The purpose of this study is to report normative data and predict thresholds for a rapid test of spatial release from masking for speech perception. The test is easily administered and has good repeatability, with the potential to be used in clinics and laboratories. Normative functions were generated for adults varying in age and amounts of hearing loss.The test of spatial release presents a virtual auditory scene over headphones with 2 conditions: colocated (with target and maskers at 0°) and spatially separated (with target at 0° and maskers at ± 45°). Listener thresholds are determined as target-to-masker ratios, and spatial release from masking (SRM) is determined as the difference between the colocated condition and spatially separated condition. Multiple linear regression was used to fit the data from 82 adults 18–80 years of age with normal to moderate hearing loss (0–40 dB HL pure-tone average [PTA]). The regression equations were then used to generate normative functions that relate age (in years) and hearing thresholds (as PTA) to target-to-masker ratios and SRM.Normative functions were able to predict thresholds with an error of less than 3.5 dB in all conditions. In the colocated condition, the function included only age as a predictive parameter, whereas in the spatially separated condition, both age and PTA were included as parameters. For SRM, PTA was the only significant predictor. Different functions were generated for the 1st run, the 2nd run, and the average of the 2 runs. All 3 functions were largely similar in form, with the smallest error being associated with the function on the basis of the average of 2 runs.With the normative functions generated from this data set, it would be possible for a researcher or clinician to interpret data from a small number of participants or even a single patient without having to first collect data from a control group, substantially reducing the time and resources needed.https://doi.org/10.23641/asha.7080878 Our goal is to report normative data and predict thresholds for a rapid test of spatial release from masking (SRM) for speech perception. SRM refers to the benefit that a listener obtains when a target sound source is at a different location in space relative to one or more masking sound sources. This benefit is reflected in the ability to understand speech in adverse environments with maskers that are much more intense than the target. Performance improvement depends on the presence of interaural differences in both time and level, both of which are less effective for listeners with increasing age and hearing loss . HoweverAnother aspect of clinical utility is the ability to be used for repeated testing. The LISN has a limited set of open-set materials that cannot be repeated an arbitrary number of times without running the risk of participants learning the materials, making it unsuitable for monitoring changes in performance across a large number of time points. https://github.com/gallunf/SR2) where anyone interested can download the MATLAB code and the wavefiles needed to run the experiments. Over the past year, in collaboration with the University of California Riverside Brain Game Center and on the basis of a grant from the National Institutes of Health, the authors have also created a version of the SR2  to be run on an iPad. Spatial Release is the same program as the SR2, takes only 5 to 7 min to complete, and is available to clinicians and the general public for free on the iTunes app store . Currently, multiple sites, both in the Veterans Administration system and at other universities, are using either the SR2 or Spatial Release to collect data on adults. The authors will continue to make the SR2 code available to anyone who is interested and are in the process of establishing the degree to which the SR2 and Spatial Release can be used interchangeably.The analyses presented below describe the calculation of normative functions for an automated CRM-based test of SRM , which was developed by SDs below the mean. One difficulty with this approach, as described in detail by Normative data are commonly collected on a sample of listeners with no known deficit and expressed as a cutoff score on the basis of the means and standard deviations of the measures recorded on this sample . TypicalWhile examining the effects of age and hearing loss on SRM, it is important to note previous studies that have also examined how age and hearing loss affect SRM for adults . Some stBelow, we present a range of normative values and the regression functions needed to incorporate age, hearing loss, or both into a comparison of SRM for a given listener with expected performance. The resulting functions predict mean and expected ranges of performance on the SR2 for listeners varying in age between 18 and 80 years and four frequency bilateral pure-tone average (PTA) of 0.5-, 1-, 2-, and 4-kHz hearing losses up to 40 dB HL. This work expands the findings of SD = 18.66). Mean bilateral PTA  was 12.48 dB relative to standard hearing level , and eight had asymmetries greater than 20 dB . None had differences exceeding 35 dB at any frequency. The full audiogram data for each participant, along with gender, hearing aid use, and calculated values of PTA and asymmetry across ears, are included in the Supplemental Material S1. All listeners were in good health with no history of otological disorders and had scores of 24 or higher on the Mini-Mental State Examination (Over the past 5 years (2012–2017), 100 participants completed at least one test session involving the SR2, and as described in ng level  with an mination  to rule As described by All testing was done in a sound-attenuated booth at the National Center for Rehabilitative Research in Portland, OR. Digitally recorded speech stimuli were processed in MATLAB (Mathworks) using the convolution described above, converted to analog signals using either a Grace m903 (Grace Design) or Lynx Hilo (Lynx Studio Technology) digital to analog converter and amplifier. Signals were presented over Etymotic ER2 earphones, and participants selected their responses using a graphical display of colored numbers presented in MATLAB on a computer touch screen monitor.Participants listened for the sentence with the predetermined call sign  and input the color–number combination following the call sign. A progressive tracking procedure was implemented to present 20 trials, two at each of 10 TMRs, starting at 10-dB TMR and ending at −8-dB TMR (decreasing in steps of 2 dB). TMR thresholds in dB were estimated by subtracting the number of correct responses from 10. For example, if a listener answered all 20 presentations correctly, the threshold would be estimated at −10 dB, whereas if all of the answers were incorrect, the estimate of TMR at threshold would be 10 dB. A listener who answered half of the presentations correctly would be assigned a threshold of 0-dB TMR. p value of .05 as long as the regression explains at least 11% of the variance. For a single predictive factor, the power is .8 if there is at least 9% of the variance explained, power of .7 if 7% is explained, and power of .5 if 5% is explained. This analysis suggests that 82 participants are sufficient to have at least a 50% chance of finding even quite small relationships.Following Supplemental Material S1. Thresholds were reduced from Run 1 to Run 2 by 0.7 dB for the colocated condition and 1.4 dB for separated condition, resulting in an increase of 0.7 dB in SRM. This is consistent with the changes observed for a subset of these participants in p value of .0033 as the criterion for significance, which corresponds to a correlation of .333 or higher. Correlations between age and thresholds in the colocated condition (r = .388) were significant, whereas correlations with hearing loss were not (r = .274 or lower). Thresholds in the separated condition were correlated with both age (r = .525) and hearing loss (r = .615 or higher) as was spatial release (r = −.402 or lower for both age and hearing loss). Age and hearing loss were significantly related, however (r = .510 or higher), suggesting that multiple regression would be a more appropriate method of exploring the relative influence of age and hearing loss on performance as this would allow the relative contribution of each to be estimated using the same equation.Supplemental Material S1. There were significant correlations between retest delay and the difference in scores for the colocated condition  and for SRM  but not for the separated condition . The effects were in the direction of reduced performance with longer delays, as would be expected. The effect sizes were small, however, with regression functions predicting increases between 0.01 dB and 0.03 dB per week of delay.As the data were collected over a range of test–retest delays, the number of days between tests (“retest delay”) was also considered and is available as an additional variable in the Supplemental Material S1. Plots of the observed and predicted values were examined to ensure linearity, and the errors were examined to ensure a normal distribution. It was clear that linearity and normality were met for the participants with PTA values of 20 dB and better, but the fact that fewer than 20 participants presented with PTAs greater than 20 dB  made it difficult to confirm or deny that the assumptions were met for those with greater hearing loss. In the absence of sufficient data to choose an alternative analysis, the linear regression was used. It is noted that future work should both examine a greater sample of those with greater hearing loss and more participants under age 50 years with greater hearing loss, and consider the possibility that nonlinear functions would be a more appropriate approach. The current data set does not allow us to reject the assumption that the linear approach is most appropriate as the predictions are quite accurate for the majority of the participants, as discussed below.The data from the average of the two runs are plotted in p values were below p < .05 for all but one of the colocated conditions  and the regression coefficients were all quite similar. This similarity of the regression coefficients is consistent with the high test–retest reliability found by Based on the correlations shown in Model error was calculated for each participant as the difference between the predicted and the observed value. As can be seen in SD above and below the mean, calculated by treating the average model error in dB as 1 SD. SD value is the model error shown in From the data shown in SDs from the predicted value for their ages and PTAs. These data are indicated with colored highlighting in the Supplemental Material S1. The records of these individuals were examined to see what factors may be predictors of poor SRM abilities. The age range of the participants was 24–74 years. Three of the participants had normal audiometric thresholds in both ears, whereas four had hearing loss in one or both ears. There was no indication that the presence of tinnitus, a history of concussion, or asymmetrical hearing loss was associated with abnormal performance. Furthermore, only one individual performed abnormally on both runs of either condition. This pattern is further evidence of the strength of the linear model and the accuracy with which the statistical description matches the observed data.Seven individuals were observed to have Z-Scores on any of the nine measures , which placed them at or beyond 2 This work describes the creation of normative functions for an automated SRM task, the SR2. The data can be applied to find Z-scores for individuals from 18–80 years of age and with PTAs of less than 40 dB HL. We examined SR2 thresholds for Runs 1 and 2 separately and the average of both. The results of multiple linear regression were similar for all three analyses and revealed (a) a significant effect of age in the colocated conditions, (b) that age and PTA were both significant predictors in the separated conditions, and (c) that for SRM, PTA was the only significant predictor. Consequently, to calculate a Z-score for the colocated condition, only the age of the participant would be needed, and for SRM, only PTA would be needed. For the separated condition, both age and PTA would need to be taken into account to generate a Z-score. The patterns found, which are consistent with those seen by The results reported here are consistent with the data that our laboratory and those of our collaborators have produced using the SR2 and the earlier measures from which the SR2 was derived e.g., . In partThe finding that age affects both the colocated condition and the separated condition and has no influence on SRM suggests that there is a small but reliable effect of age on the ability to distinguish among the talkers, regardless of the spatial cues. Perhaps, this is due to a cognitive processing ability that is more likely to be reduced in older participants, or perhaps, it is due to a reduction in sensitivity to fine timing differences in the voices of the male talkers.By calculating Z-scores, performance of individual research participants and patients can be examined in relation to the expected values, reducing the costs and effort needed to run a control group to compare to an experimental group and allowing individual patients to be characterized with reference to the expected performance for an individual of their age and hearing ability. This would have been especially helpful for those studies that, due to availability of patients and/or resources, have examined small samples of participants on the SR2. For example, were normative values available, it would perhaps have been possible to draw stronger conclusions about the influence of brain injury on SRM as measured by the SR2 . SimilarLimitations of this study include the age range of the participants, which did not include children or listeners older than 80 years, and hearing loss, which did not exceed PTAs of 45 dB HL. It is likely that the linear functions would not predict data including children due to the poorer performance of the younger children relative to the teenagers and young adults. Similarly, it is not clear whether the participants older than 80 years would be as well predicted as their younger counterparts. These tools should be used to explore these populations in the future. Hearing loss was limited in order to ensure that equal audibility would be available to all participants, but this means that the functions are most clearly valid for listeners with hearing in the normal range. As the predicted spatial release diminishes to near 0 at 50 dB HL, however, this limitation is less a characteristic of the sample and more related to the nature of spatial release. Future work should (a) examine those with greater hearing loss, however, as only four of the eight participants with thresholds above 25 dB HL achieved spatial release near the predicted values see  and (b) Translation of laboratory tests into a form suitable for clinical testing requires the transformation from tests that function well for comparing groups of participants to tests that can be used to evaluate a single individual. With the normative functions generated from this data set, it would be possible for a researcher or clinician to interpret data from a small number of participants or even a single patient without having to first collect data from a control group, substantially reducing the time and resources needed.10.1044/2018_AJA-17-0069SMS1Supplemental Material S1.Click here for additional data file.


