This study aims to analyze the feasibility and predictive value of thermal tomography (TT) for monitoring early treatment response in patients with locally advanced breast cancer (LABC) receiving neoadjuvant chemotherapy (NAC).Patients with LABC who were due to receive six cycles of NAC were examined by TT prior to NAC, the second cycle of NAC, the fourth cycle of NAC and surgery. Changes in TT parameters and ultrasonography were correlated with pathologic response to NAC, and the predictive value was assessed.P < 0.05). The best predictor of pathologic response after the 6th cycle of NAC was TT , as opposed to cross-sectional areas and the longest diameter by ultrasonography.Forty-four patients were evaluable for response (25 pathologic responders and 19 nonresponders). As early as after the first cycle of NAC, changes in the TT parameters ΔTs, ΔTn, and ΔTa correlated significantly with pathologic response (TT allows for monitoring early tumor response to NAC and can predict pathologic response in the early stages of therapy. Therefore, TT could be used as a novel imaging modality to monitor NAC treatment. Neoadjuvant chemotherapy (NAC) was originally applied in locally advanced inoperable breast cancer to enable surgical resection. Subsequently, a novel use for NAC has emerged: it is utilized to shrink tumors and facilitate breast-conserving surgery in operable breast cancer , 2. MoreCurrently, the main methods used to monitor tumor response to NAC rely on integrating clinical assessment and breast imaging modalities, such as positron emission tomography (PET), ultrasonography, or magnetic resonance imaging (MRI) , 7. HoweThermal tomography (TT) is a novel, noninvasive imaging technique that has been used for early detection of breast tumors –12. TT eIn the present study, we used TT to evaluate women receiving NAC for the treatment of LABC at regular intervals throughout the course of therapy. This study aimed to analyze the feasibility and predictive value of TT for monitoring treatment response in patients with LABC receiving NAC. Moreover, this study compares TT with ultrasonography for assessing tumor response.P < 0.05). Other characteristics, such as menopausal, ER, and PR status, did not significantly differ between the two groups. The distribution of cancer-associated characteristics, such as breast subtype, was classified following a clinical assessment and the REC of response measurements after six cycles, as shown in The study consisted of 44 females, 25 responding and 19 nonresponding patients, with an average age of 47.30±10.63 years (range 26-64) for whom complete information was available. Of the 44 breast lesions, only 1 patient achieved pCR after six cycles of NAC, and none of the patients experienced PD according to pathologic response (Miller and Payne criteria) measurements. As shown in Table A total of 44 patients received ultrasonography and thermal tomography as required. The mean ΔTs for tumors at baseline was 2.58°C for the responding group and 3.10°C for the nonresponding group. Similarly, the mean ΔTn for tumors at baseline was 2.40°C for the responding group and 3.25°C for the nonresponding group, and the ΔTa for tumors at baseline was 1.30°C for the responding group and 1.80°C for the nonresponding group. These differences were not significant. According to the characteristics of the q-r curve, blood vessels and isothermals, the images of tumors were characteristic of malignancy.P < 0.05). The q-r curve relative to baseline also indicated a significant decrease. Due to their subjective nature, it is difficult to evaluate diagnosis based on blood vessels and isothermals, though changes can be assessed from TT images. Figures P < 0.05; Table Furthermore, the values relative to baseline were adopted to intuitively assess trends. However, based on pathologic criteria, the relative ΔTs, ΔTn and ΔTa values shown in Table The ROC curves shown in Figure Given the increasing use of NAC treatment, innovative imaging modalities that can improve the early predictive value of tumor response are needed to identify patients who may benefit from treatment. Specifically, early detection of nonresponding tumors could prevent unnecessary NAC treatment or help select suitable NAC regimens. The current study demonstrated the clinical use of TT for monitoring treatment response to NAC in patients with LABC. TT monitoring revealed a significant difference in the thermal parameters of tumors between responding and the nonresponding groups as early as after the first cycle of NAC. Moreover, the predictive value of TT for determining tumor response corresponded to that of ultrasonography SM after three cycles of NAC and was superior to the predictive value of REC.TT revealed significant decreases in ΔTs, ΔTn, and ΔTa values of tumors that responded to NAC. Due to individual differences, the absolute quantities of ΔTs, ΔTn, and ΔTa varied greatly and may have been influenced by many factors, such as the environmental temperature. This large inter-individual variability obscured significant differences between the groups. Therefore, we regarded them as standardization with respect to baseline, directly reflecting the effects of NAC. Furthermore, the q-r curves showed decreases from 30-45°C, which represents a malignant tumor, to 15-30°C and even 0-15°C. As indicated in previous studies, malignant tumors are characterized by a temperature of 30-45°C, which is significantly higher than the temperature of benign tumors and normal tissues . The shrBlood vessel scans are promising biomarkers for treatment response because tumor growth strongly depends on angiopoiesis, especially for larger tumors. Patients receiving NAC often harbor much larger tumors, and these tumors consequently contain more blood vessels with quicker blood flow to fulfill the demands of tumor growth. TT can clearly visualize the spread and morphology of blood vessels. In the present study, the blood vessels of malignant tumors were enlarged and centralized yet were reduced in size and became discontinuous in the responding group after three or six cycles of NAC. Corroborating our results, Mei-Lin et al. reported that changes in the micro-vessel functionality of breast cancer blood vessels, as depicted by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), soon after starting anthracycline-based NAC can predict the final clinical and pathologic response . Blood vTraditionally, tumor size measured by ultrasonography is an imperfect assessment of response to NAC because it can both over- and underestimate the histologically defined tumor size. However, in our study, normalized tumor cross-sectional areas (SM) decreased from the baseline values after six cycles of NAC. The predictive power of ultrasonography measurements after six cycles decreased because nonresponding tumors based on MP criteria showed no contrast enhancement after six cycles. Moreover, the inclusion of 11 HER2-/ER+ breast cancers affected the accuracy. As previously mentioned, MRI volume measurements were able to accurately assess response to NAC, and the AUC was 0.97 after three cycles and 0.71 after six cycles . Indeed,This study has several limitations. TT could not accurately localize a tumor; therefore, localization was partly based on conventional ultrasonography and mammography data acquired before therapy. The inability of TT to localize the tumor may result in undervaluation of tumor changes and consequently decrease the precision of the technique. Moreover, TT blood vessel scans may be influenced by many factors, such as tumor size. However, neovascularization was minimal or absent in small tumors, and dysplastic blood vessels were difficult to observe. Thus, merging molecular biomarkers and TT may improve assessment and provide more functional parameters to further increase the accuracy of assessment. In addition, the relatively small number and heterogeneity of the patients included may have resulted in bias.In conclusion, TT allows for monitoring early response of tumors to NAC and can distinguish responding tumors from nonresponding tumors during the early stages of therapy. Therefore, TT could be used as a novel imaging modality to monitor NAC treatment and assist in individualized patient care.From January 2014 to July 2016, patients received six cycles of NAC over three weeks consisting of TAC  or EC-T (doxorubicin and cyclophosphamide in three cycles over three weeks and docetaxel in three cycles over three weeks). The method used to monitor tumor response to NAC depended on the clinical assessment before each cycle and three ultrasonography examinations . In addition, four examinations by TT were planned for each patient . TT was executed using an available breast imaging system , and TT theory has been described previously –12. The Tumor response to NAC was assessed by ultrasonography based on the response evaluation criterion of the longest diameter of the enhancing tumor target lesion (REC). A complete reduction of the target lesion was defined as complete response (CR). A reduction in tumor size by at least 30% was defined as partial response (PR). Stable disease (SD) was defined as an increase in tumor size less than 20% or a decrease less than 30%. Progressive disease (PD) was defined as an increase in tumor size of at least 20%. The tumor cross-sectional area on ultrasonography images was calculated using dedicated software. All measurements were performed by an experienced sonographer.Standard pathology analysis of excised tissues was used to assess subject outcomes. The excised specimens were fixed, embedded in paraffin as tissue blocks and then serially sectioned at a thickness of approximately 5 mm before being stained with hematoxylin and eosin (HE). The final pathology examinations were evaluated by an experienced pathologist for grading of pathologic tumor response of the primary breast lesion using Miller and Payne criteria (MP criteria) .The thermal tomography system was applied by Prof. Kaiyang Li , as described previously , 12. TheThe parameters of TT were recorded, including the ΔTs , ΔTn , ΔTa , q-r curve, blood vessels and isothermals. The heat distribution in the human body and metabolism are reflected by the shape of the q-r curve. A flat surface was created to signify the distribution and trend of the q-r curve, and this surface was segmented into four sections with three straight lines, with included angles with the horizontal axis of 45°C, 30°C and 15°C. The q-r curve of most female breast tumors is at 30-45°C, whereas that of most benign tumors is at 15-30°C; the q-r curve lies at 0-15°C for most hyperplastic or normal tissues, as described previously. These colored images consisted of color pixels that each reflect a single temperature measurement. These measurements can vary from black (coolest) to white (warmest), with 14 “intermediate” colors . The bloP value < 0.05 was considered to indicate a significant difference.The main goal of this study was to assess the feasibility of TT for the early assessment of tumor response to NAC based on pathologic response after the first cycle. All subjects were divided in two subgroups based on MP criteria: nonresponding  and responding (MP 2-5) groups. For TT, changes in all parameters relative to baseline were compared between the two groups and analyzed for significant differences using the Mann-Whitney U test. The classifier was evaluated by estimating the ROC. The final predictive value of the evaluation was calculated based on the average ROC. All statistical analyses and all charts were generated using SPSS 19.0 . A two-sided t-test. Moreover, the predictive value of ultrasonography was evaluated.Compared with REC, the sensitivity and specificity of both TT and SM for predicting tumor response to NAC were assessed. The assessment based on TT criteria was numerically ordered as follows: nonresponding - 0 and responding - 1. The REC and SM after three and six cycles were normalized to the respective baseline values. The subjects were divided into two groups based on MP criteria, nonresponding and responding, and normalized cross-sectional areas after three and six cycles were compared between the groups. The significance of these differences was assessed using the The predictive value was estimated based on the classification results, which are a trade-off between the desired fraction of true positives (nonresponding classified as nonresponding) and the accepted fraction of false positives (responding classified as nonresponding). The area under the curve (AUC) is a measure of the predictive power and is defined as 1.0 for a perfect classifier, 0.5 for a random classification, and 0 for a completely incorrect classification.


