), standard protein (600 pg), for western blot analysis. 2O. Dilute the protein to a concentration of 1,200 pg/µL.Dissolve the standard protein with ddHDilute the concentrated protein from the prepared samples in section 1.4 to 4 µg/µL.2O. Dilute the protein to a concentration of 1,200 pg/µL and 4 µg/µL.Dissolve the BSA with ddHTable 1 and Table 2.A series of dilution of standard protein and prepared samples were prepared with the guidance of Mix 10 µL dilution standard protein or dilution prepared samples and 10 µL 2x loading buffer  together.Table 1. Dilution Scheme for standard protein curve.Table 2. Dilution Scheme for prepared samplesHeat the mixtures at 85 °C for 5 min.Sample applicationSupport the QDB plate to avoid the bottom of the plate touching the surface of the table. For instance, use an empty pipette tip box as support.Load up to 2 µL of the sample to the center of the membrane at the bottom of the individual unit of the QDB plate.Drying the plateLeave the loaded QDB plate for 1 h at room temperate (RT) or as an alternative leave the loaded plate at 37 °C for 15 min in a well-ventilated space.Blocking the plateDip the QDB plate in the transfer buffer  and gently shake the plate for 10 s.Rinse the QDB plate gently with TBST  three times, and then wash the plate for 5 min in TBST under constant shaking.Block the QDB plate with blocking buffer (5% nonfat milk in TBST (100 µL/well) for 1 h under constant shaking.Primary antibody incubationDilute the primary antibody in the blocking buffer at a chosen concentration (from 1: 500 to 1:5 000), and add 100 µL to each individual well of an ordinary 96 well plate. Insert the QDB plate into the 96 well plate and incubate the combined plates either for 2 h at RT or overnight at 4 °C under constant shaking.Alternatively, place the QDB plate inside a box, and fill the box with blocking buffer 2 to 3 mm above the membrane portion of the plate if the same antibody is used for the whole plate. Add the primary antibody at the chosen concentration, and incubate the plate either for 2 h at RT or overnight at 4 °C by under constant shaking.Rinse the plate gently with TBST for three times before it is washed with TBST for three times, each time for 5 min under constant shaking.Secondary Antibody incubationDilute the secondary antibody in the blocking buffer at the chosen concentration , and either aliquot 100 µL/well into a 96 well plate or in a box as described in section 4.4.2, and then incubate the QDB plate inside either the loaded 96 well plate or the box for 1 h at RT under constant shaking.Rinse the QDB plate gently 3 times with TBST, then wash the plate for 3 times, 5 min each with TBST under constant shaking.QuantificationPrepare enhanced chemiluminescence substrate (ECL) by following the manufacturer's instructions.Aliquot ECL substrate into a 96 well plate (100 µL/well) and insert the QDB plate inside the 96 well plate for 2 min under constant shaking.Remove the QDB plate from the 96 well plate and shake briefly to remove the excess liquid. Place the plate onto a white microtiter plate.Note: Make sure to use the white, non-transparent microtiter plate as an adaptor to avoid any interference from the plate. Make sure to choose "plate with cover" to avoid jamming the machine when combined plates (QDB plate+ 96 well plate adaptor) are placed inside the microplate reader.Turn on the microplate reader, and select "plate with cover" on the user interface before placing the combined plates (QDB plate + white plate adaptor) inside the microplate reader for quantification. The determination of the absolute amount of any protein including the CAPG protein in mouse tissue requires both a specific antibody and a purified protein as standard. The linear range of the analyses of both the protein standard and the lysates also needs to be established prior to any large-scale analysis. The linear range of the antibody is highly dependent on the antibody per se, and the appropriate dilution range of a specific antibody need to be verified by each individual user.A). The anti-CAPG antibody was specific against lysates from mouse spleen, heart, muscle, and prostate (one band detected). Non-specific bands were observed in lysates from kidney and liver. However, in kidney lysate, the non-specific bands were significantly weaker than the specific band, which suggests the antibody is suitable for kidney tissue analysis. Next, the linear range of the protein standard and the amount of lysates for analyses were determined by two side by side dose curves. A commercially available CAPG protein was serially diluted as indicated in Table 1 based on our past experiences, and the tissue lysates of the mouse kidney, spleen and prostate were also diluted based on the amount of total protein in the lysates determined by a BCA total protein determination kit. Two dose studies were