Vitamin D deficiency (VDD) is associated with an increased risk for lifestyle-related diseases. In Japan, VDD is quite prevalent in all age groups, with its high risk in young women. Furthermore, its association during pregnancy with gestational hypertension and low birth weight has also been reported. VDD can be diagnosed by serum 25-hydroxyvitamin D [25(OH)D] levels, which, however, is not suited for screening. Therefore, we will create a predictive model for serum 25(OH)D concentration and prevalence of VDD based on such data as region, sun exposure habit, and vitamin D intake in young women.From 2020 to 2022, we conduct a cross-sectional study of 600 young women in four regions of Japan, identify the indices associated with serum 25(OH)D concentrations such as sun exposure habits, habitual vitamin D intake, ultraviolet-B irradiation, seasons (summer and winter) and latitude, and construct prediction models for serum 25(OH)D concentrations and VDD risk. This study has been registered with UMIN-CTR (ID: UMIN000041527).One hundred and fifteen subjects have been collected from 6 institutions in winter as of May 2021. When data from more than 200 subjects have become available, we will conduct the interim analysis, summarize the data by region and facility, review the inclusion criteria for analysis, and check for missing values and outliers. Prediction models for serum 25(OH)D concentration and VDD will be determined in the final analysis when all cases have been collected.A screening tool for VDD risk to be developed in our study based on the predictive model would help the public and medical professionals prevent lifestyle-related diseases through improving VDD. Additionally, the results may serve as the scientific basis for determining the appropriate vitamin D intake and sun exposure standards. Vitamin D insufficiency/deficiency increases the risk of various diseases, such as osteoporotic fracture, infectious diseases, diabetes mellitus, cardiovascular diseases, and cancer, and their primary prevention through improving vitamin D status can play pivotal roles . AlthougConsidering the high prevalence of vitamin D insufficiency/deficiency in almost all age groups in Japan –8, its iSerum 25(OH)D concentrations reflect not only the vitamin D intake but also the production in the skin by UV irradiation . Since JMaternal vitamin D status has been reported to be significantly associated with fetal and neonatal vitamin D status and can affect the outcomes during pregnancy and neonatal and infant development . For exaBased on such background, the present study will collect data on the seasons, lifestyle, and dietary habits associated with serum 25(OH)D concentrations in 600 young women from four different latitudes to identify predictors of serum 25(OH)D concentrations and develop a predictive model for vitamin D deficiency.This is a cross-sectional study. Because of the potential influence of latitude in predicting serum 25(OH)D concentrations, we plan to recruit 600 healthy women between the ages of 18 and 40 years living in Japan, who participate in the study from November 2020 to September 2022 from four regions: Hokkaido/Tohoku, Kanto, Chubu/Kinki/Shikoku, and Kyushu/Okinawa. The prefectures and their latitudes included in this study are shown in Women aged 18 and 40 years at the study entry and who have given voluntary written consent to participate in this study are being enrolled. Participants with comorbidities such as chronic kidney disease, autoimmune disease, cancer, liver disease, diabetes mellitus, and those under sun exposure restriction due to such diseases as xeroderma pigmentosum are excluded. Those with chronic gastrointestinal diseases such as celiac disease, chronic pancreatitis, inflammatory bowel disease, and cystic fibrosis are also excluded considering its possible interference with vitamin D absorption.This study is conducted in two seasons: summer (June to September) and winter (December to February), each corresponding to the highest and lowest serum 25(OH)D values, respectively ,20.The participants are asked to fill out a questionnaire about their dietary history and habitual lifestyle during the week prior to their blood draw. Participants’ blood samples are collected in the morning and stored at 4 to 10°C in the refrigerator before centrifugation. Biochemical tests are performed on the next day, and then the residual serum samples are stored at -80°C for the measurement of serum 25(OH)D concentration. The study schedule is shown in Both a comprehensive self-administered diet history questionnaire (DHQ) and the Lifestyle Questionnaire assess habitual food intake and sun exposure over the past month, based on that serum 25(OH)D concentrations reflect the vitamin D status for approximately one month.This study conforms to the Ethical Principles for Medical Research Involving Human Subjects issued by the Ministry of Health, Labour and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology in Japan. This research protocol has been approved by the ethics committees at institutions written below;1. The committee on research ethics, The Graduate School of Comprehensive Rehabilitation Osaka Prefecture University, August 3, 2020 , 2020–302 , 2. The ethical committee in Osaka Shoin Women’s University, Feb 10, 2021 , 20–15 , 3. The research ethics review committee of Kagawa Nutrition University, November 18, 2020 , 308 , 4. The ethics committee of human research of Kobe Women’s University, December 4, 2020 , 2020-17-2 , 5. The research ethics committee of Mukogawa Women’s University, November 25, 2020 , 20–64 , 6. The committee on research ethics, University of Nagasaki, October 7, 2020 , 439 , 7. The committee on research ethics, Kagoshima Women’s College, October 1, 2020 , R2-1 , 8. The human research ethics committee of Nakamura Gakuen University, December 4, 2020 , 20–012 , 9. The ethics committee of Tenshi College, October 12, 2020 , 2020-21.This study has been registered with UMIN-CTR (ID: UMIN000041527).The sample size required for this study was calculated to be 600 cases; based on a previous study showing that 52% of females aged 17–18 years had VDD (serum 25(OH)D level below 20 ng/mL) [A dietary assessment is performed. Trained research staffs check the participants’ answers to a comprehensive self-administered diet history questionnaire (DHQ) and resolve the ambiguity. Vitamin D intake is calculated by the revised comprehensive self-administered DHQ, which is a semi-quantitative questionnaire assessing habitual dietary intake over the past month, and already validated in previous studies –23. VitaThe questionnaire on habitual lifestyle is studied as in “Indices obtained from a questionnaire response” of 2D, respectively. Total serum 25(OH)D level is calculated by their summation. VDD refers to 25(OH)D level less than 20 ng/mL. Serum intact parathyroid hormone (iPTH) is measured by electro chemiluminescnce immunoassay at SRL, Inc. .Serum vitamin D metabolites are measured by liquid chromatography tandem mass spectrometry (LC-APCI-MS/MS) as previously described with some modification , i.e. deE(λ) for wavelength region between 280 and 4,000 nm [We calculate the ultraviolet-B (UV-B) radiation flux density as detailed below, using a radiative-transfer code called ‘SMARTS2 ’ developed by Gueymard to calculate the flux density of solar radiation on the ground surface λ at the horizontal plane surface is expressed as follows;In particular, for wavelengths from 280 to 1,700 nm, calculations can be made at 1 nm intervals. UV spectrum of wavelength E (λ): UV flux density of the wavelength, λ,Ebn (λ): UV spectrum of the direct component of solar radiation,θz: solar zenith angle,Id (λ): UV spectrum of the diffusive component of solar radiation.Ebn (λ) and Id (λ) at the ground surface for a given season, time, and place under a cloudless sky. We apply this calculation to the sites where subjects are recruited at each participating university or college in the study. The UV flux reaching the top of the atmosphere from the sun is corrected for the distance between the earth and the sun for each day of the year. Total ozone at each site is given by measurements of NASA’s OMI [α and β. The data for aerosols for Tsukuba is used when there are no data available for aerosols at study sites. Rayleigh scattering is related to the number of air molecules that may generate atmospheric pressure. UV spectra have been observed by the Japan Meteorological Agency, every hour on a daily basis with Brewer spectra available for every 0.5 nm interval [This equation allows us to calculate the UV spectrum SA’s OMI  and OMPSSA’s OMI  satellitinterval .The continuous variable will be expressed as mean ± standard deviation or median (interquartile range), and a categorical variable as frequency (percentage). Mann-Whitney U test and Kruskal-Wallis test will be used for the comparison between two groups and more than two groups, respectively. The chi-squared test will be used for categorical data.An interim summary will be performed when the number of registered cases exceeds 200. The data will be summarized by region and institution. The inclusion criteria for analysis will be reviewed. We will also check for missing values and outliers. Predictors and prediction models will be determined in the final analysis when all cases have been collected.Multivariate linear and logistic regression models are used to determine the predictors and predictive models for serum 25(OH)D concentration and VDD as outcomes, respectively. The regression coefficients and odds ratios will be calculated, and their 95% confidence intervals and p-values by Wald test will be calculated. Candidate predictors include age, residential area, skin type, smoking status, medication history, habitual drinking, physical activity, the season of blood collection, habitual sun exposure time and frequency, sunscreen use, the experience of suntan within the past 12 months, vitamin D supplement use, the habitual avoidance of sun exposure with parasol use or wearing long sleeve clothes, weekly frequency of vitamin D abundant fish consumption, and cumulative ambient UV-B irradiation.Two-thirds of the randomly selected data will be used to build the predictive model, and the remaining data will be used to validate the built model. In the test set, variables shown to be significant in the univariate analysis and previously reported predictors are inserted into the multivariate model to construct the predictive model. In the validation set, the predictive performance of the prediction model is checked with the degrees of freedom-adjusted R-squared or AIC for linear regression analysis, and with the c-index for logistic regression analysis. In addition, as model calibration, the predicted and observed values are compared using Cook’s D for linear regression analysis and Hosmer-Lemeshow test for logistic regression analysis. Reporting on the construction of the predictive model will be done following the TRIPOD statement .Statistical significance will be set at a two-tailed p-value < 0.05 for all analyses. All statistical analyses will be performed according to the statistical analysis plan decided before the analysis.We plan to register 150 cases each in the summer and winter quarters of 2021–2022. Case registration has been delayed due to the spread of coronaviruses, and 115 cases