General practitioners (GPs) provide primary care and advise their patients on which diagnostic and therapeutic pathways they judge most appropriate. For patients with functional neurological disorders (FND), receiving a proper explanation of diagnosis by their GP from the very beginning may drastically improve prognosis. Novel approaches to the diagnosis and treatment of FND have important implications for effective management. The aim of this study was to investigate Italian GP opinion and knowledge about FND in light of new approaches to the illness. To do this, we evaluated the responses to a 13-item web-based survey completed by 133 GPs practicing in northern Italy. Psychological terms to describe FND were more frequently used than functional neurological disorder and mental illness was considered an important predictor of diagnosis. Referral to a neurologist rather than to a psychiatrist was largely preferred, while physiotherapy consultation was seldom recognized as a valuable approach to treating FND. Overall, the survey findings suggest that knowledge about novel approaches to FND is somewhat lacking. Currently, GPs appear to be transitioning from a classical psychological view of the disorder toward a more modern conceptualization, in which neurobiological, psychological, and social factors all play an important role. Professional education during this transition would be an advantageous way to optimize physician management of FND and to enhance diagnosis, explanation, and management across primary and secondary care pathways. General practitioners (GPs) often encounter patients presenting with symptoms unexplained by organic damage . In patiOur understanding of FND has made considerable advances, with implications for diagnosis and treatment. Research into the pathophysiology of FND has challenged old assumptions about the disease as a primarily psychological illness . It has Nonetheless, many health care professionals, including GPs, find FND difficult to manage , 11. StuExploring GP attitudes toward FND deserves particular attention since they play a pivotal role in health care . They arad hoc online survey. The items were focused on areas  that have been consistently reworked in light of the novel approach to FND. We also explored GP attitudes toward FND, their role in diagnosis and treatment, and their interest in improving their understanding of the disease.With this exploratory study we investigated the opinion, knowledge, and clinical experience of a cohort of Italian GPs regarding FND. To do this, we developed an ad hoc quantitative exploratory web-based cross-sectional survey are reported according to the Checklist for Reporting Results of Internet E-Surveys (CHERRIES) guidelines  and professional characteristics (number of years practiced as GP and number of patients).Section 2: Opinion, knowledge, and clinical experience. The first of seven items investigated exposure to FND . The second was a multiple-choice question about the terms used to name FND. The third focused on predictors of diagnosis. The respondents rated the extent to which they felt certain criteria were predictive for a diagnosis of FND as “not predictive at all,” “not very predictive,” “somewhat predictive,” “very predictive,” “extremely predictive,” or “I don't know.” The fourth item investigated opinions about the adequacy of specialist consultation/treatment. The respondents rated the degree of adequacy  of four specialist consultations and five types of treatment for FND. The fifth regarded the management strategies the respondents used to deal with their patients. The respondents stated the extent to which they agreed  with options for initial intervention in FND. The sixth item was a multiple-choice question on the role of GPs in FND diagnosis and treatment. The final item investigated the level of satisfaction on an 11-point scale from 0 (no satisfaction) to 10 (high satisfaction) in dealing with patients with FND. The items in this section are reported in Section 3: Interest in FND. The third section consisted of two dichotomic items investigating interest in FND.non-organic neurological disorders. The neutral term non organic was used to avoid connotations with presumed pathophysiological mechanisms underlying the disease . An example of what we meant by a “non-organic” neurological disorder  was also given to exclude potential bias due to misleading terminology. The invitation contained the survey link, and personal data handling (anonymity). Also, GPs were informed that the survey took about 5 min to complete. Four e-mail reminders were sent by SIMG at 2, 4, 8, 12, and 16 weeks after the initial mailing. Response to all survey items was mandatory. If a response to an item was missed, an alert appeared on the screen and the respondent had to respond to the item in order to proceed to the next one. The study was approved by the ethics committee of the University of Verona and was conducted in accordance with the Declaration of Helsinki. Response to the survey was assumed to grant consent  – Section of Verona, and the Federazione Italiana Medici di Medicina Generale (FIMMG). Only society members practicing in Verona were invited to take part at the survey. The Google Forms online tool was used . Data were collected over a 18-weeks period (30 October 2017–7 March 2018). An e-mail from the Verona chapter of SIMG (SIMG -Verona) inviting participation in the study was sent to the 397 SIMG and FIMMG members practicing in Verona. The e-mail explained that the study investigated GP opinions, knowledge, and clinical experience with  consent .p < 0.05.Data were downloaded from the Google Forms onto Excel sheets and reviewed for accuracy. Analysis was performed using SPSS software (version 19). Responses were analyzed with descriptive statistics, expressed as the mean (±standard deviation [SD]) and percentage for continuous and categorical variables, respectively. Data from Section 2 were analyzed in depth after converting responses to predictors of diagnosis, specialist consultation, treatment adequacy for FND, and management strategies into a 5-point Likert scale from 1  to 5 . The distribution average of the responses to each item  was compared using the Wilcoxon signed-rank test. Spearman's correlations were performed to investigate potential correlations between respondent age and years of medical practice. The difference in the number of male and female GPs was analyzed using the chi-squared test. Statistical significance was set at n = 133). Most respondents were men   and >51 years old  . The majority had been practicing for more than 20 years  , with a catchment area of more than 1,000 patients   .n = 78, 59%) reported they believe that <10% of their patients might present neurological symptoms without an organic cause. Many  estimated a higher proportion of these disorders among their patients (10–25%). Few  believed that 25–50% of their patients were likely to be affected by FND, while none reported that more than half of their patients might have FND. Very few were unable to estimate how many of their patients might have FND   .n = 6), with the majority of respondents  selecting more than one response. “Functional neurological disorders”  and “Somatization disorder”  were the two most frequent responses (n = 3), “Somatization disorders” (n = 21), “Depression” (n = 6), “Unspecific anxious syndrome” (n = 9), “Psychogenic disorder” (n = 9), “Medically unexplained syndrome” (n = 1), “Non organic neurological disorder” (n = 12). The term “Somatization disorder” was selected 9 times alone and 49 times together with “Functional neurological disorders” (n = 21), “Conversion disorder” (n = 3), “Depression” (n = 15), “Unspecific anxious syndrome” (n = 21), “Stress related disorder” (n = 20), “Psychogenic disorder” (n = 8), and “Non organic neurological disorder” (n = 5).Respondents could choose from a list of 10 terms they usually used to describe neurological symptoms without an organic cause. More than one response was allowed for this item. A total of 259 responses were collected, including the free-text responses  than the novel term “Functional neurological disorders” , indicating that a psychological view of FND is still widely held by this sample of GPs. Terms related to uncertain etiology  were less often selected   were selected more often  , suggestn = 2), intentional tremor (n = 1), fibromyalgia (n = 1), neurological disease under assessment (n = 1), cognitive deficit due to side effect of statins in patients with genetic predisposition (n = 1).Finally, free-text responses were included to capture any other terms in use; for example: symptom description (p < 0.01). “Litigation” was rated lowest among all options (2.72 ± 1.15) : either “not predictive at all” (n = 23) or “not very predictive” (n = 21) (p > 0.09) or years of practice .When asked to judge the extent to which certain diagnostic criteria were predictive for FND  , on aver(n = 21) . The degp < 0.038). “Psychotherapy consultation” (3.31 ± 1.03) was rated higher than “Physiotherapy consultation” (2.92 ± 1.00) . “Psychiatric consultation” (3.12 ± 0.96) was rated equally adequate for FND as both “Psychotherapy consultation”  and “Physiotherapy consultation”  (p > 0.30) or years of practice (p > 0.21).When asked to judge the extent to which certain specialist consultations were adequate for FND  , the res = 0.14) . These rp > 0.07), suggesting that they believed them to be equally adequate for treating FND. Moreover, “Psychotherapy without antidepressant or anxiolytic medications” (3.16 ± 0.80) was rated lower than “Rehabilitation”  and “Educational interventions” . Finally, “Pharmacological treatment” (2.89 ± 0.79) was rated lowest among all treatments , indicating that the pharmacological approach was judged the least adequate for treating FND , suggesting that the younger the respondent, the higher that psychotherapy without pharmacological treatment was judged adequate for treating FND. Significant correlations were also found between years of practice and two treatments: “Psychotherapy without antidepressant or anxiolytic medication”  and “Rehabilitation services” , indicating that the fewer the years of practice, the higher the adequacy of these two treatments was rated.When the GPs were asked to indicate the degree of adequacy of five different treatments for FND  , on averting FND . Age sigp > 0.208). These interventions were more often selected than “Referral to a psychiatrist (2.83 ± 0.96), “Pharmacological prescription” (2.73 ± 0.87), and “Referral to another specialist” (2.19 ± 0.90) . “Referral to a psychiatrist” and “Pharmacological prescription” (p = 0.435) were rated higher than “Referral to another specialist”   or years of practice .When asked to indicate their level of agreement (from “extremely disagree” to “extremely agree”) with management strategies that they would adopt as a first step when dealing with FND , the res< 0.001) . The degn = 93, 70%) rated their level of satisfaction between 5 and 10. The satisfaction level did not correlate with age  and years of practice .When asked to rate the degree of satisfaction in managing FND on an 11-point scale from 0  to 10 (extremely satisfied), on average, the respondents stated they were satisfied with how they dealt with FND (5.48 ± 2.16). The majority  gave more than one response, with “Following-up the treatment together with the specialist who delivered the diagnoses”  the most frequent one; “Make the diagnosis and recommend the most adequate treatment”  and “Educational intervention for patients and their families”  were also frequently given. “Referral to the specialist most appropriate for the patient's medical condition” was selected by 32 GPs (24%). Finally, some believed that their role was “Make the diagnosis and personally follow-up the patient” ” ” .n = 119, 89%) or by taking part in professional