performed side by side and plotted together in Figure 1B. The appropriate amount of lysates of mouse kidney, spleen and prostate were chosen based on the QDB signals measured by the microplate reader in arbitrary unit. The original reading for this experiment is shown in Table 3.We first determined the specificity of the CAPG antibody in mouse kidney, spleen and prostate tissues using western blot analysis with the negative control (IgG free BSA) and positive control  A. The anC with the original reading shown in Table 4.In the last step, the serially diluted CAPG protein standard and the lysates from mouse kidney, spleen and prostate were loaded onto the QDB plate. In this case, we chose to load 0.3 µg/sample for lysates from mouse kidney and spleen and 1 µg/sample for lysates from mouse prostates analysis, as at these levels, the QDB reading was at least 20-fold over the background while well within the linear range of the analysis based on the dose curve of both the lysates and the protein standard. The plate was subjected to the described QDB protocol before the plate was quantified directly by the microplate reader. Using the serially diluted purified CAPG protein, we could establish a dose-response curve, the equation, and R2 by simple regression analysis using available software (e.g. Microsoft office excel). The QDB signals from lysates of mouse kidneys, spleens and prostates were converted to the absolute amount of CAPG protein in these lysates using the established equation, and the results were corrected by the total protein amount, in this case, 0.3 µg for lysates from mouse spleens and kidneys, and 1 µg for lysates from mouse prostates, for the final concentration of CAPG protein in these tissues as pg/µg. The results are shown in Figure 1. Representative result of a QDB analysis to determine the absolute CAPG levels in three mouse tissues . A. Examining the specificity of rabbit anti-CAPG antibody using mouse tissue lysates prepared from Spleen, Heart, Muscle, Kidney, Liver, and Prostate using western blot analysis with the negative control (IgG free BSA) and positive control . B. defining the linear range of QDB analyses of rabbit anti-CAPG antibody using lysates prepared from prostate, kidney, spleen, and using purified recombinant CAPG protein standard. The results were an average of triplicates. C. Absolute determination of CAPG levels in lysates prepared from mouse kidneys, spleens, and prostates. Each bar represents one tissue from an individual mouse. The results were the average of triplicate. Please click here to view a larger version of this figure.Table 3: Result from microplate reader of the dose studies using both serially diluted, pooled lysates from mouse spleens, kidneys, and prostates and purified recombinant CAPG protein.Table 4: Result from microplate reader of the QDB analysis of the absolute CAPG level in mouse kidneys (8), spleens (4) and prostates (5) using a recombinant CAPG protein as standard. Among all the current available immunoassay techniques of protein analysis except ELISA, QDB analysis is the only method to achieve absolute content of a specific protein at cellular and tissue levels in a high throughput format. Although with stable isotope-labeled standard, mass spectrometry is able to achieve absolute quantification of a few proteins, this method is not yet designed for high throughput performance. In this study, we demonstrated the process of QDB analysis to achieve absolute determination of CAPG protein level in mouse tissues including kidney, spleen, and prostate. CAPG levels were found to be between 200 to 360 pg/g in mouse kidney tissues, 460 to 650 pg/g in mouse spleens, and 330 to 390 pg/g in mouse prostate tissues.Compared to ELISA, QDB analysis requires minimum efforts to be developed in a regular lab. First, the QDB plate is a based on a nitrocellulose membrane to eliminate the coating step in ELISA. Second, QDB analysis only requires one instead of two specific antibodies in a sandwich ELISA. Third, the high binding capacity of nitrocellulose membrane as compared to the ELISA plate surface also allows the membrane to withstand the stringent washing steps typically used in the immunoblot analysis to reduce the background interference. This feature is very useful in analyzing complicated lysates prepared from cells and tissues. In contrast, due to the relatively low binding capacity of ELISA plates, the reduction of the background when analyzing complicated cellular and tissue lysates becomes a real challenge in the developmental process.A, CAPG antibody is specific when analyzing lysate from mouse kidney, spleen, and prostate, and yet becomes non-specific when analyzing mouse liver. In fact, we routinely found one antibody to be specific for one tissue type, but not always specific to other tissue type from the same species. Thus, prior western