were collected from 6 institutions by March 2021. We plan to conduct an interim analysis when the number of registered cases reaches 200 or more, and we plan to complete case registration and conduct the final analysis by around September 2022.The purpose of this study is to determine how serum 25(OH)D concentration in young women is affected by such factors as latitude, season, diet, and lifestyle, and build a prediction model to predict serum 25(OH)D concentrations. The results of this study may provide a scientific basis for the forthcoming Dietary Reference Intakes and other various guidelines. We also plan to develop a tool to screen the risk of VDD and make it available through our website so that individuals can monitor their vitamin D status and easily assess their risk of vitamin D deficiency or insufficiency, which will lead to the prevention of lifestyle-related diseases by improving VDD.This study is characterized by collecting data on UV-B exposure and lifestyle habits from four different latitudes (seven sites). Although previous reports are available in Japan on the circulating 25(OH)D levels at different latitudes ,11, theyThere have been some previous papers reporting serum 25(OH)D concentrations in summer and winter, which has yielded conflicting results such as a significant correlation of serum 25(OH)D with UV exposure time and UV energy only in summer ,20, and A previous study has reported that the use of sunscreens and sun protection tools did not affect serum 25(OH)D concentrations in multivariate regression models . This paIndividual UV irradiation dose can be evaluated either by direct measurement or estimation. As the former, the use of wearable devices is useful enabling a more accurate assessment of individual UV doses, which, however, is a burden to the participants. Then, we have not adopted such devices, and estimate the UV exposure for each region. The association of estimated UV irradiation dose and serum 25(OH)D levels has previously been reported ,13. For Much more strong association between the dietary vitamin D intake and serum 25(OH)D levels could be expected than that in European and American studies, with the possible bases described below. Previous European reports on the ultraviolet effects on vitamin D status had a methodological flaw; no data on dietary vitamin D intake ,13. AnotThere are some obvious limitations in this study. First, since the study subjects are limited to young women, caution is needed in the generalization of the current results. Second, all participants are undergraduate nutrition students, likely with a higher level of knowledge about nutrition than the general population, leading to more accurate responses to the DHQ. Hence, the possibility of selection bias cannot be ruled out. Third, since this is a cross-sectional study, a causal relationship between serum 25(OH)D level and the predictors cannot be definitely concluded.In this study, we will investigate various indices related to VDD in young Japanese women, construct a model for predicting VDD, and develop a screening tool to determine the risk of VDD, which will be useful for disease prevention through the improvement of VDD.S1 FileA questionnaire used in the present study (in Japanese) was described. The English translation is also shown.(DOCX)Click here for additional data file.


CBFV responses were population mean normalized from a 30-s baseline period prior to task initiation. A significant increase in bilateral CBFV response was observed at the start of all paradigms and provided a similar pattern in most responses, irrespective of complexity or duration. Although significant inter-hemispherical differences were found during performance of R-word and all serial subtraction paradigms, no lateralisation was observed in more complex naming word tasks. Also, the effect of duration was manifested at late stages of 100–7, but not for other paradigms. CBFV responses could not distinguish different levels of complexity or duration with a single presentation of the cognitive paradigm. Further studies of the ordinal scalability of the NVC response are needed with more advanced modelling techniques, or different types of neural stimulation.Cognitive paradigms induce changes in cerebral blood flow (CBF) associated with increased metabolic demand, namely neurovascular coupling (NVC). We tested the hypothesis that the effect of complexity and duration of cognitive paradigms will either enhance or inhibit the NVC response. Bilateral CBF velocity (CBFV) in the middle cerebral arteries (MCAs) via transcranial Doppler ultrasound (TCD), blood pressure (BP), electrocardiogram (ECG) and end-tidal CO Actilaxation –5. CBF claxation , positrolaxation , single-laxation , near inlaxation , laser Dlaxation  and tranlaxation . MRI, PElaxation . fTCD halaxation , ischaemlaxation  or haemolaxation  stroke, laxation , intraoplaxation , cerebralaxation , and coglaxation , includilaxation  and neurlaxation . The qualaxation , 22–25. laxation , 26–28, laxation , 29, 30 laxation , 31, 32 scalability, that is, to demonstrate the sensitivity to reflect differences in cognitive load, or its complexity. In the case of fTCD, this has been shown to be the case with visual stimulation [In addition to detecting differences in lateralisation, a diagnostic tool to assess cognitive function needs to satisfy the condition of mulation , 33, witmulation , 34, 35.mulation , but doemulation –39. Prevmulation , 40, 41.mulation , 41 and mulation –39.In summary, we tested the hypotheses that the complexity and duration of stimulation of NW and SS tasks can: 1) induce either larger or smaller magnitude of NVC response, and 2) lead to inter-hemispherical differences that could be the consequence of either enhanced or inhibitory mechanisms.Sixteen healthy participants (eight women) without history of cardiovascular, neurological, or respiratory conditions, including metabolic abnormalities, inflammatory abnormalities, and severe somatic  or psychiatric , were recruited from the University of Leicester as students or staff. The Research Ethics committee approved the study (ref: 19452-rp9-ls:cardiovascularsciences), and fully informed, written consent was signed by each participant. Only adults 18 years old or older, with good insonation of bilateral temporal windows were suitable for study inclusion. The Edinburgh inventory was used for assessment of handedness .2 (EtCO2), using small nasal cannula  with a capnograph (Capnocheck Plus).All participants abstained from caffeine, alcohol, vigorous exercise, heavy meal, and smoking for at least 4 hrs before attending a temperature (22–24°C) and light controlled laboratory. All measurements were undertaken with participants in the seated position. To avoid any artefacts due to movement, participants were asked to sit comfortably and avoid any movement, and all signals were checked prior to recording. The 2MHz TCD probes (Viasys Companion III device) were securely placed over the temporal windows using a head frame, and bilateral CBFV was monitored continuously in the middle cerebral artery (MCA) with the depth of insonation at 45–55 mm. Arterial volume clamping of the left middle finger digital artery was used for continuous beat-to-beat BP measurement , and automated brachial BP was also intermittently recorded in the right arm to calibrate the Finometer before each recording, using a validated OMRON (UA767) device. Continuous measurements were recorded for heart rate (HR), with a three-lead electrocardiography (ECG), respiratory rate and end-tidal COPrior to the tests, the paradigms were described in detail, and participants were encouraged to provide their best mental effort when providing their answers in a low voice. Each paradigm was recorded during 60s with subjects asked to keep an ‘empty mind’, Tasks were given verbally to participants during recording before task initiation. Thereafter, paradigms were performed over one of three different periods of duration stimulation, with a 60s recovery phase. Naming words (e.g. P-words) and serial number subtraction  paradigms have been applied routinely for diagnosing mild cognitive impairment (MCI) and dementia, and have been increasingly adopted as equivalent cognitive paradigms for assessment of NVC and also functional cerebral lateralization , 41. HenREF) and serial subtraction (SSREF), respectively. Paradigms of different complexity were randomized and presented during 60s. For changing the duration of presentation, the NWREF and SSREF paradigms were also randomised and then interrupted after 5, 30 or 60s, without participants’ prior knowledge. The 60 s has been the traditional duration of NW and SS tasks [2 supply. The beginning and end of stimulation were marked with an on/off electrical switch to identify the duration of the paradigm and allow synchronization for coherent averaging of CBFV responses. All physiological signals were sampled at 500 Hz and recorded in the PHYSIDAS data acquisition system  for offline analysis.Subjects rested for 15 min and a resting baseline recording was performed for 5 min . Two sepSS tasks , 41, but2 synchronized to the end of each cardiac cycle. Beat-to-beat data were spline-interpolated with a third order polynomial and resampled at 5 Hz to obtain signals with a uniform time base.BP was calibrated at the beginning of each recording, and all signals were visually inspected to identify artefacts, with narrow (<0.1s) spikes, which were linearly interpolated. Bilateral CBFV signals were passed through a median filter with a window width of five samples to remove random noise. All signals were low-pass filtered by a zero-phase Butterworth filter with a cut-off frequency of 20 Hz. RR interval was automatically marked from the ECG, and HR was continuously plotted against time and manually corrected under visual inspection in case of missed markings. Mean values of CBFV and BP were calculated for each cardiac cycle. Linear interpolation was applied to obtain estimates of EtCOEach variable was synchronized by the start of each paradigm, to obtain the coherent average of CBFV for each subject. Bilateral CBFV were expressed in percent (%) by normalization by the 30s of baseline before the paradigm activation. The synchronization placed the beginning of each paradigm at 60s, within a window of 180s duration. Only bilateral CBFV signals were averaged for 10s during five time segments, with the beginning of stimulation starting at 60s: baseline (T1) at 40-50s, (T2) at 60-70s, (T3) at 110-120s, (T4) at 120-130s, and (T5) at 160-170s. Therefore, T1 represents baseline values before stimulation, T2 the immediate response to stimulation, T3 the sustained response, T4 near the end of the response (for 60 s duration of stimulation) and T5 during recovery. All timepoints were fixed for all paradigms, independently of the duration of presentation.th and 75th percentile  for indicating extreme values beyond 1.5 times (above or below) of the IQR as outliers which were modified by replacing the second smallest or largest values in observations to the threshold boundaries as recommended in Winsorization [All data were tested for normality using Shapiro-Wilk test. Continuous data with a Gaussian distribution are presented as mean (SD). Outliers of parameters were determined by using the boxplot with interquartile limits between 25rization . Paired 