courses on these disorders .The majority in our sample were interested in improving their knowledge of FND through access to more information  views of these disorders clearly emerged when the GPs were asked about the terms they usually adopt for symptoms without an organic cause. Use of terminology reflects the many different ways of conceptualizing and approaching the problem of patients with symptoms that are unexplained by the disease . In our This emerged also from responses to the items about predictors of diagnosis, specialist consultations, treatment, and management strategies. Although the term “psychological stressors” has been removed from the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), previous mental illness or psychological stress was rated among the most predictive criteria and often ranked as “very predictive” or “extremely predictive.” This shows that many still hold a psychological view of FND and lack knowledge about new diagnostic criteria. Another observation is that litigation was rated as the least predictive factor for a diagnosis of FND. Since litigation has long been linked to feigned symptoms or malingering, our results suggest that the respondents felt patient-reported symptoms to be generally genuine and not faked to obtain secondary gain, with implications also for forensic medicine.per se to distinguish functional symptoms from their organic counterpart . Recent evidence has shown the effectiveness of psychological therapy  for seizure reduction, improved quality of life, and better global functioning in patients with dissociative seizures . MoreoveRemarkable findings also regarded the pharmacological approach to FND, which was considered less useful compared to rehabilitation and psychotherapy. Accordingly, pharmacological prescription was rarely endorsed by the GPs as the first approach to the disorder, suggesting their awareness that drug therapy is not particularly effective.Our findings highlight that the respondents were, on average, generally pleased with how they managed patients with FND. The majority indicated a high level of satisfaction when dealing with these disorders. This contrasts with evidence from previous surveys that found negative attitudes toward FNDs among specialists  , 6, 34. The study findings provide a picture of the current knowledge base, opinions, and clinical experience of a sample of Italian GPs about novel approaches to FND. Several limitations need to be acknowledged. The study sample was small, limiting the generalizability of our findings. Nonetheless, the response rate was in line with previous studies , 12, 24 Our survey findings suggest that GPs need professional education about FND. Up-to-date guidance about appropriate terminology to use, mechanisms leading to FND, evidence-based treatments, and stages of the stepped care approach should be provided with the overarching aim of ensuring that patients receive consistent diagnosis, explanation, and management across primary and secondary care pathways. Steps in this direction have been undertaken by the study Authors, who have conducted specialist educational courses for GPs about the novel approach to FND and created an illustrative leaflet about recent advances in FND. These interventions need to be extended to a wider population of Italian GPs in order to increase the effectiveness of primary care for patients with FND.The original contributions generated for this study are included in the article/The studies involving human participants were reviewed and approved by Ethics committee of the University of Verona. The patients/participants provided their written informed consent to participate in this study.AM, MF, and MT: conception, design, organization of the research project, and interpretation of data. IF and CF: organization of the research project and acquisition of the data. AM: organization and acquisition of the data, design and execution of data analysis, writing the first draft and revision of it based on the other authors' critique. MF, IF, CF, GD, and MT: revised the work for critique. All authors approved the final version to be published.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.


Oncofertility has evolved over the years, with a prodigious amount of research documenting the importance of fertility for young patients with cancer, and the potential impact that fertility impairments due to cancer treatments has on their Quality of Life (QoL). Multiple professional bodies and scientific societies have included fertility as an integral part of clinical management. Clinical guidelines advocate that health professionals have the duty to discuss the risk of infertility and fertility preservation options as early as possible and refer to fertility specialists when appropriate. Collectively, fertility decisions are regarded as difficult for both patients and providers. Since providing fertility‐related information is vital for better decision making, researchers and policy makers have concentrated their efforts in developing educational tools to aid decisions and guidelines to optimize the delivery of this information, focusing mainly on patients‐providers and largely neglecting the role and influence that partners play in this process. Here, we reflect on the importance of partners in fertility decisions, with a focus on the provision of fertility‐related information that is also geared towards partner. We highlight the need to involve partners in fertility discussions, and that their needs should be taken into account in both clinical guidelines and in the development of educational tools, for an optimal decision‐making process. There is a need to involve partners in fertility discussions. Partners' needs should be taken into account in both clinical guidelines and in the development of educational tools, for an optimal decision‐making process Over the years, the potential struggle that young patients diagnosed with cancer may face when confronted with the real possibility of losing their childbearing capabilities due to cancer treatments has been extensively documented. Despite a cancer diagnosis, many young patients dream of creating their own family or of extending their existing family in the future. The importance of fertility among young patients and the potential implications of its impairment on their quality of life (QoL) is well established. The use of assisted reproductive technology (ART) has become a hope to help men and women become parents in the future. For female patients, cryopreservation of oocytes, embryos, ovarian tissue, ovarian suppression, and transposition are available fertility preservation procedures. For men, cryopreservation of semen is the established method for fertility preservation. Adoption and third‐party reproduction are other options for patients who were not able to use fertility preservation techniques prior to the start of cancer treatments or for whom techniques were unsuccessful. The need to include fertility as an integral part of the clinical management of these young patients has been validated by several professional bodies and scientific societies by recognizing the significance of providing information and discussing potential fertility loss and offering fertility preservation to people diagnosed with cancer.Given the differences between female and male infertility risks, fertility preservation options, and the nature of the techniques and time required to implement them, this manuscript will focus solely on young women diagnosed with cancer.Significant and rapid advances have been made in oncofertility, a discipline that merges oncology with fertility,This narrative review addresses the importance of partners in the process of oncofertility decision making. Firstly, we provide current evidence on the importance of the partner during the decision‐making process. Next, we focus on the vital role of the inclusion of partners in fertility‐related information. This information includes the development of clinical and educational evidence‐based resources, such as decision aids and clinical practice guidelines for fertility preservation in women with cancer.2Deciding on fertility preservation options is already an emotional‐charged processResearch on the process of fertility‐related decision making in the context of cancer places the interaction between the patient and the health‐care professional  at the cornerstone of the decision. However, and without taking any legitimacy of value to the key players identified above, research has consistently documented that a significant number of participants in the studies are, in fact, women who are married or in a committed relationship.,35 Supporting this view, another study showed that women expressed that the ideal setting for fertility preservation discussions to occur were in the presence of their partner before the cancer treatment started.Culturally, partners have a strong influence on women's decisions to become a mother and, in general, the decision to initiate or extend the family concerns both members of the partnership. Such decisions are based on a consensus between the couple about both having a child and sustained by their commitment to endure the fertility treatment together.3A vital component of any decision‐making process in medical settings is the provision of patient information that is relevant to their individual needs.Beyond the possible emotional support that partners may provide to patients during the decision‐making process, for example, by accompanying them to the consultations; it is relevant to assume that partners are also active beneficiaries of the all processes, with justifiable informational needs in order to contribute to an informed fertility decision. For example, a couple may need to deal with the legal complexities involving some fertility decisions which may impact each partner, or have to adapt to a new reality as third‐party reproduction becomes the only available option. One might therefore also expect to see partners taken into account as key stakeholders, beneficiaries, and players during the fertility decision‐making process acknowledged in the development of fertility‐related informational tools and guidelines. It is worth examining partners’ acknowledgement in the development of these clinical and educational evidence‐based resources, designed to educate health professionals about the need to include fertility discussions into clinical management and aid patients in optimal idiosyncratic fertility decisions, respectively.3.1Decision Aids (DAs) are educational materials designed to assist with treatment decision making by addressing individual values and preferences,One of the first fertility‐related DAs for young breast cancer patients was developed and validated by Peate in Australia.3.2Clinical practice guidelines are intended to guide health‐care professionals and patient decisions regarding appropriate, safe, and cost‐effective fertility care for women who desire biological children after a cancer diagnosis,4Insights from cancer research allow us to discern that during the process of fertility discussions, which may include the use of educational resources, partners seem to play a valuable and influential role during the fertility decision‐making process. This reflects the uniqueness of fertility decisions, which contrary to many other shared medical decisions, such as treatment for a chronic condition, are characterized by a distinctive complex triadic interaction between the health‐care professional, the women, and their partner.The authors made no disclosures.VG conceived the idea, conducted the literature search, and wrote the original and revised versions of the report. PF participated in manuscript revisions. GQ conceived the idea and participated in manuscript planning and revisions. All authors approved the final version of the manuscript.