Cerebrospinal fluid cytology is performed by operator-dependant light microscopy as part of the routine laboratory work-flow diagnosis of meningitis. We evaluated operator-independent lens-free microscopy numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis. In a first step, prospective optical microscopy counts of leukocytes done by five different operators yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm adapted for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes was modified step-by-step in the prospective analysis of 215 cerebrospinal fluid specimens. The definite algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology-confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories. Performing the cytological analysis of the cerebrospinal fluid (CFS) and enumerating leukocytes and erythrocytes is a routine first step in the laboratory diagnosis of meningitis123572 and the image of every single cell present in the sample can be retrieved through the computation of a phase retrieval algorithm111314Therefore, we aimed to develop an alternative, operator-independent method for counting CSF leukocytes and erythrocytes, which would require a CSF volume <50 μL, and would be suitable for connection to the laboratory’s information systems and thus, which would be better adapted to the POC laboratoryWe thus evaluated the ability of lens-free microscopy to perform CSF cytology to contribute to establishing an early detection of infectious meningitis, in three successive steps. In the first step, we studied the inter-individual variability of CSF cytology performed by the reference standard optical microscopy. In the second step, we set-up a lens-free microscopy algorithm adapted for counting CSF cells and capable of discriminating leukocytes from erythrocytes based on the prospective analysis of 215 CSF specimens. In the third step, we established a proof-of-concept that lens-free microscopy and the algorithm created allowed for the rapid cytology-based laboratory diagnosis of meningitis. This proof-of-concept has been demonstrated through the blind assay of 116 further CSF specimens.In the first step, the inter-operator variability of optic-microscopy was assessed on 72 CSF samples that were prospectively, independently, and blindly observed by five different operators using optical microscopy . Of the Z from the sensor plane , in which the leukocyte optic-microscopy count was, respectively, six and seven leukocytes/μL, were well below the limit of 10 cells/μL used for the early diagnoses of meningitis  prevented an automated analysis based on image processing. It was known that sub-pixel super-resolution techniques could be applied to improve the spatial resolution down to ~1 μm1819The various existing lens-free microscopy techniques able to count leukocytes and erythrocytes cannot usefully be applied to cell counts in CSF. The first method consists in analyzing the holographic pattern acquired by the CMOS sensor15Institut Fédératif de Recherches 48, Marseille.CSF leftovers were used, so that no additional sampling was specifically done for this study, which was approved by the 3. The gold-standard working volume of CSF is 10 μL and 6.6 μL are deposited by capillarity into the counting slide. The optical microscope used in the study was an Olympus CX41 (USA), with a magnification of ×40 to ×100. The results of the different counts for each of the CSF specimens were registered in a dataset by an independent operator. The individual variability was determined by a comparative statistical analysis of ANOVA type of variations.A prospective blind inter-operator variability study was conducted for two months. It consisted of optical microscopy cell-counting conducted by five different operators on 35 consecutive and independent CSF specimens. The five operators were laboratory technicians and residents in medical biology. Each operator manually registered the count results in a table that was not accessible to the other operators. The cell counts were performed on KOVA Glasstic 10 counting slides with grids , performing the count of leukocytes and erythrocytes in the nine cell segmentations corresponding to the count per mmet al.2. Hence the volume of CSF specimen analyzed with one lensfree acquisition is about 3 μl (29.44 mm2  * 0.1 mm). The Countess cell counting device features two chambers, allowing duplication of the reading if necessary. The illumination source was about 5 cm away from the chamber, and was based on a Cree MC-E Color quadrant RGB LED emitting respectively at 620–630 nm, 520–535 nm, and 450–465 nm. This was coupled to a 150 μm pinhole through a 60° diffuser. The distance between the quadrant RGB LED and the diffuser was approximately 2 mm, in order to merge the three colored lights into a single-point source. The acquisition was sequential, and each red, green and blue LED was lit up one after the other, while the three corresponding wide-field holograms were acquired by the CMOS sensor . The lens-free optical setup did not acquire magnified images of the cells, but rather, jus the holographic interference patterns from the cells. It is therefore necessary to use a holographic reconstruction algorithm to recover the phase and module images of the cells. The holographic reconstruction consists first in back-propagating the light intensity as recorded by the sensor using the Fresnel function. The recovered complex image, however, is strongly affected by the so-called ‘twin image’, an artefact that results from a lack of phase information in the acquisition process. To retrieve the phase in the sensor plane and consequently diminish the ‘twin-image’ artefact in the reconstructed images, we used a phase-retrieval algorithm. The purpose of such an algorithm is to recover the complex image of the sample from the phaseless recorded holographic imageZ-axis profiles were calculated on either side of the object plane over 1000 μm at 20 μm intervals, and provided an optical signature for every single cell present in the CSF specimen. Now the analysis of these Z-axis profiles allowed us to discriminate efficiently between the cells and other micro-objects, e.g. particles present in the CSF specimen, or at the surface of the fluidic chamber. Next, a second algorithm would perform the automatic count on the leukocytes and erythrocytes present in the CSF clinical specimen to establish an early diagnosis of meningitis. These calculations can be performed on a laptop computer  at a speed of ~35 seconds per analysis based thanks to a GPU implementation, which is fast enough for a point of care application.A total of 215 CSF specimens were prospectively analyzed in order to optimize the lens-free microscopy acquisition and algorithm. Our system was based on lens-free microscopy with the optical setup described by Ozcan In order to establish the proof-of-concept, 116 lens-free additional acquisitions were prospectively performed using the method developed in the second step of this study. CSF cytology was performed in parallel by optical microscopy and by lens-free microscopy using the algorithm previously developed. These CSF specimens were collected in cases of clinical suspicion of meningitis as a part of routine work-flow of the laboratory. All specimens were unique, and differed from those used in the second step of this study. The algorithm developed through acquisitions in the second part of the study was applied to these acquisitions, without being modified. After the processing the acquisitions using the software, the results for meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off were compared to the actual diagnosis determined for every patient. We then determined the sensitivity and specificity of the lens-free microscopy.How to cite this article: Delacroix, R. et al. Cerebrospinal fluid lens-free microscopy: a new tool for the laboratory diagnosis of meningitis. Sci. Rep.7, 39893; doi: 10.1038/srep39893 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Purpose: Recognising that one way to address the logistical and safety considerations of research conducted in humanitarian emergencies is to use internet communication technologies to facilitate interviews online, this article explores some practical and methodological considerations inherent to qualitative online interviewing. Method: Reflections from a case study of a multi-site research project conducted in post-conflict countries are presented.  Synchronous online cross-language qualitative interviews were conducted in one country.  Although only a small proportion of interviews were conducted online (six out of 35), it remains important to critically consider the impact upon data produced in this way. Results: A range of practical and methodological considerations are discussed, illustrated with examples.  Results suggest that whilst online interviewing has methodological and ethical potential and versatility, there are inherent practical challenges in settings with poor internet and electricity infrastructure.  Notable methodological limitations include barriers to building rapport due to partial visual and non-visual cues, and difficulties interpreting pauses or silences. Conclusions: Drawing upon experiences in this case study, strategies for managing the practical and methodological limitations of online interviewing are suggested, alongside recommendations for supporting future research practice.  These are intended to act as a springboard for further reflection, and operate alongside other conceptual frameworks for online interviewing. Unstable settings, such as those where humanitarian emergencies occur, give rise to logistical considerations when designing and planning research, including restricted access to sites and populations  experience of conducting online cross-language qualitative interviews as part of a multi-site study. Incorporating online synchronous interviews was necessary due to security considerations preventing travel to one of the research sites to conduct in-person interviews. Although in this multi-site case study only a small proportion of interviews were conducted online, the impact upon data produced in this way needs to be critically considered. To this end, a range of practical and methodological considerations are identified and discussed, illustrated with examples and quotes. This small case study of online interviews is valuable for the methodological reflections and learning that can be applied and extended through further research. The reflections and considerations raised in this article are intended to complement wider literature that presents in-depth discussions of the methodological, practical and theoretical issues presented when conducting social science research online Hine, . SpecifiThe case study from which this article is drawn empirically examines how “ethics” is defined, understood, applied and managed by mental health researchers working in post-conflict settings, focusing upon the interaction between constructions of procedural and in-practice ethics , with an interpreter involved when required. Interviews followed a semi-structured topic guide that explored participants’ perspectives of the construction and management of procedural and in-practice ethics  was approved by the relevant country and Liverpool ethical review committees. All participants provided voluntary written informed consent prior to interviews. To protect confidentiality, data has been fully anonymized. This includes the use of pseudonyms for each participant, and the replacement of all potentially identifying information with fictitious country/place/organization names, denoted by {} brackets.The physical separation of researcher and interpreter from the participant raises both practical and methodological considerations. To ensure transparency regarding the reliability and rigour of qualitative interviews, it is important to explore the consistency of online and offline interviewing with underlying research epistemology, application of methods, and how these ensure the desired research outcomes are attained  (Ask her)I: #37.24–37.28#P: #37.29–37.30#R: (.3) Oh you’ve muted it again, hang on, I’ll unmute it.I: #37.38–37.41#R: (.2) ((to I)) I can do it for her, ((to P)) I don’t want