2), and completed all the NW and SS paradigms of variable duration and complexity. A majority of participants (n = 14) were right-handed and two subjects were ambidextrous, following assessment with the Edinburgh handedness inventory. Half of all participants had Asian background, six were Caucasian, and two subjects had Caribbean and African ancestry, respectively. Eight people abstained from alcohol and two subjects were current smokers. Absolute mean (SD) values of baseline CBFV did not show a significant difference between right hemisphere (RH) and left hemisphere (LH) at 66.8 (11.4) and 64.8 (11.4) cm/s, respectively. Baseline values for mean BP, HR and EtCO2 were 89.7 (9.4) mmHg, 70.8 (8.1) bpm and 38.5 (3.1) mmHg, respectively.Sixteen healthy participants (eight women) were recruited  (REF (100–7) tasks   and SSRE7) tasks . A steepIncreases in the complexity of NW and SS tasks induced similar temporal CBFV changes during neural activation as no obvious difference was seen in its effect on CBFV responses within each time period of stimulation. REF and SSREF tasks induced a similar pattern of temporal CBFV responses (REF and SSREF (REF in both RH (p = 0.002) and LH (p = 0.0057). As expected, due to the reduced duration of paradigms, significant differences were seen for the SSREF with increasing durations in the RH (p = 0.02) at T3 and T4 and borderline significant in the LH (p = 0.059)  , but notREF and SSREF were not seen at each time segment during 05s and 30s durations.Overall, there were contradictory responses between RH and LH regarding the dominance of CBFV responses during SS and NW tasks, respectively. Significant interhemispheric differences were observed for RH with SS and LH for NW tasks but only for the timepoints T3-T5 . In paraAs a non-invasive tool, fTCD has often been used to detect changes in cerebral haemodynamic responses during neural activation using cognitive paradigms , 21, 23.et al. (2014) and Szirmai et al. (2005) proposed that fast-neurogenic regulation, with a more attention-induced arousal reaction to mental activations, resulted in the rise of perfusion pressure rather than representing a vasodilatory response to meet metabolic demand [et al. (2001) suggested that the initial fast response at T2 resulted from direct neurogenic vasodilation resulting from free circulating acetylcholine, activation of both muscarinic and nicotinic cholinergic receptors in the cortex, and involvement of NO release from nitric oxide synthase cells such as interneurons or endothelial cells [The highly significant bilateral CBFV differences observed across time Tables  & 2, conc demand , 49. Howal cells , which ial cells . Neuroge2 concentration [For the second peak, a gradual CBFV increase was seen as the sustained response (T3), with differences related to the complexity of NW and SS, except for SS (100–2). This slow phase of the CBFV response could reflect metabolic regulation that induced decreases in local microvascular resistance depending on the actual level of tissue COntration . Howeverntration , combineet al. (2016), using different levels of challenge in visual search paradigms, it was possible to discriminate ΔCBFV responses in the posterior cerebral artery (PCA), but ΔCBFV changes to visual paradigms of different complexity could not be detected in the MCA [n-back paradigms compared to less demanding tasks [Paradigms of different complexity induced CBFV responses with similar temporal patterns, and without significant differences in the amplitude of the CBFV changes, or evidence of lateralization . Althoug the MCA . Similar the MCA . With mong tasks . Also, tng tasks , 59, 60 ng tasks .Noteworthy, the majority of previous studies used repeated presentations of the same paradigm to improve signal-to-noise ratio (SNR) of intra-subject averages of the ΔCBFV response. As seen in previous studies, presentation of complex tasks was repeated a variable number of times, including < 5 , 5 to 10compensation-related utilization of neural circuits hypothesis (CRUNCH) to recruit more neural activations matching with load increases [There are potential additional explanations for the lack of significance in our results to the effects of cognitive load with NW and SS paradigms. First, the differences in complexity of paradigms in the current study might not have been sufficient to induce differential CBFV responses, as proposed in terms of the ncreases , 64. As ncreases , arithmencreases  or visuancreases , 65 parancreases , this doSecondly, the phenomenon of ‘central command’ anticipation of the brain when preparing to receive an increased demand for oxygen may be important. To our knowledge, the role of the ‘central command’ during cognitive paradigms in fTCD studies has received very little attention in the human NVC literature . Previouet al. (2010) highlighted a shift in the process of brain response from prefrontal cortex to the bilateral posterior parietal cortices, including additional brain areas (caudate nucleus and cerebellum) during complex mental arithmetic [2 with hypocapnia-induced hyperventilation or hypercapnia-induced breath holding can also accompany CBFV alterations during cognitive tasks [2 to CBFV changes following cognitive tasks has been confirmed in previous fTCD studies [Thirdly, cognitive stimulation of some brain areas leads to inhibition of other cortical centres . Despiteithmetic . It is pithmetic , 73. Theve tasks , 74. Furve tasks , resulti studies . HoweverREF and SSREF, temporal CBFV responses showed similar patterns at the first peak of initial stimulation (T2), without significant differences in the amplitude of CBFV or lateralized dominance. Only CBFV responses to the longer (60s) stimulations led to a second peak for SSREF  demonstrated a significant decrease of CBFV at sustained response during a language task in Alzheimer’s disease but not in MCI when compared with healthy controls [This is the first systematic study to determine fTCD-measured NVC responses to NW and SS paradigms of varying complexity  and duration  by modifying well known paradigms used for clinical assessment of cognitive performance –39, 41. controls . Althougcontrols , 84, comcontrols . These a2 and BP [et al. (2015) showed differences in fTCD responses to the joint fast paced presentation of rhyme and line array judgement tasks [There are a number of limitations to our study. First, TCD reflecting CBFV changes in the MCA, can be directly proportional to changes in absolute CBF with the assumption that the diameter of insonated MCA remains constant despite small changes in CO2 and BP , 87. Sec2 and BP . One int2 and BP , 89, altnt tasks . It is pnt tasks . Fourth,t al. 201 showed dnt tasks . AlthougREF for the effects of different task durations, indicating a complex interaction between NVC response and duration of stimulation. Further studies, on the sensitivity of the fTCD response to stimulation of varying complexity and duration, is needed by performing similar studies in the influence of ageing, patients with early signs of MCI, Alzheimer’s disease or other types of dementia, exploring alternative cognitive paradigms. Also, a more integrated approach as described previously [2, to identify parameters of the NVC response that could be scalable, ideally in a linear fashion.We have demonstrated that complexity and duration of NW and SS paradigms provided similar patterns and amplitudes of CBFV responses, without the possibility of making a scalable metric for assessment of NVC in healthy subjects. Nevertheless, some significant changes were seen in comparison with SSeviously , 24, 92


However, the pandemic cannot be ended overnight and more social distancing and other self-care measures are needed to protect our communities. Therefore, people´s awareness, knowledge, attitudes, and appropriate behaviors are instrumental to containing the pandemic. The aim of this study was to determine COVID-19 awareness, knowledge, attitudes, and related behaviors in Libya.th to November 10th, 2020 in 24 cities in Libya. The participants were non-medical professionals who were living in Libya for at least 2 years and were at least 18 years old.a cross-sectional online survey was conducted from October 10a total of 1018 participants completed the questionnaire, with ages ranging from 18-74 years (Mean ± SD = 33.49±13.24); nearly two-thirds were < 40, and 68.2% were females. Almost half of the participants considered the potential threat of COVID-19 to be low, and one in five reported that they were “Not worried at all” about getting COVID-19. In multivariate analyses, participants who were 40-49 years old, had master´s degrees or higher, and worked in the private sector reflected high mean scores for both knowledge and attitudes, while those who lived in the Eastern or Southern regions had lower mean attitude scores.the low levels of awareness as well as the attitudes and behaviors among the public in Libya are worrisome. This study highlighted profound gaps that may put Libyan communities at high risk of a COVID-19 explosion. Therefore, immediate action is needed to address public awareness and attitudes and to improve COVID-19 related behaviors among the Libyan public. The acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was acknowledged as a pandemic by the World Health Organization (WHO) in March, 2020 . At the Nonetheless, this unprecedented global crisis has also been marked by miscommunication regarding the imminent threat of COVID-19, leading to public confusion and inaction. As of today, we are unaware of any available effective treatment for COVID-19 , and witAwareness, knowledge, attitudes, and practices people hold and adhere to towards the disease play an integral role in determining a society's readiness to accept health authorities´ measures and determining a path through the pandemic. Unfortunately, poor awareness and harmful practices can lead to severe consequences affecting the population's health. This was confirmed by a study conducted in Saudi Arabia  that shoStudy design and participants: a cross-sectional online survey was used to gather details on the Libyan public´s awareness, knowledge, attitudes, and behaviors associated with the COVID-19 pandemic. In order to fulfill the objective of the study while restricting physical contact with the respondents and obtain their responses as quickly as possible during the outbreak in Libya, we used an online Google form. To be included, participants had to be Libyan citizens or foreigners who had lived in Libya for at least 2 years , be 18 years of age or older, and be able to read and understand the Arabic language. All questionnaires that did not meet the above criteria were excluded from the study. Additionally, we excluded all healthcare professionals , as we assumed that their awareness, knowledge, and behavior about COVID-19 would be higher than those of the general public.Sample size calculation: the sample size was estimated by using Epi Info web-based software, version 3. Since the literature search did not identify similar studies conducted among the general public in Libya, we hypothesized the probability of awareness and knowledge about COVID-19 in the Libyan public as 50%, the power is 99%, precision of ±5%, with a design effect of 1.0, and the minimum required sample size was initially estimated to be 664 participants. Additionally, we