Distributed temperature sensing (DTS) systems can be used to estimate the temperature along optic fibers of several kilometers at a sub-meter interval. DTS systems function by shooting laser pulses through a fiber and measuring its backscatter intensity at two distinct wavelengths in the Raman spectrum. The scattering-loss coefficients for these wavelengths are temperature-dependent, so that the temperature along the fiber can be estimated using calibration to fiber sections with a known temperature. A new calibration approach is developed that allows for an estimate of the uncertainty of the estimated temperature, which varies along the fiber and with time. The uncertainty is a result of the noise from the detectors and the uncertainty in the calibrated parameters that relate the backscatter intensity to temperature. Estimation of the confidence interval of the temperature requires an estimate of the distribution of the noise from the detectors and an estimate of the multi-variate distribution of the parameters. Both distributions are propagated with Monte Carlo sampling to approximate the probability density function of the estimated temperature, which is different at each point along the fiber and varies over time. Various summarizing statistics are computed from the approximate probability density function, such as the confidence intervals and the standard uncertainty (the estimated standard deviation) of the estimated temperature. An example is presented to demonstrate the approach and to assess the reasonableness of the estimated confidence intervals. The approach is implemented in the open-source Python package “dtscalibration”. Temperatures can be measured with distributed temperature sensing (DTS) along optical fibers that may extend to several kilometers with a sub-meter resolution ,2. The aDTS systems that estimate temperature from Raman-backscatter measurements shoot a laser pulse through a fiber-optic cable, which is scattered back to the DTS system by inhomogeneities in the fiber. Most of the backscattered laser has the same wavelength as the emitted laser (Rayleigh scattering), but a small fraction has different wavelengths (Raman scattering). The detectors in DTS systems measure the intensity of the backscatter at two distinct wavelengths: Stokes (-Raman) and anti-Stokes (-Raman) scatter. The temperature at the point of reflection is estimated from these two types of scatter. Stokes scatter has a longer wavelength than the laser and its intensity does not vary much with temperature, while anti-Stokes scatter has a shorter wavelength than the laser and its intensity varies significantly with temperature. The location of the measurement along the fiber is estimated from the time between sending the laser pulse and receiving the scatter. Temperature along the fiber is estimated from the measured intensities of the Stokes and anti-Stokes scatter by calibrating to reference sections with a known temperature. In practice, these fiber sections are submerged in water baths of which the temperature is continuously measured with a separate temperature sensor. The water can be mixed with small pumps in an attempt to equalize the temperature of the water in the baths. Sequential temperature measurements require continuous calibration due to varying gains and losses in the DTS system. Detailed calibration procedures are available in the literature ,12,13,14Attenuation of light propagating through a fiber depends on its wavelength and therefore is different for Stokes and anti-Stokes scatter. The key in DTS calibration is to differentiate the attenuation from the temperature effects at the point of reflection. There are two types of setups to estimate temperature from Stokes and anti-Stokes scatter: single-ended and double-ended. In single-ended setups, only one end of the fiber is connected to the DTS system, and the difference in attenuation between the Stokes and the anti-Stokes scatter is approximated as constant over the fiber. However, this difference in attenuation is also affected by sharp bends, connectors, and bad splices, which may result in a shift in the temperature if not accounted for. In double-ended setups, measurements are carried out from both ends of the fiber to estimate the difference in attenuation between the Stokes and anti-Stokes scatter at each point along the fiber. In such setups, measurements from both ends of the fiber must be combined, including their uncertainty.In this paper, a new calibration procedure is presented for the temperature estimates for single-ended and double-ended setups, including estimates of the uncertainty in the form of confidence intervals. First, a brief review is given of how temperature can be estimated from Stokes and anti-Stokes intensity measurements. Next, the calibration steps are outlined for single-ended and double-ended setups. The spatial and temporal variability in the uncertainty of the temperature are demonstrated by an example of the double-ended calibration procedure. The new calibration procedure is implemented in the open-source Python package “dtscalibration”. This article concludes with a conclusion and a discussion with practical tips to reduce the uncertainty in the temperature.v, through the fiber, during which it attenuates with S is the scattering-loss coefficient for the scattering at the wavelength of the laser pulse, and R is the capture fraction, which depends on the fiber composition . The bagiven by  Selengths , λ is thx at the Stokes and anti-Stokes wavelengths. Most DTS systems use the ratio of Equation =1xαl+α+dxP−=1quations  are subsquation P−quation =−CI, if the terms C, and The temperature along the fiber can now be estimated from the Stokes and anti-Stokes intensity measurements, The differential attenuation, In single-ended setups, Stokes and anti-Stokes intensity is measured from a single end of the fiber. The differential attenuation is assumed constant along the fiber so that the integrated differential attenuation may be written as :(11)∫0xΔThe temperature can now be written from Equation  as:(12)TC must be estimated for each time and is constant along the fiber. Confidence intervals for the estimated temperature are discussed in The parameters x (m) is defined here positive in the forward direction, starting at 0 where the fiber is connected to the forward channel of the DTS system; the length of the fiber is L. Consequently, the backward-channel measurements are flipped and shifted to align with the forward-channel measurements. Alignment of the measurements of the two channels is prone to error because it requires the exact fiber length [In double-ended setups, Stokes and anti-Stokes intensity is measured in two directions from both ends of the fiber. The forward-channel measurements are denoted with subscript F, and the backward-channel measurements are denoted with subscript B. Both measurement channels start at a different end of the fiber and have opposite directions, and therefore have different spatial coordinates. The first processing step with double-ended measurements is to align the measurements of the two measurement channels so that they have the same spatial coordinates. The spatial coordinate r length . DependiEquation ). The cax along the fiber. Location Parameter quations  and 14)A(x) is iA must be estimated for each location and is constant over time. The calibration procedure is discussed in Parameters The Stokes and anti-Stokes intensities are measured with detectors, which inherently introduce noise to the measurements. Knowledge of the distribution of the measurement noise is needed for a calibration with weighted observations  and to pThe variance of the noise in the Stokes (quations  and 5)  σP+2) anquations  and (5) Values for quations  and 21)G(t) and quations . G(t) anquations  and (21)C, where C needs to be estimated for each time step. The parameters are estimated from the reference temperature at M locations along the reference sections and at N times. Equation  is solvm, time n, is a function of the variance of the noise in the Stokes and anti-Stokes intensity measurements σImThe variance of the noise in the Stokes and anti-Stokes intensity measurements is estimated directly from Stokes and anti-Stokes intensity measurements using the steps outlined in The sum of the squared weighted residuals may be minimized with a standard routine. In this paper, a custom sparse implementation of the weighted least squares routine of the Python package Statsmodels is used , which rA at that location equals 0 17). The Equation ). There quations  and (17)m are given by:The weights of the observations at location Equation  applied M locations are gathered in a single set of equations given by Equations M locatioquations –39). Th. ThM locEquation , where WEquation  with EquEquation  for Wm.A for the locations along the reference sections only. An estimate of A and its variance for locations outside the reference sections are required to estimate the temperature outside of the reference sections. An estimate of A for location p and time n outside the reference sections is obtained by setting The set of estimated parameters contains estimates of quations  and 17)A for thep is obtained using the inverse temporal-variance weighted mean: The variance of The number of reference sections required for calibration is discussed in The uncertainty in the estimated temperature varies along the fiber as the laser pulse attenuates when propagating through the fiber, and varies over time due to varying gains and losses in the DTS device. The two sources that contribute to the uncertainty in the temperature estimate are the uncertainty in the calibrated parameters and the uncertainty associated with the noise in the Stokes and anti-Stokes intensity measurements. The former dominates the uncertainty in the estimated temperature for measurements with longer acquisition times, while the latter dominates measurements with shorter acquisition times. Other sources of possible uncertainty are not taken into account here. These include the uncertainty introduced by the model that relates measured Stokes and anti-Stokes intensities to temperature, and the uncertainty in measured temperatures obtained with external sensors. The latter is generally much smaller than the uncertainty in the DTS temperature from the noise in the Stokes and anti-Stokes intensity measurements.Estimation of the confidence intervals of the temperature starts with estimating separate probability density functions for the Stokes and anti-Stokes intensity measurements and the calibrated parameters. The probability density functions are propagated through the model using a Monte Carlo sampling procedure following the steps from JCGM , JCGM 2. This prEstimation of the confidence intervals for the temperatures measured with a single-ended setup consists of five steps. First, the variances of the Stokes and anti-Stokes intensity measurements are estimated following the steps in A for each location outside of the reference sections. These distributions are centered around i the temperature Double-ended setups require four additional steps to estimate the confidence intervals for the temperature. First, the variances of the Stokes and anti-Stokes intensity measurements of the forward and backward channels are estimated following the steps in quations  and 45)A for eacquations  and 17)A for eacThe best estimate of the temperature Alternatively, the best estimate of https://github.com/dtscalibration/python-dts-calibration, together with installation instructions, examples, and documentation. The package reads DTS measurement files into an object, which has several calibration and plotting methods. Calibration and calculation of confidence intervals can be conducted within 10 lines of Python code, as is demonstrated in the examples of The presented calibration procedure is implemented in the Python package “dtscalibration” . It is oAn example of a double-ended setup is presented to demonstrate the spatial and temporal variability in the uncertainty of the temperature. This example attempts to estimate the uncertainty of the temperature along the entire fiber for a given acquisition time. The uncertainty in the estimated temperature can easily be reduced by increasing the acquisition time, but that limits the ability to observe temporal variation in temperature. Alternatively, the uncertainty can be reduced by increasing the sampling distance, but that limits the ability to observe spatial variation in temperature.A