Classical antiretroviral agents may acutely impact on metabolic, mitochondrial, renal and hepatic function in HIV-infected and uninfected persons. Fusion and integrase inhibitors are supposed to be safer, but have been scarcely investigated. To avoid any interference with HIV or other antiretrovirals, we assessed markers of these toxicities in healthy adult volunteers treated with Enfuvirtide (T20) or Raltegravir (RAL).Twenty-six healthy participants were randomized to T20/90mg vs. placebo (n = 12) or RAL/400mg vs. placebo (n = 14) every 12h in two 7-day periods separated by a 4-week washout period. Major end-points were changes in lipid profile -cholesterol, low-density-lipoprotein (LDL)-cholesterol, triglycerides), insulin resistance (glucose) and mitochondrial toxicity . Renal and hepatic toxicity , alanine aminotransferase (ALT), bilirubin and total plasma proteins) and overall safety were also analysed. Effect of period, treatment, and basal measures were evaluated for each end-point.Neither T20-administration nor RAL-administration yielded to any statistic significant change in the markers of metabolic, mitochondrial, renal or hepatic toxicity assessed. No symptoms indicative of drug toxicity were neither found in any subject.In absence of HIV infection, or concomitant treatment, short-term exposure to T20 or RAL in healthy adult volunteers did not lead to any indicative changes in toxicity markers thus presuming the safe profile of both drugs. A combination of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either a boosted protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) has constituted the standard highly active antiretroviral treatment (HAART) for many years –4. DespiEnfuvirtide (T20) is the first entry inhibitor available for salvage combination therapy ,27. LocaOn the other hand, raltegravir (RAL) is the first integrase inhibitor available . The mosControversially, post-marketing data have recently raised concerns on the safety of these new antiretroviral drugs. Interestingly, this is the case for RAL and other integrase inhibitors, as associated weight gain has been reported in cohort studies .Importantly, for both RAL and T20 medications there are no studies assessing changes in insulin resistance and mtDNA parameters  and, in all cases, all were assessed in concomitant HAART schedules, after drug discontinuation, or in the presence of HIV infection acting as a confounder ,34.Although the target and the chemical structure of both T20 and RAL are different from those classical antiretrovirals, it is unknown whether T20 and/or RAL have any early effect in metabolic, mitochondrial, renal and hepatic toxicity markers. All these reasons provided the basis for performing post-commercialization Phase I studies of T20 and RAL administration in healthy volunteers to assess univocal causality of drug safety/toxicity without the interference of previous/concomitant medication or HIV infection . We desith October 2008). In all cases specific legal and institutional requirements at the time of the studies were followed.The present work includes two different studies to assess T20 and RAL toxicity performed at the Hospital Clinic of Barcelona  at different time periods. Clinical Research Ethics Committee of our hospital approved T20 and RAL studies in June 2005 and November 2008, respectively. Potential candidates were recruited in 2006 for T20 and 2011 for RAL, following Helsinki guidelines. Ongoing and related trials for this drug/intervention are registered. Since both studies were performed at different timing, they were subjected to different legal registry requirements. According to legal obligations, in 2006 we performed the registry of T20 study (14th April 2008) after patient inclusion (March 2006). Conversely, by the time we performed the inclusion of patients for RAL trial (March 2011), we were required to prior register the study  vs. placebo-T20 (n = 6) or RAL-placebo (n = 7) vs. placebo-RAL (n = 7). The study was performed at the Phase I Unit of Hospital Clínic .Eligible volunteers were required to be healthy males between 18 and 45 years, with normal blood biochemical parameters, BMI between 19–24.9 kg/mAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.The study was designed as a single-centre, randomized, cross-over, double-blind, placebo-controlled trial in healthy adult volunteers . Randomization was performed with the Glaxo software: MAS V 2.1 Glaxo Wellcome 2001 by the CTU  of the Clinical Pharmacology Service of the Hospital Clínic of Barcelona. The Trials Agency of the Hospital Clínic was provided by the CTU with the random assignment list so that they can proceed with the preparation of the medication for the study, which was administered following a double-blind scheme. It was also provided, in a sealed envelope, with the code of random assignment of each participant to the Principal Investigator.In order to balance all patients into comparable basal conditions and avoid food interferences, participants were instructed to follow a specific diet within the World Health Organization standards one week prior to the initiation of the trial and during the periods of medication administration. A simple randomization was performed. For both T20/RAL studies, volunteers were scheduled to a 1-week period of either T20/RAL or placebo followed by a 4-weeks washout period and finally a 1-week period of placebo or T20/RAL, respectively. Washout period was set in order to avoid carry-over effect . Fig 1 aT20  was subcutaneously administered at 90 mg/mL doses. As placebo we used 0.9% saline solution . Both T20 and placebo were prepared for administration at the hospital pharmacy. Subcutaneous syringes with either 1mL of T20 or placebo were wrapped with aluminium foil for blinding and kept in a temperature-controlled refrigerator for a maximum of 24 hours.RAL, formulated as Isentress , with 400 mg of active substances and 26.06 mg of lactose-monohydrate. Placebo was also formulated for oral intake (Merck Sharp & Dohme).Medication was administrated every 12 hours for 7 consecutive days. All medication was administered by one of the study investigators  at the Phase I Unit, and volunteers were directly monitored for potential immediate adverse effects for at least one hour after subcutaneous administration. Volunteers were routinely asked about potential adverse events prior to each dose of study medication.Major end-points were changes in lipid profile -cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides), insulin resistance (glucose) and mitochondrial toxicity . Renal and hepatic toxicity markers , alanine aminotransferase (ALT), bilirubin and total plasma proteins were also analysed. Moreover, data regarding overall safety was recruited. The effects of period, sequence (carry-over), and treatment were evaluated for each end-point from absolute values and results were expressed as percentage of change.Venous blood sampling was withdrawn in the morning after at least 12-hours overnight fasting at the beginning and the end of each 1-week period . A urineBlood samples were tested for metabolic markers of lipid metabolism and insulin resistance toxicity. Total cholesterol and triglycerides levels were determined using enzymatic reagents  for a Cobas Mira automated analyser . The HDL-cholesterol level was determined after precipitation with phosphotungstic acid and magnesium chloride, LDL-cholesterol was directly measured. Plasma glucose was measured by the glucose hexokinase method .MtDNA content was measured in PBMCs as a marker of mitochondrial toxicity. PBMC were isolated by Ficoll-Histopaque technique and stored at -80 ± 5°C until analysis. MtDNA was obtained by standard phenol-chloroform extraction and amplified by quantitative real time PCR . Results were expressed as mean ratio between ND2-mitochondrial-encoded gene respect to 18SrRNA nuclear-encoded gene copies .Creatinine levels were measured using high-resolution liquid chromatography with ultraviolet-visible detection in the concentration range of 0.1 g/l urine 4.0 g/l urine. Transaminases values were determined using the malate dehydrogenase (MDH)-NADH reaction measured at 340 nm . Bilirubin determination was made by the diazotized sulfanilic acid reaction and posterior photocolorimetry measured at 530 nm. Total plasma proteins were measured according to the Biuret method.Safety end-points included abnormal laboratory results or any clinical adverse events. The adverse events were graded according to the World Health Organization score. Injection site reactions were assessed according to persistent pain and discomfort at local level, size of the indurations and erythema.Sample size was calculated based on total cholesterol measurement, established as one of the major endpoints of the study.α+Zβ)2*S2/d2, where Zα = 1.64 (one-sided α = 0.025), Zβ = 0.84 where the effect size d was set to 0.07 and the variability S to 0.10. Thus, we assumed a minimum sample size per group of 12 volunteers to detect significant increases in fasting plasma total cholesterol.According to our previous experience, as well as other investigations , the intDue to the small sample size, non-parametric test (Wilcoxon test for paired data) was used to evaluate the effects of the period, treatment (differences between Phase 1 and Phase 2), as well as any potential differences in the basal measurements . Results were expressed as mean values or percentage of change . To assess the direction and the magnitude of change and variation of results, Standard Error of the Mean (SEM) values were used. All statistical tests were performed by the Statistical Package of Social Sciences . Significance level was set at p value of 0.05.http://hdl.handle.net/2445/131036).According to the lack of legal or ethical restrictions for data sharing, we have submitted the anonymous data of both clinical trials into the University of Barcelona repository  mainly mild injection site reactions. 