to do it and then you. (.8) Okay I can hear you, oh no hang on, don’t touch anything, let me do it.I: #38.00–38.02#R: (.7) You’re touching it, so you’re doing it at the same time. Okay we seem to have fixed it. Okay ((R laughs)), sorry.Fernanda It is important to note that AC is familiar with the use of online communication tools including regular use of Skype™ for meetings as well as personal use, and has used the Adobe Connect™ platform for teaching and meetings, ensuring familiarity with its features for application to this study. All research participants mentioned their familiarity with online platforms—notably Skype™—for both professional and personal communication. However, to the researcher’s knowledge only one participant had prior experience of Adobe Connect™. This lack of familiarity meant that for interviews conducted at the hospital a participant supporting the study’s logistical arrangements and oriented to Adobe Connect™ by the researcher prior to study commencement was present at the start of each interview to set up the online space. Similarly, for the participant at home, the researcher provided guidance on setting up the connection prior to commencing the interview. This process of establishing a connection and introducing participants to the online space lasted around 10 minutes. Despite these brief orientations, there were instances during interviews where unfamiliarity with the software led to accidental muting of the microphone:Consequently, whilst the researcher, interpreter and participants all had prior experience of online synchronous communication technologies for professional and/or personal purposes, the use of Adobe Connect™ put the researcher in a position of power due to her familiarity with the software. Equally, to the researcher’s knowledge none of the participants had engaged in interviewing or being interviewed via online platforms, and neither the researcher nor the interpreter had conducted qualitative interviews online before. Therefore, in engaging in online interviews the researcher, participant, and interpreter were drawing upon personal and professional micro-cultures that shape understandings of the personal and professional use of online communication tools Conscious construction of an online secure password-protected site ensured the researcher was able to achieve her ethical responsibility to ensure the privacy of information exchanged online , or “Door opens in room P is in, can see her eyes go up to see who is coming in. Some background talking, then door opens and closes again—assume they left the room” . These were kept alongside general reflections about the interview environment from the researcher and interpreter, documented immediately after each interview.Due to the impossibility of predicting connection quality in advance of interviews, flexibility in responding to the availability/unavailability of video was necessary. Once interviews move online, the ability to project and negotiate role performance is restricted, particularly in the absence of video. This includes limited access to cues regarding background demographics such as age; self-presentation—for example, though clothing; and subtle cues such as smiling, frowning or nodding. Additionally, in the context of this cross-cultural study conducted in South Asia, the availability of facial expressions could not be assumed as the research participants’ cultures include females wearing veils that cover their face. This impacted upon the availability of non-verbal cues such as smiles, and occasionally the clarity of verbal utterances.the video conversation is very important because she wanted to know that who is Anna and how she looks like that er, that who is involved in {Rudo} programme so she just wanted to meet you so that’s like. It’s good.I: … Fernanda When working in cross-cultural contexts, restricted visual cues, coupled with the involvement of an interpreter, reinforced the distance between the white, Western, English-speaking researcher and local interpreter and participants. This was reflected by participants who commented on the advantages of being able to see the researcher, to “meet” whom they were talking to:This quote illustrates the importance the participant attached to “meeting” the researcher, emphasizing the desire for in-person interaction. Whilst the extent to which this is achieved via the online format remains limited, it does offers a substitute to in-person interactions where required.P: … actually it depends upon the person … how much another person is comfortable while dealing with a new person …. [N]ormal level of anxiety is definitely there.Leslie The researcher had prior relationships with some participants that were felt to aid online interviews, allowing the researcher and participant to build upon previous interaction dynamics. Furthermore, conducting the group informed consent session had enabled all participants to be introduced to the researcher and interpreter in advance of interviews. Having a prior relationship with the researcher, alongside familiarity and comfort with online formats, was identified by a participant as a key factor in influencing the extent to which video supported rapport building and facilitated interview conduct:As this indicates, a range of factors affect building relationships between researcher and participant. Of the interviews conducted in the third country, six were online and two were in person. When reflecting upon the difference between the online and in-person interviews with participants the researcher had not met before, the suggestion that being comfortable with interacting with someone new is more influential than the interview format is supported. However, it is difficult to isolate factors that may have influenced this. It is possible the gender difference between researcher and participant may have been the critical factor influencing rapport building because the two in-person interviews were with males.In interviews conducted with an interpreter, the presence of an additional unknown third party may also have impacted upon rapport building, as conversations and therefore connections between the researcher and participant are mediated by a third party. In this country, the interpreter was male whilst the majority of participants interviewed online were female; therefore, patriarchal gender norms in the setting may potentially have influenced narratives. Equally, the researcher found that the relationship with a male interpreter led to a different style of interviewing than was experienced in the other countries when interviewing with female interpreters. It is accepted that the impact of gender norms and interaction dynamics between the researcher and interpreter may in turn have impacted upon efforts to build rapport with participants who may have sensed an awkwardness to the researcher/interpreter relationship. Therefore, the relationship between the researcher and interpreter may also have influenced rapport-building between the participant and researcher/interpreter dyad.Online interviews without video have been characterized as disembodied, with the removal of non-verbal cues acting to limit interview contextualization and potentially reduce the impact of the interviewer on the interview encounter ), so we will probably take notes whilst you’re talking, so if you see us looking down that’s what we are doing” . Furthermore, the researcher in subsequent interviews narrated what was happening during silences or pauses—including when the video was on. For example:R: … Um, you’ve given me so many extra questions I want to ask you, er just give me a second to have a think.P: Okay.Margareta In an attempt to address this rush to the next question, the expectation of pauses in conversation was established at the outset of the interview when the researcher explained: R: P: #53:47-55:32#R: (.4) Okay he’s just finishing writing.P: Okay.R: Okay.I: She said that ….Tanika The researcher would also clarify when the interpreter was finishing writing notes prior to translating what a participant had said:This approach was effective in providing the space for more considered questioning and probing. Despite these efforts, the length and frequency of pauses as well as the depth of probing were felt to be less than occurred in in-person interviews, where the researcher can sense how comfortable a participant is with natural pauses in conversation. This approach also increased the sense of interview as performance, with the researcher providing cues akin to stage directions to ensure the participant remained informed about interactions that were out of sight. Whilst these responses were effective in this study, it is noted that, where possible, researchers should conduct practice interviews to explore and select their preferred online interviewing platform, as well as to rehearse strategies to manage foreseeable differences in the experience of online interviewing, such as the unavailability of non-verbal cues and impact of interview disembodiment Hine, .When interviewing with an interpreter, the time required for interviews necessarily increases, with interviews across the 3 countries involved in this study lasting on average 90 minutes. Online interviews brought additional considerations that impacted upon interview length, chiefly setting up the conversation at the outset, and interruptions to audio such as fading out or overlapping speech. When recording conversation within the Adobe Connect™ platform, as a result of the time-lag overlapping speech was a significant problem, leading to some lost sections of interview data where it is impossible to distinguish what is being said. By listening back to check recording quality this issue was quickly identified and addressed by using a Dictaphone to double record interviews.P: Um (.2) then the er (.3) consent, confidentiality, er patient comfort. I mean all these are everything.R: Patient comfort, what does that mean?P: Yeah.R: What does patient comfort mean?P: Sorry?R: What do you mean by patient comfort?P: It means that er patientTanika Within the language-processing loop it is recognized that meaning can be lost, misheard or misinterpreted Frisoli, . DifficuR: … how you found it in relation to the, the online setup?P: Er actually I’m used to it before also but er sometimes, just like today a little exhausting because of the Internet connection.Leslie As a result of these difficulties, for all interviews conducted online the researcher had a heightened awareness of timing than with in-person interviews. For example, one interview conducted online involved 22 minutes of recording in Adobe Connect™, during which 8 minutes of conversation took place; followed by a 1-minute 15 Skype™ conversation before the connection went; and finally a 51-minute conversation in Adobe Connect™. At the end of this interview the participant reflected upon the frustrations that could arise as a result of technological difficulties:The potential for frustration due to repeated connection issues led to a concern to keep interviews shorter both to limit the burden upon the participant and to limit interpreter fatigue and potential impact upon translation quality. This resulted in interviews conducted online being shorter and therefore more limited in their depth than those conducted in person.Physical safety is contested in unstable and unpredictable research environments Hanna, . In thisP: … um keeping in mind the availability and our own problems etcetera. So at times this kind of interaction is also okay.Leslie This asymmetry in the relative safety of the researcher and interpreter versus that of the participant brings an additional dimension to the site of interviews (Karray et al., This construction, normalizing an unstable context as “our own problems”, frequently arose during interviews that considered the impact post-conflict settings may have upon the application of ethics. In settings that are unstable, the concept of researchers protecting participants becomes less applicable, with the assumptions of ideal field sites where researchers are the ones in a position of control no longer holding true (Kovats-Bernat, In this section, suggestions for managing key logistical and methodological considerations that arise when conducting interviews online will be made, drawing upon experiences in this case study see . These sIn order to validate or refine these suggestions, continued documentation and sharing of experiences of conducting interviews online is encouraged, supporting future researchers who choose this interview format (Ferrante et al., As a result of the shift to online interviewing, this study entailed methodological messiness where the researcher was learning the research process alongside generating data (Rossman & Rallis, The reflections in this article have identified a range of practical and methodological considerations that arose in the conduct of a cross-language qualitative research study that involved online interviewing. Notably, the challenges of gaining depth of data collected via online interviews are a central consideration when using this interview format.Online interviewing presents methodological and ethical potential and versatility, but should not be viewed as an easy option (James & Busher,