schematic representation of the setup is shown in The DTS system used in this example is a Silixa Ultima-S DTS system  that measures fibers up to 2 km with a factory-reported spatial resolution of approximately 30 cm. The system is configured to measure the Stokes and anti-Stokes intensity with an acquisition time of 2 s in the forward direction and 2 s in the backward direction, every 12.7 cm along 100 m of fiber . The sysThe variance of the noise in the Stokes and anti-Stokes intensity measurements of the forward and backward channels were estimated using measurements from the two reference sections , and fiber sections with a different differential attenuation are not accounted for . This includes sharp bends in ‘bend-tolerant’ fiber ,31. The The proposed calibration procedure to estimate the integrated differential attenuation in double-ended setups differs from the two-step procedure of van de Giesen et al. . They esThe presented calibration procedure can lead to better temperature estimates compared to existing calibration procedures. Parameters that are time invariant are kept constant and are estimated from all available data, which improves their estimation. By weighing Stokes and anti-Stokes measurements with their uncertainty, the estimation of parameters is less affected by measurement noise along reference sections with a low signal strength, i.e., at the end of the fiber.Furthermore, weighted averaging of the temperature from the forward and backward-channel measurements results in better temperature estimates than arithmetic averaging or using either of the individual channel measurements. Consider the synthetic temperature measurement of a double-ended setup shown in The presented calibration procedures require either one or two reference sections with a different temperature. In single-ended setups, two reference sections are needed to differentiate between All existing calibration procedures rely on the temperature of reference sections measured with external sensors. Any deviation of the fiber temperature from the temperature measured with an external sensor introduces errors. Problems that are commonly occurring with DTS calibration are ill-defined positions of the reference sections and non-uniform temperature of the water baths. Both can be discovered at different stages of the calibration procedure. They may introduce a bias in the estimated temperature, which is not accounted for in the estimation of the standard uncertainty and confidence intervals. Therefore, an additional external temperature sensor is recommended for identification of a bias in the reference temperature. A bias may then be identified by, for example, composing a figure of the time-averaged difference between the estimated temperature and the reference temperature, similar to quations  and 37)Xm in Eququations  and 21)Xm in Eququations . A non-uA new approach is presented to calibrate temperature from Stokes and anti-Stokes intensity measurements and to provide a confidence interval for the estimated temperature. The uncertainty in the estimated temperature is caused by the noise from the Stokes and anti-Stokes detectors, and the uncertainty in the calibrated parameters that relate the Stokes and anti-Stokes intensity measurements to temperature. Estimation of the confidence interval for the estimated temperature requires an estimation of the distribution of the noise from the Stokes and anti-Stokes detectors and a multi-variate distribution of the parameters that relate the Stokes and anti-Stokes intensity measurements to temperature. All these distributions are propagated with Monte Carlo sampling to approximate the probability density function of the temperature, which is different at each location and varies over time. Various summarizing statistics can be computed from the approximated probability density function, such as standard uncertainties and confidence intervals.Several improvements were made to existing calibration procedures to reduce the uncertainty in the estimated temperature. The integrated differential attenuation differs per setup and must be experimentally estimated to differentiate it from a shift in temperature. The integrated differential attenuation for double-ended setups is formulated such that it can be used directly in linear regression and the uncertainty in the parameter estimation is reduced. The parameter estimation is further improved by making use of the fact that some parameters are time-invariant, and to use all available data to estimate their value. The linear regression accounts for spatial and temporal variation in the signal intensity, so that large-intensity measurements have a larger weight in the parameter estimation than the small-intensity measurements.In double-ended setups, the temperature is estimated from measurements made from both ends of the fiber. Close to the ends of the fiber, the difference in Stokes and anti-Stokes intensity between the forward and backward measurements is large, resulting in a large difference in the uncertainty of the temperature estimated from the forward and backward measurements. The estimated temperature is a weighted average of the temperatures estimated from the forward and backward-channel measurements. Compared to unweighted averaging, this reduces the uncertainty in the estimated temperature. The uncertainty of the temperature that is estimated with the proposed calibration procedure is assessed in an example. The estimated temperature and 95% confidence intervals adequately represent the temperature of the reference sections.https://github.com/dtscalibration/python-dts-calibration. The package contains the new calibration procedures for both single-ended and double-ended setups, can compute confidence intervals of the estimated temperature, and includes several routines for visualization of the results.https://doi.org/10.4121/uuid:71b5c3c2-4105-4f4f-bd1e-d7c56732a665Dataset: Dataset license: GPL-3.0-or-laterThe calibration procedure is implemented in “dtscalibration”, an open-source Python package that is freely available under the BSD 3-or-later license from


S)-UA is less abundant in nature than (R)-enantiomer, it is difficult to source, thus precluding a deeper investigation. Among the lichens producing UA, Cladonia foliacea is a valuable (S)-UA source. In the present work, we report on a rapid HPLC-UV/PAD-CD protocol suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea extract. Best results were achieved using XBridge Phenyl column and acetonitrile and water, which were both added with formic acid as mobile phase in gradient elution. By combining analytical, spectroscopical, and chiroptical analysis, the most abundant analyte was unambiguously identified as (S)-UA. Accordingly, a versatile microwave-assisted extractive (MAE) protocol, assisted by a design of experiment (DoE), to quantitatively recover (S)-UA was set up. The best result in terms of UA extraction yield was obtained using ethanol and heating at 80 °C under microwave irradiation for 5 min. Starting from 100 g of dried C. foliacea, 420 mg of (S)-UA were achieved. Thus, our extraction method resulted in a suitable protocol to produce (S)-UA from C. foliacea for biological and pharmaceutical investigation or commercial purposes.During the years, many usnic acid (UA) conjugates have been synthesized to obtain potent endowed with biological properties. Since ( R)-configured enantiomer of UA is the most abundant in nature, and Cladonia arbuscula, C. mitis, Ramalina boninensis, R. pacifica, R. roesleri, R. farinacea, Usnea diffracta, U. longissima, U. hirta, U. steineri, Flavoparmelia caperata, and Xanthoparmelia chlorochroa were identified as valuable source for its extraction. Conversely, (S)-UA was isolated as an exclusive enantiomer in few species of Cladonia  and Alectoria (A. lata and A. ochroleuca) D25 value of the standard (R)-enantiomer reported in the datasheet of the supplier . By comparison of the absolute value of the optical rotation, we can state that the optical purity of the herein extracted UA is c.a. 99%  and that no racemization occurred during the extraction procedure rocedure .C. foliacea. The fully characterized (S)-UA and was used as standard in further experiments.To conclude, the (S) absolute configuration can be undoubtedly assigned to the UA extracted from To assess the experimental conditions suitable to extract exhaustively (S)-UA, a design of experiments (DoE) methodology was applied ,58. BrieIn contrast with the classic “one factor at a time” approach, DoE permits the study of the influence of multiple factors simultaneously. Specifically, two responses were studied varying three different parameters: solvent, extraction time, and temperature. Acetone, ethyl acetate, and ethanol characterized by different polarity and dielectric constant have been selected as extraction solvent, since it is well known that the microwave heating response depends on the solvent used.1) and the (-)-(S)-UA % recovered (Y2).We considered the total extraction yield percentage (Y7x − (3 ± 2) × 105). The statistical control of the method was assessed by performing three injections of external standard at the beginning of every measurement session on different days. The limits of detection and quantification were also estimated during the validation procedure using the S/N ratio. S was at least 3N in the peak region of the chromatogram for the limit of detection (LOD), whereas S > 10N for the limit of quantification (LOQ). The values resulted in being 0.0063 mg/mL and 0.0022 mg/mL, respectively, and were also experimentally confirmed. The method repeatability and accuracy were assessed by performing three injections on different days and assessing the UA recovery by a double-point standard addition method, spiking two aliquots of 0.30 g of sample with 12 mg and 9 mg of (-)-(S)-UA. Good recovery has been obtained, being the comparison between the spiked amount of UA and the amount found in the non-spiked samples as high as 99%. To sum up, the method is suitable for (S)-UA quantification in C. foliacea extracts. Results are reported in The quantitative UA determination was performed by HPLC-UV/PAD using the same protocol exploited for the identification of UA in the lichen dry extract. The (S)-enantiomer of UA herein isolated was used as an external standard and six-point calibration curves, each replicated three times, were determined. The method response function was linear with a good correlation coefficient (R) of 0.9996 (y = (2.58 ± 0.04) × 102), whose quantification is the primary goal of the present study, were obtained using ethanol, applying two cycles of microwave heating of 5 min each at 80 °C -(S)-UA . The extracted (-)-(S)-UA was analyzed by HPLC-UV/PAD-CD, the optical rotation was measured, and the analytical data were compared with those of the commercial (R)-UA standard and the (S)-UA standard previously prepared and characterized as described above. Based on the results obtained, we can state that the applied extraction protocol does not induce racemization.To obtain an insight into thermal (S)-UA degradation, an additional MAE experiment  was carried out, adding known amounts of (S)-UA to the natural matrix. The good recoveries obtained (about 90%) confirmed the chemical stability of UA under the experimental conditions. With the extraction method in hand, the extraction procedure was scaled up. In detail, 100 g of dried C. foliacea were harvested from a lowland dry grassland located at Bosco della Ghisolfa  in Spring 2018.Thalli of Plant material was reduced to a homogenous powder by grounding it with a blade-mill  just before performing the extractions.Formic acid and deuterated solvents were purchased from Sigma-Aldrich .HPLC-grade solvents were supplied by Honeywell , while analytical grade solvents were supplied by PanReac .Standard (R)-UA was purchased by Carbosynth .The evaporation procedures were performed under reduced pressure using a Heidolph Laborota 4000 instrument . Analytical thin-layer chromatography (TLC) was carried out on silica gel pre-coated glass-backed plates . The detection was conducted with UV light (λ = 254 nm). Flash chromatography was performed with silica gel 60 (particle size 230–400 mesh) purchased from Nova Chimica . Separations were carried out at room temperature, and separations were obtained using two different columns from Waters , namely a Symmetry C-18, 5 μm, 150 × 3.9 mm and an XBridge Phenyl 5 μm, 4.6 × 150 mm. Analyses were carried out using the following