blot analysis is necessary to ensure the specificity of the QDB analysis. In fact, the relative specificity of the antibody may be the cause of false results often associated with ELISA analysis, as no matter how much effort the manufacturing company may have spent to ensure the specificity of the assay, they cannot exhaust all the possible types of samples the users may choose to analyze using their ELISA products.QDB analysis can be adopted easily in any lab with the access of a specific antibody. However, it is important to mention that the specificity of the antibody is a relative term, limited by factors including the species, the tissue types and cell types. As shown in Figure 1As with all immunoassays, a potential problem with the QDB analysis method is the lack of consistency of the quality of the commercial antibody. Even if the apparent quality of an antibody from the same company  is used, there could be large differences from one purchase to another due to batch-to-batch variability. Therefore, unless full confidence is attained in the quality of the antibody available, re-characterizing the antibody upon every purchase is important.In summary, we provide here a detailed protocol and an example when using the QDB analysis method to achieve absolute quantitative determination of a specific protein at the tissue level. We show that the QDB analysis method is a convenient tool for anyone who is interested in high throughput, quantitative immunoblot analysis. Its ability to achieve absolute determination of the content of a specific protein at a cellular and tissue level also distinguish this technique from traditional immunoblot techniques. This feature allows for the combination and comparison of results from multiple analyses, a step necessary especially in larger population studies, and the realization of relevant association studies at the protein level in the near future.The authors Yunyun Zhang, Wenfeng Zhang and Jiandi Zhang are employees of Zestern Biotechniques that produces QDB plates used in this Article. Wenfeng Zhang and Yunyun Zhang declare conflict of interests, and Jiandi Zhang has filed patent applications. The others claim no competing interests.


Bacillus and Candida were identified as the dominant bacteria and fungi genus, respectively. Furthermore, nineteen strains were selected from the dominant species of Douchi samples, including 14 Bacillus strains, 2 Staphylococcus strains, 1 Enterococcus strain and 2 Candida strains, and their BAs formation and degradation abilities were evaluated. B. subtilis HB-1 and S. pasteuri JX-2 showed no BAs producing ability, and B. subtilis GD-4 and Candida sp. JX-3 exhibited high BAs degradation ability. Finally, fermented soybean model analysis further verified that B. subtilis HB-1 and S. pasteuri JX-2 could significantly reduce BAs. This study not only contributed to understanding the BAs formation mechanism in Douchi, but also provided potential candidates to control the BAs in fermented soybean products.Biogenic amines (BAs) have been reported to threaten the Douchi safety, while the BAs formation mechanism and corresponding control method have not been clarified for Douchi. The present study aims to investigate the microbial contribution to BAs in Douchi, and to find the beneficial strain for BAs control. Firstly, the BAs profiles of 15 Douchi samples were analyzed, and common 6 kinds of BAs were detected from different samples. All the samples showed the total BAs contents within the safe dosage range, while the histamine concentrations in 2 samples and β-phenethylamine in 6 samples were above the toxic level. Then, the bacterial and fungal communities were investigated by high-throughput sequencing analysis, and  BAs can be categorized into aliphatic (putrescine and cadaverine), aromatic (tyramine and β-phenethylamine) and heterocyclic amines (histamine and tryptamine) based on their chemical structures3. Recently, BAs have been broadly reported in various fermented foods, such as cheese, beer, wine and fermented soybeans, which shows potential harmful risk for human health10. BAs at low concentrations are essential for many physiological functions, while high concentrations of BAs can cause some deleterious effects11. Histamine can cause adverse neurological, gastrointestinal, circulatory and respiratory symptoms, such as headache, nausea, hot flushes, skin rashes and intestinal problems12. Tyramine, tryptamine and β-phenylethylamine have been confirmed to be implicated in hypertensive crises and dietary-induced migraine13. Therefore, evaluation and control of BAs are highly important to guarantee the safety of fermented food.Biogenic amines (BAs) are organic nitrogenous compounds with low molecular weight, and they are formed during normal metabolic processes in various organisms16. The BAs have been reported as potential