12% had other than injection site reactions, including headache, odinophagia and other minor flu-like symptoms. None of these events required any intervention or led to drug discontinuation.Metabolic, mitochondrial, renal and hepatic toxicity together with overall clinical safety issues have been attributed to antiretrovirals used for HIV management, including PIs, NRTIs and NNRTIs, due to off target interferences with physiological pathways ,16,39–41in vivo studies [Entry and integrase inhibitors are supposed to be safer and free of these toxicities, however there is lack of evidence of  studies . Additio studies . Moreove studies ,34 for e studies , or prev studies .In the present study, there were no changes indicative of lipid disturbances, insulin resistance, hepatic or renal toxicity were evident after T20 or RAL administration. Considering mitochondrial toxicity, no significant changes were detected in RAL treatment nor after T20 administration. Given the fact that mitochondrial toxicity and primary mitochondrial disorders are associated with a decrease in mtDNA content, none of these treatments should be associated to potential mitochondrial detrimental side effect that may lead to adverse clinical manifestation . FinallyOur data are in accordance with registries of adverse-drug reactions in HIV-infected patients suggesting null or minimal toxicity for T20 or RAL . Moreovea priori in selected patients with extensive viral resistance who are unable or unwilling to inject T20 subcutaneously [Importantly, some studies have shown that continuous intravenous administration of T20 may be considered aneously . Other saneously , suggestOur studies had limitations. First, previous studies have shown the safety profile of both T20 and RAL in HAART experienced patients –50 whichIn summary, the administration of short-term therapy with T20 or RAL in healthy volunteers was not presumably associated with either: (i) significant changes in metabolic parameters; (ii) decrease of mtDNA levels suggestive of mitochondrial toxicity; (iii) apparent changes in renal or hepatic toxicity markers and; (iv) significant clinical safety issues other than local injection site reactions in the case of T20 administration.Further studies with bigger sample size and longer antiretroviral exposure should be conducted to confirm herein reported conclusions and elucidate toxic effects of antiretrovirals without HIV or previous ARV interference. Finally, in clinical settings, the assessment of drug toxicity in the context of concomitant HAART medication and HIV infection should be tested, in parallel, to strengthen conclusions and safety for T20 and RAL administration and offer new therapies free of secondary effects for HIV management.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 FileFile containing SPSS output for descriptive data and Wilcoxon test for the differences between PRE values and the change.(PDF)Click here for additional data file.S5 FileFile containing SPSS output for descriptive data and Wilcoxon test for the differences between PRE values and the change.(PDF)Click here for additional data file.


The biomarkers were measured in rectal biopsies at baseline and after one year of follow up, using automated immunohistochemistry and quantitative image analysis. In the vitamin D plus calcium group relative to control, in the crypt differentiation zone (upper 40% of crypts), mib-1 expression decreased 24% (P = 0.28); p21 expression alone and relative to mib-1 expression increased 29% (P = 0.06) and 73% (P = 0.06), respectively; and bax expression relative to mib-1 expression increased 58% (P = 0.21). The estimated vitamin D alone treatment effects were similar but of lesser magnitudes, and those for calcium alone were mixed. All estimated treatment effects on bcl-2 expression were close to the null. These pilot study results support further investigation of whether 1) vitamin D and calcium promote colorectal epithelial cell differentiation, reduce proliferation, and promote apoptosis in the normal-appearing human colorectal mucosa, 2) vitamin D and calcium act as chemopreventive agents against colorectal neoplasms, and 3) mib-1, p21, and bax are potential “treatable”, pre-neoplastic, biomarkers of risk for colorectal neoplasms.To clarify the roles of vitamin D and calcium as potential chemopreventive agents against colorectal cancer in humans, and to develop “treatable”, pre-neoplastic, phenotypic biomarkers of risk for colorectal neoplasms, we estimated the effects of supplemental vitamin D Colorectal cancer remains the second leading cause of cancer death in the United States, and mortality has only modestly declined in recent years despite advances in screening, treatment, and prevention . FurtherThe efficacy of calcium and vitamin D as chemopreventive agents against colorectal cancer is supported by strong biological plausibility and animal experimental and human evidence –5. Propo3 (800 IU/day [20 μg/day]) over six months increased the expression of p21 (biomarker of differentiation) D) concentrations have been consistently associated with lower risk for colorectal neoplasms –5, and ctiation)  and bax tiation)  in the ntiation) . Inconsicurrence . Howevereoplasms . All of 3, alone and in combination, over one year on markers of proliferation, differentiation, and apoptosis in the normal colorectal mucosa.To help clarify these issues, as reported herein, in the same subset (n = 104) of participants in the recent, third adenoma recurrence trial noted above , we asse3, alone and in combination, over 3–5 years on colorectal adenoma recurrence (NCT00153816) ) for mib-1, p21, and bcl-2, and a polymer system (Envision Plus Rabbit System [DAKO K4003]) for bax.WAF1/Cip1 antibody; clone ID—SX118; clonality—monoclonal; target antigen—CDKN1A human; vendor/catalog number—DAKO M7202; and host organism—mouse. For bcl-2: antibody ID—AB_626733; antibody name—Bcl-2 (100) antibody; clone ID– 100; clonality—monoclonal; target antigen—human bcl-2; vendor/catalog number—Santa Cruz Biotechnology sc-509; and host organism—mouse. For bax: antibody name—rabbit anti-human bax antibody; clonality—polyclonal; target antigen—synthetic peptide corresponding to amino acids 43–61 of the human bax protein; vendor/catalog number—DAKO A3533; and host organism—rabbit. The antibodies were used in the following concentrations: mib-1–1:350; p21–1:40; bax– 1:200; and bcl-2–1:100).Commercially-available antibodies were selected based on previous literature and comparing the staining patterns found in positive and negative control tissues and in normal colorectal mucosa with those reported in the literature in which antibody specificity was validated. Information on the antibodies is as follows. For mib-1: antibody ID—AB_2142367; antibody name—Ki-67 antibody; clone ID—mib-1; clonality—monoclonal; target antigen—human recombinant peptide corresponding to a 1002 bp Ki-67 cDNA fragment; vendor/catalog number—DAKO M7240; and host organism—mouse. For p21: antibody ID—AB_2077700; antibody name—p21The baseline and follow-up biopsy slides from each given participant were included in the same staining batch, and each batch included slide sets from a balance of participants from each treatment group, along with positive and negative control slides. A Leica CV5000 Coverslipper  was used to coverslip the slides, which were not counterstained.A quantitative image analysis procedure (“scoring”) was used to describe and measure the detected biomarkers in colon crypts . The uniThe major equipment and software used for the image analysis procedures were: Scanscope CS Digital scanner , computer, digital drawing board, MatLab Software , CellularEyes Image Analysis Suite , and MySQL . Throughout the scoring procedures, standardized settings were used for all equipment, and negative and positive control slides were checked for staining adequacy before analysis.First, digital images of the slides were acquired using the Aperio Scanscope CS digital scanner, and then the CellularEyes program was used to review the electronic images and identify colon crypts acceptable for analysis. The technician was blinded to treatment assignments and used a strict protocol to select “scorable” hemicrypts for analysis as previously reported . The borThe treatment groups were assessed for comparability of participant characteristics at baseline using the Fisher’s exact test for categorical variables and ANOVA or the Student t-test for continuous variables.h) –[reference group follow-up—reference group baseline]). Since the biomarker measurements were in optical density, to provide perspective on the magnitudes of the estimated treatment effects, relative effects ([treatment group follow-up / treatment group baseline] / [reference group follow-up / reference group baseline]) were taken from the model. Relative effects are interpreted similar to odds ratios .Participants were retained in their originally assigned treatment group, regardless of adherence to study treatment and procedures. All statistical analyses were conducted using SAS 9.4 statistical software and SAS University Edition statistical software . A two-sided P-value <0.05 was considered statistically significant.3). There were differences across the treatment groups in several participant characteristics as described three paragraphs above.Selected baseline characteristics of the study population are presented in 3) among participants assigned to the vitamin D treatment groups, and decreased by 1.69 ± SD 9.45 ng/mL (4.22 ± SD 23.62 mmol/L or 0.0000169 ± SD 0.0000945 kg/m3) among those who received vitamin D placebo.Among the adjunct biomarker study participants, 76% reported taking ≥80% of their assigned study tablets during the one-year treatment period. The mean serum 25(OH)D increased from baseline to 1-year follow-up by 9.17 ± SD 9.68 ng/mL  the distribution of proliferating cells in colorectal crypts, and increase colorectal epithelial cell differentiation and apoptosis, especially in relation to proliferation. Our findings also suggest that, consistent with our hypotheses and previous findings, calcium may increase differentiation; however, in contrast with our hypotheses and previous findings, our findings for calcium were consistent with it modestly increasing proliferation and decreasing apoptosis.Our findings in this pilot, adjunct biomarker study suggest that, in sporadic colorectal adenoma patients, daily vitamin D3 1,000 IU/day (25 μg/day) over 3–5 years on colorectal adenoma recurrence [As noted in the Introduction and reviewed extensively elsewhere, the potential efficacy of vitamin D and calcium as chemopreventive agents against colorectal cancer is supported by strong biological plausibility and animal experimental and human evidence –5. Calcicurrence .in vitro, calcium reduces cell proliferation and increases cell differentiation; these effects may be mediated via calcium binding with the calcium sensing receptor, which downregulates pro-proliferative β-catenin and upregulates the cell adhesion molecule E-cadherin [Calcium and vitamin D are thought to protect against colorectal carcinogenesis via multiple mechanisms. If the amount of calcium consumed exceeds that which binds with phosphate in the gut lumen and is absorbed for maintaining calcium homeostasis, the remaining free calcium can bind the bile acids and fatty acids resulting from fat digestion, thus protecting colonocytes from oxidative damage to DNA  and to ccadherin , 7, 8. Vcadherin , 7. Vitacadherin , 7. Thus0 phase, cells [waf1/cip1, a cyclin-dependent kinase inhibitor, has important roles in cell cycle