systems. HPLC-UV/PAD-CD: a Jasco  system consisting of a PU-1580 pump and a MD-1510 photodiode array (PDA) detector. Chromatogram acquisitions and elaborations were performed using the ChromNAV software . HPLC-UV/PAD-ESI/MS: Finnigan LCQ fleet ion trap system, controlled by Xcalibur software 1.4 . Optical rotation was recorded using a Jasco photoelectric polarimeter DIP 1000. Electronic circular dichroism (ECD) spectra were recorded on a Jasco J-1500 Circular Dichroism Spectrophotometer from 400 to 240 nm. Nuclear magnetic resonance spectra were recorded on Bruker Avance 400 spectrometers operating at 400 MHz. Chemical shifts (δ) are reported in parts per million with the solvent reference relative to tetramethylsilane (TMS) internal standard.v/v) added with formic acid ), (−)-(S)-UA as yellow powder and 3 mg of fumarprotocetraric acid have been isolated.A preliminary extract was prepared by applying an MAE methodology on 10 g of natural matrix using acetone as a solvent  for three cycles of 5 min each. Then, the extract was filtered, and the solvent was evaporated under reduced pressure. The green syrup so obtained was suspended in dichloromethane (DCM). The suspension was filtered, and the solid residue was washed two times with DCM, thus obtaining atranorin as white powder, as confirmed by NMR analysis. Then, the combined organic phases were concentrated under reduced pressure and purified by L/L extraction (water/hexane). The aqueous phase was discarded, and the combined organic phases were concentrated under reduced pressure. After flash chromatography : 10.57 , 6.82 , 6.62 , 6.62 , 5.28 , 2.46 , 2.42 .Fumarprocetraric acid, 5 mg, mp 257°C, 1H-NMR in CDCl3, δ (ppm): 12.59 , 12.53 , 11.98 , 10.40 , 6.55 , 6.44 , 4.02 , 2.72 , 2.58 , 2.13 .Atranorin, 2mg, mp 190°C, m/z 375; D25= −476°, c 0.5% CHCl3, 1H-NMR in CDCl3 δ (ppm): 18.86 , 13.33 , 11.05 , 6.00 , 2.70 , 2.68 , 2.13 , 1.78  (see (-)-S-UA, 15 mg, mp 204 °C, m/z 344, [α]A proper high-performance liquid chromatography-electrospray-tandem mass spectrometry (RP-HPLC-UV/PAD-ESI/MS) method was set up, analyzing the crude extract, atranorin, (-)-(S)-UA and fumarprotocetraric acids isolated, as described in v/v) of formic acid. The eluent was applied onto the column in gradient mode from 75% to 100% B in 7 min, which was followed by an isocratic elution step for 1 min. The column was reconditioned by eluting from 100% B to 75% B in 1 min and with a final 4 min isocratic elution at the initial conditions. Mass spectra were generated in positive and negative ion mode : ion spray voltage 3 kV, capillary voltage 10 V, aux gas flow rate 10, sheath gas flow rate 20, and tube lens voltage 75 V for positive mode and ion spray voltage 5 kV, capillary voltage −45 V, aux gas flow rate 10, sheath gas flow rate 20, and tube lens voltage −125 V for negative mode. MS/MS data were acquired in dependent scan mode (full-scan MS followed by MS/MS of the most intense ion). The identification of (S)-UA was unequivocally accomplished by comparing its HPLC retention time, UV, and MS spectra with those of the standard analyzed in the same conditions.The best results in terms of time of analysis and peak resolution were obtained at 1 mL/min flow rate at room temperature using an XBridge Phenyl column . The mobile phase consisted of water (A) and acetonitrile (B), which were both added with 0.1%  and (+)-(R)-UA (c: 1.37 × 10−6 M) were analyzed in a nitrogen atmosphere with an optical pathway of 1 cm. ECD spectra were scanned at 200 nm/min with a spectral bandwidth of 1 nm and a data resolution of 1 nm. For each measurement, 10 scans were taken and averaged, considering both enantiomers. ECD spectra of the solvent in the same experimental conditions were subtracted. Data are reported in Δε versus λ (nm) from knowledge of the cell path length and solution concentration (The chloroform solutions of (–)-(S)-UA - and (S)-UA were performed with Merck Molecular Force Field (MMFF) by using Maestro 10.3 of the Schrödinger Suite. The lowest energy conformers of (R)- and (S)-UA were subjected to ECD calculation, which was carried out with the B3LYP/6-31G level of time-dependent density functional theory (TDDFT). The polarizable continuum model (PCM) was used to take into account the solvent effects of chloroform. The calculated ECD spectra of (R)- and (S)-UA were the result of a weighted average of the calculated ECD spectra of each conformer based on Boltzmann distribution.7 x −(3 ± 2)×105). The statistical control of the method was assessed by performing three injections of external standard at the beginning of every measurement session on different days. The method accuracy was evaluated by assessing the UA recovery by a double-point standard addition method, spiking two aliquots of 0.30 g of sample with 12 mg and 9 mg of (−)-(S)-UA, respectively. Each experiment has been repeated in triplicate. The recovery found by comparing the spiked amount of UA to the amount found in the non-spiked samples was 99.3 ± 0.2% for samples spiked with 12 mg of UA and 99.1 ± 0.3% for samples spiked with 9 mg of UA.Quantitative determination of (−)-(S)-UA was performed using the (−)-(S)-enantiomer isolated in the preliminary part of this work as an external standard, exploiting the HPLC/UV-PAD protocol used for the UA identification in the dry extract. The calibration curve was built through six points, each replicated three times. The limits of detection (LOD) and quantification (LOQ) were estimated using the S/N ratio. S was at least 3N in the peak region of the chromatogram for the LOD, whereas S > 10N for the LOQ. The values resulted in being 0.0063 mg/mL and 0.0022, respectively. These data were also experimentally confirmed. However, the calibration curve was studied in the range of 0.75–0.1 mg/mL. The method response function was linear with a correlation coefficient (R) of 0.9996 (y = (2.58 ± 0.04)×101) and the yield of (−)-(S)-UA extracted per unit amount of source material (mg (−)-(S)-UA/g of lichen, Y2).The experimental design of the extraction procedure was aimed at the screening of the factors that influenced two responses, namely the total extraction yield , acetone , and ethanol .Three process factors were considered. These parameters are all known to strongly influence the extraction procedures and are also strictly related to microwave power. The first factor was the extraction solvent. Three solvents were selected having different responses to microwave heating, namely ethyl acetate .The second factor was the time of extraction .The third factor was the extraction temperature  under m2·3 experimental design selected counted twelve experiments. One additional test experiment was performed in three independent replicates and kept apart from those used to build the model  results were modeled by the equationThe (−)-(S)-UA yield  shows a good fit (r = 0.935), the model for (−)-(S)-UA yield (Y2) shows limited fitting ability (r = 0.661). However, the model for Y2 provides sufficient evidence of a linear dependence on the parameters X2, time of extraction, and X3, temperature, and no significant dependence on the interaction term X2 X3 accounting for the combined effect of extraction time and temperature.Both models predict reliably the experimental results with the predicted data, which refer to the experiment with ethanol irradiated with two cycles of 5 min each, at 70°C, i.e., in the test point having coded coordinates S1 and Y2 validated. The difference between the predicted data and the experimental data is within the uncertainty limits associated with the used experimental procedure.Based on these results, we can consider the models for Y1) and for UA recovery (Y2), while extraction time (X2) and temperature (X3) affect the responses to a lower extent. In particular, high temperature (80°C) and low extraction times (2 cycles x 5 min each) enhance (−)-(S)-UA recovery (Y2) and do not affect the extraction yield (Y1).The results of our experiments showed that the proposed models could predict the trend of the expected extraction yield and (−)-(S)-UA recovery. According to our results, ethanol is the best solvent both for the extraction yield , spectroscopical , and chiroptical analysis  and by comparison with the corresponding commercially available (R)-enantiomer used as standard, the UA produced by the C. foliacea was unambiguously identified as (S)-UA with an optical purity > 99%. Moreover, the herein proposed HPLC-UV/PAD protocol is suitable for the quantitative determination of UA in lichen extracts, and the association of an in-line CD allows us to obtain at the same time information about the stereochemistry of the UA, thus discriminating between the two enantiomers of UA that the different lichen species may produce.To sum up, in the present work, a suitable microwave-assisted extraction (MAE) coupled with HPLC-UV/PAD-CD protocol was set up for the stereochemical identification of UA content in the lichen extracts and to achieve a complete recovery of UA from C. foliacea quantitatively. The best result in terms of UA extraction yield was obtained using ethanol and heating twice at 80°C under microwave irradiation for 5 min. The herein proposed procedure is simple, rapid, low cost, and applicable to evaluate the content of UA also in other lichen extracts. Of note, the optimized protocol requires less than 20 min in total to perform (−)-(S)-UA extraction and quantification. The optimized MAE protocol was assessed for the scale-up extraction of UA, and it represents a suitable procedure to produce (S)-UA for biological or pharmaceutical studies or commercial purposes.Afterward, a versatile microwave-assisted extractive (MAE) protocol, assisted by a design of experiment (DoE), was optimized to recover (S)-UA from


Serological tests have been widely used for detecting human T-cell lymphotropic virus type 1/2 (HTLV-1/2) antibodies in the endemic areas, but their performance in low-risk populations is rarely reported. The aim of this study was to evaluate the performance of four HTLV-1/2 screening assays and to discuss a strategy for diagnosis of HTLV-1/2 infection in a non-endemic area. At the present study, 1546 specimens repeatedly reactive (RR) by one screening ELISA were collected from blood centers/banks from January 2016 to April 2019. Avioq-ELISA, Murex-ELISA, Roche-ECLIA and Fujirebio-CLIA were independently performed on each plasma sample and compared to WB and LIA confirmatory tests. Positive or indeterminate specimens with blood available were quantified by qPCR. The results showed that 48 samples were finally confirmed as HTLV-1 positive, 13 were HTLV positive, 151 were indeterminate, and 387 were negative. All the WB-positive samples were also LIA-positive. Roche-ECLIA showed the highest sensitivity that was able to detect 91.8% positives and combined with the Murex-ELISA would significantly increase the positive detection rate (98.4%). In addition, LIA yield more indeterminate and HTLV-untyped results than WB (152 vs. 27), but was able to resolve infection status of some individuals with an indeterminate WB. Besides, 3 WB indeterminate and 1 LIA-untyped samples were confirmed as HTLV-1 positive by qPCR. Based on these findings, we put forward a proper test strategy for HTLV-1/2 diagnosis in low-prevalence areas. If possible, the Roche-ECLIA with the highest sensitivity is suggested as a second screening assay in primary labs. If not, all RR specimens are recommended to be firstly retested by Roche-ECLIA and Murex-ELISA in the reference lab. Secondly, samples reactive to any one of the two tests were quantified by qPCR, and then the NAT-negatives were furtherly submitted to LIA for confirmation. Thereby, the cost can be reduced and the diagnostic accuracy would be improved. Human T-lymphotropic virus (HTLV) was the first RNA retrovirus to be associated with cancer . To dateCurrently, initial diagnosis of HTLV-1/2 infection is mainly based on detecting specific antibodies in plasma or serum using enzyme-linked immunosorbent assay (ELISA) and CLIA or ECLIA. Several commercial kits based on recombinant and/or synthetic peptide antigens alone or in combination with viral lysates have also been adapted for large-scale screening of HTLV-1/2 antibodies. However, the kits fail to differentiate between HTLV-1 and HTLV-2 infections because the two types share a high homology between them . BesidesWestern blot is most frequently used to confirm the presence of anti-HTLV-1/-2 antibodies. The FDA-licensed MP Diagnostics HTLV Blot 2.4 uses a