toxic substances in commercial Douchi19, thus evaluation and control of the BAs are valuable for the safety of Douchi. The BAs are mainly generated through decarboxylation of amino acids by various microbes, and putrescine, cadaverine, tyramine, β-phenethylamine, histamine and tryptamine were generated from ornithine, lysine, tyrosine, phenylethylamine, histidine and tryptophan, respectively20. Moreover, putrescine can also be formed through deimination of agmatine21. Different microbes may excrete different amino acid decarboxylases to produce specific BA, and some can also produce amine oxidases to degrade BAs25. Hence, the relations between BAs and microbes are complicated. Using the high BAs-degrading strains or the low BAs-producing strains as the starter cultures will be beneficial to control the Douchi BAs.Douchi is a traditional fermented soybean product commonly consumed as flavouring agents in China, Japan, and other Asian countries16. The microbial diversities in commercial Douchi have been investigated using traditional culture method, denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing28, and the Bacillus species were identified as the dominant bacteria in Douchi27. However, the microbial contributions on BAs formation in Douchi have not been clarified, and the efficient starter cultures for BAs control have not developed. In this study, BAs contents and microbial communities in Douchi samples from different regions were investigated, and the BAs producing and degradation abilities of representative strains were evaluated to elucidate the microbial contribution and select beneficial candidate for BAs control.Generally, Douchi is produced by spontaneous fermentation with an uncontrolled manner, and various microbial communities are formed at different environment conditions2HPO4 2 g/L, MgSO4 0.2 g/L, MnSO4 0.04 g/L, Tween 80 1 g/L, pH = 5.7), and SDB medium , and 1.5% agar was added to prepare solid medium.Fifteen fresh Douchi samples were purchased from 15 different regions in China, named JXJJ (Jiangxi), GXWZ (Guangxi), GDYJ (Guangdong), SXHZ (Shanxi), YNKM (Yunnan), GZTR (Guizhou), HNLY (Hunan), HBYC (Hubei), CQ (Chongqing), GSLN (Gansu), SCCD (Sichuan), TJ (Tianjing), ZJJS (Zhejiang), JSXZ (Jiangsu) and HNLB (Henan). All samples were collected in three replicates, and stored at 4 °C for further analysis. The media used this study included LB medium , MRS medium  and 806 R (GGACTACHVGGGTWTCTAAT) were designed according to the V4 region of bacterial 16S rRNA gene, and ITS1 (TCCGTAGGTGAACCTGCGG) and ITS2 (GCTGCGTTCTTCATCGATGC) were designed based on the ITS1 region of the fungal internal transcribed spacer (ITS).The total DNA was extracted from each Douchi sample by a SDS-based DNA extraction method previously describedet al. and Kozich et al.31, respectively. The bacterial 16S rDNA and fungal ITS genes fragments were amplified using the total DNA, primers, and Thermo Scientific® Phusion High-Fidelity PCR Master Mix , purified by the QIAquick PCR Purification Kit . Then, purified amplicons were employed for DNA library construction using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina). At last, DNA libraries were evaluated using Qubit@ 2.0 Fluorometer (Thermo Scientific) and real-time PCR, then sequenced on the MiSeq platform at Beijing Novogene Bioinformatics Technology Co., Ltd. All experiments were conducted in three replicates.The Miseq sequencing for bacteria and fungi followed the protocols described by Caporaso 32, and low-quality sequences were discarded using QIIME. Clean paired sequences retained for each sample were analyzed using the UPARSE pipeline to generate operational taxonomic units (OTUs) and select representative sequences at 97% similarity33. PyNAST alignment and ribosomal database project (RDP) assignment were carried out based on the latest Green genes database34. Resampling was performed according to the minimum sequence numbers across all samples before the downstream analysis. In addition, the community compositions were provided at different taxonomic levels.Raw sequences generated through MiSeq sequencing were merged using fast length adjustment of short reads (FLASH)3 and SDB plates added with 50 mg/L rifampicin, respectively. The LB and SDB plates were cultured at 37 °C and 30 °C to isolate the dominant bacteria and fungus strains, respectively, and the MRS plates were incubated at 37 °C in a vacuum bag to select the lactic acid bacteria.Douchi samples (5 g) were added into 45 mL sterile water, rotated at 37 °C and 140 rpm for 40 min. The liquid mixtures were diluted, and spread onto the LB plates, MRS plates added with 20 g/L CaCO29. The 16S rDNA fragment was amplified through the universal primers of 27f (AGAGTTTGATCMTGGCTCAG) and 1492r (CTACGGCTACCTTGTTACGA), and the ITS fragment was amplified with the universal primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (GCATATCAATAAGCGGA). The PCR protocol was set as: 95 °C, 5 min; 95 °C, 45 s, 55 °C, 1 min, 72 °C, 1 min, 32 cycles; 72 °C, 10 min; and 4 °C for 10 min. The PCR is conducted in the 25 μL reaction system consisting of 2 μL genomic template, 2.5 μL Easy-Taq Buffer, 2 μL primers, 2.5 μL dNTPs, 0.3 μL Easy-Taq enzyme and 15.7 μL nuclease-free water. The DNA fragments were sequenced by Tsingke Biological Technology Co., Ltd. , and the sequence identity was analyzed using the Blastn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The bacterial genomic DNA was extracted by Gen-EluteTM Kit  following the manufacturer’s protocol, and the fungal genomic DNA was extracted using a SDS-based DNA extraction method previously describedBacillus and Staphylococcus), MRS (Enterococcus) or SDB (Candida) medium added with 1 g/L of histidine, tyrosine, tryptophan, phenylalanine, ornithine monohydrochloride, lysine or agmatine sulfate salt. After incubating for 48 h, the resulting BAs were determined. To measure the BAs-degrading ability, the cells were cultured and collected by centrifugation at 6000 × g for 5 min. After washing with 0.05 mol/L phosphate buffer (pH = 7), the cell pellets were diluted to OD600 = 0.4 in phosphate buffer (0.05 mol/L) containing 100 mg/L of histamine, tyramine, tryptamine, β-phenethylamine, putrescine and cadaverine, cultured for 48 h to detect the residual BAs, and the blank phosphate buffer without cell pellets was applied as the control. The BA-degradation rate was calculated according to the equation i.e., M = [(A − B)/A] × 100%, where M means the BAs degradation percentage, A and B indicate the initial and residual contents of BAs, respectively20.The BAs production ability was assessed by culturing the strains in 5 mL LB  were inoculated into the soaked soybeans (30 g) with the inoculum size of 5% (v/w), incubated for 7 days, and the total BAs contents were measured by HPLC. The soybeans were fermented by inoculating with 20. Briefly, 1.5 mL of 0.4 M HClO4 was added into 0.5 g solid sample or 0.5 mL liquid sample, extracted for 1 h. After centrifugation at 12,000 × g for 10 min, the supernatant (250 μL) was added with 25 μL of 2 M NaOH and 75 μL of saturated NaHCO3, then reacted with 500 μL of 5 mg/mL dansyl chloride at 50 °C for 45 min. After that, the reactant was mixed with 25 μL of 25% NH4OH and incubated at 50 °C for 15 min to remove the residual dansyl chloride. Then the mixture was adjusted to 1.5 mL with acetonitrile and centrifuged at 2500 × g for 5 min. The supernatant was filtered through a 0.22 μm membrane for HPLC (high-performance liquid chromatography) analysis, which was carried out using an Agilent 1260 HPLC system with an Agilent column Zorbax Eclipse XDB-C18  at 30 °C. The separation was achieved using a linear gradient of mobile phase A (acetonitrile) and B (H2O) at a flow rate of 1 mL/min. The solvent gradient was as follows: 50% A (0–3 min), 50–90% A (3–20 min), 90% A (20–29 min), 90–50% A (29–32 min), 50% A (32–35 min). The detection was carried out at 254 nm. Each BA in the samples was quantified with a calibration curve generating by analyzing standard BA solution. Typical chromatograms are shown in Figure The BAs contents were detected based on a previous reportAlpha diversity including Chao 1, Shannon diversity indices and Goods coverage were subjected to statistical analysis using Qiime (Version 1.7.0). The community structure was statistically analyzed at different classification levels. The significance was analyzed by one-way ANOVA using the statistical software SPSS 20.0, and means were compared by Duncan’s multiple range test at 5%.et al. detected six BAs in fermented soybean products, natto4, and Gong et al. reported that tryptamine, putrescine and cadaverine were detected in Douchi samples19. In this study, six common BAs with different contents were found among the Douchi samples  in the total content level, and as-detected samples were in the relative safe level on the whole. However, different BA showed different toxic level for human, thus each BA is needed to be further analyzed for comprehensive safety assessment.BAs were widely detected in various soybean foods. Kim 13. And 100–800 mg/kg of tyramine and 30 mg/kg of β-phenylethylamine have been reported to be the toxic doses in food37. Although most of the Douchi samples contained tyramine, no sample showed the amount above the hazardous level (100 mg/kg). Tryptamine was detected in six samples, and their concentrations were under 100 mg/kg. Though no β-phenethylamine was detected in three samples, it was noteworthy that the concentrations of six products were higher than the toxic dose (30 mg/kg). While Gong et al. detected no histamine, tyramine and β-phenethylamine in Douchi samples19. The differences in distribution and content of BAs could be attributed to