A novel electromyography (EMG) signal decomposition framework is presented for the thorough and precise analysis of intramuscular EMG signals. This framework first detects all of the active motor unit action potentials (MUAPs) and assigns single MUAP segments to their corresponding motor units. MUAP waveforms that are found to be superimposed are then resolved into their constituent single MUAPs using a peel-off approach and similarly assigned. The method is composed of six stages of analytical procedures: preprocessing, segmentation, alignment and feature extraction, clustering and refinement, supervised classification, and superimposed waveform resolution. The performance of the proposed decomposition framework was evaluated using both synthetic EMG signals and real recordings obtained from healthy and stroke participants. The overall detection rate of MUAPs was 100% for both synthetic and real signals. The average accuracy for synthetic EMG signals was 87.23%. Average assignment accuracies of 88.63 and 94.45% were achieved for the real EMG signals obtained from healthy and stroke participants, respectively. Results demonstrated the ability of the developed framework to decompose intramuscular EMG signals with improved accuracy and efficiency, which we believe will greatly benefit the clinical utility of EMG for the diagnosis and rehabilitation of motor impairments in stroke patients. Electromyography (EMG) signals carry information regarding the motor unit action potential trains (MUAPTs) generated by the motor units (MUs) that are recruited during muscle contraction. Each MUAPT is made of a series of intermittent discharges that take the form of spatially dispersed individual motor unit action potentials (MUAPs). Intramuscular EMG is commonly acquired by means of indwelling needles or fine wire sensors that provide direct and targeted contact with the musculatures. Clinically, intramuscular EMG is used as a routine method for the electrophysiological examination of neuromuscular symptoms.EMG decomposition reverses the process of signal generation by separating the de-noised EMG signal into its constituent MUAPTs. This process is accomplished by identifying MUAP waveforms generated by the MUs adjacent to the detection surface and assigning these MUAPs to their corresponding MUAPTs. Characteristic properties of a decomposed MUAP, such as wave shape and firing pattern , can proIn this paper, we propose a novel intramuscular EMG decomposition framework by advancing the completeness and accuracy of MUAP decomposition. This framework is realized through six stages of analytical procedures: (1) EMG signal de-noising, (2) MUAP segmentation and extraction, (3) MUAPs alignment, feature extraction, and similarity measurement, (4) MUAP clustering and cluster refinement, (5) supervised classification, and (6) superimposed waveform resolution. Following this framework, we have attempted to improve the decomposition performance in four ways. First, we utilized a modified segment extraction scheme that is capable of detecting complete MUAP sets by incorporating amplitude threshold detection and resting segment recognition techniques. Second, single and overlapped MUAP waveforms were identified based on a phasic detection scheme, where phase templates were chosen based on the neurological condition of the tested muscle. The single MUAP segments of each MU underwent a clustering process that markedly reduced the buffer size and processing time required for this task. Third, all recognized single MUAP segments were aligned by centering their main peaks (regardless of polarity). Finally, we resolved superimposed waveforms using a peel-off approach based on measurements of pseudo-correlation (PsC). The performance of the proposed decomposition framework was evaluated using both synthetic EMG signals and real recordings obtained from healthy and stroke participants. Results demonstrated the favorable performance of the developed framework in decomposing intramuscular EMG signals with improved accuracy and efficiency.Twenty healthy subjects  participated in our data collection. No subject reported any history of neuromuscular diseases. Eight subacute hemiparetic stroke subjects  were recruited from the Third Xiangya Hospital of Central South University in China. The research protocol was approved by the local research ethics committee. All subjects were informed about the purpose and details of the experiment prior to the data collection.All EMG signals were recorded from the biceps brachii muscle. Subjects were seated in a chair with either the right forearm  or the affected forearm (for the stoke patients) supported by a horizontal table. Subjects were then asked to maintain elbow flexion at a 90° angle with their palms facing upward. A conventional needle electrode  was inserted into the muscle belly at a depth of approximately 1 cm. Each subject was then asked to perform 10-s constant-force isometric contractions by resisting a load with pre-trained force. Each subject performed three contractions at both mild (3–4 MUs detected) and moderate (6–8 MUs detected) force levels. A 3-min break was provided following each contraction to avoid muscle fatigue. Signal quality and force level were monitored on a real-time display screen with audio feedback. All clinical procedures were performed by an experienced physician (Xuhong Li). The frequency band of the standard EMG instrument was set to 2 Hz–10 kHz. All signals were sampled at 48 kHz and stored for off-line decomposition using an EMG workstation .Synthetic EMG signals are valuable for evaluating decomposition results as, unlike real recordings, the exact firing patterns and waveform templates of the synthetic MUs are known. The use of synthetic EMG signals thereby represents the only way to assess the sensitivity of decomposition algorithms to different parameters. In this study, 5-s segments of EMG signals (sampled at 30 kHz) were generated based on a model proposed by Farina et al. , where eSignal preprocessing followed the methods described in our previous publications , 26. Brik multiplied by the estimated noise power, k was selected by the investigator according to the force level, with a range of 5–8.RL is the length of a window and the sEMG[k] is the discrete EMG signal. All active MUAP segments were first identified using a modified segmentation scheme. A detection window of 1.25 ms was shifted through the entire EMG signal. A resting segment was recognized if the absolute values of the signal within the window were continuously lower than the pre-set amplitude threshold. Whenever two or more successive resting epochs were detected, the signals spanning between these epochs were extracted as the active segments, in which the signal exceeded this pre-set amplitude threshold. The boundaries of identified active epochs were then spatially expanded by at least 0.2 ms to ensure that the whole MUAP waveform was preserved. This amplitude threshold level was defined as Extracted MUAP-containing segments can be either isolated or overlapped. Isolated MUAPs that discharge multiple times can be easily recognized and labeled using clustering methods. Conversely, overlapped MUAP waveforms are created by the partial or full superposition of two or more single MUAPs discharging simultaneously, making the constituent waveforms much more difficult to parse. According to Thornton and Michell , MUAPs fExtracted active segments with only one phase or a MUAP duration shorter than 1.5 ms were regarded as invalid and excluded from the detected MUAP set. Finally, the remaining single MUAP segments were retained as a valid set for the following alignment. The beginning points of these active segments, representing the onsets of the MU firing instances, were assembled into a separate one-dimensional array.At this stage, all of the detected MUAP waveforms were aligned with their main peaks (either positive or negative) at the spatial center and shorter waveforms were zero padded so that all segments were of equal length. This alignment scheme can enhance the sensitivity in discerning and grouping MUAPs into their MU origins.Wavelet-domain features have been shown to improve stability when analyzing EMG signals that are contaminated by high frequency background noise or baseline drift , 16. As s1(n) and s2(n) are the two active segments to be compared and e(n) is their error signal. The distance measure defined by Eq. After feature extraction, the distance matrix was calculated based on the variance of the error normalized by the sum of the RMS values for the paired segments . This isd=The MUAP set was partitioned into its constituent MUAPTs based on the similarity measure presented above. To do this, we utilized a single-linkage hierarchical clustering algorithm that permits a simple graph-theoretical interpretation, namely the MST method. The MST method, considered best suited for EMG clustering , is ableA subsequent cluster refining procedure was performed to verify if any potential class should be deleted or subdivided. Clusters with at least three templates were chosen as potential MUAP classes, while those with less than three templates were regarded as invalid MU clusters and excluded. All MUAP segments belonging to these deleted clusters were moved to an unclassified set for subsequent supervised classification. At times, two different MU clusters can be incorrectly assigned to the same cluster due to similarities in their characteristic waveforms. In these cases, the mis-clustered MUs should also be subdivided based on the MST method. MUAPT templates were then calculated as the mean waveforms of each MUAP cluster. After clustering, we obtained the initial sets of MU clusters and the unassigned MUAPs were set aside to be classified in the next step.At this stage, we used the supervised minimum distance classifier, which is based on measurements of Euclidean distance, to classify the MUAP waveforms in the unassigned candidate set. The classification program was based on the wavelet coefficient features and valid clustering results. During classification, the threshold was set to the lowest mean value obtained from the inter-class distances. Signal instability and electrode movement can cause MUAP shapes to vary from discharge to discharge. Therefore, a weighted averaging technique reported by Zennaro et al.  was utilDuring muscle contraction, a portion of the entire MU pool is recruited and the intermittent firing pattern of each recruited MU can be extracted as its MUAPT. Multiple MUs that discharge simultaneously or within a very short interval will results in the superposition of MUAP waveforms. Resolving these waveforms is the process of identifying the overlapped MUAP segments and splitting them into their constituent single MUAPs. In our study, a peel-off approach based on PsC was adopted to resolve the superimposed waveforms after the isolated MUAP segments had been successfully classified.kk, PsC, is defined  or the number of MUs manually obtained by a neurophysiologist —and NMdetected is the number of MUAP segments detected.The detection ratio (DR%) was used to measure the rate of the successful detection of active MUAP segments. The DR% is