added 10% to the sample size to account for possible non-responses. Therefore, the final minimum sample size was estimated to be 730 participants.Data collection tool: the questionnaire was designed based on previous studies [ studies ,10. The The questionnaire consisted of four parts, as follows. The first part collected the sociodemographic information of the participants such as place of residence, gender, age, marital status, education, number of family members, occupation, and income . The secThe last part of the questionnaire focused on collecting data about COVID-19-related behaviors. Participants were asked about any changes to their daily routines due to the COVID-19 pandemic, with possible answers being “no change at all”, “changed a little bit”, “somewhat changed”, or “changed a lot”. Participants also answered questions about how often they wear masks in public places . Procedure: the final version of the questionnaire was circulated through several online platforms, including social media and the official pages of community organizations, sports clubs, colleges and universities, and the official websites of 24 municipalities across the country representing the four geographical regions of Libya: the Western, Middle, Eastern, and Southern regions. The questionnaire was made accessible over a period of one month, from October 10 to November 10, 2020.Study variables: the sociodemographic characteristics of the respondents such as place of residence, gender, age, marital status, education, number of family members, occupation, and income were considered as explanatory/independent variables. Other study variables  were considered as response/dependent variables. The responses for knowledge and attitude were scored as 1 = correct answer, and 0 = wrong or do not know. The total score was the sum of all correct answers, which ranged from 0-18 (for knowledge) and 0-14 (for attitudes).Data analysis: after checking for missing data, all completed questionnaires were imported, coded, and analyzed using the Statistical Package for Social Sciences (SPSS) version 26.0, and descriptive statistics were calculated for all the participants´ characteristics and survey responses. Dichotomous variables were expressed in frequency and percentage, while continuous variables were expressed as mean ± standard deviation. Bivariate and multivariate analyses were used as appropriate in order to test the significant associations between the participants´ characteristics and responses regarding COVID-19 awareness, knowledge, attitude, and behavior. An alpha level below 0.05 was considered for statistical significance (2-sided tests).The questionnaire was accessed 1423 times, with 1018 completed questionnaires (response rate = 71.5%). In the following section, the respondents´ sociodemographic characteristics as well as the findings regarding the study variables, i.e., awareness, knowledge, attitudes, and COVID-19-related behaviors, are presented.Sociodemographic characteristics: a total of 1018 participants completed the questionnaire, with the respondent´s ages ranging from 18-74 years (Mean ± SD = 33.49±13.24). The respondents were young overall; nearly two-thirds were > 40 years old, and 68.2% were female. The sample was representative of the four geographic regions of Libya, with nearly half of the respondents (47%) living in the middle region. Nearly half of the participants were socioeconomically disadvantaged, with monthly incomes of less than 2,000 Libyan dinars (LD). More than half the respondents had at least a bachelor´s degree, and a little more than one-third (39.4%) were living with at least five family members , through coughing or sneezing (94.7%), and when using an infected person personal item (86.5%). In terms of COVID-19 symptoms, almost all the respondents agreed that fever (95.6%), cough (86.7%), and breathing difficulty (94.8%) were among the common symptoms of COVID-19 infection , were reported regarding closing unnecessary stores , closing the borders (91.4%), and closing the airports (95.1%) to control the spread of COVID-19. In terms of school and university closures, about 88.4% of the respondents agreed that this was necessary to contain the pandemic (pandemic .COVID-19-related behaviors: about one in five of the respondents (22.7%) reported that the COVID-19 pandemic had changed their daily routine a lot. A total of 36.8% of the study sample reported that they would leave their house only once a day, whereas 33.4% reported that they would leave their home multiple times a day. In contrast, only 3.9% said that they never left their homes. We also asked questions to investigate the public´s level of preparedness for other outbreaks and their trust in the current government to prevent them. One in five respondents (19.5%) reported that they were “not prepared at all”, and more than half the respondents (52.6%) reported that they were “a little prepared” for another wave of COVID-19. The majority of respondents (89.1%) were “not confident at all” or “not very confident” that the Libyan government could prevent further outbreaks of the COVID-19 virus , which might be due to gender differences in responding to online surveys, as reported in previous studies . A littlet al. [et al. [Overall, the findings show that public awareness about COVID-19 in Libya is moderate, as the majority of respondents perceived the threat of a COVID-19 outbreak to be moderately serious on a scale of 1 to 10 (Mean score ± SD = 5.28±2.76). A study carried out by Wolf et al. [et al. [However, our respondents showed high levels of knowledge regarding the sources of infection and symptoms of COVID-19. For example, 94.7% of the respondents knew that the COVID-19 virus can be transmitted through coughing and sneezing, 85% knew that the COVID-19 virus can be transmitted through touching and hand-shaking, and 86.5% knew that the COVID-19 virus can be transmitted when using an infected person´s personal items. A study done in Ghana by Serwaa  [et al.  reportedThe high knowledge percentages reported in our study may, however, be due to the timing of our survey, since the above-mentioned studies were conducted at the beginning of the COVID-19 outbreak, while our study was conducted in October . Therefore, people in Libya had a better chance to learn more about the virus and gain more knowledge about its transmission and the common symptoms of the infection. In this study, regression analyses were also conducted in order to examine whether public knowledge and attitudes varied based on sociodemographic characteristics. In our population, we found a significant positive correlation between COVID-19-related knowledge and the variables for age, education, and occupation . RespondIn terms of attitudes towards the COVID-19 pandemic, we found that the mean attitude score was significantly correlated with geographic location, education, and occupation. People who lived in the Middle and Western regions showed slightly higher attitude mean scores  than those who lived in the Eastern and Southern regions. In Libya, most of the Southern region, for instance, comprises rural areas (mostly in the Sahara) that lack appropriate infrastructure to keep track of the continuous updates about the pandemic. This poor attitude may also be connected to the cultural and ethnic diversity in those regions, which needs further attention. The findings of this study are of utmost importance, however, since they may provide data on implementing programs that target people who might be unable to access useful educational platforms due to financial or technical reasons.It is clear that the evolving COVID-19 outbreak requires social distancing and other measures to protect public health; however, recommendations in Libya have been inconsistent, unclear, and often confusing to the public. This was confirmed by our study, as only a little over half of the participants considered the potential threat of COVID-19 to be high . MoreoveAt the individual level, several self-care practices have been recommended by the WHO to control for the spread of COVID-19 in the community. For instance, wearing masks, washing hands, physical distancing, and self-isolation when feeling unwell are among the common practices that have been in place and have been supported by the literature. In our study, the majority of respondents agreed that wearing masks, for instance, is a good strategy to combat the virus; however, only 39.4% of them said they wore masks in stores, and one in ten said that they never wore them at all. This finding is disturbing, since wearing masks in public places like grocery stores is among the best possible strategies to control the pandemic [et al. [In Libya, the situation has been exacerbated by the intermittent military conflicts in many parts of the country for nearly 10 years. In June 2020, the WHO urged all armed groups in Libya to enable humanitarian organizations to access healthcare facilities, especially in the south, to help combat the increase in the country's COVID-19 cases. As of December 2019, more than 300,000 people had been displaced because of the war [et al. , the heaet al. . TherefoA few limitations of this study should be noted. First, the data used for this study were collected solely online, which might have recruited only participants who are more interested or concerned about COVID-19 than the general public. Second, the number of female participants was almost twice that of their male counterparts. In fact, this is a shared limitation among many studies that have used the same methodology ,23, whicAlmost a year has passed since the first case of COVID-19 was announced in Wuhan, China; however, the awareness, knowledge, and behaviors among the general public in Libya are worrisome. Our study highlights profound gaps that may put Libyan communities at high risk of a COVID-19 explosion. The COVID-19 pandemic has the potential to put more pressure on the exhausted healthcare system in Libya due to poor awareness and inadequate preventative behaviors among the public. Our study also provides evidence on those who are socioeconomically disadvantaged and live with limited resources. Immediate actions are needed now to protect Libya from this deadly virus, and the first step toward containing the pandemic is by addressing awareness, knowledge, and research-based behaviors among the public, especially in vulnerable populations. As the COVID-19 graph for Libya is ascending as more cases are confirmed every day and the second wave of the virus is near, with new traits being confirmed in many parts of the world, it is imperative to take tangible steps at personal and community levels to increase the awareness, knowledge, and appropriate behaviors to fight the pandemic.There is miscommunication regarding the imminent threat of COVID-19, leading to public confusion and inaction;Self-care strategies are the only option available now to prevent the spread of this deadly virus;Poor awareness, knowledge, and harmful practices regarding COVID-19 can lead to severe consequences affecting population health.Libyan people´s awareness about COVID-19 is moderate;Respondents agreed that wearing masks, physical distancing and hand-washing were protective measures against COVID-19;Individuals who live in rural areas have lower attitude scores overall regarding the COVID-19 pandemic.