combination of recombinant HTLV-1/2 proteins and HTLV-1 viral lysate to improve sensitivity. This assay also uses HTLV type-specific recombinant envelope protein (gp46-1 and gp46-2) to discriminate viral types . HoweverMolecular assay is another method used for HTLV confirmation. Real-time PCR or quantitative PCR (qPCR) have been used to determine the real status of HTLV infections and quantify the proviral load (PVL). However, these molecular assays have had low sensitivity in special population groups, such as individuals infected with HIV or HTLV-2 .Although several commercially available HTLV-1/2 diagnostic assays have been used worldwide, their performance in endemic and non-endemic areas varies greatly. Moreover, few studies have been conducted to compare the performance of LIA and WB in low-prevalence populations. Therefore, the objective of this study was to evaluate several FDA- or CE-licensed HTLV-1/2 assays to diagnose the specimens that were RR to one ELISA, and to discuss strategies for the diagnosis of HTLV-1/2 infection in a non-endemic area.All specimens were collected from eligible donors from blood banks/centers in 26 provinces in China between January 2016 and April 2019. The RR samples were delivered at 2–8°C or frozen to the NCCL for confirmation. The RR samples were also HBsAg, anti-HCV, anti-HIV, and anti-TP negative by both two screening ELISAs in the enrolled blood centers. According to the confirmatory process , all RR Samples with limited plasma volumes that were not enough for all assays were excluded from our study. Enrolled samples with a positive result either by WB or LIA were classified as positive. Positive or indeterminate specimens with whole blood available were further quantified by qPCR in our lab. In addition, blood donations with an indeterminate result were recollected from the same donor, and retested by the same detection process.Three different kinds of commercially available ELISA assays  were used for screening HTLV1/2 antibodies at all participating blood centers/banks in China. Reactive specimens were retested by the same assay and any one of two-round retests found to be reactive were defined as RR specimen.Further detection was done in the NCCL using Elecsys HTLV-I/II , Lumipulse G HTLV-I/II  and two kinds of ELISA assays  and Avioq HTLV-I/II Microelisa System . The key features of the evaluated assays are summarized in In the LIA, the strips contained recombinant proteins  for confirmation and synthetic peptides  for differentiation of HTLV-1 and HTLV-2 antibodies. Samples were considered HTLV positive if they were reactive with at least two confirmation bands, including rgp21 I/II. The positive samples were typed as HTLV-1 if the intensities of gp46 I and p19 I bands were equivalent to or higher than that of the gp46 II band. HTLV-2 positivity was defined if the gp46 II band were more intense than the gp46 I and p19 I bands and if no discriminate bands appeared, an untypable HTLV result was denoted. A specimen was classified as indeterminate if it were reactive with one band (rgp21 I/II) or two bands (except rgp21 I/II). Samples were considered negative if a single rp24 I/II, rp19 I/II, gp46 I, or gp46 II band or no bands appeared.WB is most frequently used as a supplemental assay for confirming and differentiating the presence of antibodies against HTLV. Results of MP HTLV Blot 2.4 were interpreted according to the stringent criteria provided by the manufacturer. Briefly, HTLV-1 seropositivity was defined as reactivity to gag (p19 with or without p24) and two env (GD21 and rgp46-I). HTLV-2 seropositivity was defined as reactivity to gag (p24 with or without p19) and two env (GD21 and rgp46-II). Samples that were reactive to both gag (p19 and p24) and env (GD21) were defined as HTLV seropositive but were considered untypable. Any other patterns of specific bands that did not meet the above criteria were considered as indeterminate. Absence of bands or appearance of non-specific band patterns were interpreted as negative.DNA was extracted from whole blood of each specimen using the Tiangen Magnetic Blood Genomic DNA Kit . HTLV-1 proviral DNA was detected and quantified by qPCR using a set of primer pairs and a TaqMan probe targeting the HTLV-1 pol region. The RPPH1 gene was also amplified simultaneously as an internal control. The primers and probes are shown in t-test. p < 0.05 was considered statistically significant.The PPVs were calculated by SPSS version 21.0 . Descriptive statistics are presented as geometric means + standard deviations. Results were analyzed with A total of 1546 RR samples with enough volumes were included in the final sample count in our study. Of the 1546 samples, 555 showed discordant results and 991 showed consistent results across the four assays. Of the 991 consistent samples, 44 were reactive to all four assays while the rest were negative in all assays. Finally, 599 samples that were reactive to at least one assay were confirmed by LIA and WB. Of these samples, 73.29% (439/599) showed consistent results in the two confirmatory tests, including 44 HTLV-1 positive samples, 8 indeterminate samples and 387 negative samples . As sampAs shown in the p < 0.0001) and negative samples (p < 0.0001) (p < 0.05), indicating that reactivity indexes may correlate with the confirmatory results.In each assay, positive samples confirmed by LIA and WB displayed a significantly higher reactivity index than indeterminate ( 0.0001) . MoreoveTo determine the relationship between PPVs and reactivity indexes on four evaluated assays, we analyzed the results of the assays at different cut-off reactivity indexes for the four assays . We founSeventeen samples that were finally defined as HTLV-1 or HTLV positive showed discrepant results between INNO-LIA and WB , 3. Of tAfter combining the results confirmed by LIA, WB and qPCR, 5 samples were classified as HTLV-1 positive and 12 samples were HTLV positive but untypable . The aveOf the 151 samples which were finally interpreted as indeterminate, 130 were LIA ± / WB-, 15 were LIA-/WB ± and 6 were indeterminate by both tests , 5. MostThirteen indeterminate samples were recollected from the same donors no less than 8 weeks after last donation and retested by all assays . SeroconCurrently, screening for HTLV-1/2 antibodies in healthy blood donors is not done routinely in China. A systematic review revealed that HTLV-1/2 prevalence in the coastal areas of China, and especially in Fujian province, is higher, compared to the low average HTLV-1/2 prevalence in the mainland . HoweverIn our study, almost one-third samples displayed discordant results among the four evaluated assays, which may due to the differences in the combination, source and quantity of HTLV-1/2 antigens used by different manufacturers. None of the evaluated assays could detect all the true HTLV positives when using LIA as a standard. In contrast, the Roche-ECLIA was a relatively more sensitive test that was reactive to 91.8% confirmed positive samples. This assay was especially sensitive to the untyped positives . Moreover, Roche-ECLIA could detect more indeterminate samples than the other three assays, indicating it was a sensitive method for screening HTLV-1/2 antibodies. From the perspective of blood banks/centers, sending RR samples to a reference lab is costly and time-consuming. Therefore, we propose that, where possible, such samples could be retested by the Roche-ECLIA and the positives subjected to subsequent confirmatory tests. Our data suggest that this would reduce the need for confirmation by about two thirds.The present study indicated that most untypable HTLV infections had relatively low S/CO or COI values or were misdiagnosed by some screening tests, indicating a high risk of missed detections when only one screening assay is used in the reference labs. In our study, the combination of Roche-ECLIA and Murex-ELISA performed relatively well, detecting 98.4% true positives when LIA was the standard. We detected 100% positive samples when Murex-ELISA was combined with Roche-ECLIA and Fujirebio-CLIA but this strategy would be costly, time-consuming and labor intensive for reference labs.HTLV-1-associated diseases develop in only a small proportion of infected individuals and slowly progress to disease relative to the average life span of humans. Consequently, after balancing screening/conformational costs and the threat of HTLV infections to public health especially in the non-endemic area, we recommend the use of a combination of Roche-ECLIA and Murex-ELISA in reference centers to retest initially reactive blood samples in low-risk areas. Since Roche-ECLIA is more sensitive than Murex-ELISA, it is best to test RR samples with Roche-ECLIA first and then the negatives by Murex-ELISA. Therefore, the number of test would be reduced by about 12% compared with test simultaneously.Our results also indicate that higher S/CO or COI values were associated with higher diagnostic reliability. As demonstrated in this study, samples with S/CO values over 2.5, 11.0, 67.2 for Avioq-ELISA, Murex-ELISA, and Roche-ECLIA, respectively, or COI value over 28.0 for Fujirebio-CLIA, were rarely negative. Hence, samples with values above these thresholds can be interpreted as HTLV-1/2 positive, decreasing the demand and cost of confirmatory tests. Accordingly, if Murex-ELISA and Roche-ECLIA were combined for sample retesting in reference centers, the number of samples needing confirmation would reduce by 14.7%. Nevertheless, molecular assays or other supplementary tests would be necessary to determine PVL or HTLV infection types.WB-indeterminate and/or untypable variants remain a serious problem worldwide. It has been found that the frequency of WB-indeterminate samples reactive to HTLV-1 ELSIA varies among different cohorts, but is much higher in low-risk blood donors . In our Indeterminate WB patterns indicating true or false virus infection have been reported previously . In our Although LIA reduced the number of WB-indeterminate samples, the assay yielded more indeterminate  and untypable  results than WB. Most of the samples expressed a single gp21 I/II pattern , the largest of which had faint intensity . We propose several reasons for these inconclusive results, including low HTLV proviral loads, mutations in the provirus (defective particles), seroconversion period or cross-reactivity with other antigens or viruses. In our study, 30 LIA-indeterminate and 5 LIA-untyped samples were tested by qPCR, but only one LIA-untyped sample was confirmed HTLV-1 positive with a PVL of 0.009 copies/100 PMBC, indicating that samples with very low PVL may be hard to identify. Although the qPCR method used in our study did not detect all the specimens, our results indicated that qPCR was useful for resolving inconclusive confirmatory tests. Due to the large numbers of uncertain results and the high cost of WB and LIA assays, we propose employing qPCR first, followed by WB or LIA to test any qPCR-negatives. Other studies also recommend this strategy, which has been shown to reduce costs and improve the accuracy of HTLV-1/2 diagnosis .Without a prophylactic vaccine and with limited available treatment options for HTLV-related diseases, recommendations for the deferral and reentry of blood donors are necessary, especially in China where HTLV-1/2 screening is underdeveloped. Based on our study, we suggest that donors with a final positive interpretation by a confirmatory test should be deferred permanently but donors returning a negative result should be reentered. Additionally, donors with indeterminate results should be followed-up, and recollected specimens should be confirmed through the same supplemental test.In this study, we compared the performance of four commercially available kits for screening anti-HTLV-1/2 antibodies and used WB and LIA as confirmatory tests. We also proposed a new testing algorithm for screening and confirming HTLV-1/2 in a low prevalence region . We recoThe original contributions presented in the study are included in the article/This study was approved by the institutional review boards in every participating blood bank. Written informed consent are routinely completed at the time of donation from blood donors.HJ, LC, andLWdesigned the study and wrote the manuscript. HJ, LC, XJ, HS, and FG performed the experiments. HJ, LC, YY, and LW analyzed the data. All authors participated in critical revision of the manuscript and approved the final version.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.