defined asunassigned is the number of MUAP segments not assigned by the EMG decomposition framework and NMdetected the total number of MUAP segments detected.The assignment ratio (AR%) measures the rate of MUAP assignment using the proposed EMG decomposition framework. It is defined ascorrect (the number of correctly decomposed MUAPs) to NMdetected :The correct classification rate (CCR%) assesses the performance of the whole EMG decomposition system. It is defined as the ratio of NMIn our study, the DR% of the MUAP detection program for all real and generated EMG recordings reached 100% by using the novel MUAP segment detection scheme.The complete decomposition of the superimposed waveforms resulted in a marked improvement in the assignment ratio. In this paper, the average assignment ratios (AR%s) of our decomposition system were 99.79% for the synthetic signals, and 98.66 and 91.52% for real recordings from healthy and stroke participants, respectively (listed in Table Figures Figure Figure Table According to Table Accurate clustering results are critical to the decomposition performance. In our study, we performed a cluster refining step to improve the accuracy of results. Cluster refinement in this case included deleting invalid clusters and subdividing one incorrectly identified cluster into two or more clusters. Figure The whole analysis process was conducted using a custom MATLAB script and performed on 2.5 GHz Intel i7 desktop computer. The average processing time for decomposing a 10-s-long EMG data was approximately 15–20 min.EMG decomposition has been widely employed to provide information of alterations in motor unit characteristics in stroke patients . AchieviA high DR% value is critical, as the successful extraction of MUAPs greatly impacts subsequent decomposition procedures and, consequently, the AR% and CCR%. All active MUAP segments and resting segments comprise the whole EMG signal. Due to signal disparities, direct identification of MUAP segments is more difficult than the identification of the resting epochs. Thus, we utilized a wavelet hard-threshold estimation technique to attenuate instrumental and background noises, then applied a novel segmentation scheme based on resting segment detection. All active MUAP segments were further detached by subtracting rest segments from the original signal. The employment of a modified segmentation scheme greatly enhanced the performance, achieving a DR% value of 100% and demonstrating the complete detection of all active MUAP segments. Conventional segmentation methods often fail to identify some active MUAPs, even when using different rigid detection thresholds, because of the abnormal waveform complexity . TherefoMotor unit action potential segments detected in the aforementioned manner can be either isolated MUAP segments or superimposed MUAP waveforms. Then isolated/overlapped MUAP segments were separated based on either tetra- or hexaphasic waveform recognition methods. Segments with more than four MUAP phases for healthy subjects or six phases for stroke subjects were generally recognized to contain superposed MUAPs and separated for further analysis to resolve the superposition. Thus, by grouping segments based on their isolated MUAP characteristics in the first instance, the efficiency of the decomposition system was improved greatly. The implemented main peak alignment method further served to improve the methodical distinguishability of MUAPs originating from different MU clusters, resulting in more accurate and efficient results.k-means algorithm is based on the minimization of a global cost function, which is related to its classification ability. It is our ongoing effort to further integrate these two clustering algorithms and, in doing so, achieve optimal clustering results.In our study, the MUAP waveforms were clustered using a single-linkage hierarchical clustering algorithm. This technique is suitable for the clustering of MUAPs with slow variation and does not depend on the presentation order of the samples. However, the results of clustering are very sensitive to the value of the discriminatory threshold. Clustering results were, therefore, verified through visual inspection. Invalid clusters were excluded or subdivided again using the MST algorithm to ensure that all final clusters are valid. In addition, the fuzzy To obtain a thorough EMG decomposition, superimposed waveforms need to be resolved into their constituent MUAPs. This stage is the most time-consuming and critical procedure in the whole EMG decomposition framework. As it is essential to obtain a high-level AR% and complete information, the consistent achievement of AR%s over 90% by the new framework represents a marked improvement over the 67% classification rate achieved by our previous methods . Two typIn summary, an effective EMG decomposition framework was developed. First, we utilized a novel MUAP segment extraction method to detect all active MUAP segments. This procedure was based on amplitude threshold detection and resting segment recognition. We then grouped the MUAP segments into single and overlapped waveforms using tetraphasic or hexaphasic detection schemes to save buffer size and improve computational efficiency. Third, all recognized single MUAP segments were aligned with the main peak at the center for the effective assessment of waveform similarities. Finally, we resolved superimposed waveforms using a peel-off approach based on a measure of PsC. By incorporating multiple analytical approaches, this developed EMG decomposition framework achieves accurate and complete results without hampering computational speed, which we believe will greatly benefit clinical EMG utilities.The study protocol was approved by the West China ethics committee of Sichuan University and the Institutional Review Board of Third Xiangya Hospital, Central South University.XR led the development of the proposed decomposition algorithm, participated in healthy subject experiment, data acquisition and analysis, and paper writing. CZ participated in stroke patient experiment, data acquisition, and paper writing; XL participated in stroke patient recruitment, data acquisition, and analysis; and GY participated in algorithm development, data acquisition, and data analysis. TP participated in data analysis and the article polish. YZ participated in study design, algorithm development, data acquisition and analysis, and paper writing.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.


A gastric adenosquamous carcinoma (ASC) that produces granulocyte-colony stimulating factor (G-CSF) is an uncommon malignancy with a poor prognosis. Due to the rarity of this lesion, a standard treatment for the disease has not been established.We describe a case of a 66-year-old male with a G-CSF-producing gastric ASC who presented with severe anemia and leukocytosis. A radical resection was performed, followed by a course of adjuvant chemotherapy. Histopathologic examination revealed that the tumor consisted of areas of both squamous cell carcinoma and adenocarcinoma. Immunohistochemical staining with an anti-G-CSF antibody was also positive. He was started on adjuvant capecitabine and oxaliplatin (CapeOX) 6 weeks after surgery. The patient stopped treatment after 3 months due to his own preference. Eight months following surgery, the patient was found to have diffuse lymph node, liver, and peritoneal metastases.G-CSF-producing gastric ASC is a rare and aggressive tumor. Because patients are usually diagnosed at an advanced stage, multidisciplinary evaluation and innovative treatments are needed. The rarity of this disease, with its aggressive features, poses a significant challenge in its treatment. In this brief case report, we summarize the management and outcomes of G-CSF-producing gastric ASC. Gastric cancer is the fourth most prevalent cancer in the world and the second most common cause of cancer-related deaths . GastricExpression of granulocyte-colony stimulating factor (G-CSF) and the G-CSF receptor (G-CSFR) by gastric cancers rarely occurs. G-CSF is a pro-inflammatory cytokine that stimulates myeloid stem cell maturation, proliferation, and migration into the circulation . DespiteG-CSF-producing gastric ASC has two poor prognostic factors, namely, the squamous cell component of the tumor and the G-CSF production. Although the incidence of G-CSF-producing gastric tumors is approximately 0.5% of all pure gastric cancers, it has a very poor prognosis . InteresA 66-year-old male with a history of type 2 diabetes, chronic hepatitis, and 2 years of anemia despite iron supplementation, presented with a generalized and worsening fatigue over a 1-month period, which necessitated an emergency department visit. The patient also experienced unintentional weight loss of 10 kg over 3 months. He had a history of smoking and drinking for 30 years. He had a benign physical exam. On laboratory evaluation, he had severe anemia (hemoglobin 4.7 g/dl) and leukocytosis . Serum C-reactive protein (CRP) was elevated to 1.61 mg/dl .A computed tomography (CT) scan of the abdomen and pelvis demonstrated irregular thickening of the antrum wall with surrounding para-aortic lymph node enlargement Fig. . There wCoincidentally, the CT scan also demonstrated irregular thickening of the colon wall, in two separate areas within the descending and sigmoid colon with surrounding lymph node enlargement Fig. . A colonWe planned to resect both gastric cancer and the two colon cancer foci simultaneously. Because some para-aortic lymph nodes were only slightly enlarged, we suspected that these nodes represented a reactive, inflammatory process as opposed to obvious metastases from either the gastric or colon cancers. Although a PET-CT scan was considered, it was not completed since we did not have access to a PET-CT scanner in our institution at that time. The patient underwent an open subtotal gastrectomy accompanied by a D2 lymphadenectomy (including superior mesenteric vein lymph node resection and middle colic artery lymph node resection), a gastrojejunal Roux-en-Y anastomosis, and a left colectomy accompanied by extended lymphadenectomy (D3)—including para-aortic node lymph node sampling—to address the slight preclinical suspicion of lymph node involvement from either the gastric or colonic primary.An 8.0 × 8.0 × 6.0 cm type 2 tumor was identified in the antrum Fig. . A 6.5 ×Although preoperatively the patient had a leukocytosis, this normalized after surgery Fig.. SimilarGastric ASC consists of a mixture of adenocarcinoma and squamous cell carcinoma components . In rareThese aspects of G-CSF-producing ASC, together with the very low incidence of this disease, pose challenges for treatment. There are few reports on G-CSF-producing ASC and no established standard treatment for this very rare disease. Takahashi et al. reported that chemotherapy (S-1 and CDDP) treated the adenocarcinoma component but not the squamous component and therefore suggested that sensitivity to chemotherapy may differ between adenocarcinoma and squamous cell carcinoma .The most common sites of metastases in gastric ASC are the liver and intra-abdominal lymph nodes . In JapaDue to the complicated management of this disease, multidisciplinary therapy that includes surgery and chemotherapy is often considered. There are few reports of chemotherapy in G-CSF-producing gastric ASC, and the outcomes of G-CSF-producing gastric ASC with surgery and adjuvant chemotherapy are suboptimal. Table Saito et al. reported treatment of their G-CSF-producing gastric ASC case using a combination of chemotherapy with docetaxel, cisplatin, and S-1 (DCS). Interestingly, an early antitumor effect was observed that correlated to the normalization of the WBC count. However, the tumor relapsed after the cessation of chemotherapy. In our case, the WBC count also normalized after surgery. Thus, the WBC count may serve as a potential biomarker for G-CSF-producing gastric ASC, though the number of cases is low to make this a strong claim.G-CSF-producing gastric ASC is a rare and aggressive tumor. Because these patients are usually diagnosed at an advanced stage, multidisciplinary evaluation and innovative treatment approaches are needed to improve their long-term outcomes.