regulation, and p21 expression downregulation is a common occurrence in colorectal cancer [in vitro [The cell cycle biomarkers used in our study are well recognized in the literature. Ki-67 (reliably detected via an antibody to its mib-1 epitope), is a non-histone protein located in the nucleus of proliferating, but not quiescent Ge, cells . p21waf1l cancer . Bax prol cancer , while bl cancer . The baxl cancer . Evidencin vitro  and animin vitro , 25 studin vitro , 27.The effects of vitamin D and calcium, separately and combined, on cell cycle markers in the crypts of the normal-appearing rectal mucosa of sporadic colorectal adenoma patients were estimated in a previous pilot, 2x2 factorial design chemoprevention trial (n = 92)  , 12, andh of crypts, a finding that was statistically significant for the 2.0 g calcium group  in the CCECP trial.Across the three trials, the estimated treatment effects of calcium, vitamin D, and calcium plus vitamin D on markers of proliferation in the whole crypt, were, for the most part, variable and close to the null, and none was statistically significant; however, in all treatment groups  there was an estimated decrease in the ϕh of crypts is not in line with those in the previous trials may be attributable to chance and other limitations of the present study as discussed further below. The results of the two factorial design trials also do not support a role for vitamin D in reducing the overall proliferation rate, but are consistent with vitamin D, alone or combined with calcium, in normalizing the proliferation zone.Thus, none of the three trials supports that calcium substantially affects the overall proliferation rate, but the previous trials—especially the full-scale trial—support that calcium helps maintain the normal distribution of the proliferation zone. That the finding in the present study for the effect of calcium alone on the ϕIn the present study and the CaD v MAP trial, p21 expression was estimated to increase in every treatment group, and the findings for calcium and vitamin D alone were statistically significant in the latter trial. In the present trial, the findings for the p21/mib-1 ratio were not substantially different from those for p21 alone.Thus, the findings from the present and CaD v MAP trials support that calcium and vitamin D alone or combined may promote colorectal crypt epithelial cell differentiation. Further investigation is needed into the p21/mib-1 ratio as an indicator of differentiation relative to proliferation in colorectal crypts.In the CaD v MAP trial, there were estimated substantial increases in bax expression in all treatment groups, especially the vitamin D alone group, for which the finding was statistically significant, whereas in the present study, bax was estimated to increase only in the calcium + vitamin D group. In the present trial, the findings for the bax/mib-1 ratio were not substantially different from those for bax alone. Across the two pilot factorial design trials, the estimated effects of all active treatments on bcl-2 in the whole crypt, were, for the most part, variable and close to the null, and none was statistically significant; however, in the CaD v MAP trial, there were estimated substantial increases in the bax/bcl-2 ratio in all three active treatment groups—especially in the calcium + vitamin D group (an estimated 71% increase)—whereas in the present study, the findings were closer to the null, except for an estimated 15% increase in the calcium + vitamin D group.Thus, the findings from the present and CaD v MAP trials suggest that, at least calcium and vitamin D combined may promote apoptosis in crypts in the normal-appearing colorectal mucosa of sporadic adenoma patients, but the two studies differed in the estimated effects of calcium and vitamin D alone, with the CaD v MAP trial suggesting substantial apoptosis-promoting effects, and the present trial suggesting essentially no effects. Possible reasons for the discrepant findings are discussed below. The results from the two studies do not support substantial calcium and/or vitamin D effects on bcl-2 expression, nor advantages of the bax/bcl-2 ratio over bax expression alone when using our measurement methodology. Further investigation of the effects of calcium and/or vitamin D on bax expression or on other biomarkers of apoptosis is needed.As described above, the findings across the present and previous trials are mostly concordant, but there are some differences for which there are several possible reasons. The present and CaD v MAP studies had relatively small sample sizes, suggesting that chance may have played a role. The present study’s calcium dose was smaller than the CaD v MAP trial’s dose, suggesting that a larger dose may be more efficacious. Also, the present study had some unique limitations. Participants in the present study were recruited from the larger adenoma recurrence trial without knowledge of their treatment group assignments. Consequently, the sample sizes for the treatment groups were unequal, and the small sample sizes for some of the treatment groups necessitated combining groups for analysis, making for a less “clean” analysis. Also, the small double-placebo group (n = 12), perhaps due to chance, was particularly influential in our results, especially those for the calcium only group and for bax, both of which were the most discordant from our previous findings. Second, the treatment groups were not balanced on various participant characteristics, which, even though adjustment for them in our analyses did not materially affect our results, leaves the potential for treatment effect confounding by unmeasured factors. It is also possible that study population differences affected treatment responses. Support for this is that calcium reduced adenoma recurrence in two previous, large trials, but not in the trial in which the present study was nested.3, alone or combined with calcium, modified APC, β-catenin, and E-cadherin  expression in directions hypothesized to reduce risk for colorectal neoplasms [Although the parent trial in which the present study was nested found no overall effects on adenoma recurrence, the present study’s findings are relevant. As noted above, the biological plausibility for calcium and vitamin D, the animal experimental evidence, the extensive human observational literature, and the two previous adenoma recurrence trials support that calcium and vitamin D may reduce risk for colorectal neoplasms , 28, 29.eoplasms . CollectAn issue with adenoma recurrence trials is that it is commonly thought that it takes many years to develop a macroscopic adenoma from a completely normal mucosa, and a substantial proportion of persons diagnosed with adenomas develop subsequent adenomas . This indicates that a substantial proportion of persons who have adenomas removed may have fairly committed clones of cells in the “pipeline” to become macroscopic adenomas in the ensuing few years. So, if a preventive agent is primarily effective in preventing the very earliest stages of colon carcinogenesis, an effect on preventing recurrent adenomas may not be detectable until enough years  elapse for the committed clones to have progressed to a detectable stage. The pattern of 3- vs. 5-year findings in the parent trial, and the long-term follow up of participants in the previous calcium and adenoma recurrence trial  also are consistent with this .While the present study had the above-noted limitations, it also had several strengths, including the double-blind, placebo-controlled design, high protocol adherence by study participants, automated immunohistochemistry, novel quantitative image analysis software, and high level of biomarker measurement reliability. Also, to our knowledge, this was the first human study to investigate the effects of vitamin D and calcium supplementation on differentiation and apoptosis relative to proliferation in the normal-appearing human colorectal mucosa.In summary, the results from this pilot clinical trial, taken together with previous literature, suggest that 1) calcium and vitamin D, alone or together, while they may not affect the overall proliferation rate, may normalize the distribution of the crypt proliferation zone and increase differentiation; and 2) that vitamin D combined with calcium may increase apoptosis in the normal human colorectal epithelium. Also, the collective findings do not support substantial calcium and/or vitamin D effects on bcl-2 expression, nor advantages of the bax/bcl-2 ratio over bax expression alone when using our measurement methodology. The collective findings also indicate that further investigation of the following is needed: the p21/mib-1 ratio as an indicator of differentiation relative proliferation in colorectal crypts, and the effects of calcium and/or vitamin D on the expression of bax or other biomarkers of apoptosis. Finally, based on our findings, literature review, and discussion, calcium and vitamin D remain viable chemopreventive agents against colorectal neoplasms, and mib-1, p21, and bax expression in the normal-appearing colorectal mucosa remain viable, treatable pre-neoplastic biomarkers of risk for colorectal neoplasms.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