Under severe damage leading to the loss of conventional ISCs, quiescent stem cells and even differentiated cells can be reactivated into stem-cell-like cells with multi-potency and contribute to the reconstruction of the intestinal epithelium. This process requires fine-tuning of the various signaling pathways, including the Hippo–YAP system. In this review, we summarize recent advances in understanding the correlation between Hippo–YAP signaling and intestinal homeostasis, repair, and tumorigenesis, focusing specifically on ISC regulation.Despite endogenous insults such as mechanical stress and danger signals derived from the microbiome, the intestine can maintain its homeostatic condition through continuous self-renewal of the crypt–villus axis. This extraordinarily rapid turnover of intestinal epithelium, known to be 3 to 5 days, can be achieved by dynamic regulation of intestinal stem cells (ISCs). The crypt base-located leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5 The small intestinal epithelium consists of single-layered, well-organized structures called the crypt–villus unit ,2. Intes+ ISCs via cellular reprogramming of differentiated cells or reactivation of quiescent stem cells, implying that the superior regenerative potency of the intestine rather than other adult tissues is exerted by dynamic regulation of IECs in a context-dependent manner [ISCs replenish all intestinal epithelial cells (IECs) throughout the intestinal tract. For instance, at least six different epithelial lineages comprising the small intestine can be derived from the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-positive ISC pool ,8. Theset manner .The development of cell-type-specific promoter-based lineage-tracing techniques and the emergence of single-cell RNA sequencing analysis provides an in-depth understanding of the characteristics of IECs and regulatory-signaling pathways ,12,13. F+ ISCs for lineage tracing and found that CBCs are genuine stem cells that can replenish the entire crypt–villus axis in the intestinal tract. Lgr5 is a Wnt target gene and when it binds to its ligand, R-spondin, it is internalized with Wnt-degrading E3 ubiquitin ligase RNF43 and ZNRF3, which potentiate Wnt signaling [The presence of continuously-dividing stem cells in the crypt base was first described in the 1970s . These cignaling . CBCs arignaling ,21, whilignaling . This joignaling . These fignaling .+ cells are less dependent on the Wnt pathway and resistant to radiation injury than CBCs [+ CBCs both in vivo and in vitro, confirming their multi-potency as stem cells [The +4 cells are located between the CBC-Paneth cell-rich crypt zone and the TA zone, usually just above the uppermost Paneth cell . Besideshan CBCs . In addiem cells . Howeverem cells , the debem cells ,38, sugg+ ISC-depleted mice can be recovered from experimental colitis due to the contribution of Atoh1+ secretory progenitors to IEC regeneration. Considering their position, Dll1-high or Atoh1+ secretory precursors might belong to the LRC population. Indeed, LRCs share the transcriptional phenotype of secretory lineages including Mmp7, Kit, Chga, Gip, and Pax6 [+ enterocytes after the depletion of Lgr5+ cells and unexpectedly found that these cells migrated downwards to the crypt base and de-differentiated into multiple cell types of cells, including stem cells and secretory cells [Despite the genetic ablation or injury-associated loss of CBCs, the intestinal epithelium can be completely regenerated; thus, many efforts have been made to define the alternative cellular source of conventional ISCs during the regenerative procedure using the lineage-tracing technique. Since the first evidence was presented in 2012 , secretoand Pax6 ,43. In aand Pax6 , Paneth and Pax6 ,45, and and Pax6 . Specifiry cells , implyin+ ISC-associated signature genes become open chromatic states during the de-differentiation process [+ cells [Collectively, it is now widely accepted that cellular reprogramming of differentiated cells into ISCs is one of the primary strategies for intestinal recovery; however, to date, the underlying molecular mechanisms that regulate cellular plasticity remain obscure. It is reported that these LRC-like secretory precursors are under the dynamic modulation of chromatin accessibility and that Lgr5 process . Others [+ cells ,48. A re[+ cells . NotablyDrosophila melanogaster. In Drosophila, the core components of the Hippo pathway, including Hippo (Hpo) [The Hippo pathway is an evolutionarily-conserved signaling pathway involved in various cellular functions. The pathway was initially described through genetic screening for tumor suppressors involved in regulating tissue growth in po (Hpo) ,51,52,53po (Hpo) , Warts (po (Hpo) , Mats [5po (Hpo) , Yorkie po (Hpo) , and Scapo (Hpo) ,59, coorThe core kinase cascade and downstream effectors of the Hippo pathway discovered in flies are highly conserved in mammals. The mammalian sterile 20-like 1 and 2 (Mst 1 and 2) are the first kinases of the mammalian Hippo pathway and are homologues of Hpo . ActivatThe Hippo pathway is regulated by variable upstream inputs or cross talk with other signaling pathways. So far, the Hippo pathway does not have specific receptors or relevant ligands, unlike other well-known classical signaling pathways. Instead, upstream components involved in polarity, morphology, and adhesion of cells have been reported to regulate the Hippo pathway ,79,80,81Drosophila epithelium, apical membrane-localized Merlin (Mer), Expanded (Ex), and kidney- and brain-expressed protein (Kibra) constitute a complex to cooperatively activate the Hippo pathway by binding to Sav, Hpo, and Wts [Drosophila, the transmembrane cadherins Fat (Ft) and Dachsous (Ds) form a complex and activate the Hippo pathway through the induction of Wts degradation [Most cells show polarity, represented by apical–basal polarity and planar cell polarity observed in epithelial cells. Both types of polarity can regulate the Hippo pathway. The apical–basal cell polarity is established by protein complexes that make up cell–cell junctions, including adherent and tight junctions. In the  and Wts ,86,87. I and Wts . Nf2 is  and Wts . Planar radation ,91. Althradation ,93.Several groups have demonstrated that structural proteins of tight junction and adherens junctions can interact with YAP. A tight junction protein, angiomotin (Amot) can inhibit YAP by physically interacting and transferring YAP from the cytoplasm to tight junctions or actin filaments, lessening the phosphorylation status of YAP . In addiYAP activity is also sensitive to mechanical cues from neighboring cells or the ECM. Indeed, ECM stiffness, cell tension, cell spreading, and cell attachment or detachment were proven to modulate the Hippo pathway ,80,81. F12/13, Gαq/11, or Gαi/o by coupled ligands including lysophosphatidic acid, sphingosine 1-phosphate, and thrombin receptor agonists can stimulate YAP and TAZ, whereas Gαs activation with glucagon and epinephrine can suppress YAP and TAZ [Recently, several studies have suggested that extracellular ligands can regulate the activity of YAP and TAZ for G-protein-coupled receptors (GPCRs). GPCR signaling can either activate or inactivate YAP and TAZ, in a manner dependent on the class of G proteins. For instance, the activation of Gα and TAZ ,105,106.Moreover, other signaling pathways such as Wnt, Notch, Sonic hedgehog (Shh), and epithelial growth factor receptor (Egfr)/Kras, have been shown to cross talk with the Hippo pathway ,108,109.The Hippo pathway also has cross talk with Notch and Shh signaling pathways. Deletion of Mst 1/2 or overexpression of YAP has been shown to induce Notch activation ,117. In Recent studies have shown that YAP protein can regulate the Egfr/Kras signaling pathway. For example, it was found that EGFR-activated Ras during tumorigenesis up-regulates the level of cytoplasmic YAP protein and unphosphorylated YAP, which in turn, can induce Egfr transcription to create a positive feedback loop . MoreoveAs observed in other organs, the Hippo signaling pathway is indispensable during the steady state of the mammalian intestinal epithelium. IEC-specific Mst1/Mst2 double knockout mice exhibit a short life span (13 weeks in median) with severe wasting signs and disorganized crypt–villus structure due to the hyperplasia of ISCs and undifferentiated cells are found in their intestinal tract, leading to adenoma formation . A simil+ ISCs in both the in vivo and in vitro organoid culture systems [The nuclear accumulation of YAP in the IECs is dynamically regulated along the crypt–villus axis. In general, a high level of nuclear YAP is detected at the ISC-rich crypt base (except for Paneth cells), while it tends to be translocated into the cytoplasm and diminished in the upper region of the villi ,122,124. systems . In cont systems . Both mi systems ,122,124; systems . They fu systems . Organoi+ single-cell-derived intestinal organoid development. They compared the trajectory of ISC dynamics between normal budding organoids and spheroid-like enterocysts in a time-dependent manner and found that YAP1 and its target genes are transiently activated for the induction of a symmetry-breaking process. Interestingly, homogenous YAP activation prevents, as observed in the constant YAP-OFF state, the formation of budding structure, and further maturation of the organoid. They further revealed that uneven YAP activity induces Paneth cell generation by generating differences in cell-to-cell availability for Notch ligand Dll1, suggesting the contribution of YAP activity to the formation of an epithelial niche.These contradicting observations regarding the role of YAP in intestinal homeostasis might be derived from the differences