Objective: Due to the extensive use of diazepam worldwide, self-induced intoxication is very common, yet rarely fatal. Nevertheless, the management of intoxication caused by extremely high doses of diazepam is not known, as well as the effectiveness of flumazenil, a specific benzodiazepine (BDZ) antagonist. Here we present the first report on the enhanced elimination (clearance) of diazepam using the Molecular Adsorbents Recirculating System (MARS) following autointoxication with an extremely high dose as part of a suicide attempt.Case: A 44-year-old male patient was admitted to the ICU because of impaired consciousness following the ingestion of 20 g of diazepam. Blood and urine samples revealed high benzodiazepine levels. Repeated doses of flumazenil were without effect on consciousness. Following deterioration of the patient's clinical condition, including unconsciousness, hypoventilation, and decreased SpO2 (88%), the patient was intubated and mechanically ventilated. On the fourth day after admission, the patient was unresponsive, with no attempt to breath spontaneously. The plasma level of benzodiazepines was 1,772 μg/l. The elimination of benzodiazepines by MARS was attempted, continuing for 5 days, with one session per day. Five sessions of MARS effectively enhanced benzodiazepine elimination. After the first MARS treatment, the plasma level of benzodiazepines dropped from 1,772 to 780 μg/l. After the final MARS treatment on the eighth day, the patient was weaned from mechanical ventilation and extubated. Two days later, the patient was discharged to the internal medicine department and subsequently to the psychiatry department.Conclusions: To the best of our knowledge, this is the first case reporting successful treatment of diazepam intoxication using MARS. In severe cases of diazepam intoxication, with prolonged unconsciousness and the necessity of mechanical ventilation, we suggest considering the use of MARS elimination therapy together with the monitoring of the BDZ plasma level. Because of the extensive use of diazepam worldwide, self-induced intoxication is highly prevalent , 2. WhilA 44-year-old male was admitted to the ICU because of impaired consciousness, with a Glasgow Coma Score of 11 , after reported ingestion of diazepam. The intoxication occurred at the pharmacy during the evening hours. The patient was found unconsciousness by an employee, next to an emptied pharmaceutical powder canister that had contained diazepam.2. The patient had no renal or liver dysfunction. Plasma analyses revealed no changes in urea (3.27 mmol/L), creatinine (52.9 μmol/L), bilirubin (6.39 μmol/L), AST (0.37 ucat/l), ALT (0.21 ucat/l), and AMS (1.38 ucat/l). Further physical examination revealed no abnormalities. Blood and urine samples were taken for toxicology investigation. According to protocol, the patient was administered 75 g of activated charcoal and 20 g of MgSO4 by nasogastric tube every 6 h. I.v. flumazenil (0.5 mg) administration was without effect. Because of agitation, intermittent i.v. administration of propofol (50 mg) was used. BDZ intoxication was confirmed from blood and urine samples 5 h after admission, with plasma concentration above the upper detection limit of 2,000 μg/l. Plasma and urine BDZs were measured using DRI® Benzodiazepine Serum Tox Assay and Emit® II Plus Benzodiazepine Assay for urine. Because of hypoventilation and irregular breathing, decreased SpO2 (to 88%) and impaired GCS (to 3), the patient was intubated and mechanically ventilated starting from 5 h after admission without the need for additional sedation. Subsequent repetitive administration of flumazenil  was without effect.Attempted suicide was suspected because of a letter left by the patient. Circumstances thus indicated that the patient had ingested 2,000 mg  of diazepam, some 2–4 h prior to presentation at the ICU. The patient has no relevant medical, family, or psychosocial history, as well as no past interventions. No medical report about depressive disorder or previous suicide attempts exists. The patient worked as an economist in the pharmacy. On ICU admission, the patient's blood pressure was 110/70 mmHg, heart rate 90/min, with regular rhythm. The SpO2 was 90%, and the patient was hypothermic (35.5°C). The patient's weight was 80 kg, height 175 cm, and BMI 26.12 kg/mDuring the subsequent days, the patient remained unconsciousness with an absence of spontaneous breathing. Plasma BDZ remained above the upper detection limit, which prompted the start of MARS on the fourth day of admission and its continuation for 5 days, one session per day. Informed consent was obtained from the patient's relatives.Throughout the course of MARS, plasma levels of BDZ were measured twice daily—at 6:00 (before the MARS session) and at around 14:00 (after the MARS session). The first MARS session lowered the plasma level of BDZ from 1,772 to 780 μg/L, and subsequent sessions further lowered the BDZ levels , Table 1As a result of MARS, plasma BDZ levels were effectively lowered by a similar percentage at each session, averaging 45% , Table 1Plasma BDZ levels during the last measurements, when MARS had been stopped, allowed calculation of the elimination half-life . During the first time interval, the BDZ level dropped from 191 to 161 μg/l in 17 h; consequently the calculated elimination t1/2 was 69 h. During the last 24 h of measurements, the BDZ level dropped from 161 to 111 μg/l; therefore, calculated t1/2 was 45 h. Overall, calculation of the elimination half-life over the last three time points, when the plasma BDZ level dropped from 191 to 111 μg/l in 41 hours, rendered a similar result, with a t1/2 of 52 h.MARS is a system of extracorporeal albumin dialysis that is mostly used in patients with established liver damage. The concept of albumin dialysis depends on the elimination of protein-bound molecules and can also be used with intoxication in order to remove toxicants. To the best of our knowledge, this is the first case reporting accelerated elimination of diazepam using MARS.in-vivo and in-vitro evidence on the impact of MARS on BDZ elimination is scarce. Specifically, one in-vitro study focuses on the impact of albumin stabilizers  , 4, it wbilizers  and anotbilizers , rather  failure . This exOur data show that MARS efficiently lowers plasma BDZ levels, as single sessions resulted in an average drop in plasma BDZ of 43%. However, we also observed a significant rebound of plasma BDZ levels following the initial MARS sessions, which may have been the result of accumulated drug recirculation from plasma protein binding sites or redistribution from peripheral compartments, such as adipose and muscle tissue. Interestingly, absolute plasma BDZ levels increased similarly after the three initial MARS sessions, resulting in an increasing percentage of nightly plasma level rebound. This likely represents a release of accumulated drug from peripheral compartments at zero-order kinetics. This novel finding not only reflects the release mechanism of diazepam from tissue but may also have clinical implications, with the cessation of the rebound increase in BDZ plasma level being a useful indicator of the end of intoxication. However, up to our best knowledge, we cannot compare this finding with any literature. Based on this observation, we recommend routine plasma level sampling both prior to and after MARS.The calculated patient's BDZ elimination half-life of 52 h, based on the last time points without MARS, is well in line with half-lives reported for diazepam, which range from 20 to 56 h. Based on this calculation, elimination of BDZ without MARS would take 16 days; therefore, MARS shortened the recovery of patient for 7 days. This would shorten the stay at ICU, decreased the risks of infections, ventilation-associated lung damage, etc.However, diazepam is metabolized into the active metabolites nordiazepam and oxazepam, which have substantially longer elimination half-lives of more than 200 h and high protein binding , 9. ConsTo the best of our knowledge, our case represents the highest dose of diazepam intoxication ever reported. Greenblatt et al. reported a diazepam poisoning with a 10-fold lower dose, that is, 2,000 mg (Our study has strengths but also limitation: Measurements of plasma BDZ concentration was assessed prior to and following every MARS session, allowing us to show clearly the beneficial effect of MARS. Based on our further calculation  moreoverCollectively, based on the observations and favorable outcome of the current case and in absence of official guidelines for severe flumazenil irresponsive BDZ poisoning, we suggest considering the use of MARS elimination treatment with daily sessions together with twice daily monitoring of BZD plasma levels.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.AD, AG, AY, and RH made substantial contributions to the conception or design of the manuscript. PV and DV-Y to the acquisition, analysis, and interpretation of the data. RH revised it critically. All authors participated in drafting the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wheat blast (WB) is a destructive disease in South America and its first outbreak in Bangladesh in 2016 posed a great risk to food security of South Asian countries. A genome wide association study (GWAS) was conducted on a diverse panel of 184 wheat genotypes from South Asia and CIMMYT. Phenotyping was conducted in eight field experiments in Bolivia and Bangladesh and a greenhouse experiment in the United States. Genotypic data included 11,401 SNP markers of the Illumina Infinium 15K BeadChip and four additional STS markers on the 2NS/2AS translocation region. Accessions with stable WB resistance across experiments were identified, which were all 2NS carriers. Nevertheless, a dozen moderately resistant 2AS lines were identified, exhibiting big variation among experiments. Significant marker-trait associations (MTA) were detected on chromosomes 1BS, 2AS, 6BS, and 7BL; but only MTAs on 2AS at the 2NS/2AS translocation region were consistently significant across experiments. The resistant accessions identified in this study could be used in production in South Asian countries as a preemptive strategy to prevent WB outbreak. Bread wheat grown in tropical and subtropical regions is subjected to a range of diseases, among which is wheat blast (WB). This is an emerging disease with growing impacts on wheat production in South America, South Asia, and Africa, and is a potential threat to other major wheat producers like India, United States and China . The disTriticum pathotype of the ascomycete fungus Magnaporthe oryzae (MoT), which is closely related to the notorious pathogen of rice blast, the Oryzae pathotype of M. oryzae, but cross infection normally does not happen  into wheat genome to utilize rust resistance genes Lr37, Sr38, and Yr17 , and RmgGR119 , India (40), Bangladesh (19), and Nepal (28). In the last three groups, there are genotypes directly introduced from CIMMYT due to extensive traditional utilization of CIMMYT germplasm in these countries.The field experiments took place in three locations, Quirusillas and Okinawa in the Department of Santa Cruz, Bolivia, and Jashore in the Division of Khulna, Bangladesh. Field trials were performed in the 2017–2018 and 2018–2019 cropping seasons in Quirusillas, the 2018 cropping season in Okinawa, and the 2017-18 cropping season in Jashore. Two sowings of approx. 