CMT1A patients showed significantly decreased temporal and spectral resolution (both p < 0.05). Also, auditory temporal processing of CMT1A patients was correlated with speech perception in a noisy background  and median motor conduction velocity . Therefore, we assumed that demyelination of auditory nerve in CMT1A causes defective cochlear neurotransmission, which reduces temporal resolution and speech perception in a noisy background. Because the temporal resolution test was well correlated with the degree of demyelination in auditory and peripheral motor nerves, temporal resolution testing could be performed as an additional marker for CMT1A.The aim of this study was to investigate hidden hearing loss in patients with Charcot-Marie-Tooth disease type 1 A (CMT1A), a common inherited demyelinating neuropathy. By using pure-tone audiometry, 43 patients with CMT1A and 60 healthy controls with normal sound detection abilities were enrolled. Speech perception in quiet and noisy backgrounds, spectral ripple discrimination (SRD), and temporal modulation detection (TMD) were measured. Although CMT1A patients and healthy controls had similar pure-tone thresholds and speech perception scores in a quiet background, CMT1A patients had significantly ( CMT type 1 A (CMT1A) is the predominant subtype, accounting for an estimated 50% of CMT cases. CMT1A is a demyelinating peripheral neuropathy characterized by distal muscle weakness, sensory loss, areflexia, and slow motor and sensory nerve conduction velocities4. CMT1A is associated with chromosomal duplication of 17p12 including peripheral myelin protein 22 (PMP22) gene2.Charcot-Marie-Tooth (CMT) disease, the most common hereditary peripheral neuropathy, affects 1 in 2500 people3. Hearing is usually assessed in the clinic by using pure-tone audiometry which measures the smallest detectable levels of pure tones from 0.125 to 8 kHz. However, audiograms do not predict selective neural loss which does not affect sensitivity to weak sounds5. Thus, CMT1A patients could have normal audiograms even though they have difficulty hearing in their daily lives.Because the cochlear nerve is a peripheral nerve, CMT patients can exhibit sensory peripheral nerve deficit. Hearing impairment is a relatively common symptom of CMT1A7. The relationship between auditory deficit and disease progression of CMT1A remains elusive. The objective of this study was to evaluate hidden hearing loss and determine diagnostic values of new auditory tests by comparing auditory measures and disease progression in CMT1A patients.Hidden hearing loss is recently described as auditory neuropathy believed to contribute to speech discrimination and intelligibility deficits in people with normal audiogramsp > 0.05, two-tailed t-test). Percentage of females did not differ significantly between the CMT1A group (53.5%) and the control group (51.7%) either . The side of tested ears did not differ significantly between the two groups either . Clinical information on CMT1A patients is shown in Table 8. Also, no CMT1A patients showed absent distortion product otoacoustic emission (DPOAE). Motor nerve conduction velocities (MNCV) of the median nerve in all CMT1A patients were decreased (ranging from 12 to 38 m/s) compared to normative data 9. However, compound muscle action potential (CMAP) of the median nerve was decreased only in 7 CMT1A patients compared to normative data .Ages did not differ significantly between the CMT1A group  and the control group  . Thus, CMT1A patients had more difficulty understanding speech in a noisy background compared to controls. Spectral and temporal resolutions for CMT1A patients were also significantly worse those than for healthy controls . Mean TMD threshold was −14.73 ± 5.43 dB for the CMT1A group and −17.53 ± 2.78 dB for the control group .Auditory outcomes of tested ears and subjective hearing disabilities measured with APHAB and K-HHIE questionnaires for CMT1A patients and healthy controls are shown in Table ls Table . The mear = 0.411, p < 0.05) and noisy backgrounds . However, ABR inter-peak latency, ABR amplitude, or spectral resolution did not correlate with any other auditory measures. Thus, reduced temporal resolution could lead to impaired speech perceptions for CMT1A patients with normal hearing.We conducted correlation analysis to investigate relationships among auditory neural conduction time (ABR wave I-V latency), ABR amplitude (ABR V/I ratio), spectral resolution (SRD), temporal resolution (TMD), and speech perception in quiet and noisy backgrounds (HINT). Findings for CMT1A patients with normal hearing are shown in Table r = −0.329, p < 0.05), disease duration , and median nerve conduction velocity . ABRs, spectral resolution, or speech perception did not correlate with any factors related to disease progression. This result indicated that temporal resolution was significantly decreased if CMT1A was developed earlier, the duration of CMT1A was longer, or the conduction velocity of median motor nerve was delayed.Results of correlation analysis between auditory measures  and disease progression  of CMT1A patients are shown in Table r = 0.447, p < 0.01). Speech comprises dynamic spectral and temporal modulations that can change depending on utterance. Spectral and temporal modulation sensitivities are important factors affecting speech perception11. In our study, psychoacoustic tests revealed that CMT1A patients could not distinguish subtle differences in spectral shape  or rapid fluctuations in the envelope  compared to controls , disease duration , and median MNCV . Therefore, hearing measurements can be used to track neural damage as the disease progresses. CMTNS has been used as a major outcome measure for CMT1A. However, CMTNS has limited sensitivity in CMT1A patients with mild clinical symptoms19. CMT1A is a slowly progressive neuropathy. Thus, detecting the disease progression and evaluating the efficacy of interventions are difficult20. Because temporal resolution test (TMD test) was associated with hidden hearing loss (reduced speech perception in a noisy background) and neural deficit (delayed median MNCV), temporal resolution test can be used to detect disease progression in CMT1A patients with mild clinical symptoms.The sensitivity of the auditory system to neural damage in patients with CMT1A indicates that hearing measurements can be used as clinical indicators of disease progression. Because both peripheral and central auditory physiology could change with age, we conducted a correlation analysis adjusted for age when evaluating the relationship between auditory measures and disease progression in patients with CMT1A Table . Our res3, this study has several strengths. Our study had large number of participants with normal sound detection across all ages. Detailed psychoacoustic tests (SRD and TMD) were conducted to evaluate auditory processing associated with auditory neuropathy. We also analyzed auditory processing after controlling for age in participants with the same hearing configuration.Although auditory function in children with CMT1A has been reportedResults of this study showed that CMT1A patients had hidden hearing loss compared to healthy controls, although all participants had normal audiogram. Their difficulty understanding speech in a noisy background might be due to impaired ability to handle temporal envelope clues. We assumed that demyelination of auditory nerve cause defective cochlear neurotransmission and this cochlear neuropathy could be consider as different type of hidden hearing loss. Because auditory temporal envelope processing was related to median MNCV, temporal processing ability might be a suitable marker of disease progression. Therefore, temporal resolution testing could be performed in the clinical setting to detect hidden hearing loss and monitor the degree of neural damage. Future longitudinal studies are needed in this direction to assess the role of auditory measures as biomarker in CMT1A patients with mild to severe symptoms.All participants were recruited and tested in the Hearing Laboratory at Samsung Medical Center. Every participant provided written informed consent to participate in this study. The study protocol was approved by Samsung Medical Center Institutional Review Board (IRB No. 2015-04-075). This study was carried out in accordance with approved guidelines.21. Age at onset was determined by asking patients their age when symptoms first appeared. To determine physical disability, two scales were used: CMT neuropathy score (CMTNS)22 and functional disability scale (FDS)23.Forty-three CMT1A patients  and 60 healthy controls  were enrolled. On pure-tone audiometry, all study populations had an average hearing threshold of less than 25 dB at 0.5, 1, 2, and 4 kHz in both ears. Pure-tone thresholds of both groups are shown in Fig. For neurophysiologic study, auditory brainstem responses (ABRs), distortion product otoacoustic emission (DPOAE), and nerve conduction of median nerves were recorded in CMT1A patients. The equipment used to record brainstem auditory evoked potentials was Nicolet Viking Select . Click stimuli at 80 dB nHL were transmitted via inserted earphone. Absolute latency (stimulus to peak) of each peak  and inter-peak latencies  were measured. DPOAE was recorded using an ILOV6 OAE analyzer . Primary signals f1 and f2, with f2/f1 = 1.22, generated with test frequencies ranging from 1001 Hz to 6006 Hz with a frequency resolution of one DPOAE per octave was used. Two levels were chosen: L1 = 65 dB SPL, L2 = 55 dB SPL. Response parameters to consider DPOAE as present included DP amplitude and SNR. A peak at 2f1-f2 in the spectrum was accepted as a DPOAE if it was 3 dB above the noise floor. Nerve conduction studies were performed by placing surface electrodes on median nerves. Motor nerve conduction velocities (MNCVs) for median nerves were determined by stimulating at the elbow and wrist while recording compound muscle action potentials (CMAPs) over the abductor pollicis brevis muscle.24 using two different audiometers . Word recognitions were measured at the most comfortable level. Sentence recognition tests were performed in quiet and noisy backgrounds with a Korean version of Hearing in Noise Test (HINT) sentences25.Audiograms were used to assess pure tone threshold, speech perception threshold (SRT), and word recognition score (WRS). Pure-tone thresholds were obtained under headphones using pure-tones ranging from 250 Hz to 8 kHz. They were measured with ascending 5 dB method (modified Hughson-Westlake method)26 and hearing handicap inventory for the elderly (HHIE)27. Everyday listening and communication were investigated with the APHAB questionnaire. This 24-question metric explores four aspects of auditory function: communication difficulty, effect of background noise, effect of reverberation, and aversion to loud sounds. Effects of hearing impairment on emotional and social adjustment were investigated with the HHIE questionnaire, with higher scores for the questionnaire indicating greater discomfort related to hearing.Hearing disability was assessed with questionnaires such as the abbreviated profile of hearing aid benefit (APHAB)Psychoacoustic tests consisted of spectral ripple discrimination (SRD) and temporal modulation detection (TMD) tests. A custom-made MATLAB®  graphical user interface was used to present acoustic stimuli to subjects for SRD and TMD tests. The stimuli were presented monoaurally through an insert earphone  at an average of 65 dBA.30. Three rippled noise tokens with a 30-dB peak-to-trough ratio, two with a standard ripple phase, and one with an inverted ripple phase were created from 2,555 tones. The spectral modulation starting phase of the full-wave-rectified sinusoidal spectral envelope was set to zero radian for standard ripple stimuli. The spectral modulation starting phase of the inverted spectral-ripple stimulus was set to π/2 radians. The bandwidth of the rippled spectrum was 100–5000 Hz. The stimulus duration was 500 ms. The order of presentation of the three tokens was randomized and the subject’s task was to select the “oddball” stimulus. To measure SRD thresholds, a three-interval, three-alternative forced choice (3-AFC) paradigm with an adaptive two-up, one-down procedure was used. Ripple density varied between 