Botrytis cinerea on apple fruit. Apples treated with thyme and savory essential oils showed significantly lower gray mold severity and incidence. Thyme essential oil at 1% concentration showed the highest efficacy, with lower disease incidence and smaller lesion diameter. The expression of specific pathogenesis-related (PR) genes PR-8 and PR-5 was characterized in apple tissues in response to thyme oil application and B. cinerea inoculation. After 6 h of pathogen inoculation, thyme essential oil induced a 2.5-fold increase of PR-8 gene expression compared to inoculated fruits. After 24 h of inoculation, PR-8 was highly induced (7-fold) in both thyme oil-treated and untreated apples inoculated with B. cinerea. After 48 h of inoculation, PR-8 expression in thyme-treated and inoculated apples was 4- and 6-fold higher than in inoculated and water-treated apples. Neither thyme oil application nor B. cinerea inoculation markedly affected PR-5 expression. These results suggest that thyme oil induces resistance against B. cinerea through the priming of defense responses in apple fruit, and the PR-8 gene of apple may play a key role in the mechanism by which thyme essential oil effectively inhibits gray mold in apple fruit.The efficacy of thyme and savory essential oils were investigated against  Botrytis cinerea, which causes gray mold, is one of the main pathogens causing postharvest losses of apples, especially for its conidial germination and mycelium growth at storage temperatures as low as 0 °C [Apple is one of the most important fruits in international trade; however, it can suffer from severe postharvest losses during long distance transport and storage.  as 0 °C .Botrytis populations [Gray mold control strategies mainly rely on chemical treatments; however, the use of synthetic fungicides is limited by the emergence of resistant strains. Following intensive use, resistance to thiabendazole has been reported in ulations . In addiulations ,4.Plant essential oils are gaining interest due to their apparently safe nature and their potential effectiveness as biopesticides for crop protection. On pome and stone fruit, as on table grapes, many plant essential oils have been effective against brown rot and gray mold rot ,6. ThymeOther studies described their role in the induction of host resistance against pathogens. Induced resistance is a promising strategy to enhance a host’s defense capacity after treatment with biotic- or abiotic-inducing agents, which provides long-term systemic resistance to a broad spectrum of pathogens and pests ,11. UponB. cinerea in stored apple fruit, and then to investigate whether the efficacy of the oil application is associated with the priming of defense responses in apples.The objectives of this work were to evaluate the efficacy of important plant essential oils against Malus x domestica Borkhausen ‘Red Fuji’) were harvested from a commercial orchard in Piedmont, Italy. Fruits were surface-sterilized and punctured with a sterile plastic tip. A strain of Botrytis cinerea Persoon was isolated from rotten apples and tested for its virulence by inoculation in artificially wounded apples. The strain was maintained on PDA  slants at 4 °C and the spores were harvested after the pathogen was incubated on PDA in Petri dishes at 25 °C for 7 days. The strain was used throughout the experiments at a concentration of 105 conidia/mL determined by a Bürker chamber (Knittel).Apples (Satureja montana L.) and thyme (Thymus vulgaris L.) were purchased as commercial preparations with 99% purity from Soave . The compositional analyses were performed using a gas chromatograph Shimadzu  GC-2010 Plus equipped with a mass spectrometer GCMS-QP  and a split-splitless injector  [vol/vol) were prepared for each essential oil application by diluting the stock emulsion with distilled water. All the resultant emulsions were shaken for 30 s before application to ensure a homogeneous essential oil mixture.Essential oils from savory  . A 10% sB. cinerea strain was assessed as follows: 30 µL of each treatment was pipetted into apple wounds. Water served as a control. Twelve hours later, 30 μL of pathogen conidial suspension was inoculated into each wound. Also, a standard chemical , Italy; 19.7% active ingredient, a. i.) was employed at 0.3 mg a. i. mL−1. Fruits were incubated at 23 °C. Rot incidence and diameter were measured 6 days after inoculation. Each treatment contained three replicates with 10 fruits per replicate and the experiment was performed three times. Wounded apples were treated with thyme (1%) in each wound. Twelve hours later, wounds were inoculated with B. cinerea. Apples treated with thyme or inoculated with B. cinerea were included to compare PR gene expression. Sterile distilled water served as a control. Apple tissue samples (exocarp and mesocarp) were obtained from 20 fruits stored at 23 °C at 6, 24, and 48 h post inoculation (hpi).The control activity of plant essential oils against ® Extraction Kit , then treated with TURBO DNase . Absence of genomic DNA contamination was confirmed by PCR amplification of the elongation factor 1 α (EF1α) gene using a One Step RT-PCR Kit (Qiagen). First-strand cDNA was synthesized using an iScript cDNA Synthesis Kit .Total RNA was extracted from apple samples using an RNeasyΔΔCT, where ΔΔCT represents ΔCTsample—ΔCTcontrol [Reverse transcriptase (RT)-qPCR was performed in triplicate on cDNA obtained from each biological replicate using 2× Power SYBR Green Supermix (Bio-Rad). Amplification and detection were carried out in an iCycler (Bio-Rad), set up with initial denaturation at 95 °C for 10 min followed by 40 cycles comprising a denaturation step at 95 °C for 15 s and an annealing step at 52 °C for PR-8 and at 60 °C for PR-5 for 1 min. Primers PR8-F (5′-GCCACTGCAACCCCGCTAGT-3′), PR8-R (5′-GCGGGCGCGAATCTGACTGA-3′), PR5-F (5′-CAAGCAGCTTCCCTCCTCGGC-3’), and PR5-R (5′-GCCCCAGAAGCGACCAGACC-3’) were used to optimally amplify PR-8 and PR-5 gene sequences. Transcript levels of the elongation factor 1 (EF1α) served as an internal standard, and the primers used were EF1α-F (5′-GACATTGCCCTGTGGAAGTT-3′) and EF1α-R (5′-GGTCTGACCATCCTTGGAAA-3′) . The expTcontrol , and valStatistical analysis was performed with SPSS version 20.0 . Data obtained in all experiments were analyzed using analysis of variance (ANOVA). Means of treatment results were separated at the 5% significance level by using Duncan’s multiple range tests. Values are presented as the mean ± SD (standard deviation of the mean). Results are the mean of three independent experiments.B. cinerea rots were measured in ‘Red Fuji’ apples treated with two plant essential oils , especially thyme, which showed a disease incidence and rot diameter of about 85% and 21 mm, respectively, compared to 100% and 38 mm of the inoculated control.Disease incidence and the diameter of lesions of ial oils . Six dayB. cinerea on different apple cultivars was reported by Lopez-Reyes et al. [Thyme essential oil, and its major components thymol and carvacrol, showed antifungal activity against plant pathogenic fungi . Moreoves et al. , who cons et al. . A certas et al. .B. cinerea apple inoculation. However, additional molecular studies were accomplished to prove this hypothesis.In the present study, the efficacy of thyme essential oil could also be related to the induction of host resistance, since the oil was applied 12 h before In order to analyze the molecular mechanisms involved in thyme-induced resistance in apple, the expression of the two defense-related genes, PR-8 and PR-5, was analyzed in treated apples at different time points .After 6 h of pathogen inoculation, thyme essential oil induced a 2.5-fold increase of PR-8 gene expression compared to the negative control (wounded fruits treated with water), while no significant upregulation of PR-8 expression was observed in inoculated fruits.B. cinerea. The early induction of PR-8 expression was no longer significant in thyme oil-treated fruit at 36 h after the essential oil treatment.After 24 h of inoculation, PR-8 expression in thyme-treated apples was similar to the negative control, whereas PR-8 was highly induced (7-fold) in both thyme-treated and untreated apples inoculated with B. cinerea mycelium. In a recently performed experiment on the germination of B. cinerea conidia on apple at 20 °C [B. cinerea conidia formed appressorium-like terminal thickenings within 4 h of incubation, indicating the initiation of penetration [At this time point, PR-8 was induced similarly in inoculated untreated fruit and in inoculated treated fruit. This could be related to the early response of apple, which tries to counteract the penetration of at 20 °C , the timetration . The fruetration .B. cinerea could produce elicitors either directly or indirectly that are able to trigger a defense response strong enough to significantly increase the resistance of the fruit to a subsequent pathogen attack [Therefore, after penetration into the host tissue during the first 24 h after inoculation, n attack .B. cinerea is significantly higher.After 48 h of inoculation, PR-8 expression in apples treated with thyme oil and inoculated with the pathogen was 4- and 6-fold greater than that in inoculated and water-treated apples. This is the most significant effect, as 48 h after pathogen infection inoculated fruit no longer show an increase of PR-8 expression, while the response in fruits treated with oil and inoculated with B. cinerea and treated with thyme essential oil.Thyme essential oil influenced PR-5 expression during the trial, as shown in The treatment of plants and fruits with different inducers, such as cell walls, plant extracts, essential oils, compounds of biological origin, and synthetic chemical compounds, was shown to trigger resistance to pathogen attack locally and systemically . There aB. cinerea since it favored the accumulation of the pathogenesis-related protein PR-8, which codes for a class III chitinase.In our study, thyme essential oil significantly induced apple resistance against Candida oleophila was reported to elicit a long-lasting activation of PR-8 at 24 and 48 h, but a high level of PR-8 protein was present even at 8 h. PR-8 expression was higher than that in fruit inoculated with B. cinerea, and it increased during the trial. The rapid host response to C. oleophila could be due to the general activation of apple fruit defense when exposed to microbes [After 6 h of pathogen inoculation, thyme caused a low direct induction of resistance (PR-8 expression level was 2.5-fold higher than that in water-treated fruits) since this signal activated the plant to a state of alertness. After pathogen infection, fruits strongly and efficiently responded, showing a long-lasting period of protection (PR-8 expression level was 6-fold greater than