14 days apart were adopted in each trial, totally making eight experiments, which were named as per the location , cropping season , and sowing (“a” for the first sowing and “b” for the second). For example, Quir18b represents the second sown experiment in the 2017–2018 cycle conducted in Quirusillas.The cropping cycle runs from December to April in Quirusillas and Jashore, and from May to September in Okinawa. Field experimental units were 1-m double rows separated by 20-cm spacing in all three locations, and no replication was set within each sowing. A field misting system was equipped at each site to create a micro-environment favorable for WB infection. The system worked from 8am to 7pm in Quirusillas and Okinawa and from 9am to 5pm in Jashore, with a 10-min-misting per hour, during the WB development period. Field inoculation was done twice in all the experiments, with the first at anthesis and the second at two days after anthesis, using a backpack sprayer. Inoculum was a mixture of locally collected MoT isolates with high pathogenesis, which included isolates QUI1505, QUI1601, QUI1612, OKI1503, and OKI1704 in Quirusillas and Okinawa, and BHO17001, MEH17003, GOP17001.2, RAJ17001, CHU16001.3, and JES16001 in Jashore. The first three letters of the isolate names indicate the place of collection, followed by two digits for the year of collection and the rest digits as isolate identifiers. The MoT isolates were cultured on oatmeal agar medium following the protocol by WB evaluation was performed on 10 spikes that had been marked at anthesis at 21 days after the first inoculation, for which the total and infected number of spikelets were recorded. Incidence was derived from the proportion of spikes with blast infection and severity was calculated as the averaged percentage of infected spikelets. WB index was calculated with the formula WB index = incidence × severity, which was used in all subsequent analysis. In addition, days to heading (DH) and plant height (PH) were scored in all experiments.In 2017, a greenhouse experiment was conducted in the biosafety level-3 laboratory at USDA-ARS, Foreign Disease-Weed Science Research Unit, Fort Detrick, MD, United States (nominated as US17 in this study). The accessions were sown into 15-cm-diameter pots containing a commercial potting medium , which were arranged in an incomplete randomized block design with one replication. Wheat varieties Urubo and Glenn were used as resistant and susceptible checks, respectively. A MoT isolate B-71 collected in Okinawa (Bolivia) in 2012 was used. Inoculation production was carried out on homemade oatmeal agar according to Ventriup-LN2 reported by WGGB156 and WGGB159 by cslVrgal3 that was derived from a follow-up study of The panel was genotyped with Illumina Infinium 15K BeadChip at Trait Genetics GmbH, Germany. Markers with missing data points more than 10% or minor allele frequency less than 5% were excluded from further analysis. Four STS markers in the 2NS/2AS region were applied to evaluate their association with WB index and suitability for marker-assisted selection (MAS), including 2 among the markers was calculated using TASSEL 51, and LD was visualized with R2 plotted against the physical distances. A kinship matrix and clusters among individual genotypes was generated with all the SNP markers.The linkage disequilibrium (LD) parameter RH2 = y + s +sy) for experiments in Quirusillas and H2 = s) for those in Okinawa and Jashore, where y for the number of years, and s for the number of sowing. Principal component analysis (PCA) on phenotypic data was conducted with the PAST software ver. 3.01  was performed with the PROC GLM module in SAS program ver. 9.2, and the results were used for calculating the heritability estimates, using the formula er. 3.01 .p-values, additive effects and percentage variation explained by each marker were obtained and Manhattan plots with the −log10 p-values of the markers were plotted using the ‘R’ package CMplot. The Bonferroni method for multiple testing correction at an α level of 0.20 was used to declare significance of the markers.Marker-trait association (MTA) tests were carried out in TASSEL 5 using the mixed linear model (MLM)  with ther values ranging from 0.27 to 0.72. Among the experiments, Jash18a that exhibited the lowest disease pressure showed generally poor correlation with others, whereas other experiments, especially those in Bolivia, exhibited better correlations . A CIMMYT breeding line Kachu/Solala  has been released in Bangladesh as BARI Gom 33 and in Bolivia as INIAF Okinawa, while another CIMMYT accession BORLAUG100 F2014 (CIM-35) has been released in Bangladesh as WMRI Gom 3, in Bolivia as INIAF Tropical, and in Nepal as Borlaug 2020. However, all these accessions are 2NS carriers, indicating the limited genetic variation in WB resistant sources, which needs to be complimented with non-2NS resistance. The implication for wheat breeding in the WB vulnerable regions is to combine different resistant sources to achieve a better and durable resistance, just like other wheat diseases  and HD 3171 (IND-13) have been recommended to the farmers of WB prone areas in West Bengal that borders to Bangladeshdiseases . To achiThe CIMMYT breeding line Milan (CM-34) is a WB resistance donor that has been widely used in South American, leading to many WB resistant varieties with Milan in their pedigree. Such varieties include Sausal CIAT that was released in Bolivia, CD 116 in Brazil, Caninde#1 in Paraguay, etc. . Howeverp values was identified, probably being caused by the lower mapping resolution. Therefore, larger GWAS panel or specifically designed fine mapping populations will be needed to better map the underlying gene. An additional observation was that STS markers Ventriup-LN2 and cslVrgal3 predicted the presence of 2NS better than WGGB156 and WGGB159 in Indian germplasm (WGGB156 and WGGB159 were linked with the resistance allele of the WB QTL.Despite the attempts on different GWAS algorithms, MTAs on the 2NS/2AS region were the only stably expressed QTL across experiments, and MTAs in other chromosome regions were hardly repeatable and have not been reported in other genetic studies . This coermplasm , which wermplasm . A possiPhenological traits like PH and DH are often associated with disease resistance under field conditions, as frequently reported for Fusarium head blight FHB, , spot blhttps://hdl.handle.net/11529/10548559.The data presented in the study are deposited in the Dataverse repository and can be accessed via the link PS, RS, and AC conceived and designed the experiments. MK, KR, RI, FM, XH, and PS performed field trials. GP conducted the greenhouse experiments. GS, AJ, and RS provided germplasm. XH and PJ analyzed the data. XH wrote the first draft of the manuscript. All authors contributed and approved the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over the study period, the mean age at death was 55.3 years , with differences depending on the period analyzed. The age-standardized mortality rate was 0.13/million person-years and was not different between men and women. When SD was the UCD, the most frequent associated causes were cardiovascular diseases , infections , and blood disorders , including six cases (54%) with macrophage activation syndrome. As compared to the general population, SD decedents aged <45 years were more likely to die from a cardiovascular event ; decedents at all ages were more likely to die from infection .Still’s disease (SD) is often considered a benign disease, with low mortality rates. However, few studies have investigated SD mortality and its causes and most of these have been single-center cohort studies. We sought to examine mortality rates and causes of death among French decedents with SD. We performed a multiple-cause-of-death analysis on data collected between 1979 and 2016 by the French Epidemiological Center for the Medical Causes of Death. SD-related mortality rates were calculated and compared with the general population . A total of 289 death certificates mentioned SD as the underlying cause of death (UCD) ( Still’s disease (SD) encompasses adult-onset Still’s disease (AOSD) and systemic-onset juvenile idiopathic arthritis (sJIA). SD is a rare multisystemic autoinflammatory disorder, characterized by a symptomatic triad associating high spiking fever, arthralgias, and evanescent skin rash ,2,3. BioMost of the time, SD is seen as a benign disease, with low mortality rates estimated at between 2 and 3% ,10,11,12Centre d’épidémiologie sur les causes médicales de décès, CepiDC) to evaluate SD-related mortality using MCOD analysis and compare it to that of the general population.Studies investigating SD mortality are scarce and most of these focus on specific complications or are single-center cohort studies, limiting the generalization of the results. Multiple-cause-of-death (MCOD) analysis provides comprehensive data at a country level with complete coverage of all deaths over selected periods of time and has been used successfully to study mortality in rare chronic diseases such as inflammatory or autoimmune conditions ,18,19. Whttps://www.insee.fr/fr/statistiques/4204054?sommaire=4204068, accessed on 24 August 2021).For this MCOD analysis, we used the same