0.125 and 11.314 ripples per octave (rpo) in equal ratio steps of 1.414 in an adaptive manner with 13 reversals that converged to the 70.7% correct point31. A level attenuation of 1–8 dB (in 1 dB increments) was randomly selected for each interval in the three-interval task. The SRD threshold for each adaptive run was calculated as the mean of the last eight reversals. The SRD threshold for each subject was the mean of three adaptive runs. Higher spectral-ripple thresholds indicated better discrimination performance.SRD thresholds were determined with stimuli based on a previously established technique32. The stimulus duration was one second for both modulated and unmodulated signals. For modulated stimuli, sinusoidal amplitude modulation was applied to a wideband noise carrier. For unmodulated stimuli, continuous wide band noise was applied. A modulation frequency of 10 Hz was tested. Modulated and unmodulated signals were gated on and off with 10-ms linear ramps. They were concatenated without gap between signals. The TMD threshold was measured with a two-interval, two-alternative adaptive forced choice  paradigm. One interval consisted of modulated noise while the other consisted of unmodulated white noise. Subjects were asked to identify the interval that contained the modulated noise. A two-down, one-up adaptive procedure was used to measure TMD threshold, starting with a modulation depth of 100% and decreasing in 4-dB steps from the first four reversals and 2-dB steps for the next 10 reversals. For each testing run, the final 10 reversals were averaged to obtain the TMD threshold. TMD thresholds in decibels relative to 100% modulation . Independent t-tests were performed to assess significant differences in auditory tests between CMT1A patients and control participants. Binary logistic regression analyses were performed to assess related auditory processing in CMT1A patients compared to controls. Each auditory measure was adjusted for age and sex. Correlation coefficient was calculated to investigate whether auditory measures were correlated among themselves or with age. Correlation coefficients between auditory and non-auditory measures were calculated after controlling for age.


There were no significant differences between predicted and measured RMR using the new equations for females and males. Of the pre-existing equations, Schofield’s is the most suitable for Chinese mainland adults. However, the two new equations developed in this study seem to be more effective for predicting the RMR of Chinese mainland adults, and need to be validated by a larger independent sample with different physiological and anthropometric characteristics.Accurate measurement of the resting metabolic rate (RMR) is necessary when we make energy requirements and nutrition suggestions in clinical. However, indirect calorimetry is not always available. The objectives of this study were to make a comparison between RMR measured by indirect calorimetry and RMR predicted by different kinds of equations, and to develop new predictive equations for Chinese mainland adults. In this study, 315 Chinese mainland adults from different provinces all over China were recruited. Subjects underwent half a day of testing, which consisted of anthropometric assessment and RMR measurement. Measured and predicted RMR were compared; new optimal equations for Chinese mainland adults were developed and tested by splitting the subjects into a development and validation group. The measured RMR was in the range of 831–2776 kcal/day . Our findings indicated that, except for the Harris–Benedict and Schofield equations, three Chinese equations and two fat-free mass (FFM) modeling equations all significantly underestimated RMR compared to the measured value (all  Resting metabolic rate (RMR) is typically defined as the energy required when we are in a resting condition , which iIndirect calorimetry, which is considered to be the clinical gold-standard measurement of RMR, is a metabolic measurement system that tests oxygen consumption and carbon dioxide production to calculate energy expenditure . HoweverComparisons have been made between various equations and measured RMR in several studies. Most of these studies have shown that overestimations or underestimations are found when the equations are used in different populations . ConsequIn the United States, Australia, and several European countries, the Harris–Benedict equation  and the Therefore, the first objective of this study was to compare measured RMR with predicted RMR by using (a) internationally used equations, (b) Chinese-specific predictive equations, and (c) FFM modeling equations; the second goal was to develop new equations, if possible, body composition modeling equations were preferred from our data applicable for Chinese mainland adult populations without age limits and body-mass-index (BMI) limits.2; male: n = 127, female: n = 188), volunteered to take part in this study. Healthy adults (age range: 18–70 years) with no disease history were eligible for this study. Subjects who had diseases that affect body energy metabolism, such as asthma, chronic obstructive pulmonary disease, hyperthyroidism, diabetes mellitus, and hypertension were excluded from this study. Three hundred and fifteen healthy Chinese subjects from different provinces of mainland China, aged 18–67 years .All subjects were asked to take off shoes and heavy clothing before any measurement; we took all measurements between 08:00 and 10:00 to avoid diurnal variation differences. Before the resting-metabolic-rate measurements, all participants underwent anthropometric measurements and demographic data collection.The same trained tester performed all anthropometric measurements including body height, weight, and composition. A bioelectrical impedance analysis composition analyzer  was used to evaluate body composition, which was validated as having a high correlation with dual-energy X-ray absorptiometry (DXA) in estimating body-fat percentage in Chinese subjects . We reco2, 5% CO2) and dried atmospheric air . Measurement was carried out in a quiet room with dim lighting and controlled environment temperature (22–25 °C) and humidity (40–50%). Subjects were asked to lie in a supine position, and stay quiet and awake during the measurement. The volume of oxygen consumption and carbon dioxide production was measured for a period of 30 min, and then the RMR was calculated by using the VO2 and VCO2 of the steady periods for about 20 min according to the Weir formula [Resting metabolic rate was measured with a ventilated mask by one trained tester in the morning using indirect calorimetry . Before measurement, subjects were told to have 6–8 h sleep, to not undertake intense physical activity in the previous 24 h, and to fast overnight before arriving at the laboratory. Calibration of flow and gas analyzers was done before each measurement according to the manufacturer’s instructions. Flow calibration was performed, and gas analyzers were calibrated with a standard gas mixture  and Schofield (1985); the Chinese equations of Liu (1995), Yang (2010) and the Singapore equation (2016); and the FFM modeling equations of Cunningham (1980)  and Wangn = 95, female: n = 140, age: 20–67 years, BMI: 16.7–38.1 kg/m2) and a 25% validation subsample .Finally, Present 1 and Present 2 were developed for Chinese mainland adults . To testr) was used to examine the relationship between predicted RMR and measured RMR. Bland–Altman plots [p < 0.05 was defined as statistically significant.All results are shown as mean ± standard deviation. The SPSS statistical package  was used to analyze the data. Comparisons of differences at the group level were done between the calculated RMR from the equations and the measured RMR via indirect calorimetry by using paired t-tests. The average percentage bias between predicted and measured RMR was used to estimate accuracy at the group level. The Pearson correlation coefficient (an plots  were crean plots . The new2 (16.7–38.2 kg/m2).Comparisons of the predicted RMR from all equations with the measured RMR are presented in r = 0.244, p < 0.01), Schofield equation , Liu equation , Yang equation  and the Singapore equation ; the Cunningham equation , and Wang equation ; and Present equation 1 ; Present equation 2   .The mean difference, limits of agreement, and 95% confidence interval for the bias between measured RMR and predicted RMR are presented in Hundreds of studies suggest that one equation cannot be accurately used for subjects whose age, sex, race, and physical status is different to those used to formulate the original equation . The purThe data suggested that most equations, except for the Schofield equation, could not accurately predict RMR in this study group. Previous published studies have indicated that, whatever the race of the subjects, most equations overestimate the RMR of both Caucasian and Asian populations ,27,28. HOur data showed that the Harris–Benedict equation significantly overestimated the RMR of females at the group level, but significantly underestimated the RMR of males at the group level; accuracy at the individual level was low 37.1%) in total. The Harris–Benedict equations were developed from Caucasian subjects only, so overestimation and underestimation is to be expected .1% in to. The SchIt has been reported in previous studies that the RMR value was mainly influenced by fat-free mass ,23,30,31Hence, the current study developed two kinds of Chinese-specific equations, one of which was based on easily accessible variables and one based on body-composition variables. We tested the internal validity of the newly derived equations using validation groups, and found that our newly derived equations showed no significant differences with measured RMR. Additionally, they displayed the smallest mean differences and bias at the group level and showed a high accuracy rate at the individual level compared to the other equations.We found that the Harris–Benedict and the two FFM modeling equations may not be appropriate for predicting the RMR in Chinese mainland adults. The equations of Liu, Yang, and Singapore significantly underestimated the RMR of subjects in this study. Of all the pre-existing equations we tested, Schofield’s equation proved to be the most appropriate for predicting the RMR of Chinese mainland adults; however, new equations developed in this study seem to perform better than any of the pre-existing equations. Taken together, we conclude that we can use the new RMR equations in cases where RMR cannot be measured by direct methods. In subsequent studies, the equations developed in this study should be tested and validated with a larger independent sample with different physiological and anthropometric characteristics.