between YAP activation (systemic vs. IEC-specific), the complexity of ISC-regulating signaling pathways, the possible contribution of TAZ for YAP activity ,128, and+ CBCs, followed by a robust regeneration.To study the nature of intestinal regeneration, various experimental conditions such as irradiation exposure, chemotherapeutic 5-Fluorouracil injection, and administration of colitogenic agent dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) are used ,132,133.To date, growing evidence has proven the involvement of Hippo and YAP signaling in tissue regeneration . In gene+ cells in the crypt revealed that mature Paneth cells re-enter the cell cycle, divide actively, and re-differentiate into other cell types after irradiation [+ ISCs, confirming their clonogenic potential. Importantly, distinctive nuclear translocation of YAP precedes this process, implying its involvement in Notch-signaling-dependent reprogramming of Paneth cells. Indeed, a simple increment of Wnt signaling achieved by constitutive β-catenin induction cannot reproduce this phenomenon, but forced expression of the Notch signaling activator NICD1 induces de-differentiation of Paneth cells. Meanwhile, the role of YAP activation in the emergence of repairing-associated cells has been suggested [+ cells hardly proliferate or differentiate under homeostasis; during the repair phase, on the other hand, they replace lost CBCs and re-constitute the entire epithelial structure as shown by crypt–villus ribbon formation. Strikingly, the gene signature of the Clu+ cluster of the damaged crypt is similar to the YAP-associated gene signature [During epithelial regeneration, cellular reprogramming such as cell fate conversion and de-differentiation occurs under the control of Hippo and YAP signaling. First, lineage-tracing analysis of Lyzadiation . These iuggested . After tuggested . Using aignature  and Lats2 (PGE2) is regarded as a master regulator of YAP-induced intestinal cellular reprogramming. The therapeutic impact of PGE2 on intestinal repair and regeneration has been well established in various experimental models [2-mediated epithelial regeneration and that YAP plays a major role in this context [2 to the recovery of DSS-induced colitis by showing the increased epithelial regeneration of PGE2-degrading enzyme 15-PGDH-null mice. Upon binding the EP4 receptor, PGE2 increases the transcription of YAP and their target genes via the cyclic adenosine monophosphate response element-binding protein (CREB) pathway. Intact YAP signaling is critical for PGE2-derived therapeutic effect against DSS-induced colitis because either the deletion of one copy or complete loss of YAP in 15-PGDH-null mice significantly reduces regenerative efficacy. A recent study provides a more comprehensive and detailed perspective on how the PGE2–YAP axis regulates intestinal regeneration [2 to the intestinal epithelium. These fibroblasts, named as rare pericryptal Ptgs2-expressing fibroblasts (RPPFs) by the authors, support the generation of Ly6a+ RSCs in the organoid culture system through the EP4 receptor. In line with the previous observation [2-EP4 signaling turns on the YAP pathway via nuclear translocation and, in turn, induces YAP-associated transcriptional program including RSC signature genes such as Clu and Ly6a in organoids. Indeed, ISCs isolated from the YAP-deficient intestine do not respond to PGE2 treatment and fail to acquire an RSC-like phenotype. Collectively, these results indicate the critical role of YAP signaling in PGE2-induced cellular reprogramming during intestinal regeneration.In addition, one of the arachidonic acid derivatives prostaglandin El models ,144,145. context . First, neration . Single-ervation , PGE2-EPDuring inflammation or regeneration in the intestine, genetic mutations can occur and lead to malignant transformation . AlthougThe oncogenic characteristics of YAP and TAZ in CRCs have also been supported by the results of analyzing patient tumor samples or human CRC cell lines. Indeed, the expression of YAP and TAZ was reported to have a positive correlation with poor prognosis in CRC patients, implying that the levels of YAP and TAZ might be prognostic indicators for CRC ,153,154.Drosophila was found to trigger the nuclear translocation of Yki, which further activates JAK/STAT signaling to increase cell proliferation [As explained in the previous section, the Hippo pathway interacts with the Wnt/β-catenin signaling pathway, which plays a crucial role in the initiation of CRC. Loss-of-function mutation in APC gene has been frequently observed in CRCs . APC mutferation . HoweverThe intestinal epithelium exhibits a superior self-renewal potency based on stem-cell plasticity, and recent findings strongly imply the involvement of the Hippo–YAP pathway in this phenomenon. In the homeostatic intestine, active Hippo signaling is required to suppress YAP activity and in turn prevent uncontrolled overgrowth of the crypt. On the contrary, YAP signaling must be activated to induce the regeneration-associated alternative stem cells upon injury and dysregulated, and constant-activation of YAP leads to tumor initiation. In addition, the Hippo–YAP pathway seems to regulate the lineage specification of IECs, since YAP activation prevents Paneth cell induction while it drives Goblet cell differentiation. Depending on the context, Hippo–YAP signaling is involved in various pathways such as Wnt, Notch, and EGF. Inflammatory signals as well as alterations in the mechanical force due to ECM remodeling also regulate Hippo–YAP signaling in the intestinal epithelium .Since the Hippo–YAP pathway can intervene in both intestinal regeneration and tumorigenesis, several attempts have been conducted to evaluate the therapeutic potential of Hippo–YAP modulatory drugs in these context. For instance, targeting the Hippo pathway by small molecule could stimulate the nuclear accumulation of YAP, which in turn promoted the intestinal repair process . In thisAlthough our understanding of both physiological and pathological roles of Hippo–YAP signaling in the intestine has been developed considerably, several questions remain to be addressed. There are still controversial observations regarding the impact of YAP activity in the homeostasis as well as disease progression. For instance, YAP activity is highest in the intestinal crypt and lowest in the villi, given the nuclear localization pattern of YAP; however, YAP signaling must be suppressed to control ISC proliferation. In addition, both consistent activation and suppression of YAP signaling impair crypt integrity. Hence, complicated cross talk with other pathways and how the duration and/or timing of YAP activation is regulated should be studied in detail. Finally, the possible contribution of Hippo–YAP signaling in the mesenchymal niche or other compartments of the intestinal microenvironment should be elucidated. Indeed, a recent work indicated the detrimental action of macrophage-derived YAP during IBD development . Therefo


Bone morphogenetic proteins (BMPs) comprise a major subgroup of the transforming growth factor (TGF)-β superfamily. They play pivotal roles in embryonic development and tissue homeostasis in adults. Deregulation of BMP and TGF-β signaling contributes to developmental anomalies and multiple diseases. In this mini-review, we focus on BMP signaling in inflammatory disorders of the pancreas, acute and chronic pancreatitis, in contrast to TGF-β signaling. We then discuss molecular mechanisms that interact with and connect between the BMP and TGF-β signaling pathways. Lastly, we review potential implications of these molecular mechanisms for therapeutic development. In summary, BMP signaling pathway plays different roles during pancreatitis disease development, and the antagonism between BMP and TGF-β signaling can be manipulated for therapeutic development against pancreatitis. Bone morphogenetic proteins (BMPs) constitute a major subgroup of the transforming growth factor (TGF)-β superfamily, comprising 18  out of the total 33 members . The TGFLines of evidence demonstrate that BMP signaling has pro-inflammatory properties in bronchial epithelial cells during airway inflammation , in actiIn recent years, our group has focused on the role of BMP signaling in acute (AP) and chronic pancreatitis (CP), the highly debilitating and painful inflammatory diseases of the pancreas. While a majority of AP cases self-resolve, approximately 16% progress to CP after repeated episodes of AP (RAP), mainly in patients with risk factors like alcohol and tobacco use ,18. CP iTo understand the role of BMPs in AP, we utilized experimental animal and cell models, and revealed that BMP/Smad1/5 signaling is activated; inhibition of BMP signaling attenuates the disease severity, indicating a pro-inflammatory role of BMP signaling in AP . The rolSeveral studies from our group have demonstrated opposing roles of BMP and TGF-β in CP. For instance, in pancreatic stellate cells, the key executive cells in pancreatic fibrosis, BMP2 alone does not induce extracellular matrix (ECM) expression but inhibits TGF-β induced-ECM production . KnockouTo search for molecular links between BMP and TGF-β, we focus on Gremlin1 (Grem1), a BMP antagonist, which has reported pro-fibrogenic function in several organs ,27,28. WOverall, temporal changes of the TGF-β/BMP signaling molecules and Grem1 during disease progression from AP to CP ,24,29,30Studies on the different roles and the reciprocal regulation of BMP and TGF-β not only advance our knowledge on how the same superfamily members regulate each other’s functions in diseased states, but also provide insights on how the antagonistic roles of BMP and TGF-β signaling can be modulated as therapeutic approaches.