the variation of the raw materials, the microbiological compositions and the fermentation conditions.As shown in Table 7. Though putrescine was found in all Douchi samples, the concentrations were under 100 mg/kg in 14 Douchi samples. And the content of putrescine under 100 mg/kg has been usually attributed to natural occurrence of amine in soybean products7. Meanwhile, cadaverine was detected in twelve products, while the concentrations of cadaverine in two samples were higher than 200 mg/kg. The toxicity of putrescine and cadaverine is not obvious relatively, but they can interact with nitrite to form carcinogenic nitrosamine38.Putrescine and cadaverine are regarded as two typical BA reported in soybean products including sufu, soy sauce, tempe, miso and natto39, highlighted good overall sampling, with levels of 99%.As described above, BAs contents in Douchi samples showed relatively large variations, which was probably due to the different microbiological compositions. Therefore, it is important to analysis the microbial communities among the Douchi samples. By high-throughput sequencing, 3,514,240 16S rRNA (V3–V4 regions) and 3,402,964 ITS2 reads were generated from 45 sequencing samples. After quality filtering and chimera removal, 62,603 bacterial and 74,327 fungal sequences were obtained from each sample, with the average read length of 426 bases for bacteria and 211 bases for fungi. Community richness and diversity were assessed using four alpha-diversity metrics  , Bacillus methylotrophicus (2 isolates), Bacillus amyloliquefaciens (2 isolates), Bacillus sonorensis (1 isolate), Bacillus licheniformis (4 isolates), Staphylococcus carnosus (1 isolate), Staphylococcus pasteuri (1 isolate), Enterococcus faecium (1 isolate) and Candida sp. (2 isolates), respectively . The B. subtilis HB-1 and S. pasteuri JX-2 with no BAs producing ability were more beneficial to reduce the BAs accumulation in fermented soybean product, and their existence probably inhibited the growth of BAs-producing strains by competition for nutrients, which might be the main reason for the decreased BAs. Eom et al. also observed that B. subtilis isolated from a Korean traditional fermented food was able to inhibit BA production50. In addition, Staphylococcus xylosus was used as a starter culture to inhibit BA production in a salted and fermented anchovy55. Similarly, the B. subtilis HB-1 and S. pasteuri JX-2 isolated in this study can be used as potential candidates for BA control in fermented soybean.For the 19 strains, ty Table , and B. As Table . Those fBacillus and Candida were the dominant genera. Nineteen strains were isolated from Douchi samples to investigate microbial contribution to BAs accumulation. B. subtilis HB-1 and S. pasteuri JX-2 showed no BAs formation ability, and B. subtilis GD-4 and Candida sp. JX-3 revealed high BAs degradation abilities. Moreover, fermented soybean model further confirmed that B. subtilis HB-1 and S. pasteuri JX-2 would be the potential candidates for BAs control. This study not only explained the microbial contribution to BAs in Douchi, but also provided potential strains to reduce the BAs in fermented soybean products.In summary, the BAs profiles of 15 representative Douchi samples were analyzed and the correlation between microbes and BAs were also investigated. The total BAs contents of all the Douchi samples were within the acceptable dose level, while histamine and β-phenethylamine showed above the toxic level in some samples, indicating that evaluation and control of specific amine are important for Douchi safety. High-throughput sequencing showed that Supplementary Dataset 1


When medically indicated, caesarean section can prevent deaths and other serious complications in mothers and babies. Lack of access to caesarean section may result in increased maternal and perinatal mortality and morbidity. However, rising caesarean section rates globally suggest overuse in healthy women and babies, with consequent iatrogenic damage for women and babies, and adverse impacts on the sustainability of maternity care provision. To date, interventions to ensure that caesarean section is appropriately used have not reversed the upward trend in rates. Qualitative evidence has the potential to explain why and how interventions may or may not work in specific contexts. We aimed to establish stakeholders’ views on the barriers and facilitators to non-clinical interventions targeted at organizations, facilities and systems, to reduce unnecessary caesarean section.We undertook a systematic qualitative evidence synthesis using a five-stage modified, meta-ethnography approach. We searched MEDLINE, CINAHL, PsychINFO, EMBASE and grey literature databases  using pre-defined terms. Inclusion criteria were qualitative and mixed-method studies, investigating any non-clinical intervention to reduce caesarean section, in any setting and language, published after 1984. Study quality was assessed prior to data extraction. Interpretive thematic synthesis was undertaken using a barriers and facilitators lens. Confidence in the resulting Summaries of Findings was assessed using GRADE-CERQual.8,219 studies were identified. 