Escherichia coli (APEC) are economically devastating to poultry industries worldwide and are also potentially threatening to human health. Pathogens must be able to precisely modulate gene expression to facilitate their survival and the successful infection. The Cpx two-component signal transduction system (TCS) regulates surface structure assembly and virulence factors implicated in Gram-negative bacterial pathogenesis. However, the roles of the Cpx TCS in bacterial fitness and pathogenesis during APEC infection are not completely understood. Here, we show that the Cpx TCS response regulator CpxR is critical to the survival and virulence of APEC. Inactivation of cpxR leads to significant defects in the interbacterial competition activity, invasion and survival of APEC in vitro and in vivo. Moreover, activation of CpxR positive regulates the expression of the APEC type VI secretion system 2 (T6SS2). Further investigations revealed that phosphorylated CpxR directly bound to the T6SS2 hcp2B promoter region. Taken together, our results demonstrated that CpxR contributes to the pathogensis of APEC at least through directly regulating the expression and function of T6SS2. This study broadens understanding of the regulatory effect of Cpx TCS, thus elucidating the mechanisms through which Cpx TCS involved in bacterial virulence.Systemic infections caused by avian pathogenic The online version of this article (10.1186/s13567-019-0658-7) contains supplementary material, which is available to authorized users. Escherichia coli (APEC) are economically devastating to poultry industries worldwide. APEC shares a broad range of virulence factors with human extraintestinal pathogenic E. coli (ExPEC), including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Moreover, these ExPEC strains can cause cross infections, thus indicating that APEC may be a potential virulence gene reservoir for UPEC and NMEC  reported in E. coli [hcp2B gene (Figure hcp2B operon. Furthermore, we demonstrated that the lacZ transcriptional reporter fusion Phcp2B-lacZ under the control of the hcp2B promoter showed β-galactosidase activity (Additional file hcp2B promoter sequence including the potential CpxR binding site was amplified, biotin-labeled and subjected to EMSA analysis, to verify the direct binding of CpxR-P to the hcp2B promoter region. Indeed, the migration of the hcp2B probe was slowed in the presence of increasing amounts of CpxR-P. In addition, binding specificity was confirmed via a competitive EMSA, and the binding of CpxR-P to biotin-labeled probes was abolished by an excess of specific competitor consisting of unlabeled hcp2B promoter fragments. In addition, the hcp2B promoter fragments without the CpxR binding site were no longer shifted by the CpxR-P protein (Figure hcp2B operon in APEC.To investigate whether CpxR directly regulates  E. coli , 57. OneBacterial virulence is determined by the expression of virulence factors, and pathogens must be able to precisely modulate gene expression to facilitate their adaptation and survival in the local microenvironment or host cells. The infection process requires rapid adaptation to the host environment through various regulatory mechanisms. TCSs are activated in response to changing environmental cues, thus representing one mechanism enabling bacterial adaptation through transcriptional regulation of gene expression , 19. ThecpxR gene attenuates the virulence of APEC. Moreover, the complemented strain showed recovered virulence. Thus, we concluded that CpxR is necessary for full virulence of APEC. APEC initially infects poultry via the respiratory tract, then spreads systemically throughout the entire body, thus resulting in subsequent bacteremia and death. Colonization and invasion are important virulence parameters in APEC infection [fimA promoter [fimA promoter [cpx mutant strain in different Gram-negative pathogens [By using a duck systemic infection model, we demonstrated that inactivation of the nfection , 59. A ppromoter . Indeed,promoter , 60. Ourpromoter . In contathogens , 61–63.Xenorhabdus nematophila during infection through regulation of the nil locus [Pathogens have developed diverse attack strategies to efficiently compete with other bacteria for limited resources and facilitate survival and infection . Our resil locus . In addiil locus .E. coli CpxR regulon contains hundreds of genes, according to genome-wide screens for the putative CpxR binding sequence [Citrobacter rodentium [hcp2B operon. Although the fold-change in the Hcp2B protein was less than the changes at the transcription level, it was also decreased in the mutant strain ΔcpxR. Previous study showed that the hcp2B operon is necessary for the interbacterial competition activity of T6SS2 in APEC [Citrobacter rodentium [hcp2B operon, including the interbacterial effector encoding the gene xmtU, might be responsible for the decreased invasion, diminished survival and impaired competition of the mutant strain ΔcpxR. Many environmental conditions upregulate the Cpx pathway. The lipoprotein NlpE has been shown to activate the Cpx pathway when it is overproduced [hcp2B operon expression and subsequently significantly enhanced interbacterial competition. In contrast, no regulatory effect was found for the mutant strain ΔcpxR overexpressing NlpE. An in silico analysis identified a putative CpxR binding site in the hcp2B promoter region. Furthermore, we provided evidence that CpxR-P binds the promoter region of the hcp2B operon, thus suggesting that CpxR directly regulates T6SS2 hcp2B expression. Taken together, these results indicated that the regulation of T6SS2 expression and function by CpxR may contribute to the infection and pathogenesis of APEC.The sequence , 57. In odentium . T6SSs aodentium , 16. Theodentium –16, 55.  in APEC . Moreove in APEC , possiblodentium . Additioproduced , 65, 66.hcp2B (Figure In conclusion, we provided evidence that CpxR contributes to colonization, invasion and interbacterial competition fitness for APEC in vivo and in vitro. The most provocative finding of this study is that CpxR may contribute to APEC virulence by directly binding and regulating the expression and function of T6SS2 Additional file 1.The β-galactosidase activity of thelacZtranscriptional reporter fusion Phcp2B-lacZwas measured as described in “”.


Unilateral hearing loss causes difficulty in localization of sound and impairs the ability to hear in noisy settings such as a classroom. Additionally, this deficit has been associated with lower oral language scores when compared to normal-hearing counterparts.,,The global prevalence of hearing loss must be discussed to better understand the burden of this condition. Research has traditionally focused on characterizing the prevalence of disabling hearing loss (> 30 dB) – identifying 34 million children worldwide affected at this threshold or greater.Latest available global prevalence estimates of childhood hearing loss range show significant regional variability. Low- and middle-income countries in geographical areas, such as south Asia, are disparately affected: 82.5%  for mild hearing loss; 14.3% (95% CI: 7.4–29.7) for moderate hearing loss; 0.7% (95% CI: 0.3–1.4) for severe hearing loss; and 0.2% (95% CI: 0.1–0.4) for profound hearing loss, while high-income countries contribute only a small fraction of the global burden. In these countries, the prevalence for mild hearing loss is 17% (95% CI: 12.4–25); for moderate hearing loss is 2.7% (95% CI: 2.0–4.0); for severe hearing loss is 0.1% (95% CI: 0.1–0.2); and for profound hearing loss is 0.0% (95% CI: 0.0–0.1).While the current global state of paediatric hearing loss may appear dismal, promising data indicates that 60% of this hearing loss is preventable. These common preventable causes include infectious causes, birth complications, noise exposure and the use of ototoxic medications in pregnant women and children.To assess global progress towards achieving quality education for all, evidence of paediatric hearing loss being a barrier to this goal should be considered. SDG 4 designates universal education as one of our highest global priorities, specifically aiming to ensure equal access to all levels of education for the vulnerable, including persons with disabilities.Particularly within low- and middle-income countries, increased preventative efforts will be the most cost–effective and high-yield strategy to combat educationally disabling hearing loss. Reduction of paediatric hearing loss can be accomplished through expansion of basic low-cost health provisions, such as measles, mumps and rubella vaccines to prevent rubella-associated hearing loss. Another option is to provide easily accessible treatment for otitis media, to address chronic ear infections as a common etiology. Awareness raising will be the single most important aspect of addressing paediatric hearing loss on a global scale. Patients and health providers must be informed on topics such as the ototoxic effects of unregulated antibiotic use, safe and hygienic practices for labour and delivery, and potentially-dangerous exposures for mothers during pregnancy. However, education should also include expanding awareness of the widespread burden of paediatric hearing loss, highlighting common early presentations of this disease and emphasizing the potential for long-term effects if left untreated. Such awareness will be crucial in fighting the stigma associated to this disability, both encouraging schools to provide resources for inclusion of affected children, as well as enabling parents to recognize and seek treatment for children at an early age.Beyond prevention, newborn screening for early identification of hearing loss should be expanded and standardized. However, such efforts will require sufficient availability of otolaryngology and audiology resources to provide treatment following detection. This objective will require hearing health-care investment and expansion in low- and middle-income countries, where children currently have high rates of hearing loss, yet have little or no access to rehabilitation services. Although this objective would be a large-scale investment, the long-term outcomes of hearing loss detection and intervention programmes have been shown to be cost–effective because they mitigate educational deficits and lost productivity.