that in water-treated fruits after 24 and 48 h of pathogen inoculation). This effect is also known as the ‘priming effect’, which is beneficial since it is considered as a host solution to the trade-off dilemma between disease protection and costs involved in direct defense activation, and therefore the costs become affordable only when the host is exposed to pathogen attack . Similarmicrobes . The difB. cinerea or treated with C. oleophila. After 8 and 24 h, PR-5 gene expression was stable and similar to that observed in the untreated control. Only after 48 h were PR-5 levels higher in B. cinerea-inoculated apples in comparison to other treatments. However, since the expression level was even lower than that at time 0, in that case it was also difficult to attribute any biological significance to the observed pattern of expression [In contrast, a less clear response was achieved for PR-5 expression. As for PR-8 expression, the PR-5 level was also increased with respect to the untreated, inoculated or inoculated and treated fruit after 6 h. After 48 h, the only increase in PR-5 expression was obtained in inoculated fruit treated with thyme essential oil. Conversely, the presence of the pathogen did not induce any increase in PR-5 levels. Nevertheless, the magnitude of variation was very slight and it would be difficult to attribute any role of this gene to the induction of resistance under the studied conditions. Similar results were reported in literature on apples inoculated with pression .B. cinerea in stored apple. Based on gene expression results, it is postulated that one mechanism by which thyme effectively controls gray mold disease in apple fruit is through the induction of the PR-8 gene in apple fruit, besides its known antifungal activity. Deeper studies are required to understand the role of the PR-8 protein as a pathogen defense mechanism, its expression in various apple cultivars against a wide range of pathogens, and how specific pathogens may interfere with PR-8 expression.Thyme essential oil at 1% could be used as an efficient postharvest treatment against


Chiropractors are primarily concerned with musculoskeletal disorders but have the responsibility to deal also with prevention in other areas.To establish the prevalence of chiropractors who have a positive opinion on the use of primary prevention (PP), their actual use of PP, and the proportion of patients who consult for PP in relation to (i) musculoskeletal disorders, (ii) public health issues, or (iii) chiropractic treatment for wellness.A systematic search for literature was done using PubMed, Embase, Index to Chiropractic Literature, and Google Scholar and updated on February 15th 2017. Inclusion criteria were: surveys on chiropractors and/or chiropractic patients, information had to be present on PP in relation to the percentage of patients who consult for PP in chiropractic practice or in a chiropractic student clinic, and/or the percentage of chiropractors who reported using PP, and/or information on chiropractors’ opinions of the use of PP, in the English, French, or Scandinavian languages. The review followed the PRISMA guidelines. Articles were classified as ‘good’, ‘acceptable’ and ‘unacceptable’ based on scores of quality items. Results from the latter group were not taken into account.Twenty-five articles were included, reporting on twenty-six studies, 19 of which dealt with wellness. The proportion of chiropractors who stated that they had a positive opinion on PP was generally higher than the proportion of chiropractors offering PP. Most chiropractors offered some type of PP for musculoskeletal disorders and more than a half stated that they did so in the public health area but also for wellness. For all types of PP, however, it was rarely stated to be the reason for patients consulting. Regardless the type of PP, the proportion of patients who actually consulted specifically for PP was much smaller than the proportion of chiropractors offering PP.More research efforts have been put into wellness than into prevention of musculoskeletal disorders or public health-related disorders. It therefore seems that parts of the chiropractic profession are in search of an understanding of various aspects of clinical practice over and above its traditional musculoskeletal role. Interestingly, only a small proportion of chiropractic patients consult for PP, despite the readiness of the profession to offer such services. It is well accepted that non-communicable diseases, whether musculoskeletal or not, represent a social and economic burden, because they can be the source of long-term morbidity, and with increasing longevity they are expected to become increasingly common . The prePrevention can be performed at three stages of disease. Primary prevention (PP) deals with the prevention of disease in healthy people, secondary prevention is used to prevent a condition from recurring, whereas tertiary prevention is often defined as maintaining at a reasonable level a chronic condition that cannot be reversed . In thisChiropractors are recognized to be primary health care practitioners in many parts of the world, and consequently the regional Councils on Chiropractic Education state that a public health approach including health promotion should be implemented in chiropractic undergraduate programs –7. It thBack pain and extremity problems can result in reduced physical activity with secondary consequences, such as obesity and reduced cardiovascular fitness, so the role of chiropractors would extend beyond that of trying to prevent back pain. In fact, the World Health Organization supports the concept that chiropractors have a role in the prevention of musculoskeletal disorders and other public health issues by stating that “Chiropractic is a health care profession concerned with the diagnosis, treatment and prevention of disorders of the musculoskeletal system and the effect of these disorders on general health” .In addition to this, the World Federation of Chiropractic endorses and encourages chiropractors’ participation in public health promotion activities apart from musculoskeletal health . VariousThe ‘classical’ form of PP in relation to hygiene, improved working conditions, vaccinations etc. has resulted in large improvements of the public health status, but in more affluent countries and groups of people a more recent variant of PP has become apparent, that of the ‘wellness movement’. Wellness can be defined as “an active process in which an individual changes his or her behaviour in a manner which promotes health in all dimensions” . ChiroprSome chiropractors assume that a spinal derangement/dysfunction  can be reliably detected in both symptomatic and asymptomatic spines, and that the chiropractic manipulation (‘adjustment’), with or without other supportive treatments, can remove derangements and improve dysfunctions, a therapeutic approach which in turn is believed to have a favourable effect not only on present but also on future back problems. Some chiropractors also believe that this has a favourable effect on health in general, both in relation to a general feeling of well-being  and diseprevalence of chiropractors with positive opinions of the use of PP?What is the prevalence of chiropractors who use PP?What is the proportion of chiropractic patients who consult for PP?What is the Some of the above preventive activities intuitively make sense, whereas others are controversial. Therefore, we wanted to learn more about what chiropractors think and do in relation to PP and also what actually happens in their clinic. In other words, do patients consult for PP? For these reasons, we undertook a systematic review to obtain answers to the following questions:We attempted to deal with each of these questions from three angles: 1/Musculoskeletal conditions, 2/Public health issues, and 3/Wellness, which we defined as PP through chiropractic care.The AMSTAR checklist for methodological quality of systematic review  was follThe search included peer-reviewed articles in journals that could be traced through PubMed, Embase, Index to Chiropractic Literature, and Google Scholar. We searched the literature from January 2000 until February 15th 2017 to include only recent information. Search strategies were developed with a health science research librarian, using free text words.and  and (questionnaire* or survey*)”. In Embase the search strategy was: “chiropract* and  and (questionnaire* or survey*) and [embase]/lim not [medline]/lim)”. In Index to Chiropractic Literature it was: “chiropract* and  and (questionnaire* or survey*)”. In Google Scholar it was: “(chiropractic or chiropractors or chiropractor) and  and (questionnaire or questionnaires or survey or surveys)”.For Medline these were: “chiropract* A hand search was also done consulting texts and reference lists of relevant articles. We did not search the non-peer reviewed literature specifically, but would accept such texts if they were easily available.The first author (GG) selected the articles from the titles based on the inclusion and exclusion criteria. Thereafter, two authors (GG and CLY) independently screened abstracts and full texts using the inclusion and exclusion criteria.Surveys on chiropractors and/or chiropractic patients.Information had to be present on: PP in relation to information on chiropractors’ opinions of the use of PP, and/or the percentage of chiropractors who reported using PP, and/or the percentage of patients who consult for PP in chiropractic practice or in a chiropractic student clinic.Languages: English, French, Swedish, Danish or Norwegian, as these were the languages the authors could easily read.Inclusion criteria were:Articles reporting on the topics described above but on treatments not usually given by chiropractors . We also excluded articles on improvement of sport performance.If several publications existed from the same study, we would select the most relevant or complete of the publications in relation to our study objectives.Exclusion criteria were:Chiropractic students and chiropractic academic staff were not defined as ‘chiropractors’.The information in the selected articles was reviewed in relation to two elements: 1/quality  and 2/results. Three checklists were designed for those aspects. Our requirements were lenient. We did not check contents of references to trace additional or missing information. We sought our information in the methods and result sections but not from the abstract or title.A score was given to each selected article regarding various quality aspects and reported as a percentage. This score was used to determine the weak and strong points in this research field but also to classify the articles in descending order based on their individual total quality score. One point