methodology as described previously ,18,19. Bhttp://cepidc-data.inserm.fr/inserm/html/index2.htm, accessed on 24 August 2021).The number of death certificates that listed SD as the UCD and the number of death certificates that listed SD as a UCD or NUCD, whatever its place on the death certificate, were considered in the MCOD analyses. For each death certificate that listed SD as the UCD, the NUCDs were examined. For each death certificate that listed SD as an NUCD, the UCD was investigated and the observed/expected (O/E) ratios were calculated for three age groups. For each UCD, the O/E ratio was calculated based on the proportional mortality rate for the same UCD within the general French population was used for the statistical analysis; the mgcv package was used for the penalized Poisson model.Quantitative variables were described using means and standard deviations, while qualitative variables were described as frequencies and percentages of each modality. Observed mortality rates were calculated using the corresponding person-years from the French population (period 1979–2016), according to sex, age, and year. Age-standardized mortality rates  were then calculated by a direct method, using the age distribution of the 2006 general population of France as reference. In order to consider the effects of sex, year, and age on the mortality rate, we fitted a generalized additive model (GAM) with a Poisson distribution. The non-linear effects of age and year were modeled using penalized cubic splines [n = 154), 62% of the decedents had AOSD and 38% had sJIA. Twenty-two certificates mentioned more than one NUCD.Among the 20,460,583 death certificates recorded in France by the CepiDC, between 1979 and 2016, 289 certificates reported a diagnosis of SD; thus, the mean annual number of related deaths was 7.81/year. When death was directly linked to SD  among SD decedents was 0.13 per million person-years . This raTaking into account sex, year of death, and age on the mortality rate, a GAM found that the mortality rate increased from the early 1990s, peaked around 2002, and decreased, reaching a plateau for decedents before the age of 50 years, whereas it still increased for decedents after the age of 50 years (p = 0.229).The risk of death in women was multiplied by 1.16, but this was not statistically significant (n = 70) of cases aged <45 years at death, vs. 40% (n = 27) of cases aged 45–64 years at death, vs. 49% (n = 57) among those aged >65 years at death. Over the years, notification of SD as the UCD slightly but progressively decreased, whereas notification as an NUCD remained stable. Before 2000, SD notification as the UCD was higher in decedents aged <44 than in older decedents. After 2000, SD notification as the UCD was higher in decedents aged >65 than in younger decedents. The same trends were observed for the declarations of SD as an NUCD.Still’s disease was listed as the UCD on 154/289 (53%) death certificates and as an NUCD on the remaining 135/289. SD notification as the UCD was higher in the younger group of decedents; SD was reported as the UCD in 67% (n = 154), the three most frequently determined NUCDs were: cardiovascular diseases , infections , and blood disorders , which were reactive hemophagocytic syndromes  in six cases (50%) (When SD was the UCD (es (50%) . There wn = 135), the main UCDs were cardiovascular diseases  , while the third was solid malignancies .When SD was listed as an NUCD  . Infectin = 289), the main reported causes of death associated with SD were cardiovascular diseases (n = 72) and infections (n = 58). Pulmonary infections were the most frequently reported infections (n = 33). Pneumocystis pneumonia was reported in two cases.In the whole cohort (n = 23) were cardiac insufficiency , pericarditis , pulmonary embolism , or arrhythmia . In patients who died after the age of 65 from cardiovascular disease (n = 34), 82% of deaths were attributed to two causes: arterial events  and cardiac insufficiency . Pericarditis was mentioned in four cases , while endocarditis and myocarditis were mentioned in one case each. Pulmonary hypertension was notified as an NUCD in three cases (one death before age 24 and two after age 55).When a cardiovascular disease was listed as the UCD or an NUCD, the main diagnoses in patients who died before the age of 44 (Blood disorders were reported in 27 patients (9.3%). Macrophage activation syndrome (MAS) was reported in 15 of these cases (55%). In six additional cases, an MAS could be suspected but was not reported as such. In five cases with MAS, there was an associated mention of disseminated intravascular coagulation (DIC). MAS notification was homogenously distributed among the age categories. There were two notifications of thrombotic microangiopathy (not associated with MAS notification). Two lymphomas were reported , along with one report of acute myeloid leukemia. Amyloidosis was reported in eight (2.7%) patients, with 63% of the cases reported before the age of 44 and 87% of the cases reported before 2002. In 6/8 cases, only amyloidosis and SD were reported on the death certificate; in the remaining cases, there was one case of arrhythmia and one case of cardiac hypertrophy.There were six reports of pulmonary fibrosis ; all were over 45 years of age and 4/6 (75%) were over 65 years. There were also two reports of associated giant cell arteritis and one sarcoidosis .p < 0.01)  .p < 0.001).Infections caused a significant excess of mortality in all categories of age, with the highest O/E ratio observed in decedents between age 45 and 64 .A significant excess of mortality due to blood disorders was observed for cases who died before 65 years of age  preferentially affects children and young adults. This is further illustrated by the comparison with the general population, which shows that the proportion of people who died because of SD was much higher among those under 44 years of age. However, in the whole cohort, 64% of the cases were over 45 years of age at the time of death. This indicates that the analysis of death certificates has probably captured cases of prolonged SD , when most epidemiological studies show the age at onset. Indeed, a retrospective US study of 154 AOSD patients hospitalized between 2009 and 2013 revealed a mean age at death of 62.4 years . In anotThe sample size of our study may seem quite limited, but SD is a rare disorder with very low mortality ,12. MoreFor example, we found that patients mostly died from cardiovascular diseases. This specific excess mortality was only observed in young decedents, indicating that SD or its treatment could be considered as a cardiovascular risk factor. Chronic systemic inflammatory response and the prolonged use of steroids are associated with an increased risk of cardiovascular disease and atherosclerosis ,23. HoweThe second most common cause of death associated with SD was infection, which again has been poorly reported in the literature. A few studies have shown that infections are the first cause of death in SD ,27,28; an = 19) and is not repetitively alleged [In our study, fifteen deaths (5%) were directly or indirectly attributable to MAS. This finding contrasts with an earlier report, which indicated that up to 63% of SD decedents die of refractory MAS . While M alleged ,12,21. T alleged ,35,36,37In two additional cases, thrombotic microangiopathy was mentioned as an underlying cause of death. Thrombotic microangiopathy is an exceptional complication of SD, with less than 50 cases reported so far . This reOverall, a significant excess mortality due to blood disorders was observed in young decedents with O/E ratios ranging from 7.29 to 8.17. These results confirm the relationship between SD and hematological complications, along with their severity.While considered as uncommon, amyloidosis has long been reported as a possible complication driving mortality in SD ,39. In tWhile pulmonary fibrosis is a known complication of SD, the association of giant cell arteritis or sarcoidosis with SD is unusual. Unfortunately, by analyzing death certificates, it is not possible to know whether these were linked disorders, misdiagnoses, or incidental associations. As SD diagnosis is based on the exclusion of multiple differential diagnoses, some signs or symptoms associated with vasculitis may overlap with those of SD. In addition, lymph node enlargement may also challenge the diagnosis . Very reInterestingly, there was no increase in O/E ratios for solid or hematological malignancies, which can be interpreted as a reassuring signal for SD patients.The limitations of this study are mainly related to the method used. In particular, the accuracy of the completion of death certificates cannot be guaranteed. As in other studies that used a similar methodology, it can be assumed that previous or resolved SD , or quiescent/non-active SD, was not reported on the death certificates. As a result, the most severe cases of SD may be over-represented. However, this limitation is also present in retrospective cohort studies, which usually include cases referred to tertiary centers. This bias also affects inpatient cohorts.The small sample size is probably not sufficient to provide accurate estimates, but the fact that the cases are unselected and captured longitudinally does allow for some representativeness, particularly with regard to SD-specific mortality.Finally, the differentiation between the UCD and NUCD in decedents with Still’s disease might have been difficult. However, in SD, life-threatening complications are fairly well identified.Despite these limitations, this study highlights some specific medical priorities in the management of SD patients, particularly regarding cardiovascular and infectious risks. It also confirms the important association with hematological disorders such as MAS, a major factor in mortality. Finally, this study tends to confirm the decline of AA amyloidosis over time, especially since the early 2000s.The prognosis of SD has improved over the last two decades. Apart from the widely described specific causes of death, such as MAS or DIC, physicians should pay special attention to frequent and preventable causes of death, including cardiovascular disease and infections, focusing on their prevention. MAS can occur at any age and appears to be the most common hematological cause of death in SD.