Mucormycosis is a rare opportunistic fungal infection with poor prognosis. The incidence of mucormycosis has been increasing, and it is a threat to immunocompromised hosts. We present a case of gastric mucormycosis complicated by a gastropleural fistula during immunosuppressive treatment for adult-onset Still disease (AOSD).An 82-year-old woman diagnosed with AOSD who developed gastric ulcers during the administration of an immunosuppressive therapy with corticosteroids, cyclosporine, and tocilizumab complained of melena and epigastralgia. Esophagogastroduodenoscopy showed multiple ulcers covered with grayish or greenish exudates.Mucor indicus was identified using polymerase chain reaction.The patient diagnosed with mucormycosis based on culture and biopsy of the ulcers, which showed nonseptate hyphae branching at wide angles. Although liposomal amphotericin B was administered, gastric mucormycosis was found to be complicated by a gastropleural fistula. The patient died because of pneumonia due to cytomegalovirus infection, and autopsy revealed the presence of Mucorales around the fistula connecting the stomach and diaphragm.Gastric mucormycosis is refractory to treatment and fatal. Surgical resection, if possible, along with antifungal drugs can result in better outcomes. Mucormycosis is diagnosed based on histologic findings or positive culture from affected lesions owing to the lack of validated serologic biomarkers. Management guidelines for mucormycosis recommend a combination treatment with antifungal drugs and surgical resection of the devitalized tissue. Herein, we describe a case of gastric mucormycosis complicated by a gastropleural fistula during immunosuppressive treatment for adult-onset Still disease (AOSD).Mucormycosis is a rare but fatal fungal infection caused by Mucorales with an acute course. Recently, as the incidence of mucormycosis has been increasing, it has been considered an important infectious disease observed among immunocompromised patients.2 and was prescribed oral prednisolone (50 mg/d) and oral cyclosporine (200 mg/d). Her symptoms and serum ferritin levels remarkably improved on day 10. On day 36, she developed high fever with elevated CRP and serum ferritin levels. Because AOSD relapse was suspected, she was intravenously administered 400 mg tocilizumab (8 mg/kg) after methylprednisolone pulse therapy at a dose of 1 g/d for 3 days. This regimen improved her symptoms and serum ferritin levels. However, during this therapy, she complained of epigastralgia and melena, and esophagogastroduodenoscopy (EGD) showed peptic ulcers at the pylorus. Therefore, she was prescribed vonoprazan instead of lansoprazole, resulting in immediate symptomatic relief. However, epigastralgia and melena relapsed on day 47. On day 51, a repeat EGD showed multiple ulcers covered with grayish or greenish exudates spreading from the upper body of the stomach toward the fundus  was administered at a dose of 200 mg/d (5 mg/kg/d). During the antifungal therapy, oral prednisolone was carefully tapered to 17.5 mg/d, but high serum ferritin levels were sustained between 1789 and 4010 ng/mL. On day 92, EGD revealed shrinking of the ulcers  level of 28.57 mg/dL, serum ferritin level of 9899 ng/mL, and elevated liver enzyme levels. Rheumatoid factor, anticitrullinated protein antibody, and antinucleolar antibody were tested to be negative, and whole body computed tomography (CT) revealed no abnormalities. Subsequently, she was diagnosed with AOSD based on Yamaguchi criteria3We describe a case of gastric mucormycosis secondary to immunosuppressive treatment for AOSD. Although the patient was treated with antifungal drugs, the gastric ulcer developed into a gastropleural fistula and the patient died due to pneumonia. Autopsy identified the cause of the fistula to be gastric perforation due to the spread of Mucorales infection from gastric ulcers. The risk factors for mucormycosis primarily include neutropenia, diabetes mellitus , malignancies, hematopoietic stem-cell transplantation, solitary organ transplantation (SOT), corticosteroid use, iron overload, deferoxamine use, and trauma. In addition, there are several case reports on mucormycosis in patients undergoing biologic therapies,,10 for example, one report describes the presence of pulmonary mucormycosis during treatment with tocilizumab. In the case described here, the immunosuppressive therapy administered with high-dose corticosteroids, cyclosporine A, and tocilizumab is a possible risk factor for mucormycosis.Mucormycosis is an acute and fatal fungal infection with a mortality rate of 54% in the world. with the stomach being the most frequently affected organ. We reviewed 20 reports on gastric mucormycosis (Table –30 We identified common symptoms to be melena, hematemesis, and abdominal distention or pain, and most patients had predisposing factors such as DM, SOT, or corticosteroid use. The common site of infection is the upper body of the stomach, and the ulcers have been characterized as having greenish, grayish, or black exudate or necrosis during EGD.Gastrointestinal manifestations of mucormycosis account for only 7% of all cases,is Table .[12–30]  Our literature review revealed that 7 of the 9 cases with vessel invasions as per histologic examination resulted in death, suggesting that vessel invasion is a poor prognostic factor. Almyroudis et al reported that a combination of surgery with AMB therapy yielded a better outcome than AMB administration alone in SOT recipients, with mortality rates being 34.3% and 62.5%, respectively. However, during literature review, we found that 5 of 10 cases treated with antifungal drugs and surgical intervention resulted in death. Thus, it is possible that gastric mucormycosis has poor prognosis even with the combined use of surgical treatment and antifungal drug therapy. Notably, we found only 1 case report that described a gastropleural fistula secondary to gastric mucormycosis. In this report, an HIV-positive male developed gastric mucormycosis after experiencing an abdominal trauma, and a postoperative gastropleural fistula developed due to gastric perforation. In our case, the ulcers initially seemed to have improved upon treatment with an antifungal drug, but the outcome remained unfavorable. Nonetheless, it is possible that a combination of surgical resection for gastric mucormycosis and antifungal therapy during the early stages of the disease improves outcomes and prognosis.Angioinvasion is a characteristic of mucormycosis progression, which probably contributes to its hematogenous dissemination to other organs. and 10 of the 20 reported cases on gastric mucormycosis involved peptic ulcers. Possibly, gastric bleeding from peptic ulcers provides iron to Mucorales for growth and a route for angioinvasion. Additionally, hyperferritinemia is a reported poor prognostic factor for mucormycosis, and inflammation due to infection increases storage iron levels through various cytokines and hepcidin. Thus, hyperferritinemia may reflect inflammation due to mucormycosis. Meanwhile, hyperferritinemia mirrors the disease activity of AOSD and is a risk factor for macrophage activation syndrome. In our case, hyperferritinemia was sustained during the clinical course, and it was difficult to distinguish between the disease activity of AOSD and inflammation due to mucormycosis. In such cases, we are of the opinion that the significance of serum ferritin levels should be carefully judged based on clinical course.Mucorales require host iron for hyphal growth during host cell invasion,In conclusion, we reported a case of gastric mucormycosis complicated by a gastropleural fistula. In an immunocompromised host, gastric mucormycosis should be considered a cause of refractory gastric ulcers, and it is recommended to confirm Mucorales infection by gastric biopsy and culture of exudates. A mucormycosis is a fatal fungal infection, and antifungal drugs and timely surgical resection are important to manage the disease.The authors thank the patient and the family as well as the medical staff for their contribution to the case report.Investigation: Momoko Okamoto, Daisuke Yoshikawa, Tomo Mihara, Akira Kondo, Kazuhiro Kurohama, Masahiro Nakashima, Ichiro Sekine, Shigeki Nakamura.Supervision: Daisuke Yoshikawa, Akinari Mizokami, Kazuo Ohba, Yoshitsugu Miyazaki, Atsushi Kawakami.Writing – original draft: Tomohisa Uchida.Writing – review & editing: Keita Fujikawa.Keita Fujikawa orcid: 0000-0003-2907-5359.