25 studies were included, from 17 countries, published between 1993–2016, encompassing the views of over 1,565 stakeholders. Nineteen Summary of Findings statements were derived. They mapped onto three distinct themes:Health system, organizational and structural factors (6 SoFs); Human and cultural factors (7 SoFs); and Mechanisms of effect to achieve change factors (6 SoFs). The synthesis showed how inter- and intra-system power differentials, and stakeholder commitment, exert strong mechanisms of effect on caesarean section rates, independent of the theoretical efficacy of specific interventions to reduce them.Non-clinical interventions to reduce caesarean section are strongly mediated by organisational power differentials and stakeholder commitment. Barriers may be greatest where implementation plans contradict system and cultural norms.CRD42017059456PROSPERO:  This was partly due to the powerful impact of non-clinical factors, such as management processes, rules, regulations, and conflicting strategies. [“the hugely complex series of events”, contributing to high rates of intervention in pregnancy and childbirth, meant many participants reported that achieving higher rates of normal birth and lower rates of caesarean section was unlikely to be effectively addressed by the apparently simple solution of a clinical pathway. Inpathway.. In contrast, stakeholders working within organisations with low caesarean section rates valued “working together as a team, knowing that everyone’s voice will be heard, and action is taken at every level of the organization.”  Ongroup.” (:p.337)“normal”. [claimed the complications secondary to C-section are ignorable  Women also had varied views about birth method, some of which were resonant with those of health professionals. One important difference in women’s views was the embodiment of living with the health consequences of caesarean section. For example, in the context of Ghana’s subsistence culture, one woman said “the C-section itself becomes a disease.”  into gains .Global health communities have begun to mobilise to address unnecessary caesarean section. ,12,15–17Several quantitative systematic reviews, including a Cochrane Review, have previously evaluated the effectiveness and safety of interventions for reducing caesarean sections. –17,63–64To the best of our knowledge this is the first global qualitative synthesis that brings together the evidence-base of what stakeholders say are the barriers and facilitators to the implementation of non-clinical interventions to reduce unnecessary caesarean section targeted at organizations, facilities and systems. Existing studies are sparse and limited, methodologically. We were unable to undertake the sub-analyses we planned, as there were too few studies in each sub-group to do this meaningfully. The systematic methodology and GRADE-CERQual assessment we used is a strength of the review, as is the inclusion of studies from 17 countries across high-, middle- and low-income settings, including three non-English language papers. –29a priori formative research into a means of determining and accounting for local context and cultures may be of benefit in the design of multifaceted interventions in this area in the future, to ensure that likely mechanisms of effect are harnessed in the study design. Controlled studies of interventions, using adaptive designs, and including nested qualitative components that capture the nature and sustainability of local adaptation within randomised clusters of sites could add to the developing evidence base surrounding interventions to reduce unnecessary caesarean section. The use of the Robson’s 10 group classification is becoming increasingly internationally accepted as a means to monitor and compare caesarean section rates [Our findings suggest that some form of on rates ,65. Routon rates  The intrThe global concern on the unprecedented increase of caesarean section has translated into societal willingness to change this trend by implementing interventions to optimize the use of caesarean section. This systematic review presents the evidence-based for critical structural, health system and organizational factors that will require careful local consideration in the design and implementation of such interventions. We propose that these factors are investigated in-depth in local initial formative research to ensure that likely mechanisms of effect are harnessed in the design of any intervention considered at country level.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.