In conclusion, sequenced movements can modify tone and stiffness as a function of age, while age-associated changes in viscoelastic characteristics depends on pain but not on movements. The MMPs should be assessed, not only at the beginning of the physical examination at rest, but also along the patient’s follow-up, depending on their pain and age, in a clinical setting.This research aimed to identify changes in muscle mechanical properties (MMPs) when a standardized sequence of movements is performed and to determine the influence of acute low back pain (LBP) and age on the MMPs. Socio-demographic, clinical variables and MMPs were collected in 33 patients with LBP and 33 healthy controls. A 2 × 2 × 2 (group × age × time) analysis of variance (ANOVA) mixed model was used to determine the effect of the study factors on the different MMPs. There were no significant triple interactions. After the movements, tone and stiffness increased 0.37 Hz and 22.75 N/m, respectively, in subjects <35 years, independent of their clinical status. Relaxation showed differences by age in healthy subjects and creep in LBP subjects. Furthermore, elasticity was higher in <35 years ( Back pain is one of the most common musculoskeletal pain disorders, affecting 80% of the population at some point in their lives . The incThe MMPs play a central role in several physiological and pathophysiological processes, suggesting a relationship between pain and muscle stiffness in different spinal pain disorders, including LBP ,15. HoweNew technologies, such as elastography, help to study the passive mechanical behaviour of skeletal muscles . HoweverTherefore, this study aimed to identify changes in paraspinal MMPs when submitted to a standardized sequence of lumbar movements and to determine the influence of lumbar pain and age in those changes.An observational, test-retest study including subjects with LBP and healthy controls was performed. Participants were recruited with a non-probabilistic sampling of two centres, Physiobalance (private physiotherapy centre) and the Biosanitary campus of the University of Córdoba, Spain, from November 2018 to January 2021. Assessments were conducted between April 2019 and March 2021. The Córdoba Research Ethics Committee approved this project (reference number 4016/2018). All participants signed written informed consent.2), without spinal pain in the last six months or any neurological or musculoskeletal pain disorder.Sixty-six subjects of both sexes participated in this study. Thirty-three of them presented acute LBP with less than four weeks of evolution  and a paThe exclusion criteria for both groups were history of traumatic lesions at the spine, scoliosis, spinal surgery, congenital deformity, inflammatory disease, pregnancy, and receiving spinal physiotherapy treatment in the last six months.A moderate difference size (Cohen d = 0.7) , conside®  device, were assessed before and after a standardized sequence of movements. The MyotonPRO© provides measurements of five MMPs: tone or state of tension, determined by the frequency (Hz); biomechanical properties such as dynamic stiffness (N/m) and decrement, that characterizes elasticity; and viscoelastic properties such as relaxation or mechanical stress relaxation time (ms) and creep (Deborah number), which corresponds to the gradual elongation in the tissue when subjected to constant tension [® added a pulse of 15 ms and 0.40 N of mechanical force. The accelerometer at the sensor’s tip provides the data that characterizes the tissue [After signing the informed consent form, researchers collected socio-demographic  and clinical  data, respectively ). Subseq tension . The deve tissue .A clinician with more than fifteen years of clinical experience, identified the spinous process of L5 by palpatory testing and performed the tonometric test on the erector spinae, located 2.5 cm to the right and left of the spinous process. The assessment was performed with the patient in a prone position with both arms alongside the body. Muscle measurements were taken first on the left side, then on the right side, and the process was repeated. The measurement was taken during a five-second apnoea after the normal expiratory phase ,44 Figu.The standardized sequence of movements involved the maximum range of motion in frontal flexion, extension, lateral flexion, and rotation of the spine in a standing position, all routinely used in the conventional evaluation of the amount of movement of patients in a clinical setting . Each mop > 0.05 in all cases.Descriptive results for qualitative variables were expressed as frequencies and percentages. Quantitative variables were expressed as means, standard deviations, and 95% confidence interval (CI). The Kolmogorov–Smirnov test was used to assess the normality of the data distribution, with t-test between measurements on each group (p > 0.05). Consequently, pooled (mean) data from both sides were used in the main analysis.In a preliminary analysis of the data, no side-to-side differences were identified when applying Student’s A 2 × 2 × 2 mixed model (group × age × time) of analysis of variance (ANOVA) was used to determine the effect of the study factors on the different MMPs. The first factor was clinical status . The second factor was the age, with two levels (subjects <35 vs. >35 years) as previous studies with similar objectives has defined ,46,47,48p < 0.05. The analyses were carried out by IBM SPSS® Statistics version 25 .In all cases, the confidence level was established at 95%, and the statistical significance level for the tests was n = 19, >35 years n = 14) and 33 healthy controls , with a mean age of 33.3 ± 11.8 years and a BMI of 23.9 ± 2.6 kg/m2 .There was no triple interaction when analysing the pooled effect of movement, clinical status, and age on the behaviour of the MMPs  and stiffness  increased 0.37 Hz (95% CI 0.06–0.70) and 22.75 N/m (95% CI 5.83–39.67), respectively, in the younger subjects after the movements, independent of their clinical status. Only the baseline measure showed significant differences (p < 0.05) between both age groups in both MMPs. No other time-by-age interaction was identified neither for decrement , relaxation , and creep  outcomes.With borderline statistical significance found in the double interactions, frequency , with relaxation being 2.98 ms (95% CI 0.36–6.01) higher in young healthy subjects. In contrast, creep was 0.10 (95% CI 0.06–0.15) greater in subjects with LBP over 35 years. Differences between patients and controls in those over 35 were also found . Frequency , stiffness , and decrement  did not show group-by-age interaction.Likewise, the relaxation and creep showed interactions between age and clinical status of the subjects .The group-by-time interaction did not reveal any statistical significance   .The results showed that paraspinal lumbar MMPs have different behaviour under specific conditions of lumbar movements, clinical status or age. Thus, the sequenced lumbar movements protocol influenced tone and stiffness depending on age, with lower values for the MMPs in younger subjects, which were more susceptible to be influenced by movement. However, the presence of LBP did not influence MMPs. On the other hand, although with almost statistical significance, the viscoelastic state of paraspinal muscles, expressed as relaxation and creep, depends on the combination of pain and age. While healthy subjects showed a reduction in relaxation with age, these differences did not occur in individuals with LBP. The presence of pain determined the differences in creep concerning age. In this case, the performance of sequenced lumbar movements did not influence the MMPs. Furthermore, the elasticity, as the inverse of the decrement, was higher in younger subjects, without the influence of any other factor.Curiously, when the presence or absence of LBP was analysed separately, no significant differences were detected, despite the higher values of tone, stiffness, and lower values of decrement, relaxation, and creep found in LBP subjects. This was an unexpected result, since acute LBP is commonly associated to muscle spasm throughout the paraspinal muscles ,49,50. HThis study determined the interaction between the effect of sequenced movements and the advancing of age on tone and stiffness at the lumbar level, where the first measure reports a difference between age groups of 1.29 Hz that decreases to 0.88 Hz after movement. This difference indicates that younger subjects experienced a change in their muscle tone that did not occur in older subjects. Regarding stiffness, from a difference between age groups of more than 50 N/m, younger subjects increased their stiffness more than 8% after the movements, while older subjects showed a rise of only 2%. Apparently, our results would contradict the behaviour of thixotropic properties of specific tissues, which establishes the reduction of viscosity and, therefore, of muscle stiffness, during and, for some time, after movement ,21,51. HOn the other hand, the fact that only subjects under 35 years presented a significant change in muscle tone and stiffness after movement supports the relationship between age and MMPs changes ,31,32,44Our study also reported the relationship between LBP and age with relaxation, and creep, as viscoelastic characteristics, of paraspinal muscles. Relaxation was different in healthy subjects depending on age. In fact, advanced age established differences in creep between healthy subjects and patients. However, the presence of LBP inhibited this behaviour in the creep. Although there is no previous research on viscoelastic properties in acute LPB, previous studies have determined changes in MMPs in subjects with chronic mechanical and inflammatory LBP and neck pain ,36,43,58To date, no previous study has attempted to establish the adequate moment to evaluate MMPs in clinical practice based on the physical evaluation, clinical status and age. Consequently, these results will allow us to determine the effect of the therapy on the MMPs with greater precision.However, several limitations must be recognized. First, the raters were not blinded, although the assessment of the MMPs has shown high reliability and low rater dependence, which limits a negative influence on the results ,59. SecoA sequenced spinal movement protocol can modify lumbar paraspinal tone and stiffness according to the patient’s age but not according to the presence of LBP. The changes in viscoelastic characteristics of lumbar muscles depend on age and the presence or absence of pain, but not on the performance of the sequenced movements. Older subjects showed less elasticity than younger ones at the L5 spinal level, independent of their condition.The MMPs should be assessed in a clinical setting, not only at the beginning of the physical evaluation during rest, but also during the patient’s follow-up, with special attention to elder subjects and those with pain.