was given for correct answers. When the answer was incorrect or missing, it was given a score of 0. In some cases, half a score could be given. When an item was irrelevant because of the study design (e.g. no information would be available on patients if the purpose of the study was to study only chiropractors), it would be denoted as ‘irrelevant’.Target population defined: Specific subpopulations may have different practice patterns, therefore it is important to define the target population. This would give one point.Study sample: One point was given if the study sample(s) was/were described at least for age, sex, geographical distribution, or professional background.Sampling method: To avoid selection bias, the whole population, a random, or – possibly – a consecutive sample would be needed, resulting in one point, whereas a convenience sample brought 0 points. National chiropractic associations were considered whole populations and conference participants were classified as belonging to a convenience sample.Response rate: The higher the response rate, the easier to generalize the results to the underlying population. Therefore, the reader needs to be informed of the percentage of participants. One point was given for providing this information or if it was possible to calculate. Response rates in surveys are often low but, nevertheless, we considered samples of 10% or less to be unacceptable, resulting in 0 point, as it would severely limit the generalisability of the results in such cases.Response/Non response comparison: If the response rate was lower than the arbitrarily determined cut-point of 80%, we expected to find some type of responder/non-responder analysis. One point was given for this, if this comparison was needed. If it was not needed, because the response rate was above this cut-point, the response was defined as “not applicable” and given one point as well. If the response rate was not given but a response/non response comparison done, one point was given for the latter but not for the former.The first checklist refers to the representativeness of study samples Table . Points Definition/explanation of PP: PP must be well defined or at least explained in order to show that the authors have a clear understanding of which concept they are studying. However, it was not considered reasonable to expect authors to define every aspect of a study with multiple outcome variables. Therefore, this definition was required only if prevention was the main topic of the study (one point if there was a definition in the introduction or method in articles having prevention as main topic).Relevant questions or questionnaires available for the reader: Questions and/or questionnaires must be appropriate, for which reason it is important to make them accessible in the article or available on request, thus resulting in one point.Attempt to assure quality of survey instrument: The quality of the survey instrument was considered acceptable if questions were selected based on a thorough review of the literature, if there was a pilot study, or if the questionnaire/relevant questions had been previously tested at least for user friendliness, thus resulting in one point.Opinions to PP, as reported by chiropractors: One point was given if the reporting was anonymous, or if the confidentiality of the chiropractor was respected.Use of PP: One point was given if the reporting was anonymous or if the confidentiality of the chiropractor was respected.Reasons for consulting reported by the chiropractor: One point was given for actuarial reporting  and 0 point for approximate reporting .Reasons for consulting reported by patients: One point was given for patients providing reasons for consulting independently of the treating chiropractors (anonymously) or if it was stated that the patients’ confidentiality was respected.The second checklist deals with the validity of the results Table . Points One of the authors of this review had co-authored one of the reviewed articles, therefore a third person reviewed that article. Disagreements between the two reviewers were discussed to achieve consensus. If they could not reach agreement, the third author would be consulted.Thereafter, articles were arbitrarily classified, based on the scores of the two quality checklists. The article was classified as ‘good’ if the final score was ≥ 80%, as ‘acceptable’ if the final score was between 60 and 79%, and as ‘unacceptable’ if the final score was < 60%. This classification was partly based on the spread of data, because the difference between groups, particularly between ‘acceptable’ and ‘unacceptable’, should not depend on one single point.It was often difficult to understand how chiropractors and patients defined the three concepts of PP . In such cases, we looked for specific words in the text that could indicate the underlying meaning and classified the articles as shown in Table Assessment of the articles was done using the checklists independently by two of the authors, after which their respective checklists were compared, followed by a discussion on unclear points. Such queries were always resolved, because usually different interpretations of articles arose from difficulties in finding the relevant text.The articles were arranged in descending order in relation to their classification and their final quality score with a colour-coding of the three subgroups . Results Table  were then = 19), a description of how exactly it was perceived or dealt with, was provided only in four articles  in relation to the three types of PP  have been illustrated in Table We found it surprising that so few patients feel that chiropractors have something to offer in this area, although the chiropractic profession is encouraged to participate in preventive activities and clearly is interested to do so . The reaThe quality of studies varied. We classified ten of the studies as being of good quality. On the other hand, we removed six studies from the reporting of results, considering their findings to be uncertain because of their methodological approach. However, they are presented in the checklists, making it possible for interested readers to consult their characteristics and results. Interestingly, we did not note a gradual improvement of the quality scores by year of study, indicating that research teams did not learn from each other’s ‘mistakes’. The methodological approach seemed to be an aspect that was inherent in the individual research teams.Our review was somewhat limited from the lack of specific definitions of PP in most studies, which could have resulted in misclassifications, in particular in relation to wellness. We did not feel it fair to include a quality criterion on this issue unless the main topic of the survey was prevention, but even when this was the primary aim of the study, the descriptions of PP were vague and did not allow us to contextualise with accuracy. This could make it difficult to decide whether study subjects and/or the research teams had a clear opinion of whether they really dealt with PP  or if they mixed it up with other types of prevention, such as prevention of recurrences or perhaps even maintenance care and also whether the activity related to public health in general or not. These problems could have been resolved if survey instruments and the specific questions had been available, but this was often not the case. However, often the context and surrounding information could remedy this weakness, such as when authors mentioned that they studied the subluxation and its link to disease, which would indicate that chiropractors endorsing this concept considered it possible to perform PP through chiropractic adjustments.Another problem in the literature that made our interpretations difficult was that the response rates were (as is often the case in surveys) mainly low (below 80%) and that only few authors compared responders to non-responders. This probably (but not for sure) limits the representativeness of the study samples, assuming that there is heterogeneity among chiropractors and their patients on these issues. Although it is impossible to define a cut-point for when a response rate is too low to result in generalizability, perhaps authors and editors should consider whether surveys with response rates as low as 10% and less are worthy of reporting in the literature. Stating this, it is acknowledged that the 10% response rate cut off used in this review was arbitrarily chosen.In relation to the various methodological considerations surrounding this review, our work was guided by a modified AMSTAR checklist . One of However, it is possible that we failed to retrieve some relevant surveys on this topic. In the chiropractic field, some professional journals exist that are ‘invisible’ when searching through the usual library sources. We initially searched two medical databases (PubMed and Embase) later completed with Index to Chiropractic Literature and Google Scholar. This approach added two articles, but we could have missed out on some other relevant work, assuming that they could have been traceable through other library databases.As we did not explicitly search the grey literature, we would have missed surveys published by such media but, probably, studies not published through the peer-review process would have a relatively low methodological standard, which would limit their usefulness. For this review, we were unable to obtain three of the articles found through the literature search, which, potentially, were lost from the review. Nevertheless, it is unlikely that (at the most) three additional articles would have markedly changed our findings.Interestingly, according to this review of the chiropractic literature, more research efforts have been put into wellness than into prevention of musculoskeletal disorders or public health-related disorders such as cardiovascular disease. It therefore seems that parts of the chiropractic profession are in search of an understanding of various aspects of clinical practice over and above its traditional musculoskeletal role.Although it is possible that PP is provided as a natural element during the course of treatment – and hence not discovered through surveys asking for reason for consulting, it is clear that only a small proportion of chiropractic patients consult for PP, despite the readiness of the profession to offer such services.If chiropractors wish to provide more PP to their patients, it would be necessary to review the literature on the effectiveness of this approach in relation to musculoskeletal prevention and wellness. It is quite possible that this will reveal a dearth of relevant information, which in turn should incite interested chiropractors to encourage well designed clinical studies on these topics.